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  • 99
    Thermo Fisher rabbit anti gfp
    Rabbit Anti Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11001 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gfp
    Localization of <t>Aub</t> and Ago3 in the egg-chamber and embryos depends on Vasa A) Box plot showing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > <t>GFP-Vas</t> WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Aub positive pole plasm, as determined by immunohistochemical detection of Aub. Experiments were performed in 3 independent replicates. B) Box plot representing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Ago3 positive pole plasm, as determined by immunohistochemical detection of Ago3 protein. Experiments were performed in 3 independent replicates. C) Mass spectormetry analysis of GFP-Vas WT (left panel) and GFP-Vas DQAD (right panel) co-IPs. Comparison of the fold-change in abundance of proteins based on spectral count ratio (enrichment > 2 fold), and statistical significance (p
    Gfp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7375 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gfp tag polyclonal antibody
    Localization of <t>Aub</t> and Ago3 in the egg-chamber and embryos depends on Vasa A) Box plot showing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > <t>GFP-Vas</t> WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Aub positive pole plasm, as determined by immunohistochemical detection of Aub. Experiments were performed in 3 independent replicates. B) Box plot representing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Ago3 positive pole plasm, as determined by immunohistochemical detection of Ago3 protein. Experiments were performed in 3 independent replicates. C) Mass spectormetry analysis of GFP-Vas WT (left panel) and GFP-Vas DQAD (right panel) co-IPs. Comparison of the fold-change in abundance of proteins based on spectral count ratio (enrichment > 2 fold), and statistical significance (p
    Gfp Tag Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8757 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gfp
    Localization of <t>Aub</t> and Ago3 in the egg-chamber and embryos depends on Vasa A) Box plot showing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > <t>GFP-Vas</t> WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Aub positive pole plasm, as determined by immunohistochemical detection of Aub. Experiments were performed in 3 independent replicates. B) Box plot representing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Ago3 positive pole plasm, as determined by immunohistochemical detection of Ago3 protein. Experiments were performed in 3 independent replicates. C) Mass spectormetry analysis of GFP-Vas WT (left panel) and GFP-Vas DQAD (right panel) co-IPs. Comparison of the fold-change in abundance of proteins based on spectral count ratio (enrichment > 2 fold), and statistical significance (p
    Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 24758 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp  (Abcam)
    99
    Abcam gfp
    Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs. ( a ) A representative GSC showing PLA signals between lagging-strand-specific ligase-HA and new <t>H3-mKO</t> (top), and a representative GSC showing PLA signals between ligase-HA and old <t>H3-GFP</t> (bottom). ( b ) Quantification of the number of PLA puncta per nucleus between ligase and histones (old versus new) in GSCs and SGs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. In GSCs, PLA puncta between ligase and new H3-mKO: 26.5; ( n =35); between ligase and old H3-GFP: 18.5; ( n =53). In SGs, PLA puncta between ligase and new H3-mKO: 16.7; ( n =24); between ligase and old H3-GFP: 21.9; ( n =21) **: P
    Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 9437 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti gfp
    Interactome of <t>GFP-MKRN1</t> RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR
    Anti Gfp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 5342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson gfp expression
    Micro-CT analyses at the secondary spongiosa of the distal femur (A) or at S3 caudal vertebra (B) of recipient mice receiving either MLV- FGF2 -transduced <t>Sca-1</t> + cells (FGF-2) or MLV- <t>gfp</t> -transduced Sca-1 + cells (GFP) after 14 weeks post-transplantation. Top panels show a representative three-dimensional reconstruction of the trabecular structure each at the femur metaphysis (A) or at S3 caudal vertebrae (B) for each treatment group. Bottom bar graphs show the quantitative analyses of the three-dimensional bone parameters at each both site. N = 7 for the GFP group, and N = 4 for the FGF2 group.
    Gfp Expression, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 7008 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology gfp
    <t>Ykt6</t> effects on autophagosome/lysosome fusion depend on the phosphorylation at the evolutionarily conserved site in the SNARE domain in the absence of endogenous Ykt6. (A) Representative western blot for LC3-II from a stably transfected PC12 cell line with a doxycycline inducible shRNA targeting endogenous rat Ykt6 and infected with lentiviruses carrying <t>GFP-tagged</t> wild-type (WT) human Ykt6 and phosphomutants. Cells were treated for 2 hours (2h) with 200nM Bafilomycin A 1 (Baf-A 1 ) in growing conditions (fresh 10% FBS and 4.5% glucose growth medium) or in starvation conditions (Torin-1 250nM, 1X HBSS supplemented with 10mM HEPES). Actin serves as a loading control. (B-C) Autophagic flux (defined as [LC3-II Baf-A 1 ] – [LC3-II]) from western blot in (A) of (B) growing and (C) starvation conditions. N=5 *p
    Gfp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 6556 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    AvesLabs chicken anti gfp
    Semi-automatic quantification of seeded wtHtt ex1 aggregates. (A and B) Semi-automatic image processing workflow for high-magnification confocal z-stacks of DA1 glomeruli from 14 day-old adult males expressing Htt ex1 <t>Q91-mCherry</t> in DA1 ORNs and either (A1,2) Htt ex1 <t>Q25-GFP</t> or (B1,2) mCD8-GFP in GH146+ PNs. Raw data were preprocessed by deconvolution to reduce noise ( top panels ), segmented in the mCherry (A1 and B1) or GFP (A2 and B2) channels ( middle panels ), and filtered for co-localizing fluorescence signal in the other channel ( bottom panels ). Arrows in (A1 and A2) indicate seven Htt ex1 Q91+Htt ex1 Q25 puncta identified by this method. Scale bars = 10 μm. (C1-7) Selected single 0.35 μm confocal z-slices from the same confocal stack shown in (A1 and A2). Slice number is indicated at the top right of each image. Individual Htt ex1 Q91+Htt ex1 Q25 puncta identified by semi-automated image segmentation in (A) are indicated with arrows ( yellow in individual channels, white on merged images) in each slice. Two additional co-localized Htt ex1 Q91+Htt ex1 Q25 puncta identified by manual counting are indicated with asterisks in (A2). Scale bars = 10 μm. Insets show Htt ex1 Q91+Htt ex1 Q25 puncta at higher zoom (inset dimensions = 9.12 μm x 9.12 μm). Htt ex1 Q91-mCherry ( red ) and Htt ex1 Q25-GFP ( green ) fluorescence intensity profiles for lines indicated in merged insets are shown below images. Lines were scanned from leftmost to rightmost point. (D) Comparison of manual quantification (C) vs semi-automated segmentation approaches (A1 and A2) for 14 day-old males with the same genotype in (A and C). Data are shown as mean ± SEM; n.s. = not significant by one-way ANOVA followed by Tukey’s multiple comparisons test.
    Chicken Anti Gfp, supplied by AvesLabs, used in various techniques. Bioz Stars score: 93/100, based on 2806 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti gfp
    <t>DHHC5</t> internalization is required for activity-induced δ-catenin trafficking. ( a , b ) HEK293T cells were transfected with control shRNA (shRNA-c) or shRNA against DHHC5 (shRNA) plus the indicated HA-DHHC5 constructs (*shRNA resistance) and blots probed with the indicated antibodies ( P =0.024, F 3,8 =5.484, n =3 blots from 3 separate cultures; one-way analysis of variance (ANOVA)). ( c ) Confocal images of 14 DIV hippocampal neurons transfected with <t>GFP–δ-catenin</t> and the indicated shRNA and HA-DHHC5* constructs. Cells were stimulated with cLTP (+Gly) or control buffer lacking glycine (–Gly), fixed 20 min after stimulation and immunostained with the indicated antibodies. Scale bar, 5 μm. ( d ) The cLTP-induced increase in δ-catenin/PSD-95 co-localization is abolished in DHHC5 knockdown cells or those expressing the DHHC5 Y533E mutant ( P
    Anti Gfp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3029 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    ChromoTek gfp trap beads
    Loss of retromer leads to a pronounced shift in RAB 7 distribution Parental HeLa cells, two clonal VPS35 knockout cell lines, and one clonal VPS29 KO cell line were fixed in PFA and co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Co‐localization was analyzed across three independent experiments. A clonal RAB7a knockout cell line was mixed 1:1 with parental HeLa cells and seeded onto coverslips. Following PFA fixation, the mixed cells were stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Note that the RAB7a signal completely disappears in the cells not expressing RAB7a. Parental HeLa cells and clonal VPS35 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red, upper panel) or for endogenous RAB7a and a mCherry‐tagged ER marker (red, lower panel), and co‐localization between RAB7a and the respective marker was analyzed across two independent experiments. To show that RAB7a localizes to the ER and mitochondria, endogenous TOM20 (blue) was co‐stained in the lower panel. Parental HeLa cells and clonal VPS35 and VPS29 KO cells were transduced with a lentivirus expressing <t>GFP‐FIS1TM</t> (eGFP with a C‐terminal mitochondrial targeting sequence and transmembrane domain of the mitochondrial protein FIS1) and disrupted through a fine needle in detergent‐free sucrose buffer followed by isolation of the mitochondria from postnuclear supernatants with GFP‐trap agarose beads. The amount of <t>RAB7</t> precipitating with the mitochondria was quantified over four independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P
    Gfp Trap Beads, supplied by ChromoTek, used in various techniques. Bioz Stars score: 94/100, based on 3167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ChromoTek gfp trap
    NCDN colocalises with SNRPN and <t>SMN</t> in the cytoplasm, but not the nucleus, and is expressed in motor neurons in mouse spinal cord. (A) <t>NCDN–GFP</t> and mCherry–SNRPN colocalise in vesicle-like structures (chevron arrowheads) in neurites of SH-SY5Y cells constitutively expressing mCherry–SNRPN, and transiently expressing NCDN-GFP (left hand panels). White areas in the merged image show areas of colocalisation. Colocalisation images (bottom row) were generated in Volocity, using automatic thresholds on undeconvolved z-sections before excluding values below 0.05 (see Materials and Methods). No colocalisation is seen in the same cell line transiently expressing YFP alone (right hand panel). Triangular arrowheads show structures containing mCherry–SNRPN but not YFP. (B) mCherry–SMN and NCDN–GFP colocalise in vesicles (chevron arrowheads) in the cytoplasm of co-transfected SH-SY5Y cells (left hand panels). White areas in the merged image show areas of colocalisation. Colocalisation images (bottom row) were generated as above. No colocalisation is observed between mCherry-SMN and YFP (triangular arrowheads, right hand panels). (C) NCDN forms nuclear foci (arrows) in the nuclei of a small proportion of SH-SY5Y cells (≤2%, two independent experiments, n =100 cells per experiment). These do not colocalise with nuclear foci stained with coilin (arrowheads, left hand panels) or SMN (arrowheads, right hand panels). (D) NCDN (green) is expressed throughout the spinal cord, with increased expression in motor neurons (arrows), as identified with anti-ChAT antibody (magenta). (E) Higher magnification imaging confirms the presence of NCDN in ChAT-positive motor neurons (single deconvolved z-section). Scale bars: 7 µm (A,B); 500 µm (C,D); 10 µm (E).
    Gfp Trap, supplied by ChromoTek, used in various techniques. Bioz Stars score: 92/100, based on 2676 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc gfp
    NCDN colocalises with SNRPN and <t>SMN</t> in the cytoplasm, but not the nucleus, and is expressed in motor neurons in mouse spinal cord. (A) <t>NCDN–GFP</t> and mCherry–SNRPN colocalise in vesicle-like structures (chevron arrowheads) in neurites of SH-SY5Y cells constitutively expressing mCherry–SNRPN, and transiently expressing NCDN-GFP (left hand panels). White areas in the merged image show areas of colocalisation. Colocalisation images (bottom row) were generated in Volocity, using automatic thresholds on undeconvolved z-sections before excluding values below 0.05 (see Materials and Methods). No colocalisation is seen in the same cell line transiently expressing YFP alone (right hand panel). Triangular arrowheads show structures containing mCherry–SNRPN but not YFP. (B) mCherry–SMN and NCDN–GFP colocalise in vesicles (chevron arrowheads) in the cytoplasm of co-transfected SH-SY5Y cells (left hand panels). White areas in the merged image show areas of colocalisation. Colocalisation images (bottom row) were generated as above. No colocalisation is observed between mCherry-SMN and YFP (triangular arrowheads, right hand panels). (C) NCDN forms nuclear foci (arrows) in the nuclei of a small proportion of SH-SY5Y cells (≤2%, two independent experiments, n =100 cells per experiment). These do not colocalise with nuclear foci stained with coilin (arrowheads, left hand panels) or SMN (arrowheads, right hand panels). (D) NCDN (green) is expressed throughout the spinal cord, with increased expression in motor neurons (arrows), as identified with anti-ChAT antibody (magenta). (E) Higher magnification imaging confirms the presence of NCDN in ChAT-positive motor neurons (single deconvolved z-section). Scale bars: 7 µm (A,B); 500 µm (C,D); 10 µm (E).
    Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mouse anti gfp
    NCDN colocalises with SNRPN and <t>SMN</t> in the cytoplasm, but not the nucleus, and is expressed in motor neurons in mouse spinal cord. (A) <t>NCDN–GFP</t> and mCherry–SNRPN colocalise in vesicle-like structures (chevron arrowheads) in neurites of SH-SY5Y cells constitutively expressing mCherry–SNRPN, and transiently expressing NCDN-GFP (left hand panels). White areas in the merged image show areas of colocalisation. Colocalisation images (bottom row) were generated in Volocity, using automatic thresholds on undeconvolved z-sections before excluding values below 0.05 (see Materials and Methods). No colocalisation is seen in the same cell line transiently expressing YFP alone (right hand panel). Triangular arrowheads show structures containing mCherry–SNRPN but not YFP. (B) mCherry–SMN and NCDN–GFP colocalise in vesicles (chevron arrowheads) in the cytoplasm of co-transfected SH-SY5Y cells (left hand panels). White areas in the merged image show areas of colocalisation. Colocalisation images (bottom row) were generated as above. No colocalisation is observed between mCherry-SMN and YFP (triangular arrowheads, right hand panels). (C) NCDN forms nuclear foci (arrows) in the nuclei of a small proportion of SH-SY5Y cells (≤2%, two independent experiments, n =100 cells per experiment). These do not colocalise with nuclear foci stained with coilin (arrowheads, left hand panels) or SMN (arrowheads, right hand panels). (D) NCDN (green) is expressed throughout the spinal cord, with increased expression in motor neurons (arrows), as identified with anti-ChAT antibody (magenta). (E) Higher magnification imaging confirms the presence of NCDN in ChAT-positive motor neurons (single deconvolved z-section). Scale bars: 7 µm (A,B); 500 µm (C,D); 10 µm (E).
    Mouse Anti Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2039 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti gfp antibody chip grade
    <t>NLRP1</t> is required for HRV-induced inflammasome assembly and IL-18 secretion in primary human bronchial epithelial cells. A. HRV16 infection causes NLRP1 cleavage. HeLa-Ohio cells stably expressing NLRP1-HA and a vector control were infected with HRV16 at MOI=1 in duplicates. Rupintrivir (10 nM) was added at the time of infection and cell pellets were harvested 24 hours after inoculation. B. The effect of HRV16 infection on <t>ASC-GFP</t> speck formation in HeLa-Ohio-ASC-GFP cells expressing wild-type NLRP1 or NLRP1 Q130A . ***, p
    Anti Gfp Antibody Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 505 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc pspcas9 bb 2a gfp
    <t>NLRP1</t> is required for HRV-induced inflammasome assembly and IL-18 secretion in primary human bronchial epithelial cells. A. HRV16 infection causes NLRP1 cleavage. HeLa-Ohio cells stably expressing NLRP1-HA and a vector control were infected with HRV16 at MOI=1 in duplicates. Rupintrivir (10 nM) was added at the time of infection and cell pellets were harvested 24 hours after inoculation. B. The effect of HRV16 infection on <t>ASC-GFP</t> speck formation in HeLa-Ohio-ASC-GFP cells expressing wild-type NLRP1 or NLRP1 Q130A . ***, p
    Pspcas9 Bb 2a Gfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 1464 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Carl Zeiss gfp fluorescence
    Expression of the SIBR cassette from an intron. ( A ) Schematic diagrams of SIBR vectors: US2-SIBR ΔpA vector contains the SIBR cassette in the second exon of human <t>ubC</t> gene, under control of the human ubC promoter, but without a polyA signal. There is no coding region present in either the CS2+SIBR ΔpA or US2-SIBR ΔpA vector. UI2 vectors also use the human ubC promoter but contain the SIBR cassette in the first intron of the ubC gene, with the <t>GFP</t> or puromycin-resistance proteins expressed from the second exon. SD and SA indicate splice donor and splice acceptor, respectively. Exon and intron sizes not to scale. ( B ) Different SIBR vector designs expressing the ND1-1888 miRNA against NeuroD1 were cotransfected with the NeuroD1 3′-UTR luciferase reporter (see Figure 2 ). All four designs with ND1-1888 showed similar levels of inhibition of the reporter. Control SIBR vectors used the same designs but expressed an unrelated miRNA directed against the mouse POSH mRNA. Standard errors are indicated. ( C ) Comparable GFP fluorescence was detected 24 h after transfection with GFP expressed from ubC-based expression vectors, whether or not a functional SIBR cassette was present in the ubC intron.
    Gfp Fluorescence, supplied by Carl Zeiss, used in various techniques. Bioz Stars score: 93/100, based on 4003 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Localization of Aub and Ago3 in the egg-chamber and embryos depends on Vasa A) Box plot showing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Aub positive pole plasm, as determined by immunohistochemical detection of Aub. Experiments were performed in 3 independent replicates. B) Box plot representing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Ago3 positive pole plasm, as determined by immunohistochemical detection of Ago3 protein. Experiments were performed in 3 independent replicates. C) Mass spectormetry analysis of GFP-Vas WT (left panel) and GFP-Vas DQAD (right panel) co-IPs. Comparison of the fold-change in abundance of proteins based on spectral count ratio (enrichment > 2 fold), and statistical significance (p

    Journal: bioRxiv

    Article Title: Induction of Chk2 signaling in the germarium is sufficient to cause oogenesis arrest in Drosophila

    doi: 10.1101/611798

    Figure Lengend Snippet: Localization of Aub and Ago3 in the egg-chamber and embryos depends on Vasa A) Box plot showing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Aub positive pole plasm, as determined by immunohistochemical detection of Aub. Experiments were performed in 3 independent replicates. B) Box plot representing percentage of oocytes and embryo progeny of wild-type ( w 1118 ), vas PD/D1 , vas PD/D1 ; vas-Gal4 > GFP-Vas WT , vas PD/D1 ; vas-Gal4 > GFP-Vas DQAD , vas PD/D1 ; vas-Gal4 > GFP-Vas GNT and vas D1/D1 ; vas-Gal4 > GFP-Vas WT flies displaying Ago3 positive pole plasm, as determined by immunohistochemical detection of Ago3 protein. Experiments were performed in 3 independent replicates. C) Mass spectormetry analysis of GFP-Vas WT (left panel) and GFP-Vas DQAD (right panel) co-IPs. Comparison of the fold-change in abundance of proteins based on spectral count ratio (enrichment > 2 fold), and statistical significance (p

    Article Snippet: Western blotting was performed using antibodies against Vasa (rat; 1:3000; ( )), Aub (rabbit; 1:1000; , Ago3 (mouse; 1:500; gift of Mikiko Siomi), GFP (rabbit; 1:5000; Chemokine TP401), and Tub (mouse; 1:10000; Sigma T5168).

    Techniques: Immunohistochemistry

    Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs. ( a ) A representative GSC showing PLA signals between lagging-strand-specific ligase-HA and new H3-mKO (top), and a representative GSC showing PLA signals between ligase-HA and old H3-GFP (bottom). ( b ) Quantification of the number of PLA puncta per nucleus between ligase and histones (old versus new) in GSCs and SGs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. In GSCs, PLA puncta between ligase and new H3-mKO: 26.5; ( n =35); between ligase and old H3-GFP: 18.5; ( n =53). In SGs, PLA puncta between ligase and new H3-mKO: 16.7; ( n =24); between ligase and old H3-GFP: 21.9; ( n =21) **: P

    Journal: Nature structural & molecular biology

    Article Title: Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement

    doi: 10.1038/s41594-019-0269-z

    Figure Lengend Snippet: Proximity ligation assay shows distinct proximity between histones (old versus new) and lagging strand-enriched DNA replication machinery components in GSCs. ( a ) A representative GSC showing PLA signals between lagging-strand-specific ligase-HA and new H3-mKO (top), and a representative GSC showing PLA signals between ligase-HA and old H3-GFP (bottom). ( b ) Quantification of the number of PLA puncta per nucleus between ligase and histones (old versus new) in GSCs and SGs. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. In GSCs, PLA puncta between ligase and new H3-mKO: 26.5; ( n =35); between ligase and old H3-GFP: 18.5; ( n =53). In SGs, PLA puncta between ligase and new H3-mKO: 16.7; ( n =24); between ligase and old H3-GFP: 21.9; ( n =21) **: P

    Article Snippet: Primary antibodies used were mouse anti-Fas III (1:200, DSHB, 7G10), anti-HA (1:200; Sigma-Aldrich H3663), anti-PCNA (1:200; Santa Cruz sc-56), anti-GFP (1:1,000; Abcam ab 13970), anti-mKO (1:200; MBL PM051M), anti-mCherry (1:1000; Invitrogen ), anti-H3K27me3 (1:200; Millipore 07–449), anti-H4K20me2/3 (1:400; Abcam ab7817), anti-ssDNA (1:100, DSHB) and anti-BrdU (1:200; Abcam ab6326).

    Techniques: Proximity Ligation Assay

    Asymmetric H3 and symmetric H2A distribution on replicating sister chromatids. ( a,b ) Airyscan images of chromatin fibers labeled with EdU showing distribution of old H2A-eGFP and new H2A-mCherry (a) or old H3-eGFP and new H3-mCherry (b) on nonreplicated regions lacking EdU label (dashed line boxes) and replicating regions labeled with EdU (solid line boxes). Line-plots show old and new histone distribution across unreplicated (top) or replicated (bottom) regions. ( c ) Two-color STED image of chromatin fiber showing old H3-GFP and new H3-mKO distribution across unreplicated (dashed line box) and replicating (solid line box) chromatin regions. The transition from single to double-fiber occurs at the point where new histone incorporation begins (white arrow). Line-plots show old H3-GFP and new H3-mKO distribution across unreplicated region without new H3 (top) or on replicated region with new H3 (bottom). ( d,e ) Quantification of distribution of old H2A and H3 (d) or new H2A and H3 (e) between sister chromatids at replication regions on chromatin fibers. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. *** P

    Journal: Nature structural & molecular biology

    Article Title: Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement

    doi: 10.1038/s41594-019-0269-z

    Figure Lengend Snippet: Asymmetric H3 and symmetric H2A distribution on replicating sister chromatids. ( a,b ) Airyscan images of chromatin fibers labeled with EdU showing distribution of old H2A-eGFP and new H2A-mCherry (a) or old H3-eGFP and new H3-mCherry (b) on nonreplicated regions lacking EdU label (dashed line boxes) and replicating regions labeled with EdU (solid line boxes). Line-plots show old and new histone distribution across unreplicated (top) or replicated (bottom) regions. ( c ) Two-color STED image of chromatin fiber showing old H3-GFP and new H3-mKO distribution across unreplicated (dashed line box) and replicating (solid line box) chromatin regions. The transition from single to double-fiber occurs at the point where new histone incorporation begins (white arrow). Line-plots show old H3-GFP and new H3-mKO distribution across unreplicated region without new H3 (top) or on replicated region with new H3 (bottom). ( d,e ) Quantification of distribution of old H2A and H3 (d) or new H2A and H3 (e) between sister chromatids at replication regions on chromatin fibers. Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. *** P

    Article Snippet: Primary antibodies used were mouse anti-Fas III (1:200, DSHB, 7G10), anti-HA (1:200; Sigma-Aldrich H3663), anti-PCNA (1:200; Santa Cruz sc-56), anti-GFP (1:1,000; Abcam ab 13970), anti-mKO (1:200; MBL PM051M), anti-mCherry (1:1000; Invitrogen ), anti-H3K27me3 (1:200; Millipore 07–449), anti-H4K20me2/3 (1:400; Abcam ab7817), anti-ssDNA (1:100, DSHB) and anti-BrdU (1:200; Abcam ab6326).

    Techniques: Labeling

    Histone H4 shows asymmetric inheritance pattern during Drosophila GSC asymmetric divisions. ( a ) A cartoon depicting the experimental design. ( b ) H4 distribution in a post-mitotic GSC-GB pair labeled with EdU, showing H4-GFP is distributed asymmetrically towards the GSC, whereas H4-mKO is distributed more evenly between the GSC and the GB. ( c ) H4 distribution patterns in a post-mitotic SG pair, showing both H4-GFP and H4-mKO are symmetrically distributed between the two SG nuclei. ( d ) Quantification of H4- GFP and H4-mKO distributions in GSC-GB pairs ( n =44) and SG1-SG2 pairs ( n for additional statistical information. ( e ) An anaphase and telophase GSC showing asymmetric segregation of H4-GFP towards the GSC and H4-mKO towards the GB. Scale bars for panels b, c and e, 5μm; asterisk: hub.

    Journal: Nature structural & molecular biology

    Article Title: Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement

    doi: 10.1038/s41594-019-0269-z

    Figure Lengend Snippet: Histone H4 shows asymmetric inheritance pattern during Drosophila GSC asymmetric divisions. ( a ) A cartoon depicting the experimental design. ( b ) H4 distribution in a post-mitotic GSC-GB pair labeled with EdU, showing H4-GFP is distributed asymmetrically towards the GSC, whereas H4-mKO is distributed more evenly between the GSC and the GB. ( c ) H4 distribution patterns in a post-mitotic SG pair, showing both H4-GFP and H4-mKO are symmetrically distributed between the two SG nuclei. ( d ) Quantification of H4- GFP and H4-mKO distributions in GSC-GB pairs ( n =44) and SG1-SG2 pairs ( n for additional statistical information. ( e ) An anaphase and telophase GSC showing asymmetric segregation of H4-GFP towards the GSC and H4-mKO towards the GB. Scale bars for panels b, c and e, 5μm; asterisk: hub.

    Article Snippet: Primary antibodies used were mouse anti-Fas III (1:200, DSHB, 7G10), anti-HA (1:200; Sigma-Aldrich H3663), anti-PCNA (1:200; Santa Cruz sc-56), anti-GFP (1:1,000; Abcam ab 13970), anti-mKO (1:200; MBL PM051M), anti-mCherry (1:1000; Invitrogen ), anti-H3K27me3 (1:200; Millipore 07–449), anti-H4K20me2/3 (1:400; Abcam ab7817), anti-ssDNA (1:100, DSHB) and anti-BrdU (1:200; Abcam ab6326).

    Techniques: Labeling

    Histones H2A and H2B show symmetric distribution during Drosophila GSC asymmetric division. ( a ) Symmetric H2A inheritance pattern in a post-mitotic GSC-GB (top), mitotic GSC (middle) and post-mitotic spermatogonial (bottom) pairs. ( b ) Quantification of H2A-GFP and H2A-mKO distribution in GSC-GB pairs ( n =20) and SG1-SG2 pairs ( n =20). Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. * P

    Journal: Nature structural & molecular biology

    Article Title: Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement

    doi: 10.1038/s41594-019-0269-z

    Figure Lengend Snippet: Histones H2A and H2B show symmetric distribution during Drosophila GSC asymmetric division. ( a ) Symmetric H2A inheritance pattern in a post-mitotic GSC-GB (top), mitotic GSC (middle) and post-mitotic spermatogonial (bottom) pairs. ( b ) Quantification of H2A-GFP and H2A-mKO distribution in GSC-GB pairs ( n =20) and SG1-SG2 pairs ( n =20). Individual data points (circles) and mean values are shown. Error bars represent 95% confidence interval. * P

    Article Snippet: Primary antibodies used were mouse anti-Fas III (1:200, DSHB, 7G10), anti-HA (1:200; Sigma-Aldrich H3663), anti-PCNA (1:200; Santa Cruz sc-56), anti-GFP (1:1,000; Abcam ab 13970), anti-mKO (1:200; MBL PM051M), anti-mCherry (1:1000; Invitrogen ), anti-H3K27me3 (1:200; Millipore 07–449), anti-H4K20me2/3 (1:400; Abcam ab7817), anti-ssDNA (1:100, DSHB) and anti-BrdU (1:200; Abcam ab6326).

    Techniques:

    Stem cell potential and molecular characteristics of PDX1+ A undiff . a Isolation of PDX1+ and PDX1− A undiff from Plzf-mC/CreER; Pdx1 GFP/+ adults by flow cytometry. A undiff are mCherry+ CD9+ c-KIT−. Gates for GFP+ and GFP− cells were set according to Plzf-mC/CreER control (left profile). Percentage of cells in GFP+ gate from representative sample is shown ( n = 7). b Pdx1-GFP+ and GFP− adult A undiff fractions were transplanted into recipient testis and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative donor colonies. Panels on right show higher magnification details of indicated areas and grayscale panels show individual immunostaining. Scale bar, 100 μm. c Colony-forming efficiency of Pdx1-GFP+ and GFP− A undiff fractions in transplantation assays from b . Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 15 recipient testes for Pdx1-GFP− cells and n = 13 for Pdx1-GFP+ cells). Donor cells were pooled from a total of 7 Plzf-mC/CreER; Pdx1 GFP/+ adults. d Mean length ± s.e.m. of donor colonies was measured from tiled microscope images from experiment of c . e Mean number of GFP+ cells/donor colony ± s.e.m. were calculated for a set of whole-mount samples from transplant assay of c ( n = 58 colonies from Pdx1-GFP− cells and n = 63 from Pdx1-GFP+). f Heatmap illustrates expression of indicated genes from RNA-Seq analysis of Pdx1-GFP+ and GFP− A undiff fractions isolated as in a ( n = 4 mice). Differentially expressed genes (DEG) are in bold. Cut-off for DEG is false discovery rate (FDR)

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Stem cell potential and molecular characteristics of PDX1+ A undiff . a Isolation of PDX1+ and PDX1− A undiff from Plzf-mC/CreER; Pdx1 GFP/+ adults by flow cytometry. A undiff are mCherry+ CD9+ c-KIT−. Gates for GFP+ and GFP− cells were set according to Plzf-mC/CreER control (left profile). Percentage of cells in GFP+ gate from representative sample is shown ( n = 7). b Pdx1-GFP+ and GFP− adult A undiff fractions were transplanted into recipient testis and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative donor colonies. Panels on right show higher magnification details of indicated areas and grayscale panels show individual immunostaining. Scale bar, 100 μm. c Colony-forming efficiency of Pdx1-GFP+ and GFP− A undiff fractions in transplantation assays from b . Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 15 recipient testes for Pdx1-GFP− cells and n = 13 for Pdx1-GFP+ cells). Donor cells were pooled from a total of 7 Plzf-mC/CreER; Pdx1 GFP/+ adults. d Mean length ± s.e.m. of donor colonies was measured from tiled microscope images from experiment of c . e Mean number of GFP+ cells/donor colony ± s.e.m. were calculated for a set of whole-mount samples from transplant assay of c ( n = 58 colonies from Pdx1-GFP− cells and n = 63 from Pdx1-GFP+). f Heatmap illustrates expression of indicated genes from RNA-Seq analysis of Pdx1-GFP+ and GFP− A undiff fractions isolated as in a ( n = 4 mice). Differentially expressed genes (DEG) are in bold. Cut-off for DEG is false discovery rate (FDR)

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Isolation, Flow Cytometry, Cytometry, Expressing, Immunostaining, Transplantation Assay, Microscopy, RNA Sequencing Assay, Mouse Assay

    A undiff heterogeneity during culture. a Oct4-GFP− and GFP+ A undiff from Plzf-mC/CreER; Oct4-GFP adults placed in culture and analysed 2–3 weeks later. Right: IF of colonies ( n = 2 per fraction). Scale bar, 50 μm. b Mean colony-for ming efficiency of Oct4-GFP− and GFP+ A undiff ± s.e.m. from a ( n = 6 mice). c Cultures from Oct4-GFP− and GFP+ A undiff plated at 25 × 10 3 per well and counted at indicated timepoints. Mean recovery ± s.e.m. shown. d Cultures from Oct4-GFP− and GFP+ A undiff transplanted and analysed 8 weeks later by IF. Representative colonies shown (2 sets of lines) ( n = 11 testes Oct4-GFP− and n = 9 testes Oct4-GFP+). Scale bar, 100 μm. e Cultures from Oct4-GFP− and GFP+ A undiff sorted by GFP and plated at 25 × 10 3 per well. GFP+ cells determined by flow cytometry. Mean ± s.e.m. shown ( n = 6 cultures). f qRT-PCR of GFP+ and GFP− cells from Oct4-GFP− and GFP+ A undiff cultures. Expression corrected to β-actin and normalized so mean of GFP− or GFP+ fractions equals 1. Mean ± s.e.m. shown ( n = 4 cultures). g Representative IF of primary colonies (P0) and passage 5 (P5) cultures from Oct4-GFP− and GFP+ A undiff ( n = 4 lines). Scale bar, 50 μm. h Cultures from Oct4-GFP− and GFP+ A undiff plated at increasing densities (10 × 10 3 , 100 × 10 3 and 200 × 10 3 cells/well) and analysed 7–10 days later. Representative IF shown ( n = 4 lines). Scale bar, 50 μm. i Cultures from Oct4-GFP− and GFP+ A undiff plated at low and high densities (20 × 10 3 , 200 × 10 3 cells/well) and cultured for 2 weeks. Conditioned media was collected at indicated times (hours) after media replenishment for ELISA. Mean GDNF levels are shown as percentage of starting levels ± s.e.m. ( n = 4 cultures). j Cultures from Oct4-GFP− A undiff (20 × 10 3 cells/well) switched to media containing reduced GDNF and bFGF (1 ng/ml, left) or maintained with regular media (right) for 4 days. Representative flow cytometry shown. k Cultures from Oct4-GFP− and GFP+ A undiff sorted according to GFP and plated (10 × 10 3 cells/well) in media with GDNF but no bFGF (GDNF) or bFGF without GDNF (bFGF). Cells analysed at indicated timepoints by flow cytometry. Mean percentage of cells GFP+ ± standard deviation (s.d.) ( n = 3 replicates) from representative experiment shown. Grey plots: mean values in regular media (GDNF+ bFGF). l Cultures from Oct4-GFP− and GFP+ A undiff (20 × 10 3 cells/well) grown 3 days in regular media switched to media without GDNF or bFGF (−), bFGF without GDNF (bFGF), GDNF without bFGF (GDNF) or regular medium (GDNF+ bFGF) then analysed by IF after 2 weeks. Representative images shown ( n = 2 lines). Scale bar, 50 μm. m Plzf-mC/CreER cultures incubated in media containing indicated inhibitors (Inh) for 4 days prior to IF. Representative images shown ( n = 2 lines). Scale bar, 50 μm. Two-tailed Student’s t -test used (* P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: A undiff heterogeneity during culture. a Oct4-GFP− and GFP+ A undiff from Plzf-mC/CreER; Oct4-GFP adults placed in culture and analysed 2–3 weeks later. Right: IF of colonies ( n = 2 per fraction). Scale bar, 50 μm. b Mean colony-for ming efficiency of Oct4-GFP− and GFP+ A undiff ± s.e.m. from a ( n = 6 mice). c Cultures from Oct4-GFP− and GFP+ A undiff plated at 25 × 10 3 per well and counted at indicated timepoints. Mean recovery ± s.e.m. shown. d Cultures from Oct4-GFP− and GFP+ A undiff transplanted and analysed 8 weeks later by IF. Representative colonies shown (2 sets of lines) ( n = 11 testes Oct4-GFP− and n = 9 testes Oct4-GFP+). Scale bar, 100 μm. e Cultures from Oct4-GFP− and GFP+ A undiff sorted by GFP and plated at 25 × 10 3 per well. GFP+ cells determined by flow cytometry. Mean ± s.e.m. shown ( n = 6 cultures). f qRT-PCR of GFP+ and GFP− cells from Oct4-GFP− and GFP+ A undiff cultures. Expression corrected to β-actin and normalized so mean of GFP− or GFP+ fractions equals 1. Mean ± s.e.m. shown ( n = 4 cultures). g Representative IF of primary colonies (P0) and passage 5 (P5) cultures from Oct4-GFP− and GFP+ A undiff ( n = 4 lines). Scale bar, 50 μm. h Cultures from Oct4-GFP− and GFP+ A undiff plated at increasing densities (10 × 10 3 , 100 × 10 3 and 200 × 10 3 cells/well) and analysed 7–10 days later. Representative IF shown ( n = 4 lines). Scale bar, 50 μm. i Cultures from Oct4-GFP− and GFP+ A undiff plated at low and high densities (20 × 10 3 , 200 × 10 3 cells/well) and cultured for 2 weeks. Conditioned media was collected at indicated times (hours) after media replenishment for ELISA. Mean GDNF levels are shown as percentage of starting levels ± s.e.m. ( n = 4 cultures). j Cultures from Oct4-GFP− A undiff (20 × 10 3 cells/well) switched to media containing reduced GDNF and bFGF (1 ng/ml, left) or maintained with regular media (right) for 4 days. Representative flow cytometry shown. k Cultures from Oct4-GFP− and GFP+ A undiff sorted according to GFP and plated (10 × 10 3 cells/well) in media with GDNF but no bFGF (GDNF) or bFGF without GDNF (bFGF). Cells analysed at indicated timepoints by flow cytometry. Mean percentage of cells GFP+ ± standard deviation (s.d.) ( n = 3 replicates) from representative experiment shown. Grey plots: mean values in regular media (GDNF+ bFGF). l Cultures from Oct4-GFP− and GFP+ A undiff (20 × 10 3 cells/well) grown 3 days in regular media switched to media without GDNF or bFGF (−), bFGF without GDNF (bFGF), GDNF without bFGF (GDNF) or regular medium (GDNF+ bFGF) then analysed by IF after 2 weeks. Representative images shown ( n = 2 lines). Scale bar, 50 μm. m Plzf-mC/CreER cultures incubated in media containing indicated inhibitors (Inh) for 4 days prior to IF. Representative images shown ( n = 2 lines). Scale bar, 50 μm. Two-tailed Student’s t -test used (* P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Incubation, Two Tailed Test

    PDX1+ and EOMES+ spermatogonia during development and regeneration. a Representative whole-mount IF of tubules from WT mice of indicated ages (PND; postnatal day) ( n = 2 mice per age). Scale bars, 50 μm. b Representative IF of testis sections from WT mice of indicated ages ( n = 3 mice per timepoint). Insets show details of indicated areas. Arrowheads: EOMES+ PDX1+ cells. Asterisks: EOMES+ PDX1− cells. Scale bars, 50 μm. c , d Flow cytometry of fixed and permeabilized testis cells from WT mice of indicated ages. Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1 and EOMES are shown ± s.e.m. ( n = 3–5 mice/time point). e WT adults were treated with busulfan (10 mg/kg) and tubules analysed by whole-mount IF 14 days later ( n = 2 mice). Top panels: regenerative areas with GFRα1+ A al . Bottom: non-regenerative areas lacking GFRα1+ A al . Insets show details of indicated regions. Scale bar, 50 μm. f Regeneration assay of e 4 weeks post busulfan (2 areas shown). Arrowheads: PDX1+ A undiff . Scale bar, 50 μm. g Representative flow cytometry of fixed and permeabilized testis cells from WT adults untreated or busulfan treated as in e ( n = 4 busulfan-treated mice and n = 5 untreated). PLZF+ c-KIT− A undiff are shown. Percentage of A undiff KI67+ and EOMES+ are indicated. h Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1, EOMES and KI67 from g are shown ± s.e.m. i Representative flow cytometry of PDX1 in indicated populations from g ( n = 4 mice per condition). Percentages of cells PDX1+ are indicated. j PDX1 levels (median fluorescent intensity) in EOMES+ PLZF+ cells from i . Mean values ± s.e.m. shown. k Quantitative RT-PCR of A undiff from Plzf-mC/CreER mice of indicated ages or 10 days post busulfan as in e . Expression levels are corrected to β-actin and normalized to an adult sample. Mean values ± s.e.m. are indicated ( n = 4 PND10 and busulfan-treated, n = 5 PND20, n = 6 adults). Selected significance values are shown. l Model of A undiff functional states. Self-renewing (curved arrows) GFRα1+ A undiff adopt different states identified by variable expression of Pdx1 , Eomes and Lhx1 . A fraction of GFRα1+ A undiff lacks PDX1, EOMES and LHX1 (grey in panel) and may represent a transitional state destined to become differentiation-primed. Niche signals differentially support self-renewing states. Given dynamic niche properties, different states predominate in development and homeostatic plus regenerative testis. The state marked by PDX1, EOMES and LHX1 is specific to homeostatic testis and potentially optimized for life-long germline maintenance. Pdx1 is downregulated and Eomes plus Lhx1 upregulated in states suited for short-term expansion during development and regeneration. Oct4-GFP+ differentiation-primed A undiff convert back to a self-renewing state under optimised culture conditions or by transplantation into recipient testis with vacant niches. Significance was calculated by two-tailed Student’s t -test (* P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: PDX1+ and EOMES+ spermatogonia during development and regeneration. a Representative whole-mount IF of tubules from WT mice of indicated ages (PND; postnatal day) ( n = 2 mice per age). Scale bars, 50 μm. b Representative IF of testis sections from WT mice of indicated ages ( n = 3 mice per timepoint). Insets show details of indicated areas. Arrowheads: EOMES+ PDX1+ cells. Asterisks: EOMES+ PDX1− cells. Scale bars, 50 μm. c , d Flow cytometry of fixed and permeabilized testis cells from WT mice of indicated ages. Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1 and EOMES are shown ± s.e.m. ( n = 3–5 mice/time point). e WT adults were treated with busulfan (10 mg/kg) and tubules analysed by whole-mount IF 14 days later ( n = 2 mice). Top panels: regenerative areas with GFRα1+ A al . Bottom: non-regenerative areas lacking GFRα1+ A al . Insets show details of indicated regions. Scale bar, 50 μm. f Regeneration assay of e 4 weeks post busulfan (2 areas shown). Arrowheads: PDX1+ A undiff . Scale bar, 50 μm. g Representative flow cytometry of fixed and permeabilized testis cells from WT adults untreated or busulfan treated as in e ( n = 4 busulfan-treated mice and n = 5 untreated). PLZF+ c-KIT− A undiff are shown. Percentage of A undiff KI67+ and EOMES+ are indicated. h Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1, EOMES and KI67 from g are shown ± s.e.m. i Representative flow cytometry of PDX1 in indicated populations from g ( n = 4 mice per condition). Percentages of cells PDX1+ are indicated. j PDX1 levels (median fluorescent intensity) in EOMES+ PLZF+ cells from i . Mean values ± s.e.m. shown. k Quantitative RT-PCR of A undiff from Plzf-mC/CreER mice of indicated ages or 10 days post busulfan as in e . Expression levels are corrected to β-actin and normalized to an adult sample. Mean values ± s.e.m. are indicated ( n = 4 PND10 and busulfan-treated, n = 5 PND20, n = 6 adults). Selected significance values are shown. l Model of A undiff functional states. Self-renewing (curved arrows) GFRα1+ A undiff adopt different states identified by variable expression of Pdx1 , Eomes and Lhx1 . A fraction of GFRα1+ A undiff lacks PDX1, EOMES and LHX1 (grey in panel) and may represent a transitional state destined to become differentiation-primed. Niche signals differentially support self-renewing states. Given dynamic niche properties, different states predominate in development and homeostatic plus regenerative testis. The state marked by PDX1, EOMES and LHX1 is specific to homeostatic testis and potentially optimized for life-long germline maintenance. Pdx1 is downregulated and Eomes plus Lhx1 upregulated in states suited for short-term expansion during development and regeneration. Oct4-GFP+ differentiation-primed A undiff convert back to a self-renewing state under optimised culture conditions or by transplantation into recipient testis with vacant niches. Significance was calculated by two-tailed Student’s t -test (* P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Functional Assay, Transplantation Assay, Two Tailed Test

    Characterization of Plzf-mC/CreER transgenic mice. a , b Representative IF of adult Plzf-mC/CreER testis sections ( n = 3 mice). Tubule stages and populations are indicated. Scale bar, 50 μm. c Representative flow cytometry of fixed and permeabilized testis from Plzf-mC/CreER and wildtype (WT) adult testis ( n = 3 mice per genotype). PLZF+ cells are shown. d Plzf-mC/CreER; Z/EG mice injected daily with TAM for 5 days were harvested at indicated days after treatment. e Representative IF of testis sections from d ( n = 3 testes per time point). Insets show details of indicated areas. Scale bar, 50 μm. f Representative whole-mount IF from d . Inset shows detail of indicated area. Arrowheads: unlabelled GFRα1+ cells. Scale bar, 50 μm. g Flow cytometry of fixed and permeabilized testis cells from d . Graph indicates mean fraction of A undiff (PLZF+ c-KIT−) and PLZF+ c-KIT+ early differentiating cells expressing GFP ± standard error of mean (s.e.m.) ( n = 4 testes D3, D10 and D90, n = 6 testes D30). h Representative flow cytometry of live Plzf-mC/CreER testis cells. SSC is side scatter. mCherry+ gate was set according to WT. i Quantitative RT-PCR for spermatogonial markers from Plzf-mC/CreER cell fractions sorted as in h . mC− indicates mCherry−. Expression levels are corrected to β-actin and normalized so mean value of fraction #2 equals 1. Mean values ± s.e.m. are indicated ( n = 3 sorts, 2 mice pooled per sort). Significance vs. mCherry− cells is shown. j Violin plots of gene expression in 150 single cells of fraction #1 cells from h . Cells were gated according to Plzf and Vasa expression. k Left: mean in vitro colony-forming activity of Plzf-mC/CreER fractions ± s.e.m. isolated as in h ( n = 3 mice). mC− indicates mCherry−. Significance vs. mCherry− fraction is indicated. Right: representative IF of passaged cells from fraction #1 treated with vehicle or retinoic acid for 48 h ( n = 3). Scale bar, 50 μm. l Left: transplantation of cultured cells established from Plzf-mC/CreER fraction #1. Right: representative whole-mount IF of tubules 8 weeks post transplant demonstrating formation of mCherry+ colonies ( n = 5 recipients). Comparable spermatogenic capacity was observed upon transplantation of an independent line (3.90 colonies/10 5 cells; n = 4 recipient testes). Scale bar, 100 μm. Significance was calculated by two-tailed Student’s t -test (** P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Characterization of Plzf-mC/CreER transgenic mice. a , b Representative IF of adult Plzf-mC/CreER testis sections ( n = 3 mice). Tubule stages and populations are indicated. Scale bar, 50 μm. c Representative flow cytometry of fixed and permeabilized testis from Plzf-mC/CreER and wildtype (WT) adult testis ( n = 3 mice per genotype). PLZF+ cells are shown. d Plzf-mC/CreER; Z/EG mice injected daily with TAM for 5 days were harvested at indicated days after treatment. e Representative IF of testis sections from d ( n = 3 testes per time point). Insets show details of indicated areas. Scale bar, 50 μm. f Representative whole-mount IF from d . Inset shows detail of indicated area. Arrowheads: unlabelled GFRα1+ cells. Scale bar, 50 μm. g Flow cytometry of fixed and permeabilized testis cells from d . Graph indicates mean fraction of A undiff (PLZF+ c-KIT−) and PLZF+ c-KIT+ early differentiating cells expressing GFP ± standard error of mean (s.e.m.) ( n = 4 testes D3, D10 and D90, n = 6 testes D30). h Representative flow cytometry of live Plzf-mC/CreER testis cells. SSC is side scatter. mCherry+ gate was set according to WT. i Quantitative RT-PCR for spermatogonial markers from Plzf-mC/CreER cell fractions sorted as in h . mC− indicates mCherry−. Expression levels are corrected to β-actin and normalized so mean value of fraction #2 equals 1. Mean values ± s.e.m. are indicated ( n = 3 sorts, 2 mice pooled per sort). Significance vs. mCherry− cells is shown. j Violin plots of gene expression in 150 single cells of fraction #1 cells from h . Cells were gated according to Plzf and Vasa expression. k Left: mean in vitro colony-forming activity of Plzf-mC/CreER fractions ± s.e.m. isolated as in h ( n = 3 mice). mC− indicates mCherry−. Significance vs. mCherry− fraction is indicated. Right: representative IF of passaged cells from fraction #1 treated with vehicle or retinoic acid for 48 h ( n = 3). Scale bar, 50 μm. l Left: transplantation of cultured cells established from Plzf-mC/CreER fraction #1. Right: representative whole-mount IF of tubules 8 weeks post transplant demonstrating formation of mCherry+ colonies ( n = 5 recipients). Comparable spermatogenic capacity was observed upon transplantation of an independent line (3.90 colonies/10 5 cells; n = 4 recipient testes). Scale bar, 100 μm. Significance was calculated by two-tailed Student’s t -test (** P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Transgenic Assay, Mouse Assay, Flow Cytometry, Cytometry, Injection, Expressing, Quantitative RT-PCR, In Vitro, Activity Assay, Isolation, Transplantation Assay, Cell Culture, Two Tailed Test

    Comparative analysis of reporter gene expression in spermatogonia. a , b Representative whole-mount IF of adult (8–10 weeks post natal) Plzf-mC/CreER; Sox2 GFP ( a ) and Plzf-mC/CreER; Oct4-GFP ( b ) seminiferous tubules. Inset panels show individual immunostaining within indicated area at higher magnification. Tubule staging and select A s and A pr are indicated. Scale bars, 50 μm. c Representative flow cytometry analysis of fixed and permeabilized testis cells from 1 of 3 Oct4-GFP and wild-type (WT) control adults. PLZF+ cell population is shown. Percentages of cells contained within gates are indicated. d Quantification of flow cytometry results from c . Graph indicates percentage of A undiff (PLZF+ c-KIT−) and cells initiating differentiation (PLZF+ c-KIT+) expressing GFP in Oct4-GFP adults. Horizontal bars indicate mean values ( n = 3 mice). e Graph shows percentage of GFRα1+ and SOX3+ spermatogonia positive for GFP in whole-mount seminiferous tubules of Oct4-GFP adults. Spermatogonial identity was confirmed by SALL4 counterstain. Horizontal bars indicate mean values ( n = 3 mice, > 200 cells scored per data point). f Representative whole-mount IF of adult Oct4-GFP seminiferous tubules for indicated markers ( n = 3 mice). Select A undiff cells are indicated. Scale bar, 50 μm. g Scheme summarizing expression patterns of indicated genes and transgenic reporters plus changes in cell morphology during spermatogonial differentiation. Markers used to isolate different spermatogonial populations are indicated. h Isolation of Oct4-GFP− and Oct4-GFP+ A undiff from Plzf-mC/CreER; Oct4-GFP adults by flow cytometry. Percentage of cells in each gate from a representative sample is indicated ( n = 6 mice). i Oct4-GFP− and GFP+ adult A undiff fractions were transplanted into recipients and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative colonies. PLZF counterstain confirms A undiff and spermatogonial identity. Panels show higher magnification details of indicated areas. Scale bar, 100 μm. Graph shows colony-forming efficiency of Oct4-GFP+ and GFP− A undiff fractions. Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 7 recipient testes for Oct4-GFP− cells and n = 6 for Oct4-GFP+ cells). Donor cells were pooled from 2 Plzf-mC/CreER; Oct4-GFP adults. Significance was calculated by two-tailed Student’s t -test (* P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Comparative analysis of reporter gene expression in spermatogonia. a , b Representative whole-mount IF of adult (8–10 weeks post natal) Plzf-mC/CreER; Sox2 GFP ( a ) and Plzf-mC/CreER; Oct4-GFP ( b ) seminiferous tubules. Inset panels show individual immunostaining within indicated area at higher magnification. Tubule staging and select A s and A pr are indicated. Scale bars, 50 μm. c Representative flow cytometry analysis of fixed and permeabilized testis cells from 1 of 3 Oct4-GFP and wild-type (WT) control adults. PLZF+ cell population is shown. Percentages of cells contained within gates are indicated. d Quantification of flow cytometry results from c . Graph indicates percentage of A undiff (PLZF+ c-KIT−) and cells initiating differentiation (PLZF+ c-KIT+) expressing GFP in Oct4-GFP adults. Horizontal bars indicate mean values ( n = 3 mice). e Graph shows percentage of GFRα1+ and SOX3+ spermatogonia positive for GFP in whole-mount seminiferous tubules of Oct4-GFP adults. Spermatogonial identity was confirmed by SALL4 counterstain. Horizontal bars indicate mean values ( n = 3 mice, > 200 cells scored per data point). f Representative whole-mount IF of adult Oct4-GFP seminiferous tubules for indicated markers ( n = 3 mice). Select A undiff cells are indicated. Scale bar, 50 μm. g Scheme summarizing expression patterns of indicated genes and transgenic reporters plus changes in cell morphology during spermatogonial differentiation. Markers used to isolate different spermatogonial populations are indicated. h Isolation of Oct4-GFP− and Oct4-GFP+ A undiff from Plzf-mC/CreER; Oct4-GFP adults by flow cytometry. Percentage of cells in each gate from a representative sample is indicated ( n = 6 mice). i Oct4-GFP− and GFP+ adult A undiff fractions were transplanted into recipients and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative colonies. PLZF counterstain confirms A undiff and spermatogonial identity. Panels show higher magnification details of indicated areas. Scale bar, 100 μm. Graph shows colony-forming efficiency of Oct4-GFP+ and GFP− A undiff fractions. Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 7 recipient testes for Oct4-GFP− cells and n = 6 for Oct4-GFP+ cells). Donor cells were pooled from 2 Plzf-mC/CreER; Oct4-GFP adults. Significance was calculated by two-tailed Student’s t -test (* P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Expressing, Immunostaining, Flow Cytometry, Cytometry, Mouse Assay, Transgenic Assay, Isolation, Two Tailed Test

    Characterization of PDX1+ spermatogonia. a Representative whole-mount IF of WT (top 3 rows, n = 3 mice), Oct4-GFP and Pdx1 GFP adult tubules ( n = 2 mice per genotype). Antibodies to PDX1 and GFRα1 are goat polyclonals and distinguished by nuclear vs . cell surface staining respectively. Scale bars, 50 μm. b Mean percentage of GFRα1+ cells/chains PDX1+ ± s.e.m. from WT analysis in a ( n = 4 mice, > 250 cells/chains per sample). c Representative whole-mount IF of adult Plzf-mC/CreER; Z/EG tubules D3 post-TAM ( n = 2 mice). Arrowheads: PDX1+ A s . Inset shows detail of indicated area. Scale bar, 50 μm. d Representative IF of adult WT testis sections ( n = 4 mice). Insets: details of selected areas. EOMES+ GFRα1+ (arrowheads) and EOMES− GFRα1+ cells (bracket) are shown. Tubule stage is indicated. Scale bar, 50 μm. e Representative IF of adult Oct4-GFP testis section ( n = 2 mice). Insets: immunostaining within indicated area. EOMES+ GFP− (arrowheads) and EOMES− GFP+ (bracket) spermatogonia are indicated. Asterisks: autofluorescent interstitium. Scale bar, 50 μm. f Representative flow cytometry of fixed and permeabilized testis cells from Oct4-GFP adults ( n = 2). PLZF+ cells shown. g Representative IF of adult WT testis sections ( n = 4 mice). Insets: immunostaining within indicated area. PDX1+ EOMES+ spermatogonia are indicated (arrowheads). Tubule stage is shown. Asterisk: autofluorescent interstitium. Scale bar, 50 μm. h Representative flow cytometry of fixed and permeabilized WT adult testis cells ( n = 4). Mean numbers of EOMES+ and PDX1+ cells in PDX1+ and EOMES+ gates respectively are shown ± s.e.m. i Representative flow cytometry for KI67 in indicated PLZF+ populations of fixed and permeabilized adult WT testis cells ( n = 4 mice). j Quantification of flow cytometry from i . Percentage of indicated PLZF+ fractions KI67+ . Horizontal bars: mean values ( n = 4 mice). k Representative IF of adult WT testis sections demonstrating PDX1+ A undiff localisation (arrowheads) within tubules ( n = 4 mice). Asterisks: autofluorescent interstitium. Tubule stages are shown. Scale bar, 50 μm. l Quantification of spermatogonial localisation from k . Mean values ± s.e.m. are shown ( n = 4 mice, 56–95 tubule sections per mouse). Significance vs. tubule-tubule localisation is indicated. m Representative IF of Id4 IRES-GFP adult testis sections ( n = 4 mice). Insets show immunostaining within indicated area. Arrowheads: PDX1+ EOMES+ GFP+ spermatogonia. Asterisk: PDX1 low EOMES+ GFP+ cell. Tubule stage is shown. Scale bar, 50 μm. n Representative flow cytometry of indicated PLZF+ populations of fixed and permeabilized adult Id4 IRES-GFP testis cells ( n = 4 mice). Mean numbers of PDX1+ and EOMES+ A undiff expressing GFP ± s.e.m. o Flow cytometry of Id4 IRES-GFP testis cells as in n . PLZF+ fractions are shown. Mean numbers of PLZF+ GFP+ cells positive for EOMES and PDX1 are indicated ± s.e.m. ( n = 4 mice). Significance was calculated by two-tailed Student’s t -test (*** P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Characterization of PDX1+ spermatogonia. a Representative whole-mount IF of WT (top 3 rows, n = 3 mice), Oct4-GFP and Pdx1 GFP adult tubules ( n = 2 mice per genotype). Antibodies to PDX1 and GFRα1 are goat polyclonals and distinguished by nuclear vs . cell surface staining respectively. Scale bars, 50 μm. b Mean percentage of GFRα1+ cells/chains PDX1+ ± s.e.m. from WT analysis in a ( n = 4 mice, > 250 cells/chains per sample). c Representative whole-mount IF of adult Plzf-mC/CreER; Z/EG tubules D3 post-TAM ( n = 2 mice). Arrowheads: PDX1+ A s . Inset shows detail of indicated area. Scale bar, 50 μm. d Representative IF of adult WT testis sections ( n = 4 mice). Insets: details of selected areas. EOMES+ GFRα1+ (arrowheads) and EOMES− GFRα1+ cells (bracket) are shown. Tubule stage is indicated. Scale bar, 50 μm. e Representative IF of adult Oct4-GFP testis section ( n = 2 mice). Insets: immunostaining within indicated area. EOMES+ GFP− (arrowheads) and EOMES− GFP+ (bracket) spermatogonia are indicated. Asterisks: autofluorescent interstitium. Scale bar, 50 μm. f Representative flow cytometry of fixed and permeabilized testis cells from Oct4-GFP adults ( n = 2). PLZF+ cells shown. g Representative IF of adult WT testis sections ( n = 4 mice). Insets: immunostaining within indicated area. PDX1+ EOMES+ spermatogonia are indicated (arrowheads). Tubule stage is shown. Asterisk: autofluorescent interstitium. Scale bar, 50 μm. h Representative flow cytometry of fixed and permeabilized WT adult testis cells ( n = 4). Mean numbers of EOMES+ and PDX1+ cells in PDX1+ and EOMES+ gates respectively are shown ± s.e.m. i Representative flow cytometry for KI67 in indicated PLZF+ populations of fixed and permeabilized adult WT testis cells ( n = 4 mice). j Quantification of flow cytometry from i . Percentage of indicated PLZF+ fractions KI67+ . Horizontal bars: mean values ( n = 4 mice). k Representative IF of adult WT testis sections demonstrating PDX1+ A undiff localisation (arrowheads) within tubules ( n = 4 mice). Asterisks: autofluorescent interstitium. Tubule stages are shown. Scale bar, 50 μm. l Quantification of spermatogonial localisation from k . Mean values ± s.e.m. are shown ( n = 4 mice, 56–95 tubule sections per mouse). Significance vs. tubule-tubule localisation is indicated. m Representative IF of Id4 IRES-GFP adult testis sections ( n = 4 mice). Insets show immunostaining within indicated area. Arrowheads: PDX1+ EOMES+ GFP+ spermatogonia. Asterisk: PDX1 low EOMES+ GFP+ cell. Tubule stage is shown. Scale bar, 50 μm. n Representative flow cytometry of indicated PLZF+ populations of fixed and permeabilized adult Id4 IRES-GFP testis cells ( n = 4 mice). Mean numbers of PDX1+ and EOMES+ A undiff expressing GFP ± s.e.m. o Flow cytometry of Id4 IRES-GFP testis cells as in n . PLZF+ fractions are shown. Mean numbers of PLZF+ GFP+ cells positive for EOMES and PDX1 are indicated ± s.e.m. ( n = 4 mice). Significance was calculated by two-tailed Student’s t -test (*** P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Mouse Assay, Staining, Immunostaining, Flow Cytometry, Cytometry, Expressing, Two Tailed Test

    Identification and characterization of distinct A undiff populations. a Oct4-GFP− and Oct4-GFP+ A undiff fractions were isolated from Plzf-mC/CreER; Oct4-GFP adults for gene expression profiling by microarray. A undiff fraction is mCherry+ CD9+ c-KIT−. b Confirmation of gene expression signatures of Oct4-GFP− and Oct4-GFP+ A undiff by quantitative RT-PCR. Candidate genes were selected from microarray analysis of a . Expression levels are corrected to those of β-actin and normalized so mean value of GFP− or GFP+ fractions equals 1. Mean values from 3-5 mice ± s.e.m. are indicated. Genes enriched in Oct4-GFP− and Oct4-GFP+ populations are shown in separate groups. Control genes Plzf and Vasa are shown. Significance was calculated by two-tailed Student’s t -test (* P

    Journal: Nature Communications

    Article Title: Identification of dynamic undifferentiated cell states within the male germline

    doi: 10.1038/s41467-018-04827-z

    Figure Lengend Snippet: Identification and characterization of distinct A undiff populations. a Oct4-GFP− and Oct4-GFP+ A undiff fractions were isolated from Plzf-mC/CreER; Oct4-GFP adults for gene expression profiling by microarray. A undiff fraction is mCherry+ CD9+ c-KIT−. b Confirmation of gene expression signatures of Oct4-GFP− and Oct4-GFP+ A undiff by quantitative RT-PCR. Candidate genes were selected from microarray analysis of a . Expression levels are corrected to those of β-actin and normalized so mean value of GFP− or GFP+ fractions equals 1. Mean values from 3-5 mice ± s.e.m. are indicated. Genes enriched in Oct4-GFP− and Oct4-GFP+ populations are shown in separate groups. Control genes Plzf and Vasa are shown. Significance was calculated by two-tailed Student’s t -test (* P

    Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R & D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R & D Systems, 1:1000).

    Techniques: Isolation, Expressing, Microarray, Quantitative RT-PCR, Mouse Assay, Two Tailed Test

    Interactome of GFP-MKRN1 RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: Interactome of GFP-MKRN1 RINGmut reveals putative ubiquitylation substrates. Experiments were performed using SILAC-based MS. Asymmetrical z-scores of combined SILAC ratios (n = 3 replicates) are shown. Proteins are detected in at least two out of three replicates. ( A ) Protein interactome of GFP-MKRN1 RINGmut in HEK293T cells analysed by quantitative mass spectrometry. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,097 protein groups were quantified in at least two out of three replicates ( Supplemental Table S1 ). MKRN1 and interesting candidate ubiquitylation targets are highlighted. ( B ) Quantitative comparison of the interactome of GFP-MKRN1 wt and GFP-MKRN1 RINGmut shows that potential ubiquitylation candidates identified in ( A ) are enriched in GFP-MKRN1 RINGmut over GFP-MKRN1 wt . Comparison reveals 137 proteins to be significantly enriched (MKRN1 RINGmut over MKRN1 wt with FDR

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Mass Spectrometry, Transformation Assay

    MKRN1 binds upstream of A-stretches in 3’ UTRs. ( A ) MKRN1 predominantly binds in the 3’ UTR of protein-coding genes. Piecharts summarising the distribution of MKRN1 binding sites to different RNA biotypes (7,331 binding sites, left) and different regions within protein-coding transcripts (6,913 binding sites, right). ( B ) MKRN1 binding sites display a downstream enrichment of AAAA homopolymers. Frequency per nucleotide (nt) for four homopolymeric 4-mers in a 101-nt window around the midpoints of the top 20% MKRN1 binding sites (according to signal-over-background; see Material and methods). ( C ) MKRN1 crosslink events accumulate upstream of A-stretches. Metaprofile (top) shows the mean crosslink events per nt in a 201-nt window around the start position of 1,412 MKRN1-associated A-stretches in 3’ UTRs. Heatmap visualisation (bottom) displays crosslink events per nt (see colour scale) in a 101-nt window around the MKRN1-associated A-stretches. ( D ) MKRN1 binding site strength (signal-over-background, SOB) increases with length of longest continuous run of A’s (LCA) within the A-stretch. Mean and standard deviation of MKRN1 binding sites associated with A-stretches harbouring LCAs of increasing length (x-axis). MKRN1 binding sites without associated A-stretches are shown for comparison on the left. Number of binding sites in each category indicated as barchart above. ( E ) MKRN1 binds upstream of A-stretches in the 3’ UTR of the LARP1 gene. Genome browser view of GFP-MKRN1 iCLIP data showing crosslink events per nt (merged replicates, turquois) together with binding sites (lilac) and associated A-stretches (dark green).

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 binds upstream of A-stretches in 3’ UTRs. ( A ) MKRN1 predominantly binds in the 3’ UTR of protein-coding genes. Piecharts summarising the distribution of MKRN1 binding sites to different RNA biotypes (7,331 binding sites, left) and different regions within protein-coding transcripts (6,913 binding sites, right). ( B ) MKRN1 binding sites display a downstream enrichment of AAAA homopolymers. Frequency per nucleotide (nt) for four homopolymeric 4-mers in a 101-nt window around the midpoints of the top 20% MKRN1 binding sites (according to signal-over-background; see Material and methods). ( C ) MKRN1 crosslink events accumulate upstream of A-stretches. Metaprofile (top) shows the mean crosslink events per nt in a 201-nt window around the start position of 1,412 MKRN1-associated A-stretches in 3’ UTRs. Heatmap visualisation (bottom) displays crosslink events per nt (see colour scale) in a 101-nt window around the MKRN1-associated A-stretches. ( D ) MKRN1 binding site strength (signal-over-background, SOB) increases with length of longest continuous run of A’s (LCA) within the A-stretch. Mean and standard deviation of MKRN1 binding sites associated with A-stretches harbouring LCAs of increasing length (x-axis). MKRN1 binding sites without associated A-stretches are shown for comparison on the left. Number of binding sites in each category indicated as barchart above. ( E ) MKRN1 binds upstream of A-stretches in the 3’ UTR of the LARP1 gene. Genome browser view of GFP-MKRN1 iCLIP data showing crosslink events per nt (merged replicates, turquois) together with binding sites (lilac) and associated A-stretches (dark green).

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Binding Assay, Standard Deviation

    MKRN1 interacts with translational regulators and other RBPs. ( A ) Overlap of the 53 significant interaction partners of GFP-MKRN1 wt in human HEK293T cells with previously published interactors of MKRN1 in mouse embryonic stem cells (mESC) ( Cassar et al. 2015 ). ( B ) GO terms enriched for the 53 MKRN1 interactors. P values (modified Fisher exact test, Benjamini-Hochberg correction) are depicted for all significant GO terms (corrected P value

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 interacts with translational regulators and other RBPs. ( A ) Overlap of the 53 significant interaction partners of GFP-MKRN1 wt in human HEK293T cells with previously published interactors of MKRN1 in mouse embryonic stem cells (mESC) ( Cassar et al. 2015 ). ( B ) GO terms enriched for the 53 MKRN1 interactors. P values (modified Fisher exact test, Benjamini-Hochberg correction) are depicted for all significant GO terms (corrected P value

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Modification

    MKRN1 stalls ribosomes at poly(A) sequences. ( A ) The dual fluorescence reporter harbours an N-terminal GFP, followed by a FLAG-SR-X linker and a C-terminal RFP, which are separated by P2A sites to ensure translation into three separate proteins ( Juszkiewicz and Hegde 2017 ). The resulting GFP:RFP ratio was determined using flow cytometry. The inserted fragment K(AAA) 20 encodes 20 lysines by repeating the codon AAA. The starting vector without insert (K 0 ) served as control. Schematic ribosomes illustrate translation of the respective reporter segments. ( B ) Ribosomes are efficiently stalled at K(AAA) 20 in HEK293T cells. Median RFP:GFP ratios, normalised to K 0 , are shown. Error bars represent standard deviation of the mean (s.d.m., n = 6 replicates). P value indicated above (paired two-tailed t-test). ( C ) Ribosomes fail to stall in the absence of MKRN1. HEK293T cells were transfected with control siRNA or siRNAs targeting MKRN1 (KD1 and KD2) or ZNF598 for 24 h, followed by transfection of the reporter plasmids for 48 h. Western blots for KDs are shown in Supplemental Fig. S6B . RFP and GFP signals were analysed by flow cytometry. Median RFP:GFP ratios, normalised to K 0 in control, are shown. Error bars represent s.d.m.; P values indicated above (paired two-tailed t-test, Benjamini-Hochberg correction, n ≥ 6 replicates; ns, not significant).

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 stalls ribosomes at poly(A) sequences. ( A ) The dual fluorescence reporter harbours an N-terminal GFP, followed by a FLAG-SR-X linker and a C-terminal RFP, which are separated by P2A sites to ensure translation into three separate proteins ( Juszkiewicz and Hegde 2017 ). The resulting GFP:RFP ratio was determined using flow cytometry. The inserted fragment K(AAA) 20 encodes 20 lysines by repeating the codon AAA. The starting vector without insert (K 0 ) served as control. Schematic ribosomes illustrate translation of the respective reporter segments. ( B ) Ribosomes are efficiently stalled at K(AAA) 20 in HEK293T cells. Median RFP:GFP ratios, normalised to K 0 , are shown. Error bars represent standard deviation of the mean (s.d.m., n = 6 replicates). P value indicated above (paired two-tailed t-test). ( C ) Ribosomes fail to stall in the absence of MKRN1. HEK293T cells were transfected with control siRNA or siRNAs targeting MKRN1 (KD1 and KD2) or ZNF598 for 24 h, followed by transfection of the reporter plasmids for 48 h. Western blots for KDs are shown in Supplemental Fig. S6B . RFP and GFP signals were analysed by flow cytometry. Median RFP:GFP ratios, normalised to K 0 in control, are shown. Error bars represent s.d.m.; P values indicated above (paired two-tailed t-test, Benjamini-Hochberg correction, n ≥ 6 replicates; ns, not significant).

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Fluorescence, Flow Cytometry, Plasmid Preparation, Standard Deviation, Two Tailed Test, Transfection, Western Blot

    Interaction with PABP is required for MKRN1 RNA binding. ( A,B ) UV crosslinking experiments to measure the RNA binding capacity of GFP-MKRN1 wt and GFP-MKRN1 PAM2mut . Autoradiographs (top) and Western blots (bottom) show GFP-MKRN1/RNA complexes and GFP-MKRN1 protein, respectively, in the eluates from replicates 2 (with 4SU and UV crosslinking at 365 nm) ( A ) and 3 (with conventional UV crosslinking at 254 nm) ( B ). For calibration, input samples for GFP-MKRN1 wt were diluted to 75%, 50% and 25% prior to GFP AP. Note that samples were loaded in different order in ( B ). Quantifications are given below. Uncropped gel images are shown in Supplemental Fig. S10 .

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: Interaction with PABP is required for MKRN1 RNA binding. ( A,B ) UV crosslinking experiments to measure the RNA binding capacity of GFP-MKRN1 wt and GFP-MKRN1 PAM2mut . Autoradiographs (top) and Western blots (bottom) show GFP-MKRN1/RNA complexes and GFP-MKRN1 protein, respectively, in the eluates from replicates 2 (with 4SU and UV crosslinking at 365 nm) ( A ) and 3 (with conventional UV crosslinking at 254 nm) ( B ). For calibration, input samples for GFP-MKRN1 wt were diluted to 75%, 50% and 25% prior to GFP AP. Note that samples were loaded in different order in ( B ). Quantifications are given below. Uncropped gel images are shown in Supplemental Fig. S10 .

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: RNA Binding Assay, Western Blot

    MKRN1 is required to stall ribosomes at K(AAA) 20 in reporter assays. ( A ) Translation of dual fluorescence reporter plasmids was assessed by flow cytometry upon MKRN1 and/or ZNF598 KD. Median RFP:GFP ratios (normalised to K 0 in control KD) are shown for the reporter plasmids K 0 , K(AAA) 12 , K(AAA) 20 , and R(CGA) 10 . Error bars represent standard deviation of the mean (s.d.m., n ≥ 6 replicates; paired two-tailed t-test, Benjamini-Hochberg correction). Density plot of median RFP:GFP ratios of one replicate experiment with K(AAA) 20 with control or MKRN1 KD (two independent siRNAs, KD1 and KD2) or ZNF598 is shown on the right. KDs of MKRN1 and ZNF598 were assessed by Western blot (n = 3 replicates). Black arrowhead indicates ZNF598. Replicates 2 and 3, and uncropped gel images are shown in Supplemental Fig. S11A,B . ( C ) MKRN1 KD2 also reduces MKRN2 levels. MKRN1 KD1 and KD2 were performed for 72 h. Expression levels of MKRN1 and MKRN2 were assessed in relation to ß-actin levels by qPCR in MKRN1 KD (siRNA 1 and 2) and control KD. Error bars indicate s.d.m. (n = 2 replicates). ( D,E ) Cross-regulation of MKRN1 and ZNF598. ( D ) MKRN1 KD1 reduces endogenous ZNF598 protein levels. Effect of MKRN1 KD (KD1, siRNA 1 and KD2, siRNA 2) and ZNF598 KD for 72 h was assessed by Western blot for endogenous MKRN1 and ZNF598. Quantifications depict MKRN1 or ZNF598 expression levels in MKRN1 or ZNF598 KD over control KD condition, normalised to tubulin levels (n = 3 replicates). Replicates 2 and 3, and uncropped gel images are shown in Supplemental Fig. S11C,D . ( E ) ZNF598 overexpression reduces MKRN1 protein levels. Effect of ZNF598 and MKRN1 (wt and mutants) overexpression was tested after 48 h. Quantification as in ( D ). Uncropped gel images for all replicates are shown in Supplemental Fig. S11E,F .

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 is required to stall ribosomes at K(AAA) 20 in reporter assays. ( A ) Translation of dual fluorescence reporter plasmids was assessed by flow cytometry upon MKRN1 and/or ZNF598 KD. Median RFP:GFP ratios (normalised to K 0 in control KD) are shown for the reporter plasmids K 0 , K(AAA) 12 , K(AAA) 20 , and R(CGA) 10 . Error bars represent standard deviation of the mean (s.d.m., n ≥ 6 replicates; paired two-tailed t-test, Benjamini-Hochberg correction). Density plot of median RFP:GFP ratios of one replicate experiment with K(AAA) 20 with control or MKRN1 KD (two independent siRNAs, KD1 and KD2) or ZNF598 is shown on the right. KDs of MKRN1 and ZNF598 were assessed by Western blot (n = 3 replicates). Black arrowhead indicates ZNF598. Replicates 2 and 3, and uncropped gel images are shown in Supplemental Fig. S11A,B . ( C ) MKRN1 KD2 also reduces MKRN2 levels. MKRN1 KD1 and KD2 were performed for 72 h. Expression levels of MKRN1 and MKRN2 were assessed in relation to ß-actin levels by qPCR in MKRN1 KD (siRNA 1 and 2) and control KD. Error bars indicate s.d.m. (n = 2 replicates). ( D,E ) Cross-regulation of MKRN1 and ZNF598. ( D ) MKRN1 KD1 reduces endogenous ZNF598 protein levels. Effect of MKRN1 KD (KD1, siRNA 1 and KD2, siRNA 2) and ZNF598 KD for 72 h was assessed by Western blot for endogenous MKRN1 and ZNF598. Quantifications depict MKRN1 or ZNF598 expression levels in MKRN1 or ZNF598 KD over control KD condition, normalised to tubulin levels (n = 3 replicates). Replicates 2 and 3, and uncropped gel images are shown in Supplemental Fig. S11C,D . ( E ) ZNF598 overexpression reduces MKRN1 protein levels. Effect of ZNF598 and MKRN1 (wt and mutants) overexpression was tested after 48 h. Quantification as in ( D ). Uncropped gel images for all replicates are shown in Supplemental Fig. S11E,F .

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Fluorescence, Flow Cytometry, Standard Deviation, Two Tailed Test, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Over Expression

    MKRN1 binds at poly(A) tails. ( A ) MKRN1 binds near the polyadenylation site of the SRSF4 gene. Genome browser view as in Fig. 2E . ( B ) Unmapped MKRN1 iCLIP reads display increased A-content (more than half of all nucleotides in the read), evidencing poly(A) tail binding. Cumulative fraction of iCLIP reads (y-axis, merged replicates) that could not be mapped to the human genome (see Materials and methods) and show at least a given A-content (x-axis). iCLIP data for the unrelated RBP HNRNPH ( Braun et al. 2018 ) are shown for comparison. ( C ) MKRN1 crosslink events increase towards 3’UTR ends. Metaprofile shows the sum of crosslink events per nt in a 2001-nt window around annotated polyadenylation sites of transcripts with > 1 kb 3’ UTRs (n = 11,257). ( D ) Overall RNA binding of MKRN1 is strongly reduced when abrogating PABP interaction. Audioradiograph (left) of UV crosslinking experiments (replicate 1, with 4SU and UV crosslinking at 365 nm; replicates 2 and 3 in Supplemental Fig. S5 ) comparing GFP-MKRN1 PAM2mut with GFP-MKRN1 wt at different dilution steps for calibration. Quantification of radioactive signal of protein-RNA complexes and corresponding Western blots shown on the right. Uncropped gel images are shown in Supplemental Fig. S10 .

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 binds at poly(A) tails. ( A ) MKRN1 binds near the polyadenylation site of the SRSF4 gene. Genome browser view as in Fig. 2E . ( B ) Unmapped MKRN1 iCLIP reads display increased A-content (more than half of all nucleotides in the read), evidencing poly(A) tail binding. Cumulative fraction of iCLIP reads (y-axis, merged replicates) that could not be mapped to the human genome (see Materials and methods) and show at least a given A-content (x-axis). iCLIP data for the unrelated RBP HNRNPH ( Braun et al. 2018 ) are shown for comparison. ( C ) MKRN1 crosslink events increase towards 3’UTR ends. Metaprofile shows the sum of crosslink events per nt in a 2001-nt window around annotated polyadenylation sites of transcripts with > 1 kb 3’ UTRs (n = 11,257). ( D ) Overall RNA binding of MKRN1 is strongly reduced when abrogating PABP interaction. Audioradiograph (left) of UV crosslinking experiments (replicate 1, with 4SU and UV crosslinking at 365 nm; replicates 2 and 3 in Supplemental Fig. S5 ) comparing GFP-MKRN1 PAM2mut with GFP-MKRN1 wt at different dilution steps for calibration. Quantification of radioactive signal of protein-RNA complexes and corresponding Western blots shown on the right. Uncropped gel images are shown in Supplemental Fig. S10 .

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: Binding Assay, RNA Binding Assay, Western Blot

    MKRN1 interacts with PABP and other regulators of translation and RNA stability. ( A ) Protein interactome of GFP-MKRN1 wt in HEK293T cells analysed by quantitative MS-based proteomics. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,100 protein groups were quantified in at least two out of three replicate experiments. MKRN1 and significant interactors are highlighted (FDR

    Journal: bioRxiv

    Article Title: The RNA-binding ubiquitin ligase MKRN1 functions in ribosome-associated quality control of poly(A) translation

    doi: 10.1101/516005

    Figure Lengend Snippet: MKRN1 interacts with PABP and other regulators of translation and RNA stability. ( A ) Protein interactome of GFP-MKRN1 wt in HEK293T cells analysed by quantitative MS-based proteomics. Combined SILAC ratios (n = 3 replicates) after z-score normalisation are plotted against log 10 -transformed intensities. 1,100 protein groups were quantified in at least two out of three replicate experiments. MKRN1 and significant interactors are highlighted (FDR

    Article Snippet: Antibodies The following antibodies were used: anti-GFP (B-2 clone; Santa Cruz; sc-9996), anti-MKRN1 (Bethyl Laboratories, A300-990A), anti-PABPC1/3 (Cell Signaling, 4992), anti-Znf598 (N1N3; GeneTex; GTX119245), anti-αTubulin (Sigma Aldrich, T-5168), anti-Rabbit IgG (Cell Signaling; 7074), anti-Mouse IgG (Cell Signaling; 7076), IRDye® 680RD Goat anti-Mouse IgG (P/N 925-68070), and IRDye® 800CW Goat anti-Rabbit IgG (P/N 925-32211) (both LI-COR Biosciences GmbH).

    Techniques: qMS Based, Transformation Assay

    N-terminus of Cse4 is important for its distribution and stability. A , Schematic diagram of the domain organizations of Cnp1 N -Cse4 C (1N4C) and Cse4 N -Cnp1 C (4N1C) proteins. Full-length Cnp1, Cse4 and N-terminal deleted Cse4 (Cse4-NΔ) are also shown. B, Distribution patterns of overexpressed Cse4 N -Cnp1 C -GFP and Cnp1 N -Cse4 C -GFP. Induction times: 24 hours. Scale bar: 2 μm. C, Western blot analysis of cells expressing indicated proteins using an anti-GFP antibody. Tubulin was used as a loading control.

    Journal: PLoS Genetics

    Article Title: Heterochromatin and RNAi regulate centromeres by protecting CENP-A from ubiquitin-mediated degradation

    doi: 10.1371/journal.pgen.1007572

    Figure Lengend Snippet: N-terminus of Cse4 is important for its distribution and stability. A , Schematic diagram of the domain organizations of Cnp1 N -Cse4 C (1N4C) and Cse4 N -Cnp1 C (4N1C) proteins. Full-length Cnp1, Cse4 and N-terminal deleted Cse4 (Cse4-NΔ) are also shown. B, Distribution patterns of overexpressed Cse4 N -Cnp1 C -GFP and Cnp1 N -Cse4 C -GFP. Induction times: 24 hours. Scale bar: 2 μm. C, Western blot analysis of cells expressing indicated proteins using an anti-GFP antibody. Tubulin was used as a loading control.

    Article Snippet: A. Extracts from indicated cells expressing Cnp1-GFP were subject to immunoprecipitation with an anti-GFP antibody.

    Techniques: Western Blot, Expressing

    Cse4 preferentially targets to centromeres and functionally substitutes Cnp1 in fission yeast. A, Cells carrying pREP1-CSE4-GFP that were induced for 24 hours show a single GFP focus (short). Multiple foci (commonly 3–6) were detected for 28-hour induction (long). (Bottom) Percentage of cells containing single or multiple focus. OE, overexpression. B, The single Cse4-GFP focus colocalizes with Sad1-CFP. C, Cse4-GFP partially rescues cnp1-1 growth defects at 36°C. Serial dilutions of indicated strains were plated in minimal medium without thiamine. Dilution = 10. D, Multiple GFP foci in cells overexpressing Cse4-GFP colocalize with mCherry-Swi6. E . ChIP qPCR showing relative enrichment of Cse4-GFP to centromeric region ( cnt3 ), peri-centromeric region ( otr ) and sub-telomeric region ( subT ). ChIP was repeated in triplicate. Error bar indicates SEM. *, p

    Journal: PLoS Genetics

    Article Title: Heterochromatin and RNAi regulate centromeres by protecting CENP-A from ubiquitin-mediated degradation

    doi: 10.1371/journal.pgen.1007572

    Figure Lengend Snippet: Cse4 preferentially targets to centromeres and functionally substitutes Cnp1 in fission yeast. A, Cells carrying pREP1-CSE4-GFP that were induced for 24 hours show a single GFP focus (short). Multiple foci (commonly 3–6) were detected for 28-hour induction (long). (Bottom) Percentage of cells containing single or multiple focus. OE, overexpression. B, The single Cse4-GFP focus colocalizes with Sad1-CFP. C, Cse4-GFP partially rescues cnp1-1 growth defects at 36°C. Serial dilutions of indicated strains were plated in minimal medium without thiamine. Dilution = 10. D, Multiple GFP foci in cells overexpressing Cse4-GFP colocalize with mCherry-Swi6. E . ChIP qPCR showing relative enrichment of Cse4-GFP to centromeric region ( cnt3 ), peri-centromeric region ( otr ) and sub-telomeric region ( subT ). ChIP was repeated in triplicate. Error bar indicates SEM. *, p

    Article Snippet: A. Extracts from indicated cells expressing Cnp1-GFP were subject to immunoprecipitation with an anti-GFP antibody.

    Techniques: Over Expression, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Heterochromatin restricts access of proteasome to centromeric regions. A, Cnp1-GFP level is increased in mts2-1 cells after incubation at 37°C for 4 hours. Western blotting was performed using an anti-GFP antibody. B, The association of proteasome with centromeres is increased in clr4 Δ. ChIP assays were conducted with indicated cells expressing Mts2-myc using an anti-myc antibody. Data from ChIP with the myc antibody were normalized against those from IgG mock ChIP. cnt3 , centromeric region. n = 8, error bar represents SEM. *, p

    Journal: PLoS Genetics

    Article Title: Heterochromatin and RNAi regulate centromeres by protecting CENP-A from ubiquitin-mediated degradation

    doi: 10.1371/journal.pgen.1007572

    Figure Lengend Snippet: Heterochromatin restricts access of proteasome to centromeric regions. A, Cnp1-GFP level is increased in mts2-1 cells after incubation at 37°C for 4 hours. Western blotting was performed using an anti-GFP antibody. B, The association of proteasome with centromeres is increased in clr4 Δ. ChIP assays were conducted with indicated cells expressing Mts2-myc using an anti-myc antibody. Data from ChIP with the myc antibody were normalized against those from IgG mock ChIP. cnt3 , centromeric region. n = 8, error bar represents SEM. *, p

    Article Snippet: A. Extracts from indicated cells expressing Cnp1-GFP were subject to immunoprecipitation with an anti-GFP antibody.

    Techniques: Incubation, Western Blot, Chromatin Immunoprecipitation, Expressing

    Cse4 is subject to efficient ubiquitin-dependent degradation in the fission yeast. A, RT-PCR analysis of cells expressing Cnp1-GFP or Cse4-GFP. Total RNA extracted from cells overexpressing Cnp1-GFP or Cse4-GFP was used. Cnp1-GFP or Cse4-GFP transcripts were analyzed with primers specific for GFP. Actin was used as an internal control. B, Lysates from cells collected at indicated time points (hrs) following cycloheximide treatment were analyzed by western blotting with an anti-GFP antibody. C, Cse4 level is enhanced after proteasome inactivation in fission yeast. Cells overexpressing Cse4-GFP in wild type or mts2-1 background were incubated at 37°C for 4 hours, and were subject to western blot analysis using an anti-GFP antibody. Tubulin was used as a loading control. D, Extracts from cells expressing indicated proteins were split, and subject to TUBE pull-down and reverse pull-down assays, respectively. For TUBE pull-down assays, extracts were immunoprecipitated with tandem ubiquitin-binding entities (+TUBE), or control Argarose beads (-TUBE), followed by western blot analysis using an anti-GFP antibody. For reverse pull-down assays (right panel), extracts were immunoprecipitated with an anti-GFP antibody, then analyzed by western blotting using a pan ubiquitin antibody. Induction time: 20 hours for Cnp1-GFP; 24 hours for Cse4-GFP.

    Journal: PLoS Genetics

    Article Title: Heterochromatin and RNAi regulate centromeres by protecting CENP-A from ubiquitin-mediated degradation

    doi: 10.1371/journal.pgen.1007572

    Figure Lengend Snippet: Cse4 is subject to efficient ubiquitin-dependent degradation in the fission yeast. A, RT-PCR analysis of cells expressing Cnp1-GFP or Cse4-GFP. Total RNA extracted from cells overexpressing Cnp1-GFP or Cse4-GFP was used. Cnp1-GFP or Cse4-GFP transcripts were analyzed with primers specific for GFP. Actin was used as an internal control. B, Lysates from cells collected at indicated time points (hrs) following cycloheximide treatment were analyzed by western blotting with an anti-GFP antibody. C, Cse4 level is enhanced after proteasome inactivation in fission yeast. Cells overexpressing Cse4-GFP in wild type or mts2-1 background were incubated at 37°C for 4 hours, and were subject to western blot analysis using an anti-GFP antibody. Tubulin was used as a loading control. D, Extracts from cells expressing indicated proteins were split, and subject to TUBE pull-down and reverse pull-down assays, respectively. For TUBE pull-down assays, extracts were immunoprecipitated with tandem ubiquitin-binding entities (+TUBE), or control Argarose beads (-TUBE), followed by western blot analysis using an anti-GFP antibody. For reverse pull-down assays (right panel), extracts were immunoprecipitated with an anti-GFP antibody, then analyzed by western blotting using a pan ubiquitin antibody. Induction time: 20 hours for Cnp1-GFP; 24 hours for Cse4-GFP.

    Article Snippet: A. Extracts from indicated cells expressing Cnp1-GFP were subject to immunoprecipitation with an anti-GFP antibody.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Incubation, Immunoprecipitation, Binding Assay

    Heterochromatin promotes centromeric targeting of Cnp1 by preventing ubiquitin-mediated degradation. A, clr4 Δ colonies expressing Cnp1-GFP under its native promoter obtained by crossing exhibited a diffuse GFP signal or single focus. (Right) Percentage of colonies displaying diffuse GFP or a single focus, and quantifications were based on random colony analysis. Scale bar: 2 μm. B, Western blot analysis of clr4 Δ cells expressing Cnp1-GFP. WT was used as control. C, Stability assays for WT and clr4 Δ cells expressing Cnp1-GFP. D, Extracts from indicated cells were analyzed by TUBE assays. C,D, Induction time:20 hours for WT and 22 hours for clr4 Δ.

    Journal: PLoS Genetics

    Article Title: Heterochromatin and RNAi regulate centromeres by protecting CENP-A from ubiquitin-mediated degradation

    doi: 10.1371/journal.pgen.1007572

    Figure Lengend Snippet: Heterochromatin promotes centromeric targeting of Cnp1 by preventing ubiquitin-mediated degradation. A, clr4 Δ colonies expressing Cnp1-GFP under its native promoter obtained by crossing exhibited a diffuse GFP signal or single focus. (Right) Percentage of colonies displaying diffuse GFP or a single focus, and quantifications were based on random colony analysis. Scale bar: 2 μm. B, Western blot analysis of clr4 Δ cells expressing Cnp1-GFP. WT was used as control. C, Stability assays for WT and clr4 Δ cells expressing Cnp1-GFP. D, Extracts from indicated cells were analyzed by TUBE assays. C,D, Induction time:20 hours for WT and 22 hours for clr4 Δ.

    Article Snippet: A. Extracts from indicated cells expressing Cnp1-GFP were subject to immunoprecipitation with an anti-GFP antibody.

    Techniques: Expressing, Western Blot

    Cse4 is expressed at a significantly low level in the fission yeast. A, Distribution patterns of Cse4-GFP and Cnp1-GFP at repressed (Top) and overexpressed (Bottom) conditions (24-hour induction). B, Quantification of the percentage of cells showing a single focus when repressed (Left), and a single or multiple GFP foci ( > 3) in overexpressed condition (Right). C, Western blot analysis of cells expressing indicated proteins using an anti-GFP antibody. Tubulin was used as a loading control. D, Cells harboring pREP1-CSE4-GFP were grown in minimal medium with thiamine to suppress its expression (Left) or lacking thiamine to induce overexpression (Right). Wild type cells carrying an empty vector were used as a control. E, Overexpression of Cse4 results in minor chromosome segregation defects. Cells carrying indicated plasmids were plated in minimal medium that contains 15μg/ml TBZ but no thiamine. Dilution = 10. Ctrl, a control plate without TBZ. Scale bar: 2 μm.

    Journal: PLoS Genetics

    Article Title: Heterochromatin and RNAi regulate centromeres by protecting CENP-A from ubiquitin-mediated degradation

    doi: 10.1371/journal.pgen.1007572

    Figure Lengend Snippet: Cse4 is expressed at a significantly low level in the fission yeast. A, Distribution patterns of Cse4-GFP and Cnp1-GFP at repressed (Top) and overexpressed (Bottom) conditions (24-hour induction). B, Quantification of the percentage of cells showing a single focus when repressed (Left), and a single or multiple GFP foci ( > 3) in overexpressed condition (Right). C, Western blot analysis of cells expressing indicated proteins using an anti-GFP antibody. Tubulin was used as a loading control. D, Cells harboring pREP1-CSE4-GFP were grown in minimal medium with thiamine to suppress its expression (Left) or lacking thiamine to induce overexpression (Right). Wild type cells carrying an empty vector were used as a control. E, Overexpression of Cse4 results in minor chromosome segregation defects. Cells carrying indicated plasmids were plated in minimal medium that contains 15μg/ml TBZ but no thiamine. Dilution = 10. Ctrl, a control plate without TBZ. Scale bar: 2 μm.

    Article Snippet: A. Extracts from indicated cells expressing Cnp1-GFP were subject to immunoprecipitation with an anti-GFP antibody.

    Techniques: Western Blot, Expressing, Over Expression, Plasmid Preparation

    Micro-CT analyses at the secondary spongiosa of the distal femur (A) or at S3 caudal vertebra (B) of recipient mice receiving either MLV- FGF2 -transduced Sca-1 + cells (FGF-2) or MLV- gfp -transduced Sca-1 + cells (GFP) after 14 weeks post-transplantation. Top panels show a representative three-dimensional reconstruction of the trabecular structure each at the femur metaphysis (A) or at S3 caudal vertebrae (B) for each treatment group. Bottom bar graphs show the quantitative analyses of the three-dimensional bone parameters at each both site. N = 7 for the GFP group, and N = 4 for the FGF2 group.

    Journal: Gene therapy

    Article Title: Opposing Effects of Sca-1+ Cell-Based Systemic FGF-2 Gene Transfer Strategy on Lumbar versus Caudal Vertebrae in the Mouse

    doi: 10.1038/gt.2016.21

    Figure Lengend Snippet: Micro-CT analyses at the secondary spongiosa of the distal femur (A) or at S3 caudal vertebra (B) of recipient mice receiving either MLV- FGF2 -transduced Sca-1 + cells (FGF-2) or MLV- gfp -transduced Sca-1 + cells (GFP) after 14 weeks post-transplantation. Top panels show a representative three-dimensional reconstruction of the trabecular structure each at the femur metaphysis (A) or at S3 caudal vertebrae (B) for each treatment group. Bottom bar graphs show the quantitative analyses of the three-dimensional bone parameters at each both site. N = 7 for the GFP group, and N = 4 for the FGF2 group.

    Article Snippet: To assess enrichment effectiveness, aliquots of each cell fraction were incubated with phycoerythrin (PE)-conjugated Sca-1-specific or PE-conjugated rat isotype control antibody (Pharmingen, San Diego, CA, USA) and analyzed for Sca-1 and/or GFP-expression by fluorescence activated cell sorter (FACS) analysis with a FACSCalibur or FACSAria System (BD Biosciences, San Jose, CA, USA).

    Techniques: Micro-CT, Mouse Assay, Transplantation Assay

    Relative expression levels of FGF2 mRNA in the marrow cavity or in the entire caudal vertebra of recipient mice receiving MLV- FGF2 -transduced Sca-1 + cells (N = 20) compared to respective FGF2 mRNA expression levels at each bone site of control recipient mice receiving MLV- gfp -transduced cells (N = 4). *** P

    Journal: Gene therapy

    Article Title: Opposing Effects of Sca-1+ Cell-Based Systemic FGF-2 Gene Transfer Strategy on Lumbar versus Caudal Vertebrae in the Mouse

    doi: 10.1038/gt.2016.21

    Figure Lengend Snippet: Relative expression levels of FGF2 mRNA in the marrow cavity or in the entire caudal vertebra of recipient mice receiving MLV- FGF2 -transduced Sca-1 + cells (N = 20) compared to respective FGF2 mRNA expression levels at each bone site of control recipient mice receiving MLV- gfp -transduced cells (N = 4). *** P

    Article Snippet: To assess enrichment effectiveness, aliquots of each cell fraction were incubated with phycoerythrin (PE)-conjugated Sca-1-specific or PE-conjugated rat isotype control antibody (Pharmingen, San Diego, CA, USA) and analyzed for Sca-1 and/or GFP-expression by fluorescence activated cell sorter (FACS) analysis with a FACSCalibur or FACSAria System (BD Biosciences, San Jose, CA, USA).

    Techniques: Expressing, Mouse Assay

    Sca-1 + cell-based systemic FGF2 gene therapy promoted massive trabecular bone formation but also caused osteomalacia in recipient mice. In A, a cross-sectional slice (5 µm in thickness) of L3 lumbar vertebrae of a representative control mice receiving the marrow transplantation of MLV- gfp -transduced Sca-1 + cells at 14 weeks post-transplantation stained with the Goldner’s trichrome dye for mineralized bone. In B, a cross-sectional slice (5 µm in thickness) of L3 lumbar vertebrae of a representative mice receiving the marrow transplantation of MLV- FGF2 -transduced Sca-1 + cells at 14 weeks post-transplantation stained with the Goldner’s trichrome dye for mineralized bone. Mineralized bone is stained dark blue in color, whereas un-mineralized bone matrix is stained in red.

    Journal: Gene therapy

    Article Title: Opposing Effects of Sca-1+ Cell-Based Systemic FGF-2 Gene Transfer Strategy on Lumbar versus Caudal Vertebrae in the Mouse

    doi: 10.1038/gt.2016.21

    Figure Lengend Snippet: Sca-1 + cell-based systemic FGF2 gene therapy promoted massive trabecular bone formation but also caused osteomalacia in recipient mice. In A, a cross-sectional slice (5 µm in thickness) of L3 lumbar vertebrae of a representative control mice receiving the marrow transplantation of MLV- gfp -transduced Sca-1 + cells at 14 weeks post-transplantation stained with the Goldner’s trichrome dye for mineralized bone. In B, a cross-sectional slice (5 µm in thickness) of L3 lumbar vertebrae of a representative mice receiving the marrow transplantation of MLV- FGF2 -transduced Sca-1 + cells at 14 weeks post-transplantation stained with the Goldner’s trichrome dye for mineralized bone. Mineralized bone is stained dark blue in color, whereas un-mineralized bone matrix is stained in red.

    Article Snippet: To assess enrichment effectiveness, aliquots of each cell fraction were incubated with phycoerythrin (PE)-conjugated Sca-1-specific or PE-conjugated rat isotype control antibody (Pharmingen, San Diego, CA, USA) and analyzed for Sca-1 and/or GFP-expression by fluorescence activated cell sorter (FACS) analysis with a FACSCalibur or FACSAria System (BD Biosciences, San Jose, CA, USA).

    Techniques: Mouse Assay, Transplantation Assay, Staining

    Static bone histomorphometric parameters of L3 vertebrae of recipient mice of MLV- gfp -transduced Sca-1 + cells (green bars) or MLV- FGF2 -transduced Sca-1 + cells (yellow bars) at 14 weeks post-transplantation. %Md.Ar, % mineralized bone area; %O.Ar, % osteoid area; %L.Pm, % bone forming surface; Tb.Wi, trabecular width; and Tb.N, trabecular number. N = 7 per group. N.S. = not significant.

    Journal: Gene therapy

    Article Title: Opposing Effects of Sca-1+ Cell-Based Systemic FGF-2 Gene Transfer Strategy on Lumbar versus Caudal Vertebrae in the Mouse

    doi: 10.1038/gt.2016.21

    Figure Lengend Snippet: Static bone histomorphometric parameters of L3 vertebrae of recipient mice of MLV- gfp -transduced Sca-1 + cells (green bars) or MLV- FGF2 -transduced Sca-1 + cells (yellow bars) at 14 weeks post-transplantation. %Md.Ar, % mineralized bone area; %O.Ar, % osteoid area; %L.Pm, % bone forming surface; Tb.Wi, trabecular width; and Tb.N, trabecular number. N = 7 per group. N.S. = not significant.

    Article Snippet: To assess enrichment effectiveness, aliquots of each cell fraction were incubated with phycoerythrin (PE)-conjugated Sca-1-specific or PE-conjugated rat isotype control antibody (Pharmingen, San Diego, CA, USA) and analyzed for Sca-1 and/or GFP-expression by fluorescence activated cell sorter (FACS) analysis with a FACSCalibur or FACSAria System (BD Biosciences, San Jose, CA, USA).

    Techniques: Mouse Assay, Transplantation Assay

    Serum FGF2 (A), Serum PTH (B), Serum alkaline phosphatase (ALP) activity (C), and bone ALP activity (D) of recipient mice receiving either MLV- FGF2 -transduced Sca-1 + cells (FGF-2) or MLV- gfp -transduced Sca-1 + cells (GFP) after 14 weeks post-transplantation in the second marrow transplantation experiment. FGF2 and PTH were measured with respective commercial ELISA kits. ALP activity was assayed as described in Materials and Methods. N = 7 for the GFP group, and N = 4 for the FGF2 group.

    Journal: Gene therapy

    Article Title: Opposing Effects of Sca-1+ Cell-Based Systemic FGF-2 Gene Transfer Strategy on Lumbar versus Caudal Vertebrae in the Mouse

    doi: 10.1038/gt.2016.21

    Figure Lengend Snippet: Serum FGF2 (A), Serum PTH (B), Serum alkaline phosphatase (ALP) activity (C), and bone ALP activity (D) of recipient mice receiving either MLV- FGF2 -transduced Sca-1 + cells (FGF-2) or MLV- gfp -transduced Sca-1 + cells (GFP) after 14 weeks post-transplantation in the second marrow transplantation experiment. FGF2 and PTH were measured with respective commercial ELISA kits. ALP activity was assayed as described in Materials and Methods. N = 7 for the GFP group, and N = 4 for the FGF2 group.

    Article Snippet: To assess enrichment effectiveness, aliquots of each cell fraction were incubated with phycoerythrin (PE)-conjugated Sca-1-specific or PE-conjugated rat isotype control antibody (Pharmingen, San Diego, CA, USA) and analyzed for Sca-1 and/or GFP-expression by fluorescence activated cell sorter (FACS) analysis with a FACSCalibur or FACSAria System (BD Biosciences, San Jose, CA, USA).

    Techniques: ALP Assay, Activity Assay, Mouse Assay, Transplantation Assay, Enzyme-linked Immunosorbent Assay

    A. Relative engraftment of transplanted β-galactosidase (βgal)-expressing cells at the marrow cavity of femurs (left columns) or at the entire caudal vertebra (right columns) of recipient mice receiving MLV- FGF2 -transduced Sca-1 + cells (FGF-2, N = 20) after 14 weeks post-transplantation compared to that of control recipient mice receiving MLV- gfp -transduced Sca-1 + cells (GFP, N = 4). N.S. = not significant. B. Relative engraftment of gfp -expressing cells in the marrow cavity compared to that in the entire caudal vertebra of recipient mice receiving MLV-FGF2-transduced Sca-1 + cells. N = 20 per group. Engraftment in each panel was assessed by measuring the relative gfp mRNA levels (normalized against Ppia mRNA).

    Journal: Gene therapy

    Article Title: Opposing Effects of Sca-1+ Cell-Based Systemic FGF-2 Gene Transfer Strategy on Lumbar versus Caudal Vertebrae in the Mouse

    doi: 10.1038/gt.2016.21

    Figure Lengend Snippet: A. Relative engraftment of transplanted β-galactosidase (βgal)-expressing cells at the marrow cavity of femurs (left columns) or at the entire caudal vertebra (right columns) of recipient mice receiving MLV- FGF2 -transduced Sca-1 + cells (FGF-2, N = 20) after 14 weeks post-transplantation compared to that of control recipient mice receiving MLV- gfp -transduced Sca-1 + cells (GFP, N = 4). N.S. = not significant. B. Relative engraftment of gfp -expressing cells in the marrow cavity compared to that in the entire caudal vertebra of recipient mice receiving MLV-FGF2-transduced Sca-1 + cells. N = 20 per group. Engraftment in each panel was assessed by measuring the relative gfp mRNA levels (normalized against Ppia mRNA).

    Article Snippet: To assess enrichment effectiveness, aliquots of each cell fraction were incubated with phycoerythrin (PE)-conjugated Sca-1-specific or PE-conjugated rat isotype control antibody (Pharmingen, San Diego, CA, USA) and analyzed for Sca-1 and/or GFP-expression by fluorescence activated cell sorter (FACS) analysis with a FACSCalibur or FACSAria System (BD Biosciences, San Jose, CA, USA).

    Techniques: Expressing, Mouse Assay, Transplantation Assay

    Effects of marrow transplantation of MLV- FGF2 - or MLV- gfp -transduced Sca-1 + cells on relative levels of engraftment (A), as well as serum levels of FGF2 (B), and serum PTH (C) in recipient mice. In A, engraftment of FGF2-expressing Sca-1 + cells was assessed by measuring the relative level of human FGF2 genomic DNA content in peripheral blood cells of recipient mice of MLV- FGF2 -transduced cells (FGF2) or control MLV- gfp -transduced cells (GFP) at 6 or 14 weeks post-transplantation, respectively. N = 7 per group. In B, serum FGF2 levels of recipient mice of MLV-FGF2-transduced cells (FGF-2 mice) or the control MLV-gfp-transduced cells (GFP mice) at 14 weeks post-transplantation were assayed with a commercial ELISA kit. N = 7 per group. In C, serum PTH levels of recipient mice of MLV- FGF2 - or MLV- gfp -transduced Sca-1+ cells at 14 weeks post-transplantation were measured with a commercial ELISA kit. N = 7 per group.

    Journal: Gene therapy

    Article Title: Opposing Effects of Sca-1+ Cell-Based Systemic FGF-2 Gene Transfer Strategy on Lumbar versus Caudal Vertebrae in the Mouse

    doi: 10.1038/gt.2016.21

    Figure Lengend Snippet: Effects of marrow transplantation of MLV- FGF2 - or MLV- gfp -transduced Sca-1 + cells on relative levels of engraftment (A), as well as serum levels of FGF2 (B), and serum PTH (C) in recipient mice. In A, engraftment of FGF2-expressing Sca-1 + cells was assessed by measuring the relative level of human FGF2 genomic DNA content in peripheral blood cells of recipient mice of MLV- FGF2 -transduced cells (FGF2) or control MLV- gfp -transduced cells (GFP) at 6 or 14 weeks post-transplantation, respectively. N = 7 per group. In B, serum FGF2 levels of recipient mice of MLV-FGF2-transduced cells (FGF-2 mice) or the control MLV-gfp-transduced cells (GFP mice) at 14 weeks post-transplantation were assayed with a commercial ELISA kit. N = 7 per group. In C, serum PTH levels of recipient mice of MLV- FGF2 - or MLV- gfp -transduced Sca-1+ cells at 14 weeks post-transplantation were measured with a commercial ELISA kit. N = 7 per group.

    Article Snippet: To assess enrichment effectiveness, aliquots of each cell fraction were incubated with phycoerythrin (PE)-conjugated Sca-1-specific or PE-conjugated rat isotype control antibody (Pharmingen, San Diego, CA, USA) and analyzed for Sca-1 and/or GFP-expression by fluorescence activated cell sorter (FACS) analysis with a FACSCalibur or FACSAria System (BD Biosciences, San Jose, CA, USA).

    Techniques: Transplantation Assay, Mouse Assay, Expressing, Enzyme-linked Immunosorbent Assay

    Ykt6 effects on autophagosome/lysosome fusion depend on the phosphorylation at the evolutionarily conserved site in the SNARE domain in the absence of endogenous Ykt6. (A) Representative western blot for LC3-II from a stably transfected PC12 cell line with a doxycycline inducible shRNA targeting endogenous rat Ykt6 and infected with lentiviruses carrying GFP-tagged wild-type (WT) human Ykt6 and phosphomutants. Cells were treated for 2 hours (2h) with 200nM Bafilomycin A 1 (Baf-A 1 ) in growing conditions (fresh 10% FBS and 4.5% glucose growth medium) or in starvation conditions (Torin-1 250nM, 1X HBSS supplemented with 10mM HEPES). Actin serves as a loading control. (B-C) Autophagic flux (defined as [LC3-II Baf-A 1 ] – [LC3-II]) from western blot in (A) of (B) growing and (C) starvation conditions. N=5 *p

    Journal: bioRxiv

    Article Title: A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

    doi: 10.1101/2020.03.15.992727

    Figure Lengend Snippet: Ykt6 effects on autophagosome/lysosome fusion depend on the phosphorylation at the evolutionarily conserved site in the SNARE domain in the absence of endogenous Ykt6. (A) Representative western blot for LC3-II from a stably transfected PC12 cell line with a doxycycline inducible shRNA targeting endogenous rat Ykt6 and infected with lentiviruses carrying GFP-tagged wild-type (WT) human Ykt6 and phosphomutants. Cells were treated for 2 hours (2h) with 200nM Bafilomycin A 1 (Baf-A 1 ) in growing conditions (fresh 10% FBS and 4.5% glucose growth medium) or in starvation conditions (Torin-1 250nM, 1X HBSS supplemented with 10mM HEPES). Actin serves as a loading control. (B-C) Autophagic flux (defined as [LC3-II Baf-A 1 ] – [LC3-II]) from western blot in (A) of (B) growing and (C) starvation conditions. N=5 *p

    Article Snippet: For Western Blot the following primary antibodies were used: GFP (Santa Cruz, sc-9996), actin (Abcam, ab6276), Ykt6 (Abcam, ab236583), LC3B (Cell Signaling, #2775), p62 (Sigma, p0067), STX17 (Sigma, hpa001204), SNAP29 (Abcam, ab138500) Na+/K+ ATPase (Sigma, 06-172-I), alpha/beta-Tubulin (Cell Signaling, 2148S), and STX7 (Bethyl, A304-512A).

    Techniques: Western Blot, Stable Transfection, Transfection, shRNA, Infection

    Phosphorylation of the evolutionarily conserved site in Ykt6 SNARE domain S174 affects the specificity of its binding partners. Ykt6 spectral counts from three independent experiments. GFP-Ykt6 wild-type (WT) or GFP-phosphomutants were immunoprecipitated with GFP and subjected to the mass spectrometry.

    Journal: bioRxiv

    Article Title: A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

    doi: 10.1101/2020.03.15.992727

    Figure Lengend Snippet: Phosphorylation of the evolutionarily conserved site in Ykt6 SNARE domain S174 affects the specificity of its binding partners. Ykt6 spectral counts from three independent experiments. GFP-Ykt6 wild-type (WT) or GFP-phosphomutants were immunoprecipitated with GFP and subjected to the mass spectrometry.

    Article Snippet: For Western Blot the following primary antibodies were used: GFP (Santa Cruz, sc-9996), actin (Abcam, ab6276), Ykt6 (Abcam, ab236583), LC3B (Cell Signaling, #2775), p62 (Sigma, p0067), STX17 (Sigma, hpa001204), SNAP29 (Abcam, ab138500) Na+/K+ ATPase (Sigma, 06-172-I), alpha/beta-Tubulin (Cell Signaling, 2148S), and STX7 (Bethyl, A304-512A).

    Techniques: Binding Assay, Immunoprecipitation, Mass Spectrometry

    Evolutionarily conserved phosphorylation site within human Ykt6 SNARE domain (S174) is a critical determinant for its intracellular localization. (A) Representative immunofluorescence images of transiently transfected HeLa cells with GFP, GFP-tagged wild-type (WT), phospho-ablative mutant (S174A) and phospho-mimetic mutant (S174D) of Ykt6. Nuclei (blue) are stained with DAPI. (B) Representative Western Blot for membrane and cytosolic fractions of HEK293T cells transiently transfected as described in (A). Membrane (Na+/K+ ATPase) and cytosolic (tubulin) markers serve as controls for fractionation purity. (C) Fold change of membrane/cytosol fraction calculated as: 1) normalizing to GFP/actin (see supplemental Figure 2C ), 2) obtaining the ratios of GFP-Ykt6 fusion protein to tubulin (for the cytosolic fraction), GFP-Ykt6 fusion protein to Na+/K+ ATPase (for the membrane fraction) and 3) membrane fraction/cytosolic fraction. N=4 *p,0.05, **p

    Journal: bioRxiv

    Article Title: A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

    doi: 10.1101/2020.03.15.992727

    Figure Lengend Snippet: Evolutionarily conserved phosphorylation site within human Ykt6 SNARE domain (S174) is a critical determinant for its intracellular localization. (A) Representative immunofluorescence images of transiently transfected HeLa cells with GFP, GFP-tagged wild-type (WT), phospho-ablative mutant (S174A) and phospho-mimetic mutant (S174D) of Ykt6. Nuclei (blue) are stained with DAPI. (B) Representative Western Blot for membrane and cytosolic fractions of HEK293T cells transiently transfected as described in (A). Membrane (Na+/K+ ATPase) and cytosolic (tubulin) markers serve as controls for fractionation purity. (C) Fold change of membrane/cytosol fraction calculated as: 1) normalizing to GFP/actin (see supplemental Figure 2C ), 2) obtaining the ratios of GFP-Ykt6 fusion protein to tubulin (for the cytosolic fraction), GFP-Ykt6 fusion protein to Na+/K+ ATPase (for the membrane fraction) and 3) membrane fraction/cytosolic fraction. N=4 *p,0.05, **p

    Article Snippet: For Western Blot the following primary antibodies were used: GFP (Santa Cruz, sc-9996), actin (Abcam, ab6276), Ykt6 (Abcam, ab236583), LC3B (Cell Signaling, #2775), p62 (Sigma, p0067), STX17 (Sigma, hpa001204), SNAP29 (Abcam, ab138500) Na+/K+ ATPase (Sigma, 06-172-I), alpha/beta-Tubulin (Cell Signaling, 2148S), and STX7 (Bethyl, A304-512A).

    Techniques: Immunofluorescence, Transfection, Mutagenesis, Staining, Western Blot, Fractionation

    Ykt6 effects on autophagosome/lysosome fusion depend on the phosphorylation at the evolutionarily conserved site in the SNARE domain. Representative western blot for LC3-II from HEK293T transiently expressing GFP-tagged wild-type (WT) Ykt6 and phosphomutants. Cells were treated for 2 hours (2h) with 200nM Bafilomycin A 1 (Baf-A 1 ) in growing conditions (DMEM with 10% FBS and 4.5% glucose growth medium) or in starvation conditions (1X HBSS with 250nM Torin-1 and 10mM HEPES). Actin serves as a loading control. (B-C) Autophagic flux (defined as [LC3-II Baf-A 1 ] – [LC3-II]) from western blot in (A) of (B) growing and (C) starvation conditions. (A-C) N=6 *p

    Journal: bioRxiv

    Article Title: A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

    doi: 10.1101/2020.03.15.992727

    Figure Lengend Snippet: Ykt6 effects on autophagosome/lysosome fusion depend on the phosphorylation at the evolutionarily conserved site in the SNARE domain. Representative western blot for LC3-II from HEK293T transiently expressing GFP-tagged wild-type (WT) Ykt6 and phosphomutants. Cells were treated for 2 hours (2h) with 200nM Bafilomycin A 1 (Baf-A 1 ) in growing conditions (DMEM with 10% FBS and 4.5% glucose growth medium) or in starvation conditions (1X HBSS with 250nM Torin-1 and 10mM HEPES). Actin serves as a loading control. (B-C) Autophagic flux (defined as [LC3-II Baf-A 1 ] – [LC3-II]) from western blot in (A) of (B) growing and (C) starvation conditions. (A-C) N=6 *p

    Article Snippet: For Western Blot the following primary antibodies were used: GFP (Santa Cruz, sc-9996), actin (Abcam, ab6276), Ykt6 (Abcam, ab236583), LC3B (Cell Signaling, #2775), p62 (Sigma, p0067), STX17 (Sigma, hpa001204), SNAP29 (Abcam, ab138500) Na+/K+ ATPase (Sigma, 06-172-I), alpha/beta-Tubulin (Cell Signaling, 2148S), and STX7 (Bethyl, A304-512A).

    Techniques: Western Blot, Expressing

    The intracellular localization of Ykt6 is dependent on calcineurin activity. (A) Representative immunofluorescence images of transiently transfected HeLa cells with GFP-tagged wild-type (WT) or indicated phosphomutants of Ykt6. Cells were treated with ionomycin (1μM) and/or Tacrolimus (1μM) for 30 minutes. Nuclei (blue) are stained with DAPI. Scale bar is 10μm. (B) Quantification of cells with GFP plasma membrane localization as shown in (A). N=3 *p

    Journal: bioRxiv

    Article Title: A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

    doi: 10.1101/2020.03.15.992727

    Figure Lengend Snippet: The intracellular localization of Ykt6 is dependent on calcineurin activity. (A) Representative immunofluorescence images of transiently transfected HeLa cells with GFP-tagged wild-type (WT) or indicated phosphomutants of Ykt6. Cells were treated with ionomycin (1μM) and/or Tacrolimus (1μM) for 30 minutes. Nuclei (blue) are stained with DAPI. Scale bar is 10μm. (B) Quantification of cells with GFP plasma membrane localization as shown in (A). N=3 *p

    Article Snippet: For Western Blot the following primary antibodies were used: GFP (Santa Cruz, sc-9996), actin (Abcam, ab6276), Ykt6 (Abcam, ab236583), LC3B (Cell Signaling, #2775), p62 (Sigma, p0067), STX17 (Sigma, hpa001204), SNAP29 (Abcam, ab138500) Na+/K+ ATPase (Sigma, 06-172-I), alpha/beta-Tubulin (Cell Signaling, 2148S), and STX7 (Bethyl, A304-512A).

    Techniques: Activity Assay, Immunofluorescence, Transfection, Staining

    Phosphorylation within the evolutionarily conserved site in Ykt6 SNARE domain regulates its conformation. (A) Representative western blot for Ykt6 from GFP-purified WT and phosphomutants of human Ykt6 incubated with the indicated amounts of trypsin for 1 hour at 25°C. Arrows indicate specific cleavage products. (B) Densitometry analysis of the 15 kDa cleavage band. N=3 *p

    Journal: bioRxiv

    Article Title: A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

    doi: 10.1101/2020.03.15.992727

    Figure Lengend Snippet: Phosphorylation within the evolutionarily conserved site in Ykt6 SNARE domain regulates its conformation. (A) Representative western blot for Ykt6 from GFP-purified WT and phosphomutants of human Ykt6 incubated with the indicated amounts of trypsin for 1 hour at 25°C. Arrows indicate specific cleavage products. (B) Densitometry analysis of the 15 kDa cleavage band. N=3 *p

    Article Snippet: For Western Blot the following primary antibodies were used: GFP (Santa Cruz, sc-9996), actin (Abcam, ab6276), Ykt6 (Abcam, ab236583), LC3B (Cell Signaling, #2775), p62 (Sigma, p0067), STX17 (Sigma, hpa001204), SNAP29 (Abcam, ab138500) Na+/K+ ATPase (Sigma, 06-172-I), alpha/beta-Tubulin (Cell Signaling, 2148S), and STX7 (Bethyl, A304-512A).

    Techniques: Western Blot, Purification, Incubation

    Phosphorylation sites in Ykt6 SNARE domain are sensitive to calcineurin and evolutionarily conserved in yeast and in humans. (A) Fold phosphorylation of the indicated peptides from endogenous yeast Ykt6 detected by shotgun phosphoproteomics after correction for protein abundance from control yeast cells and yeast cells with high levels of Ca 2+ (driven by overexpression of α-syn) with either WT or knockout for calcineurin (ΔCaN) and knockout for modulator of calcineurin (ΔFKBP12). The identified phosphorylation sites are highlighted in red. Data from triplicate samples was pulled together for illustrated analysis 28 . (B) Alignment of Ykt6 SNARE domain sequences across species. Serines highlighted in pink are the identified phosphorylation site conserved across evolution. Arrows depicted represent Ykt6 0-layer arginine (white) and additional calcineurin-sensitive sites identified in phosphoproteomic screen (red is human; black is yeast). (C) Animal and fungal Ykt6 protein sequences by sequence logos obtained from Tracey database and aligned. The residues associated with evolutionary conserved positions are shown. The calcineurin-dependent phosphorylation sites retrieved from the mass spectrometry screens are shown in green. (D) Wild type yeast cells were spotted onto plates containing synthetic defined (SD)-Leu media; episomal Ykt6-Leu selective, and replica plated in five-fold serial dilutions onto episomal Ykt6-inducing plates [Galactose (SGal)-Leu; episomal-Leu selective: empty vector, Wild type (WT), phospho-ablative (S176A) and/or phospho-mimetic (S176D)]. Representative plate of n=3. (E) Ykt6 temperature sensitive yeast strain were spotted onto plates containing synthetic defined (SD)-Leu media; episomal Ykt6-Leu selective, and replica plated in five-fold serial dilutions onto episomal Ykt6-inducing plates [Galactose (SGal)-Leu; episomal-Leu selective: empty vector, Wild type (WT), phospho-ablative (S176A) and/or phospho-mimetic (S176D)]. Endogenous Ykt6 is depleted by incubating the cells at 37°C, the non-permissive temperature. Representative plate of n=3. (F) Fold phosphorylation of the indicated human Ykt6 peptides from HEK293T cells as detected by iTRAQ mass spectrometry. Prior to GFP-Ykt6 immunoprecipitation, cells were treated for 30 minutes with the Ca 2+ ionophore ionomycin (1μM) and co-treated with calcineurin-specific inhibitor Tacrolimus (1μM).

    Journal: bioRxiv

    Article Title: A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

    doi: 10.1101/2020.03.15.992727

    Figure Lengend Snippet: Phosphorylation sites in Ykt6 SNARE domain are sensitive to calcineurin and evolutionarily conserved in yeast and in humans. (A) Fold phosphorylation of the indicated peptides from endogenous yeast Ykt6 detected by shotgun phosphoproteomics after correction for protein abundance from control yeast cells and yeast cells with high levels of Ca 2+ (driven by overexpression of α-syn) with either WT or knockout for calcineurin (ΔCaN) and knockout for modulator of calcineurin (ΔFKBP12). The identified phosphorylation sites are highlighted in red. Data from triplicate samples was pulled together for illustrated analysis 28 . (B) Alignment of Ykt6 SNARE domain sequences across species. Serines highlighted in pink are the identified phosphorylation site conserved across evolution. Arrows depicted represent Ykt6 0-layer arginine (white) and additional calcineurin-sensitive sites identified in phosphoproteomic screen (red is human; black is yeast). (C) Animal and fungal Ykt6 protein sequences by sequence logos obtained from Tracey database and aligned. The residues associated with evolutionary conserved positions are shown. The calcineurin-dependent phosphorylation sites retrieved from the mass spectrometry screens are shown in green. (D) Wild type yeast cells were spotted onto plates containing synthetic defined (SD)-Leu media; episomal Ykt6-Leu selective, and replica plated in five-fold serial dilutions onto episomal Ykt6-inducing plates [Galactose (SGal)-Leu; episomal-Leu selective: empty vector, Wild type (WT), phospho-ablative (S176A) and/or phospho-mimetic (S176D)]. Representative plate of n=3. (E) Ykt6 temperature sensitive yeast strain were spotted onto plates containing synthetic defined (SD)-Leu media; episomal Ykt6-Leu selective, and replica plated in five-fold serial dilutions onto episomal Ykt6-inducing plates [Galactose (SGal)-Leu; episomal-Leu selective: empty vector, Wild type (WT), phospho-ablative (S176A) and/or phospho-mimetic (S176D)]. Endogenous Ykt6 is depleted by incubating the cells at 37°C, the non-permissive temperature. Representative plate of n=3. (F) Fold phosphorylation of the indicated human Ykt6 peptides from HEK293T cells as detected by iTRAQ mass spectrometry. Prior to GFP-Ykt6 immunoprecipitation, cells were treated for 30 minutes with the Ca 2+ ionophore ionomycin (1μM) and co-treated with calcineurin-specific inhibitor Tacrolimus (1μM).

    Article Snippet: For Western Blot the following primary antibodies were used: GFP (Santa Cruz, sc-9996), actin (Abcam, ab6276), Ykt6 (Abcam, ab236583), LC3B (Cell Signaling, #2775), p62 (Sigma, p0067), STX17 (Sigma, hpa001204), SNAP29 (Abcam, ab138500) Na+/K+ ATPase (Sigma, 06-172-I), alpha/beta-Tubulin (Cell Signaling, 2148S), and STX7 (Bethyl, A304-512A).

    Techniques: Over Expression, Knock-Out, Sequencing, Mass Spectrometry, Plasmid Preparation, Immunoprecipitation

    Ykt6 binding affinity for STX17, SNAP29 and Vamp8 is dependent on the phosphorylation at the evolutionarily conserved site in the SNARE domain. (A) Representative western blot of GFP immunoprecipitations from HEK293T cells expressing either GFP, wild-type (WT) GFP-Ykt6 or GFP-Ykt6 phosphomutants. Cells were treated for 2 hours (2h) in growing conditions (fresh 10% FBS and 4.5% glucose growth medium) or in starvation conditions (1X HBSS supplemented with 250nM Torin-1 and 10mM HEPES). (B) Quantification of STX17 and SNAP29 (A) relative to the efficiency of the pull down from each condition and normalized to WT Ykt6. N=3 *p

    Journal: bioRxiv

    Article Title: A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

    doi: 10.1101/2020.03.15.992727

    Figure Lengend Snippet: Ykt6 binding affinity for STX17, SNAP29 and Vamp8 is dependent on the phosphorylation at the evolutionarily conserved site in the SNARE domain. (A) Representative western blot of GFP immunoprecipitations from HEK293T cells expressing either GFP, wild-type (WT) GFP-Ykt6 or GFP-Ykt6 phosphomutants. Cells were treated for 2 hours (2h) in growing conditions (fresh 10% FBS and 4.5% glucose growth medium) or in starvation conditions (1X HBSS supplemented with 250nM Torin-1 and 10mM HEPES). (B) Quantification of STX17 and SNAP29 (A) relative to the efficiency of the pull down from each condition and normalized to WT Ykt6. N=3 *p

    Article Snippet: For Western Blot the following primary antibodies were used: GFP (Santa Cruz, sc-9996), actin (Abcam, ab6276), Ykt6 (Abcam, ab236583), LC3B (Cell Signaling, #2775), p62 (Sigma, p0067), STX17 (Sigma, hpa001204), SNAP29 (Abcam, ab138500) Na+/K+ ATPase (Sigma, 06-172-I), alpha/beta-Tubulin (Cell Signaling, 2148S), and STX7 (Bethyl, A304-512A).

    Techniques: Binding Assay, Western Blot, Expressing

    Semi-automatic quantification of seeded wtHtt ex1 aggregates. (A and B) Semi-automatic image processing workflow for high-magnification confocal z-stacks of DA1 glomeruli from 14 day-old adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and either (A1,2) Htt ex1 Q25-GFP or (B1,2) mCD8-GFP in GH146+ PNs. Raw data were preprocessed by deconvolution to reduce noise ( top panels ), segmented in the mCherry (A1 and B1) or GFP (A2 and B2) channels ( middle panels ), and filtered for co-localizing fluorescence signal in the other channel ( bottom panels ). Arrows in (A1 and A2) indicate seven Htt ex1 Q91+Htt ex1 Q25 puncta identified by this method. Scale bars = 10 μm. (C1-7) Selected single 0.35 μm confocal z-slices from the same confocal stack shown in (A1 and A2). Slice number is indicated at the top right of each image. Individual Htt ex1 Q91+Htt ex1 Q25 puncta identified by semi-automated image segmentation in (A) are indicated with arrows ( yellow in individual channels, white on merged images) in each slice. Two additional co-localized Htt ex1 Q91+Htt ex1 Q25 puncta identified by manual counting are indicated with asterisks in (A2). Scale bars = 10 μm. Insets show Htt ex1 Q91+Htt ex1 Q25 puncta at higher zoom (inset dimensions = 9.12 μm x 9.12 μm). Htt ex1 Q91-mCherry ( red ) and Htt ex1 Q25-GFP ( green ) fluorescence intensity profiles for lines indicated in merged insets are shown below images. Lines were scanned from leftmost to rightmost point. (D) Comparison of manual quantification (C) vs semi-automated segmentation approaches (A1 and A2) for 14 day-old males with the same genotype in (A and C). Data are shown as mean ± SEM; n.s. = not significant by one-way ANOVA followed by Tukey’s multiple comparisons test.

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: Semi-automatic quantification of seeded wtHtt ex1 aggregates. (A and B) Semi-automatic image processing workflow for high-magnification confocal z-stacks of DA1 glomeruli from 14 day-old adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and either (A1,2) Htt ex1 Q25-GFP or (B1,2) mCD8-GFP in GH146+ PNs. Raw data were preprocessed by deconvolution to reduce noise ( top panels ), segmented in the mCherry (A1 and B1) or GFP (A2 and B2) channels ( middle panels ), and filtered for co-localizing fluorescence signal in the other channel ( bottom panels ). Arrows in (A1 and A2) indicate seven Htt ex1 Q91+Htt ex1 Q25 puncta identified by this method. Scale bars = 10 μm. (C1-7) Selected single 0.35 μm confocal z-slices from the same confocal stack shown in (A1 and A2). Slice number is indicated at the top right of each image. Individual Htt ex1 Q91+Htt ex1 Q25 puncta identified by semi-automated image segmentation in (A) are indicated with arrows ( yellow in individual channels, white on merged images) in each slice. Two additional co-localized Htt ex1 Q91+Htt ex1 Q25 puncta identified by manual counting are indicated with asterisks in (A2). Scale bars = 10 μm. Insets show Htt ex1 Q91+Htt ex1 Q25 puncta at higher zoom (inset dimensions = 9.12 μm x 9.12 μm). Htt ex1 Q91-mCherry ( red ) and Htt ex1 Q25-GFP ( green ) fluorescence intensity profiles for lines indicated in merged insets are shown below images. Lines were scanned from leftmost to rightmost point. (D) Comparison of manual quantification (C) vs semi-automated segmentation approaches (A1 and A2) for 14 day-old males with the same genotype in (A and C). Data are shown as mean ± SEM; n.s. = not significant by one-way ANOVA followed by Tukey’s multiple comparisons test.

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Expressing, Fluorescence

    Gal80-mediated repression of Gal4 in glia or RNAi knockdown of drpr in PNs do not alter ORN-to-PN prion-like transfer of mHtt ex1 aggregates. (A and B) Confocal stacks of DA1 glomeruli from 10-14 day-old adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs with (A) no additional transgenes (“control”) or (B) repo-Gal80 to inhibit Gal4-mediated expression of Htt ex1 Q25-GFP in glia. (C and D) Confocal z-stacks of DA1 glomeruli from 14 day-old adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and co-expressing Htt ex1 Q25-GFP together with firefly luciferase (FFLuc; C) or dsRNA targeting draper (Drpr RNAi ; D) in GH146+ PNs. In (A-D), mCherry+ surfaces identified by semi-automated image segmentation are shown in the last panels, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (E and F) Quantification of Htt ex1 Q91 (E) and Htt ex1 Q91+Htt ex1 Q25 (F) aggregates for genotypes shown in (A-D). Data are shown as mean ± SEM; n.s. = not significant by one-way ANOVA with Tukey’s multiple comparisons tests.

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: Gal80-mediated repression of Gal4 in glia or RNAi knockdown of drpr in PNs do not alter ORN-to-PN prion-like transfer of mHtt ex1 aggregates. (A and B) Confocal stacks of DA1 glomeruli from 10-14 day-old adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs with (A) no additional transgenes (“control”) or (B) repo-Gal80 to inhibit Gal4-mediated expression of Htt ex1 Q25-GFP in glia. (C and D) Confocal z-stacks of DA1 glomeruli from 14 day-old adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and co-expressing Htt ex1 Q25-GFP together with firefly luciferase (FFLuc; C) or dsRNA targeting draper (Drpr RNAi ; D) in GH146+ PNs. In (A-D), mCherry+ surfaces identified by semi-automated image segmentation are shown in the last panels, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (E and F) Quantification of Htt ex1 Q91 (E) and Htt ex1 Q91+Htt ex1 Q25 (F) aggregates for genotypes shown in (A-D). Data are shown as mean ± SEM; n.s. = not significant by one-way ANOVA with Tukey’s multiple comparisons tests.

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Expressing, Luciferase

    mHtt ex1 aggregates generated in ORNs do not co-localize with markers of lysosomes or autophagosomes in glia. (A1-2 and B1-2) Single confocal slices from DA1 glomeruli of 4-5 day-old adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and either the autophagosomal marker Atg8a-GFP (A1 and A2) or the lysosomal marker GFP-Lamp1 (B1 and B2) in repo+ glia. Scale bars = 10 μm. Htt ex1 Q91-mCherry (red) and Atg8a- or Lamp1-GFP (green) fluorescence intensity profiles for lines indicated in (A1-2 and B1-2) are shown below images; a.u. = arbitrary units. Lines were scanned from leftmost to rightmost point. (C and D) Confocal stacks of DA1 glomeruli from 4-5 day-old adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and either Atg8a- GFP (C) or GFP-Lamp1 (D) in repo+ glia. mCherry+ surfaces identified by semi-automated image segmentation are shown in the last panels, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+GFP surfaces in yellow. Scale bars = 10 μm. (E and F) Quantification of Htt ex1 Q91 (E) and Htt ex1 Q91+GFP (F) surfaces for the same genotypes as in (C and D) and for control animals expressing mCD8-GFP in glia. Data are shown as mean ± SEM; n.s. = not significant by one-way ANOVA with Tukey’s multiple comparisons test.

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: mHtt ex1 aggregates generated in ORNs do not co-localize with markers of lysosomes or autophagosomes in glia. (A1-2 and B1-2) Single confocal slices from DA1 glomeruli of 4-5 day-old adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and either the autophagosomal marker Atg8a-GFP (A1 and A2) or the lysosomal marker GFP-Lamp1 (B1 and B2) in repo+ glia. Scale bars = 10 μm. Htt ex1 Q91-mCherry (red) and Atg8a- or Lamp1-GFP (green) fluorescence intensity profiles for lines indicated in (A1-2 and B1-2) are shown below images; a.u. = arbitrary units. Lines were scanned from leftmost to rightmost point. (C and D) Confocal stacks of DA1 glomeruli from 4-5 day-old adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and either Atg8a- GFP (C) or GFP-Lamp1 (D) in repo+ glia. mCherry+ surfaces identified by semi-automated image segmentation are shown in the last panels, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+GFP surfaces in yellow. Scale bars = 10 μm. (E and F) Quantification of Htt ex1 Q91 (E) and Htt ex1 Q91+GFP (F) surfaces for the same genotypes as in (C and D) and for control animals expressing mCD8-GFP in glia. Data are shown as mean ± SEM; n.s. = not significant by one-way ANOVA with Tukey’s multiple comparisons test.

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Generated, Expressing, Marker, Fluorescence

    mHtt ex1 aggregates do not transfer retrogradely from PN dendrites to ORN axons. Single confocal slices (A1-3 and B1-3) and confocal z-stacks (A4 and B4) of the antennal lobe from 7 (A) and 14 (B) day-old adult females expressing Htt ex1 Q91-mCherry in ∼60% of PNs using GH146-QF and Htt ex1 Q25-GFP in all ORNs using pebbled-Gal4 . Dissected brains were immunostained with antibodies against mCherry ( red ), GFP ( green ), and the neuropil marker Bruchpilot ( blue , shown in merged images). GFP+ puncta identified in single slices are indicated by open arrowheads; none of these were found to be mCherry+. Semi-automated segmentation of the Htt ex1 Q91-mCherry fluorescent signal (“merge surfaces” in A4 and B4) identified numerous Htt ex1 Q91 aggregates (graphed in C) throughout the antennal lobe neuropil and surrounding region. A small number of Htt ex1 Q91+Htt ex1 Q25 surfaces (graphed in D) were identified in these brains (arrows in A4 and B4); however, none of these were located within the boundaries of the antennal lobe. Scale bars = 10 μm; slice numbers are indicated at the top right in (A1-3 and B1-3). Quantified data in (C and D) are shown as mean ± SEM; n.s. = not significant by one-way ANOVA with Tukey’s multiple comparisons test.

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: mHtt ex1 aggregates do not transfer retrogradely from PN dendrites to ORN axons. Single confocal slices (A1-3 and B1-3) and confocal z-stacks (A4 and B4) of the antennal lobe from 7 (A) and 14 (B) day-old adult females expressing Htt ex1 Q91-mCherry in ∼60% of PNs using GH146-QF and Htt ex1 Q25-GFP in all ORNs using pebbled-Gal4 . Dissected brains were immunostained with antibodies against mCherry ( red ), GFP ( green ), and the neuropil marker Bruchpilot ( blue , shown in merged images). GFP+ puncta identified in single slices are indicated by open arrowheads; none of these were found to be mCherry+. Semi-automated segmentation of the Htt ex1 Q91-mCherry fluorescent signal (“merge surfaces” in A4 and B4) identified numerous Htt ex1 Q91 aggregates (graphed in C) throughout the antennal lobe neuropil and surrounding region. A small number of Htt ex1 Q91+Htt ex1 Q25 surfaces (graphed in D) were identified in these brains (arrows in A4 and B4); however, none of these were located within the boundaries of the antennal lobe. Scale bars = 10 μm; slice numbers are indicated at the top right in (A1-3 and B1-3). Quantified data in (C and D) are shown as mean ± SEM; n.s. = not significant by one-way ANOVA with Tukey’s multiple comparisons test.

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Expressing, Marker

    wtHtt ex1 aggregates in postsynaptic PNs are nucleated by mHtt ex1 or mHtt ex1-12 aggregates from presynaptic ORNs. (A-D) High-magnification confocal z-stacks of DA1 glomeruli from adult flies expressing Htt ex1 Q91-mCherry (A and B) or RFP-Htt ex1-12 Q138 (C and D) in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs. Raw data (A1-3, B1-3, C1-3, D1-3) are shown adjacent to surfaces identified by 3D segmentation of the red (A1’, B1’, C1’, D1’) or green (A2’, B2’, C2’, D2’) channels. Htt ex1 Q25-GFP surfaces are shown at 50% transparency in “merged surfaces” images (A3’, B3’, C3’, D3’) for visibility of co-localized Htt ex1 Q91-mCherry or RFP-Htt ex1-12 Q138 surfaces. Scale bars = 1 μm. (E1-4) A single confocal slice through the center of a Htt ex1 Q91+Htt ex1 Q25 aggregate before (E1, E2) and after (E1’, E2’) mCherry acceptor photobleaching. Data are shown as a heat map to highlight changes in fluorescence intensities after photobleaching. Scale bar = 1 μm. FRET efficiency (FRET eff ) for this aggregate is shown in (E4), and average FRET eff values for all Htt ex1 Q91+Htt ex1 Q25 aggregates tested are shown in (E3). (F) Volumes of Htt ex1 Q91 ( solid red boxes ), Htt ex1 Q91+Htt ex1 Q25 ( solid green boxes ), Htt ex1-12 Q138 ( striped red boxes ), and Htt ex1-12 Q138+Htt ex1 Q25 ( striped green boxes ) aggregates identified in the DA1 glomerulus at the indicated ages. Box widths indicate interquartile ranges, vertical lines inside each box indicate medians, whiskers indicate minimums/maximums, and “+”s indicate means for each data set. *p

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: wtHtt ex1 aggregates in postsynaptic PNs are nucleated by mHtt ex1 or mHtt ex1-12 aggregates from presynaptic ORNs. (A-D) High-magnification confocal z-stacks of DA1 glomeruli from adult flies expressing Htt ex1 Q91-mCherry (A and B) or RFP-Htt ex1-12 Q138 (C and D) in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs. Raw data (A1-3, B1-3, C1-3, D1-3) are shown adjacent to surfaces identified by 3D segmentation of the red (A1’, B1’, C1’, D1’) or green (A2’, B2’, C2’, D2’) channels. Htt ex1 Q25-GFP surfaces are shown at 50% transparency in “merged surfaces” images (A3’, B3’, C3’, D3’) for visibility of co-localized Htt ex1 Q91-mCherry or RFP-Htt ex1-12 Q138 surfaces. Scale bars = 1 μm. (E1-4) A single confocal slice through the center of a Htt ex1 Q91+Htt ex1 Q25 aggregate before (E1, E2) and after (E1’, E2’) mCherry acceptor photobleaching. Data are shown as a heat map to highlight changes in fluorescence intensities after photobleaching. Scale bar = 1 μm. FRET efficiency (FRET eff ) for this aggregate is shown in (E4), and average FRET eff values for all Htt ex1 Q91+Htt ex1 Q25 aggregates tested are shown in (E3). (F) Volumes of Htt ex1 Q91 ( solid red boxes ), Htt ex1 Q91+Htt ex1 Q25 ( solid green boxes ), Htt ex1-12 Q138 ( striped red boxes ), and Htt ex1-12 Q138+Htt ex1 Q25 ( striped green boxes ) aggregates identified in the DA1 glomerulus at the indicated ages. Box widths indicate interquartile ranges, vertical lines inside each box indicate medians, whiskers indicate minimums/maximums, and “+”s indicate means for each data set. *p

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Expressing, Fluorescence

    Controls for prion-like transmission of mHtt ex1 aggregates from presynaptic DA1 ORNs to postsynaptic PNs. (A-D) Confocal z-stacks of DA1 glomeruli from 10 day- old adults expressing (A) Htt ex1 Q25-mCherry in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs, (B) Htt ex1 Q91-mCherry in DA1 ORNs and mCD8-GFP in GH146+ PNs, (C) Htt ex1 Q91-mCherry together with Gal80 in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs, and (D) Htt ex1 Q91-mCherry in DA1 ORNs and Htt ex1 Q25-GFP together with QS in GH146+ PNs. mCherry+ surfaces identified by semi-automated image segmentation are shown in the last panels, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (E and F) Quantification of (E) Httex1Q91 and (F) Htt ex1 Q91+Htt ex1 Q25 aggregates in the DA1 glomeruli of flies with genotypes shown in (A-D). Data are shown as mean ± SEM. *p

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: Controls for prion-like transmission of mHtt ex1 aggregates from presynaptic DA1 ORNs to postsynaptic PNs. (A-D) Confocal z-stacks of DA1 glomeruli from 10 day- old adults expressing (A) Htt ex1 Q25-mCherry in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs, (B) Htt ex1 Q91-mCherry in DA1 ORNs and mCD8-GFP in GH146+ PNs, (C) Htt ex1 Q91-mCherry together with Gal80 in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs, and (D) Htt ex1 Q91-mCherry in DA1 ORNs and Htt ex1 Q25-GFP together with QS in GH146+ PNs. mCherry+ surfaces identified by semi-automated image segmentation are shown in the last panels, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (E and F) Quantification of (E) Httex1Q91 and (F) Htt ex1 Q91+Htt ex1 Q25 aggregates in the DA1 glomeruli of flies with genotypes shown in (A-D). Data are shown as mean ± SEM. *p

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Transmission Assay, Expressing

    mHtt ex1 aggregate transfer from ORNs to synaptically-connected PNs is inversely correlated with presynaptic activity. (A, B, E, F, I, and J) Confocal z-stacks of DA1 glomeruli from 10 day-old males (A and B, and E and F) or 7 day-old females (I and J) co-expressing Htt ex1 Q91-mCherry with either LacZ (A, E, and I), shi ts1 (B), TeTxLC (F), or dTrpA (J) in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs. In (A-B), flies were raised at the permissive temperature (18°C) and shifted to the restrictive temperature (31°C) upon eclosion, and in (I-J), flies were raised at room temperature (∼21°C) and shifted to 31°C upon eclosion. mCherry+ surfaces identified by semi-automated image segmentation are shown in the last panels, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (C and D, G and H, and K and L) Quantification of Htt ex1 Q91 (C, G, and K) and Htt ex1 Q91+Htt ex1 Q25 (D, H, and L) aggregates identified in DA1 glomeruli from adult males of the indicated ages co-expressing Htt ex1 Q91-mCherry with LacZ or shi ts1 using two independent QUAS-shi ts1 lines in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs (C and D), adult males of the indicated ages co-expressing Htt ex1 Q91-mCherry with LacZ or TeTxLC using two independent QUAS-TeTxLC lines in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs (G and H), and 7 day-old females expressing Htt ex1 Q91-mCherry with either LacZ or dTrpA using three independent QUAS-dTrpA lines in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs (K and L). Data are shown as mean ± SEM; *p

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: mHtt ex1 aggregate transfer from ORNs to synaptically-connected PNs is inversely correlated with presynaptic activity. (A, B, E, F, I, and J) Confocal z-stacks of DA1 glomeruli from 10 day-old males (A and B, and E and F) or 7 day-old females (I and J) co-expressing Htt ex1 Q91-mCherry with either LacZ (A, E, and I), shi ts1 (B), TeTxLC (F), or dTrpA (J) in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs. In (A-B), flies were raised at the permissive temperature (18°C) and shifted to the restrictive temperature (31°C) upon eclosion, and in (I-J), flies were raised at room temperature (∼21°C) and shifted to 31°C upon eclosion. mCherry+ surfaces identified by semi-automated image segmentation are shown in the last panels, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (C and D, G and H, and K and L) Quantification of Htt ex1 Q91 (C, G, and K) and Htt ex1 Q91+Htt ex1 Q25 (D, H, and L) aggregates identified in DA1 glomeruli from adult males of the indicated ages co-expressing Htt ex1 Q91-mCherry with LacZ or shi ts1 using two independent QUAS-shi ts1 lines in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs (C and D), adult males of the indicated ages co-expressing Htt ex1 Q91-mCherry with LacZ or TeTxLC using two independent QUAS-TeTxLC lines in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs (G and H), and 7 day-old females expressing Htt ex1 Q91-mCherry with either LacZ or dTrpA using three independent QUAS-dTrpA lines in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs (K and L). Data are shown as mean ± SEM; *p

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Activity Assay, Expressing

    mHtt ex1 nucleates prion-like conversion of wtHtt ex1 in fly neurons. (A-E) Confocal z-stacks of 14 day-old adult female brains expressing (A) Htt ex1 Q91-mCherry or (B) Htt ex1 Q25-GFP alone, or co-expressing Htt ex1 Q91-mCherry with (C) Htt ex1 Q25-GFP, (D) membrane-targeted mCD8-GFP, or (E) soluble GFP in all neurons using elav[C155]-Gal4 . Dimensions of each confocal stack are 250 x 250 x ∼60 ( xyz ) μm. Merged images include DAPI signal ( blue ) to label nuclei. Scale bars = 20 μm. (F) Colocalization of mCherry and GFP fluorescent signals calculated as Pearson’s correlation coefficients for genotypes shown in (A-E). Data are shown as mean ± SEM; ****p

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: mHtt ex1 nucleates prion-like conversion of wtHtt ex1 in fly neurons. (A-E) Confocal z-stacks of 14 day-old adult female brains expressing (A) Htt ex1 Q91-mCherry or (B) Htt ex1 Q25-GFP alone, or co-expressing Htt ex1 Q91-mCherry with (C) Htt ex1 Q25-GFP, (D) membrane-targeted mCD8-GFP, or (E) soluble GFP in all neurons using elav[C155]-Gal4 . Dimensions of each confocal stack are 250 x 250 x ∼60 ( xyz ) μm. Merged images include DAPI signal ( blue ) to label nuclei. Scale bars = 20 μm. (F) Colocalization of mCherry and GFP fluorescent signals calculated as Pearson’s correlation coefficients for genotypes shown in (A-E). Data are shown as mean ± SEM; ****p

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Expressing

    Inhibiting Shibire-mediated endocytosis increases mHttex1-expressing ORN axon volume and enhances transfer of mHtt ex1 aggregates from DA1 ORN axons to glia. (A and B) Confocal z-stacks of DA1 glomeruli from 10 day-old females co-expressing Htt ex1 Q91-mCherry, mCD8- GFP, and either LacZ (A) or shi ts1 (B) in DA1 ORNs. Flies were shifted from the permissive temperature (18°C) to the restrictive temperature (31°C) upon eclosion. Raw data are shown in grayscale, and 3D segmented surfaces are shown in red for Htt ex1 Q91 and as a heat map for mCD8-GFP to highlight differences in intensity between the genotypes. Scale bars = 10 μm. (C) Quantification of mCD8-GFP intensity (left y-axis, green ) and volume (right y-axis, black ) of DA1 glomeruli from 10 day-old adult females co-expressing LacZ or shi ts1 with Htt ex1 Q91-mCherry and mCD8-GFP in DA1 ORNs. a.u. = arbitrary units. Data are shown as mean ± SEM; ****p

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: Inhibiting Shibire-mediated endocytosis increases mHttex1-expressing ORN axon volume and enhances transfer of mHtt ex1 aggregates from DA1 ORN axons to glia. (A and B) Confocal z-stacks of DA1 glomeruli from 10 day-old females co-expressing Htt ex1 Q91-mCherry, mCD8- GFP, and either LacZ (A) or shi ts1 (B) in DA1 ORNs. Flies were shifted from the permissive temperature (18°C) to the restrictive temperature (31°C) upon eclosion. Raw data are shown in grayscale, and 3D segmented surfaces are shown in red for Htt ex1 Q91 and as a heat map for mCD8-GFP to highlight differences in intensity between the genotypes. Scale bars = 10 μm. (C) Quantification of mCD8-GFP intensity (left y-axis, green ) and volume (right y-axis, black ) of DA1 glomeruli from 10 day-old adult females co-expressing LacZ or shi ts1 with Htt ex1 Q91-mCherry and mCD8-GFP in DA1 ORNs. a.u. = arbitrary units. Data are shown as mean ± SEM; ****p

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Expressing

    mHtt ex1 or mHtt ex1-12 aggregates formed in presynaptic ORNs induce the aggregation of wtHtt ex1 expressed in postsynaptic PNs. (A) Primary structure of full-length human Htt (3144 amino acids), including HEAT repeats (HR, gray regions ) and the N-terminal variable-length polyQ region ( green/red box ), with the pathogenic threshold (∼Q37) indicated by a white dotted line. C-termini of two N-terminal mHtt fragments used in this study (Htt ex1 and Htt ex1-12 ) are indicated. (B) Overall experimental approach. In the fly olfactory system, ORNs synapse with PNs in discrete regions of the antennal lobe known as glomeruli ( gray circles ). PNs send axons into higher brain centers (i.e., mushroom body and/or lateral horn). Draper-expressing glial cells project processes in the antennal lobe, where they ensheath individual glomeruli. To monitor spreading of mHtt aggregates between synaptically-connected ORNs and PNs, we generated transgenic flies that express mHtt ex1 or mHtt ex1-12 fragments in DA1 ORNs and wtHtt ex1 in DA1 PNs. Inset: Transfer of mHtt ex1 or mHtt ex1-12 aggregates between ORNs and PNs was assessed by monitoring the solubility and colocalization of mHtt and wtHtt fluorescent signals. (C and D) Maximum intensity z-projections of antennal lobes from 7 day-old adult males expressing either Htt ex1 Q25-mCherry (C) or Htt ex1 Q91-mCherry (D) in DA1 ORNs using Or67d-QF and Htt ex1 Q25-GFP in GH146+ PNs using GH146-Gal4 . Raw data are shown in grayscale for individual channels and pseudocolored in merged images. Merged images include Bruchpilot immunofluorescence in blue to mark neuropil, which was used to approximate the boundaries of the DA1 glomerulus (white dotted lines). Scale bars = 20 μm. (E-G) High-magnification confocal z-stacks of DA1 glomeruli from 1 day-old (E), 14 day-old (F), and 21 day-old (G) adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs. Boxed regions in (F and G) are shown at higher magnification in insets. Raw data are shown in grayscale in individual channels (Htt ex1 Q91: E1, F1, G1; Htt ex1 Q25: E2, F2, G2) and pseudocolored in merged images (E3, F3, G3). mCherry+ “Htt ex1 Q91 surfaces” (E1’, F1’, G1’) and “Htt ex1 Q91+Htt ex1 Q25 surfaces” (E2’, F2’, G2’) identified by semi-automated image segmentation are shown adjacent to raw data and pseudocolored red and yellow, respectively, in the “merged surfaces” images (E3’, F3’, G3’). Arrows ( yellow on grayscale images, white on merged images) indicate Htt ex1 Q91+Htt ex1 Q25 surfaces. Scale bars = 10 μm. (H and I) Confocal z-stacks from 1 day-old (H) and 21 day-old (I) adult females expressing RFP-Htt ex1-12 Q138 in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs. Boxed region in (I) is shown at higher magnification in insets. RFP+ surfaces identified by semi-automated image segmentation are shown in the last column, with Htt ex1-12 Q138-only surfaces in red and Htt ex1-12 Q138+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (J) and (K) Numbers of Htt ex1 Q91 or Htt ex1-12 Q138 (”mHtt”) surfaces (J) and Htt ex1 Q91+Htt ex1 Q25 or Htt ex1-12 Q138+Htt ex1 Q25 (“mHtt+wtHtt”) surfaces (K) identified in adult males ( open bars ) or females ( solid bars ) expressing Htt ex1 Q91-mCherry in DA1 ORNs or adult females expressing RFP-Htt ex1-12 Q138 in DA1 ORNs ( striped bars ) at the indicated ages. Data are shown as mean ± SEM; *p

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: mHtt ex1 or mHtt ex1-12 aggregates formed in presynaptic ORNs induce the aggregation of wtHtt ex1 expressed in postsynaptic PNs. (A) Primary structure of full-length human Htt (3144 amino acids), including HEAT repeats (HR, gray regions ) and the N-terminal variable-length polyQ region ( green/red box ), with the pathogenic threshold (∼Q37) indicated by a white dotted line. C-termini of two N-terminal mHtt fragments used in this study (Htt ex1 and Htt ex1-12 ) are indicated. (B) Overall experimental approach. In the fly olfactory system, ORNs synapse with PNs in discrete regions of the antennal lobe known as glomeruli ( gray circles ). PNs send axons into higher brain centers (i.e., mushroom body and/or lateral horn). Draper-expressing glial cells project processes in the antennal lobe, where they ensheath individual glomeruli. To monitor spreading of mHtt aggregates between synaptically-connected ORNs and PNs, we generated transgenic flies that express mHtt ex1 or mHtt ex1-12 fragments in DA1 ORNs and wtHtt ex1 in DA1 PNs. Inset: Transfer of mHtt ex1 or mHtt ex1-12 aggregates between ORNs and PNs was assessed by monitoring the solubility and colocalization of mHtt and wtHtt fluorescent signals. (C and D) Maximum intensity z-projections of antennal lobes from 7 day-old adult males expressing either Htt ex1 Q25-mCherry (C) or Htt ex1 Q91-mCherry (D) in DA1 ORNs using Or67d-QF and Htt ex1 Q25-GFP in GH146+ PNs using GH146-Gal4 . Raw data are shown in grayscale for individual channels and pseudocolored in merged images. Merged images include Bruchpilot immunofluorescence in blue to mark neuropil, which was used to approximate the boundaries of the DA1 glomerulus (white dotted lines). Scale bars = 20 μm. (E-G) High-magnification confocal z-stacks of DA1 glomeruli from 1 day-old (E), 14 day-old (F), and 21 day-old (G) adult males expressing Htt ex1 Q91-mCherry in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs. Boxed regions in (F and G) are shown at higher magnification in insets. Raw data are shown in grayscale in individual channels (Htt ex1 Q91: E1, F1, G1; Htt ex1 Q25: E2, F2, G2) and pseudocolored in merged images (E3, F3, G3). mCherry+ “Htt ex1 Q91 surfaces” (E1’, F1’, G1’) and “Htt ex1 Q91+Htt ex1 Q25 surfaces” (E2’, F2’, G2’) identified by semi-automated image segmentation are shown adjacent to raw data and pseudocolored red and yellow, respectively, in the “merged surfaces” images (E3’, F3’, G3’). Arrows ( yellow on grayscale images, white on merged images) indicate Htt ex1 Q91+Htt ex1 Q25 surfaces. Scale bars = 10 μm. (H and I) Confocal z-stacks from 1 day-old (H) and 21 day-old (I) adult females expressing RFP-Htt ex1-12 Q138 in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs. Boxed region in (I) is shown at higher magnification in insets. RFP+ surfaces identified by semi-automated image segmentation are shown in the last column, with Htt ex1-12 Q138-only surfaces in red and Htt ex1-12 Q138+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (J) and (K) Numbers of Htt ex1 Q91 or Htt ex1-12 Q138 (”mHtt”) surfaces (J) and Htt ex1 Q91+Htt ex1 Q25 or Htt ex1-12 Q138+Htt ex1 Q25 (“mHtt+wtHtt”) surfaces (K) identified in adult males ( open bars ) or females ( solid bars ) expressing Htt ex1 Q91-mCherry in DA1 ORNs or adult females expressing RFP-Htt ex1-12 Q138 in DA1 ORNs ( striped bars ) at the indicated ages. Data are shown as mean ± SEM; *p

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Expressing, Generated, Transgenic Assay, Solubility, Immunofluorescence

    Draper mediates mHtt ex1 aggregate transfer from presynaptic DA1 ORNs to postsynaptic PNs and regulates neuronal mHtt ex1 aggregate size. (A and B) Confocal z-stacks of DA1 glomeruli from 13 day-old adult females expressing Htt ex1 Q91-mCherry in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs, either heterozygous (A; drpr +/- ) or homozygous (B; drpr -/- ) for the drpr Δ5 null allele. mCherry+ surfaces identified by semi-automated image segmentation are shown in the last row, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (C and D) Quantification of Htt ex1 Q91 (C) and Htt ex1 Q91+Htt ex1 Q25 (D) aggregates in DA1 glomeruli from female or male drpr +/- or drpr -/- flies at the indicated ages. Data are shown as mean ± SEM; *p

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: Draper mediates mHtt ex1 aggregate transfer from presynaptic DA1 ORNs to postsynaptic PNs and regulates neuronal mHtt ex1 aggregate size. (A and B) Confocal z-stacks of DA1 glomeruli from 13 day-old adult females expressing Htt ex1 Q91-mCherry in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs, either heterozygous (A; drpr +/- ) or homozygous (B; drpr -/- ) for the drpr Δ5 null allele. mCherry+ surfaces identified by semi-automated image segmentation are shown in the last row, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (C and D) Quantification of Htt ex1 Q91 (C) and Htt ex1 Q91+Htt ex1 Q25 (D) aggregates in DA1 glomeruli from female or male drpr +/- or drpr -/- flies at the indicated ages. Data are shown as mean ± SEM; *p

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Expressing

    Draper is required for enhanced transfer of mHtt ex1 from shi ts1 - expressing DA1 ORNs to GH146+ PNs. (A and B) Confocal stacks of DA1 glomeruli from 7 day-old drpr +/+ (A) or drpr -/- (B) adult females co-expressing Htt ex1 Q91-mCherry with shi ts1 in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs. Adult flies were shifted from 18°C to 31°C upon eclosion. mCherry+ surfaces identified by semi-automated image segmentation are shown in the last panels, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (C and D) Quantification of Htt ex1 Q91 (C) and Htt ex1 Q91+Htt ex1 Q25 (D) aggregates for the same genotypes shown in (A and B). Data are shown as mean ± SEM; ****p

    Journal: bioRxiv

    Article Title: Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

    doi: 10.1101/868448

    Figure Lengend Snippet: Draper is required for enhanced transfer of mHtt ex1 from shi ts1 - expressing DA1 ORNs to GH146+ PNs. (A and B) Confocal stacks of DA1 glomeruli from 7 day-old drpr +/+ (A) or drpr -/- (B) adult females co-expressing Htt ex1 Q91-mCherry with shi ts1 in DA1 ORNs and Htt ex1 Q25-GFP in GH146+ PNs. Adult flies were shifted from 18°C to 31°C upon eclosion. mCherry+ surfaces identified by semi-automated image segmentation are shown in the last panels, with Htt ex1 Q91-only surfaces in red and Htt ex1 Q91+Htt ex1 Q25 surfaces in yellow. Scale bars = 10 μm. (C and D) Quantification of Htt ex1 Q91 (C) and Htt ex1 Q91+Htt ex1 Q25 (D) aggregates for the same genotypes shown in (A and B). Data are shown as mean ± SEM; ****p

    Article Snippet: Primary antibodies used in this study include rabbit anti-DsRed (1:2000; #632496; Takara Bio USA, Inc., Mountain View, CA), rabbit anti-mCherry (1:500; #PA5-34974; Invitrogen, Carlsbad, CA), chicken anti-GFP (1:500; #GFP-1020; Aves Labs, Tigard, OR), chicken anti-GFP (1:1000; #ab13970; Abcam, Cambridge, UK), chicken anti-GFP (1:500; #A10262; Invitrogen, Carlsbad, CA), rat anti-HA (1:100; clone 3F10; #11867423001; Roche, Basel, Switzerland), rabbit anti-cleaved Dcp-1 (1:100; #9578S; Cell Signaling Technology, Danvers, MA), and mouse anti-Bruchpilot (1:100; clone nc82; Developmental Studies Hybridoma Bank, Iowa City, IA).

    Techniques: Expressing

    DHHC5 internalization is required for activity-induced δ-catenin trafficking. ( a , b ) HEK293T cells were transfected with control shRNA (shRNA-c) or shRNA against DHHC5 (shRNA) plus the indicated HA-DHHC5 constructs (*shRNA resistance) and blots probed with the indicated antibodies ( P =0.024, F 3,8 =5.484, n =3 blots from 3 separate cultures; one-way analysis of variance (ANOVA)). ( c ) Confocal images of 14 DIV hippocampal neurons transfected with GFP–δ-catenin and the indicated shRNA and HA-DHHC5* constructs. Cells were stimulated with cLTP (+Gly) or control buffer lacking glycine (–Gly), fixed 20 min after stimulation and immunostained with the indicated antibodies. Scale bar, 5 μm. ( d ) The cLTP-induced increase in δ-catenin/PSD-95 co-localization is abolished in DHHC5 knockdown cells or those expressing the DHHC5 Y533E mutant ( P

    Journal: Nature Communications

    Article Title: Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5

    doi: 10.1038/ncomms9200

    Figure Lengend Snippet: DHHC5 internalization is required for activity-induced δ-catenin trafficking. ( a , b ) HEK293T cells were transfected with control shRNA (shRNA-c) or shRNA against DHHC5 (shRNA) plus the indicated HA-DHHC5 constructs (*shRNA resistance) and blots probed with the indicated antibodies ( P =0.024, F 3,8 =5.484, n =3 blots from 3 separate cultures; one-way analysis of variance (ANOVA)). ( c ) Confocal images of 14 DIV hippocampal neurons transfected with GFP–δ-catenin and the indicated shRNA and HA-DHHC5* constructs. Cells were stimulated with cLTP (+Gly) or control buffer lacking glycine (–Gly), fixed 20 min after stimulation and immunostained with the indicated antibodies. Scale bar, 5 μm. ( d ) The cLTP-induced increase in δ-catenin/PSD-95 co-localization is abolished in DHHC5 knockdown cells or those expressing the DHHC5 Y533E mutant ( P

    Article Snippet: Antibodies and complementary DNA constructs Primary antibodies used were as follows: δ-catenin (1:500 western blot (WB), 5 μg immunoprecipitation (IP); BD Transduction Laboratories 611536), N-cadherin (1:500; BD Transduction Laboratories 610921), PSD-95 for immunocytochemistry (ICC; 1:500; Abcam ab2723), PSD-95 for IP and WB (5 μg, 1:500; Calbiochem CP35), Gephyrin (1:500; Synaptic Systems 147 011), GFP for IP (10 μl; Synaptic Systems 132 002), GFP for WB (1:1,000; Roche 11814460001), DHHC5 (1:500 ICC, 1:1,000 WB, 1 μg IP; Sigma Prestige HPA014670), TfR (1:500; Millipore GR09L), VPS-35 (1:500; Abnova H000055737-M02), GluA1 (1:1,000; Millipore 05-855R), haemagglutinin (HA) for ICC (1:500; Cell Signaling Technology C29F4), HA for IP and WB (5 μg, 1:500; Covance MMS-101P), VGlut1 (1:500; Millipore AB5905), Fyn for WB and ICC (1:250, 1:500; BD Transduction Laboratories 610163), Fyn for IP (5 μg; Life Technologies MA5-13134), non-phosphorylated Y420 Fyn (1:1,000; Cell Signaling Technologies 2102S), STEP61 (1:1,000; Life Technologies MA1-16746), phospho-tyrosine (phY; 1:1,000 WB, 5 μg IP; Millipore 4G10/05-321), AP2μ (1:500; Thermo Scientific PA5-20745) and β-actin (1:1,000; Sigma A1978).

    Techniques: Activity Assay, Transfection, shRNA, Construct, Expressing, Mutagenesis

    Activity enhances DHHC5 trafficking from spines. ( a ) Confocal images of 14 DIV neurons demonstrating partial co-localization of GFP–DHHC5 and PSD-95. ( b ) High-magnification confocal images of GFP–DHHC5 fluorescence (lower panels pseudocolored as a heat map) and DsRed before and after glycine stimulation. ( c ) GFP–DHHC5 fluorescence decreases transiently within spines after glycine stimulation ( P

    Journal: Nature Communications

    Article Title: Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5

    doi: 10.1038/ncomms9200

    Figure Lengend Snippet: Activity enhances DHHC5 trafficking from spines. ( a ) Confocal images of 14 DIV neurons demonstrating partial co-localization of GFP–DHHC5 and PSD-95. ( b ) High-magnification confocal images of GFP–DHHC5 fluorescence (lower panels pseudocolored as a heat map) and DsRed before and after glycine stimulation. ( c ) GFP–DHHC5 fluorescence decreases transiently within spines after glycine stimulation ( P

    Article Snippet: Antibodies and complementary DNA constructs Primary antibodies used were as follows: δ-catenin (1:500 western blot (WB), 5 μg immunoprecipitation (IP); BD Transduction Laboratories 611536), N-cadherin (1:500; BD Transduction Laboratories 610921), PSD-95 for immunocytochemistry (ICC; 1:500; Abcam ab2723), PSD-95 for IP and WB (5 μg, 1:500; Calbiochem CP35), Gephyrin (1:500; Synaptic Systems 147 011), GFP for IP (10 μl; Synaptic Systems 132 002), GFP for WB (1:1,000; Roche 11814460001), DHHC5 (1:500 ICC, 1:1,000 WB, 1 μg IP; Sigma Prestige HPA014670), TfR (1:500; Millipore GR09L), VPS-35 (1:500; Abnova H000055737-M02), GluA1 (1:1,000; Millipore 05-855R), haemagglutinin (HA) for ICC (1:500; Cell Signaling Technology C29F4), HA for IP and WB (5 μg, 1:500; Covance MMS-101P), VGlut1 (1:500; Millipore AB5905), Fyn for WB and ICC (1:250, 1:500; BD Transduction Laboratories 610163), Fyn for IP (5 μg; Life Technologies MA5-13134), non-phosphorylated Y420 Fyn (1:1,000; Cell Signaling Technologies 2102S), STEP61 (1:1,000; Life Technologies MA1-16746), phospho-tyrosine (phY; 1:1,000 WB, 5 μg IP; Millipore 4G10/05-321), AP2μ (1:500; Thermo Scientific PA5-20745) and β-actin (1:1,000; Sigma A1978).

    Techniques: Activity Assay, Fluorescence

    Phosphorylation of DHHC5 regulates its association with endocytic proteins and its subcellular localization. ( a ) Schematic depiction of DHHC5 constructs N-terminally tagged with GFP or HA (not shown here) and illustrating the approximate localization of transmembrane domains (grey boxes), the DHHC motif, a putative Fyn-binding site (dashed line; RLLPTGP), a putative AP2μ-binding site (dashed line; YDNL) and the PDZ-binding motif required for binding PSD-95 (solid line; EISV). ( b – e ) HEK293T cells were transfected with the indicated HA-DHHC5 and Fyn constructs for 36 h, lysates immunoprecipitated with an HA antibody and blots probed with the indicated antibodies. ( b , c ) Fyn binding of the DHHC5 P520,3A mutant is reduced, but not for for the Y533E mutant ( P =0.0153, F 2,6 =9.07). ( b , d ) Fyn-mediated tyrosine phosphorylation is attenuated in DHHC5 P520,3A and Y533E mutants ( P

    Journal: Nature Communications

    Article Title: Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5

    doi: 10.1038/ncomms9200

    Figure Lengend Snippet: Phosphorylation of DHHC5 regulates its association with endocytic proteins and its subcellular localization. ( a ) Schematic depiction of DHHC5 constructs N-terminally tagged with GFP or HA (not shown here) and illustrating the approximate localization of transmembrane domains (grey boxes), the DHHC motif, a putative Fyn-binding site (dashed line; RLLPTGP), a putative AP2μ-binding site (dashed line; YDNL) and the PDZ-binding motif required for binding PSD-95 (solid line; EISV). ( b – e ) HEK293T cells were transfected with the indicated HA-DHHC5 and Fyn constructs for 36 h, lysates immunoprecipitated with an HA antibody and blots probed with the indicated antibodies. ( b , c ) Fyn binding of the DHHC5 P520,3A mutant is reduced, but not for for the Y533E mutant ( P =0.0153, F 2,6 =9.07). ( b , d ) Fyn-mediated tyrosine phosphorylation is attenuated in DHHC5 P520,3A and Y533E mutants ( P

    Article Snippet: Antibodies and complementary DNA constructs Primary antibodies used were as follows: δ-catenin (1:500 western blot (WB), 5 μg immunoprecipitation (IP); BD Transduction Laboratories 611536), N-cadherin (1:500; BD Transduction Laboratories 610921), PSD-95 for immunocytochemistry (ICC; 1:500; Abcam ab2723), PSD-95 for IP and WB (5 μg, 1:500; Calbiochem CP35), Gephyrin (1:500; Synaptic Systems 147 011), GFP for IP (10 μl; Synaptic Systems 132 002), GFP for WB (1:1,000; Roche 11814460001), DHHC5 (1:500 ICC, 1:1,000 WB, 1 μg IP; Sigma Prestige HPA014670), TfR (1:500; Millipore GR09L), VPS-35 (1:500; Abnova H000055737-M02), GluA1 (1:1,000; Millipore 05-855R), haemagglutinin (HA) for ICC (1:500; Cell Signaling Technology C29F4), HA for IP and WB (5 μg, 1:500; Covance MMS-101P), VGlut1 (1:500; Millipore AB5905), Fyn for WB and ICC (1:250, 1:500; BD Transduction Laboratories 610163), Fyn for IP (5 μg; Life Technologies MA5-13134), non-phosphorylated Y420 Fyn (1:1,000; Cell Signaling Technologies 2102S), STEP61 (1:1,000; Life Technologies MA1-16746), phospho-tyrosine (phY; 1:1,000 WB, 5 μg IP; Millipore 4G10/05-321), AP2μ (1:500; Thermo Scientific PA5-20745) and β-actin (1:1,000; Sigma A1978).

    Techniques: Construct, Binding Assay, Transfection, Immunoprecipitation, Mutagenesis

    PSD-95 and Fyn control DHHC5 turnover in spine heads. ( a , c , e ) High-magnification confocal images of 14–16 DIV hippocampal neurons transfected with the indicated GFP–DHHC5, DsRed, PSD-95–RFP or Fyn constructs. GFP–DHHC5 fluorescence within a photobleached ROI (red circles) was analysed over 300 s (cells were initially photobleached at 0 s, white asterisks, within a 1-μm diameter ROI). Scale bar, 1 μm. ( b , d , f ) Relative fluorescence recovery of GFP–DHHC5. Solid lines represent single exponential fit. Points with error bars represent the mean±s.e.m. Statistical tests compare the plateau values from exponential fits±s.e.m. Neurons were obtained from three to five separate cultures. ( a , b ) Overexpression of PSD-95 significantly reduces the mobility of DHHC5 WT, but not ΔPDZb ( P

    Journal: Nature Communications

    Article Title: Activity-regulated trafficking of the palmitoyl-acyl transferase DHHC5

    doi: 10.1038/ncomms9200

    Figure Lengend Snippet: PSD-95 and Fyn control DHHC5 turnover in spine heads. ( a , c , e ) High-magnification confocal images of 14–16 DIV hippocampal neurons transfected with the indicated GFP–DHHC5, DsRed, PSD-95–RFP or Fyn constructs. GFP–DHHC5 fluorescence within a photobleached ROI (red circles) was analysed over 300 s (cells were initially photobleached at 0 s, white asterisks, within a 1-μm diameter ROI). Scale bar, 1 μm. ( b , d , f ) Relative fluorescence recovery of GFP–DHHC5. Solid lines represent single exponential fit. Points with error bars represent the mean±s.e.m. Statistical tests compare the plateau values from exponential fits±s.e.m. Neurons were obtained from three to five separate cultures. ( a , b ) Overexpression of PSD-95 significantly reduces the mobility of DHHC5 WT, but not ΔPDZb ( P

    Article Snippet: Antibodies and complementary DNA constructs Primary antibodies used were as follows: δ-catenin (1:500 western blot (WB), 5 μg immunoprecipitation (IP); BD Transduction Laboratories 611536), N-cadherin (1:500; BD Transduction Laboratories 610921), PSD-95 for immunocytochemistry (ICC; 1:500; Abcam ab2723), PSD-95 for IP and WB (5 μg, 1:500; Calbiochem CP35), Gephyrin (1:500; Synaptic Systems 147 011), GFP for IP (10 μl; Synaptic Systems 132 002), GFP for WB (1:1,000; Roche 11814460001), DHHC5 (1:500 ICC, 1:1,000 WB, 1 μg IP; Sigma Prestige HPA014670), TfR (1:500; Millipore GR09L), VPS-35 (1:500; Abnova H000055737-M02), GluA1 (1:1,000; Millipore 05-855R), haemagglutinin (HA) for ICC (1:500; Cell Signaling Technology C29F4), HA for IP and WB (5 μg, 1:500; Covance MMS-101P), VGlut1 (1:500; Millipore AB5905), Fyn for WB and ICC (1:250, 1:500; BD Transduction Laboratories 610163), Fyn for IP (5 μg; Life Technologies MA5-13134), non-phosphorylated Y420 Fyn (1:1,000; Cell Signaling Technologies 2102S), STEP61 (1:1,000; Life Technologies MA1-16746), phospho-tyrosine (phY; 1:1,000 WB, 5 μg IP; Millipore 4G10/05-321), AP2μ (1:500; Thermo Scientific PA5-20745) and β-actin (1:1,000; Sigma A1978).

    Techniques: Transfection, Construct, Fluorescence, Over Expression

    Loss of retromer leads to a pronounced shift in RAB 7 distribution Parental HeLa cells, two clonal VPS35 knockout cell lines, and one clonal VPS29 KO cell line were fixed in PFA and co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Co‐localization was analyzed across three independent experiments. A clonal RAB7a knockout cell line was mixed 1:1 with parental HeLa cells and seeded onto coverslips. Following PFA fixation, the mixed cells were stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Note that the RAB7a signal completely disappears in the cells not expressing RAB7a. Parental HeLa cells and clonal VPS35 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red, upper panel) or for endogenous RAB7a and a mCherry‐tagged ER marker (red, lower panel), and co‐localization between RAB7a and the respective marker was analyzed across two independent experiments. To show that RAB7a localizes to the ER and mitochondria, endogenous TOM20 (blue) was co‐stained in the lower panel. Parental HeLa cells and clonal VPS35 and VPS29 KO cells were transduced with a lentivirus expressing GFP‐FIS1TM (eGFP with a C‐terminal mitochondrial targeting sequence and transmembrane domain of the mitochondrial protein FIS1) and disrupted through a fine needle in detergent‐free sucrose buffer followed by isolation of the mitochondria from postnuclear supernatants with GFP‐trap agarose beads. The amount of RAB7 precipitating with the mitochondria was quantified over four independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Loss of retromer leads to a pronounced shift in RAB 7 distribution Parental HeLa cells, two clonal VPS35 knockout cell lines, and one clonal VPS29 KO cell line were fixed in PFA and co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Co‐localization was analyzed across three independent experiments. A clonal RAB7a knockout cell line was mixed 1:1 with parental HeLa cells and seeded onto coverslips. Following PFA fixation, the mixed cells were stained for endogenous RAB7a (green) and endogenous LAMP2 (red). Note that the RAB7a signal completely disappears in the cells not expressing RAB7a. Parental HeLa cells and clonal VPS35 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red, upper panel) or for endogenous RAB7a and a mCherry‐tagged ER marker (red, lower panel), and co‐localization between RAB7a and the respective marker was analyzed across two independent experiments. To show that RAB7a localizes to the ER and mitochondria, endogenous TOM20 (blue) was co‐stained in the lower panel. Parental HeLa cells and clonal VPS35 and VPS29 KO cells were transduced with a lentivirus expressing GFP‐FIS1TM (eGFP with a C‐terminal mitochondrial targeting sequence and transmembrane domain of the mitochondrial protein FIS1) and disrupted through a fine needle in detergent‐free sucrose buffer followed by isolation of the mitochondria from postnuclear supernatants with GFP‐trap agarose beads. The amount of RAB7 precipitating with the mitochondria was quantified over four independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Knock-Out, Staining, Expressing, Marker, Transduction, Sequencing, Isolation

    RAB 7 and retromer are a functional unit at the interface between endosomes and lysosomes HeLa cells fixed in methanol were co‐stained for the indicated endogenous proteins. Green signal is Alexa 488, and red signal is Alexa 594. Co‐staining of endogenous VPS35 and the late endosome marker LBPA (left) and the late endosome/lysosome marker LAMP2. Note that VPS35 seems to be less associated with LBPA‐positive domains than with LAMP2‐decorated entities. 3D reconstruction of PFA‐fixed single cells expressing GFP‐RAB5 and co‐stained for endogenous LAMP2 (Alexa 405, blue) and endogenous VPS35 or FAM21 (Alexa 594, red). HeLa cells transfected with siRNA targeting VPS35 and RAB7a were co‐stained for the endogenous glucose transporter GLUT1 (Alexa 594, red) and the lysosomal marker LAMP1 (Alexa 488, green). Efficiency of the siRNA was tested by Western blotting. Co‐localization was analyzed over two independent experiments with 10 images each. Data information: All scale bars = 10 μm besides lower panel in (A), where scale bar = 5 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: RAB 7 and retromer are a functional unit at the interface between endosomes and lysosomes HeLa cells fixed in methanol were co‐stained for the indicated endogenous proteins. Green signal is Alexa 488, and red signal is Alexa 594. Co‐staining of endogenous VPS35 and the late endosome marker LBPA (left) and the late endosome/lysosome marker LAMP2. Note that VPS35 seems to be less associated with LBPA‐positive domains than with LAMP2‐decorated entities. 3D reconstruction of PFA‐fixed single cells expressing GFP‐RAB5 and co‐stained for endogenous LAMP2 (Alexa 405, blue) and endogenous VPS35 or FAM21 (Alexa 594, red). HeLa cells transfected with siRNA targeting VPS35 and RAB7a were co‐stained for the endogenous glucose transporter GLUT1 (Alexa 594, red) and the lysosomal marker LAMP1 (Alexa 488, green). Efficiency of the siRNA was tested by Western blotting. Co‐localization was analyzed over two independent experiments with 10 images each. Data information: All scale bars = 10 μm besides lower panel in (A), where scale bar = 5 μm, all error bars = SD, and * P

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Functional Assay, Staining, Marker, Expressing, Transfection, Western Blot

    Retromer controls RAB 7 activity levels Parental HeLa cells and clonal VPS35 KO cells were infected with lentiviruses expressing inactive GFP‐RAB7‐T22N (A) or constitutively active GFP‐RAB7‐Q67L (B) and co‐stained for endogenous TOM20 (red). Co‐localization was analyzed across two independent experiments. RILP effector assay comparing VPS35 KO cells and VPS35 KO cells that had been infected with a lentivirus encoding untagged human VPS35. Immunofluorescent staining showing that lentivirally expressed VPS29‐myc restores vesicular VPS29. RILP effector assay with lysates from parental HeLa cells and VPS35 KO cells and lysates from VPS35 KO cells that were spiked with recombinant VPS35 produced in bacteria. Note that re‐addition of VPS35 has no effect on the amount of active RAB7 binding to the GST‐RILP beads. Quantitative proteomic interactome analysis of GFP‐RAB7 isolated from parental and VPS35 KO cell lines. The SILAC ratios (from two independent experiments with swapped isotope labeling) for the indicated proteins suggest that RAB7 is more active in the knockout cells, as the RAB7 effector RILP is increased in the RAB7 IP from KO cells while the RAB chaperones GDI1, GDI2, and PRA1 are strongly decreased. HeLa cells were fixed in cold methanol and stained for endogenous RAB7a and LAMP2. The same cell and same Z ‐position was then imaged at low‐laser settings (left) and at high‐laser setting on a confocal microscope. Data information: All scale bars = 10 μm, and all error bars = SD. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Retromer controls RAB 7 activity levels Parental HeLa cells and clonal VPS35 KO cells were infected with lentiviruses expressing inactive GFP‐RAB7‐T22N (A) or constitutively active GFP‐RAB7‐Q67L (B) and co‐stained for endogenous TOM20 (red). Co‐localization was analyzed across two independent experiments. RILP effector assay comparing VPS35 KO cells and VPS35 KO cells that had been infected with a lentivirus encoding untagged human VPS35. Immunofluorescent staining showing that lentivirally expressed VPS29‐myc restores vesicular VPS29. RILP effector assay with lysates from parental HeLa cells and VPS35 KO cells and lysates from VPS35 KO cells that were spiked with recombinant VPS35 produced in bacteria. Note that re‐addition of VPS35 has no effect on the amount of active RAB7 binding to the GST‐RILP beads. Quantitative proteomic interactome analysis of GFP‐RAB7 isolated from parental and VPS35 KO cell lines. The SILAC ratios (from two independent experiments with swapped isotope labeling) for the indicated proteins suggest that RAB7 is more active in the knockout cells, as the RAB7 effector RILP is increased in the RAB7 IP from KO cells while the RAB chaperones GDI1, GDI2, and PRA1 are strongly decreased. HeLa cells were fixed in cold methanol and stained for endogenous RAB7a and LAMP2. The same cell and same Z ‐position was then imaged at low‐laser settings (left) and at high‐laser setting on a confocal microscope. Data information: All scale bars = 10 μm, and all error bars = SD. Source data are available online for this figure.

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Activity Assay, Infection, Expressing, Staining, Recombinant, Produced, Binding Assay, Isolation, Labeling, Knock-Out, Microscopy

    RAB 7 localizes to mitochondria and the ER All images show formaldehyde‐fixed HeLa cells. HeLa cells stained for endogenous RAB7a (green) and endogenous LAMP2 (red). White arrows indicate sites of vesicular RAB7 that appears to be budding from a network of RAB7. Immunofluorescent co‐staining of endogenous RAB7a (green) and the mitochondria marker TOM20 (red). Co‐staining of endogenous RAB7 (green) and the trans ‐Golgi network marker TGN46 (red). Co‐staining of endogenous RAB7 (green) and the ER marker protein disulfide isomerase (PDI, red). Co‐staining of lentivirally expressed GFP‐RAB7a and endogenous TOM20 (red). Co‐staining of endogenous RAB7 (green) and TOM20 (red) in cells treated with Cas9 and a mixture of three gRNAs targeting the RAB7a locus. The Western blot of this cell population shows an almost complete loss of RAB7 in the treated cells compared to parental HeLa cells. GFP‐RAB7 was expressed in RAB7a KO HeLa cells and imaged in live cells using a spinning disk confocal microscope. Mitochondria were visualized with MitoTracker Red. Parental HeLa cells and clonal VPS29 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red), and co‐localization was analyzed across two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: RAB 7 localizes to mitochondria and the ER All images show formaldehyde‐fixed HeLa cells. HeLa cells stained for endogenous RAB7a (green) and endogenous LAMP2 (red). White arrows indicate sites of vesicular RAB7 that appears to be budding from a network of RAB7. Immunofluorescent co‐staining of endogenous RAB7a (green) and the mitochondria marker TOM20 (red). Co‐staining of endogenous RAB7 (green) and the trans ‐Golgi network marker TGN46 (red). Co‐staining of endogenous RAB7 (green) and the ER marker protein disulfide isomerase (PDI, red). Co‐staining of lentivirally expressed GFP‐RAB7a and endogenous TOM20 (red). Co‐staining of endogenous RAB7 (green) and TOM20 (red) in cells treated with Cas9 and a mixture of three gRNAs targeting the RAB7a locus. The Western blot of this cell population shows an almost complete loss of RAB7 in the treated cells compared to parental HeLa cells. GFP‐RAB7 was expressed in RAB7a KO HeLa cells and imaged in live cells using a spinning disk confocal microscope. Mitochondria were visualized with MitoTracker Red. Parental HeLa cells and clonal VPS29 KO cells were co‐stained for endogenous RAB7a (green) and endogenous TOM20 (red), and co‐localization was analyzed across two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Staining, Marker, Western Blot, Microscopy

    Hyperactive RAB 7 results in ATG 9a trafficking and autophagosome formation defects mCherry‐Parkin‐expressing parental HeLa cells, VPS35 KO cells, and RAB7 KO cells transduced with untagged RAB7‐Q67L were treated with CCCP for 5 h, fixed in PFA, and co‐stained for endogenous ATG9a (green) and the TGN marker TGN46 (blue). Co‐localization between ATG9a and TGN46 was analyzed across 14 images acquired in two independent experiments. Parental HeLa cells and VPS35 KO cells were transduced with GFP‐RAB7 and mCherry‐Parkin and treated with CCCP for 5 h. The cells were then fixed in PFA and stained for endogenous ATG9a (blue). Co‐localization between GFP‐RAB7 and ATG9a was quantified across 14 images acquired in two independent experiments. mCherry‐Parkin‐expressing parental HeLa cells and RAB7 KO cells transduced with untagged RAB7‐Q67L were treated with CCCP for 5 h (upper panel) and 8 h (lower panel), fixed in methanol, and stained for endogenous TOM20 (blue) and endogenous LC3b (green). Co‐localization between LC3b and TOM20 was quantified across 14 images acquired in two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Hyperactive RAB 7 results in ATG 9a trafficking and autophagosome formation defects mCherry‐Parkin‐expressing parental HeLa cells, VPS35 KO cells, and RAB7 KO cells transduced with untagged RAB7‐Q67L were treated with CCCP for 5 h, fixed in PFA, and co‐stained for endogenous ATG9a (green) and the TGN marker TGN46 (blue). Co‐localization between ATG9a and TGN46 was analyzed across 14 images acquired in two independent experiments. Parental HeLa cells and VPS35 KO cells were transduced with GFP‐RAB7 and mCherry‐Parkin and treated with CCCP for 5 h. The cells were then fixed in PFA and stained for endogenous ATG9a (blue). Co‐localization between GFP‐RAB7 and ATG9a was quantified across 14 images acquired in two independent experiments. mCherry‐Parkin‐expressing parental HeLa cells and RAB7 KO cells transduced with untagged RAB7‐Q67L were treated with CCCP for 5 h (upper panel) and 8 h (lower panel), fixed in methanol, and stained for endogenous TOM20 (blue) and endogenous LC3b (green). Co‐localization between LC3b and TOM20 was quantified across 14 images acquired in two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Expressing, Transduction, Staining, Marker

    RAB 7 activity state controls its localization to the ER , mitochondria, and lysosomes Mitochondria were purified from detergent‐free postnuclear supernatants using magnetic beads coated with a TOM22‐specific antibody from parental HeLa cells and from clonal VPS35 KO cells. The purified mitochondria were then subjected to a marker analysis using the indicated organelle markers. Note that only mitochondria (TOM20) and RAB7 are enriched over the inputs, with lower levels of RAB7 precipitating with the mitochondria of VPS35 KO cells. The quantification of RAB7 was done across three independent mitochondria isolations. RAB7a KO cells transduced with the indicated GFP‐RAB7a variants were fixed and co‐stained for endogenous LAMP2. Note that the inactive RAB7 (T22N) does not localize to lysosomes, whereas the hyperactive variant (Q67L) fully localizes to lysosomes. RAB7a KO cells transduced with the indicated GFP‐RAB7a variants and a mCherry‐ER marker were fixed and co‐stained for endogenous TOM20. RAB7a KO cells were infected with the indicated GFP‐RAB7 variants, labeled with MitoTracker Red, and subjected to live cell imaging using a spinning disk confocal microscope. Data information: All scale bars = 10 μm, and all error bars = SD. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: RAB 7 activity state controls its localization to the ER , mitochondria, and lysosomes Mitochondria were purified from detergent‐free postnuclear supernatants using magnetic beads coated with a TOM22‐specific antibody from parental HeLa cells and from clonal VPS35 KO cells. The purified mitochondria were then subjected to a marker analysis using the indicated organelle markers. Note that only mitochondria (TOM20) and RAB7 are enriched over the inputs, with lower levels of RAB7 precipitating with the mitochondria of VPS35 KO cells. The quantification of RAB7 was done across three independent mitochondria isolations. RAB7a KO cells transduced with the indicated GFP‐RAB7a variants were fixed and co‐stained for endogenous LAMP2. Note that the inactive RAB7 (T22N) does not localize to lysosomes, whereas the hyperactive variant (Q67L) fully localizes to lysosomes. RAB7a KO cells transduced with the indicated GFP‐RAB7a variants and a mCherry‐ER marker were fixed and co‐stained for endogenous TOM20. RAB7a KO cells were infected with the indicated GFP‐RAB7 variants, labeled with MitoTracker Red, and subjected to live cell imaging using a spinning disk confocal microscope. Data information: All scale bars = 10 μm, and all error bars = SD. Source data are available online for this figure.

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Activity Assay, Purification, Magnetic Beads, Marker, Transduction, Staining, Variant Assay, Infection, Labeling, Live Cell Imaging, Microscopy

    Control of RAB 7 activity is required for efficient removal of mitochondria through mitophagy All image panels show PFA‐fixed HeLa cells. mCherry‐Parkin‐transduced parental HeLa cells, VPS35 KO cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were incubated with CCCP over 4 h, followed by staining of endogenous RAB7a (green) and TOM20 (blue). Co‐localization between RAB7 and TOM20 as well as RAB7 and mCherry‐Parkin was quantified over two independent experiments. RAB7a KO cells transduced with mCherry‐Parkin and GFP‐RAB7 or GFP‐RAB7‐Q67L were treated with CCCP for 4 h, followed by co‐staining with endogenous TOM20 (blue). Co‐localization between the GFP‐RAB7 variants and TOM20 was quantified over two independent experiments. Parental HeLa cells, RAB7 KO cells, and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs were treated with CCCP for 16 h, followed by staining of endogenous TOM20 (blue). The residual TOM20 signal after CCCP treatment was quantified over two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Control of RAB 7 activity is required for efficient removal of mitochondria through mitophagy All image panels show PFA‐fixed HeLa cells. mCherry‐Parkin‐transduced parental HeLa cells, VPS35 KO cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were incubated with CCCP over 4 h, followed by staining of endogenous RAB7a (green) and TOM20 (blue). Co‐localization between RAB7 and TOM20 as well as RAB7 and mCherry‐Parkin was quantified over two independent experiments. RAB7a KO cells transduced with mCherry‐Parkin and GFP‐RAB7 or GFP‐RAB7‐Q67L were treated with CCCP for 4 h, followed by co‐staining with endogenous TOM20 (blue). Co‐localization between the GFP‐RAB7 variants and TOM20 was quantified over two independent experiments. Parental HeLa cells, RAB7 KO cells, and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs were treated with CCCP for 16 h, followed by staining of endogenous TOM20 (blue). The residual TOM20 signal after CCCP treatment was quantified over two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Activity Assay, Transduction, Construct, Incubation, Staining

    Control of RAB 7 activity is not required for retromer‐based sorting of integral membrane proteins All images show formaldehyde‐fixed cells. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were co‐stained for endogenous GLUT1 (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were surface‐biotinylated, followed by streptavidin isolation and Western blot‐based quantification of biotinylated surface proteins. Surface GLUT1 was quantified over four independent experiments. RAB7a knockout cells and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs cells were co‐stained for endogenous GLUT1 (red) and endogenous LAMP2 (blue), and co‐localization was quantified over two independent experiments. Parental HeLa cells, RAB7a knockout cells, and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs were co‐stained for endogenous CI‐MPR (red) and endogenous TGN46 (blue). Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Control of RAB 7 activity is not required for retromer‐based sorting of integral membrane proteins All images show formaldehyde‐fixed cells. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were co‐stained for endogenous GLUT1 (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were surface‐biotinylated, followed by streptavidin isolation and Western blot‐based quantification of biotinylated surface proteins. Surface GLUT1 was quantified over four independent experiments. RAB7a knockout cells and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs cells were co‐stained for endogenous GLUT1 (red) and endogenous LAMP2 (blue), and co‐localization was quantified over two independent experiments. Parental HeLa cells, RAB7a knockout cells, and RAB7 KO cells transduced with the indicated GFP‐RAB7 rescue constructs were co‐stained for endogenous CI‐MPR (red) and endogenous TGN46 (blue). Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Activity Assay, Transduction, Construct, Staining, Isolation, Western Blot, Knock-Out

    Additional mitophagy data Parental HeLa cells and VPS29 and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ATG16L1 (upper row) and endogenous TOM20 (lower row). Parental HeLa cells and VPS29 and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ULK1 (lower row) and endogenous TOM20 (upper row). mCherry‐Parkin‐expressing parental HeLa cells, VPS35 KO cells, and RAB7a KO cells transduced with RAB7‐Q67L were fixed and stained for endogenous ATG9a (green) and TGN46 (blue). Parental HeLa cells and VPS35 KO cells were transduced with mCherry‐Parkin and GFP‐RAB7, followed by fixation and staining of endogenous ATG9a (blue). Co‐localization of ATG9a and GFP‐RAB7 was analyzed across 32 individual cells acquired in two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Additional mitophagy data Parental HeLa cells and VPS29 and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ATG16L1 (upper row) and endogenous TOM20 (lower row). Parental HeLa cells and VPS29 and VPS35 KO cells transduced with mCherry‐Parkin were treated with CCCP for the indicated time points, followed by staining of endogenous ULK1 (lower row) and endogenous TOM20 (upper row). mCherry‐Parkin‐expressing parental HeLa cells, VPS35 KO cells, and RAB7a KO cells transduced with RAB7‐Q67L were fixed and stained for endogenous ATG9a (green) and TGN46 (blue). Parental HeLa cells and VPS35 KO cells were transduced with mCherry‐Parkin and GFP‐RAB7, followed by fixation and staining of endogenous ATG9a (blue). Co‐localization of ATG9a and GFP‐RAB7 was analyzed across 32 individual cells acquired in two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Transduction, Staining, Expressing

    Retromer controls RAB 7 activity levels and mobility/membrane turnover GDP‐locked (inactive) GFP‐RAB7‐T22N and GTP‐locked (constitutively active) GFP‐RAB7‐Q67L were lentivirally expressed in RAB7 KO cells and co‐stained with the mitochondrial marker TOM20 (red). Co‐localization was analyzed over two independent experiments with 10 images each. Lysates from parental HeLa cells and clonal VPS35 KO cells were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lysates from parental HeLa cells and clonal VPS29 KO cells were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lysates from parental HeLa cells and clonal VPS29 KO cells and VPS29 KO cells with lentivirally re‐expressed VPS29‐myc were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lentivirally expressed GFP‐RAB7 was precipitated from parental and from VPS35 KO cells, and the precipitates were analyzed for the presence of the endogenous RAB‐chaperone GDI2. Lentivirally expressed GFP‐GDI1 was precipitated from parental and from VPS35 KO cells, and the precipitates were analyzed for the presence of endogenous RAB14 and endogenous RAB7a. GFP‐RAB7 was transduced into parental HeLa cells and VPS35 KO cells and analyzed for its mobility/membrane turnover using FRAP imaging in live cells. The recovery kinetics were obtained by averaging 15 FRAP recoveries acquired in two independent experiments. GFP‐RAB7 was transduced into parental HeLa cells and VPS35 KO cells and analyzed for its mobility/membrane turnover using FLIP imaging in live cells. The depletion kinetics (in area A, as indicated) were obtained by averaging 18 FLIP depletions acquired in two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Retromer controls RAB 7 activity levels and mobility/membrane turnover GDP‐locked (inactive) GFP‐RAB7‐T22N and GTP‐locked (constitutively active) GFP‐RAB7‐Q67L were lentivirally expressed in RAB7 KO cells and co‐stained with the mitochondrial marker TOM20 (red). Co‐localization was analyzed over two independent experiments with 10 images each. Lysates from parental HeLa cells and clonal VPS35 KO cells were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lysates from parental HeLa cells and clonal VPS29 KO cells were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lysates from parental HeLa cells and clonal VPS29 KO cells and VPS29 KO cells with lentivirally re‐expressed VPS29‐myc were probed with immobilized GST‐RILP protein, and GST‐RILP‐bound (active) RAB7a was detected and quantified by fluorescent Western blotting across three independent experiments. Lentivirally expressed GFP‐RAB7 was precipitated from parental and from VPS35 KO cells, and the precipitates were analyzed for the presence of the endogenous RAB‐chaperone GDI2. Lentivirally expressed GFP‐GDI1 was precipitated from parental and from VPS35 KO cells, and the precipitates were analyzed for the presence of endogenous RAB14 and endogenous RAB7a. GFP‐RAB7 was transduced into parental HeLa cells and VPS35 KO cells and analyzed for its mobility/membrane turnover using FRAP imaging in live cells. The recovery kinetics were obtained by averaging 15 FRAP recoveries acquired in two independent experiments. GFP‐RAB7 was transduced into parental HeLa cells and VPS35 KO cells and analyzed for its mobility/membrane turnover using FLIP imaging in live cells. The depletion kinetics (in area A, as indicated) were obtained by averaging 18 FLIP depletions acquired in two independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Activity Assay, Staining, Marker, Western Blot, Imaging

    TBC 1D5 and retromer cooperate in the control of RAB 7 activity and mobility GFP‐trap IPs of the indicated GFP‐tagged VPS29 (upper panel) or TBC1D5 (lower panel) constructs confirm that the VPS29‐L152E and the TBC1D5‐L142E mutant lose binding to each other. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments. Note that re‐expression of both VPS29 variants fully restores the level of endogenous VPS35. PFA‐fixed parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments. Parental HeLa cells and VPS29 KO cells as well as VPS29 KO cells transduced with the indicated VPS29 rescue constructs were transduced with GFP‐RAB7, and RAB7 mobility/turnover was analyzed by FRAP in living cells. The displayed recovery kinetics were obtained by averaging kinetics from fifteen FRAP recoveries per condition acquired in two independent experiments. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments. Parental HeLa cells and clonal TBC1D5 KO cells and TBC1D5 KO cells transduced with the indicated GFP‐TBC1D5 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: TBC 1D5 and retromer cooperate in the control of RAB 7 activity and mobility GFP‐trap IPs of the indicated GFP‐tagged VPS29 (upper panel) or TBC1D5 (lower panel) constructs confirm that the VPS29‐L152E and the TBC1D5‐L142E mutant lose binding to each other. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments. Note that re‐expression of both VPS29 variants fully restores the level of endogenous VPS35. PFA‐fixed parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs were co‐stained for endogenous RAB7a (green) and endogenous LAMP2 (red), and co‐localization was quantified over three independent experiments. Parental HeLa cells and VPS29 KO cells as well as VPS29 KO cells transduced with the indicated VPS29 rescue constructs were transduced with GFP‐RAB7, and RAB7 mobility/turnover was analyzed by FRAP in living cells. The displayed recovery kinetics were obtained by averaging kinetics from fifteen FRAP recoveries per condition acquired in two independent experiments. Parental HeLa cells, VPS29 KO cells, and VPS29 KO cells transduced with the indicated VPS29 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. RAB7a activity was quantified over four independent experiments. Parental HeLa cells and clonal TBC1D5 KO cells and TBC1D5 KO cells transduced with the indicated GFP‐TBC1D5 rescue constructs cells were lysed, and the activity of RAB7a was analyzed with the GST‐RILP assay. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Activity Assay, Construct, Mutagenesis, Binding Assay, Transduction, Expressing, Staining

    Loss of TBC 1D5 does not perturb retromer‐mediated sorting of GLUT 1 and CI ‐ MPR HeLa, SNX5/6 double KO, and TBC1D5 KO cells were fixed in PFA and stained for CI‐MPR (red) and the trans ‐Golgi network marker TGN46. Note that knockout of SNX5/6 results in dispersal of CI‐MPR from the TGN. HeLa, VPS29, and TBC1D5 KO cells were fixed in PFA and stained for GLUT1 (green) and LAMP2 (red). Co‐localization between GLUT1 and LAMP2 was quantified over ten images for each condition. HeLa cells were transfected with a pool of three distinct CRISPR/Cas9 plasmids targeting the RAB7a gene at three sites together with a puromycin resistance plasmid. Following puromycin selection and 5 days of incubation, the RAB7a KO cells were transduced with lentiviruses expressing the indicated GFP‐RAB7 proteins. A Western blot for RAB7 confirms high knockout efficiency and demonstrates that the GFP‐RAB7 variants are expressed at low endogenous levels. Parental HeLa cells and clonal VPS29 and VPS35 KO cells transduced with GFP‐LC3b were starved in EBSS for 4 h without (EBSS) or with addition of bafilomycin A1 to the EBSS (EBSS + Bafi) to assess autophagic flux. Autophagic flux was calculated from the increase in lipidated GFP‐LC3 in the EBSS + bafilomycin samples compared to the EBSS‐only sample. The quantification was done across four independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Journal: The EMBO Journal

    Article Title: Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

    doi: 10.15252/embj.201797128

    Figure Lengend Snippet: Loss of TBC 1D5 does not perturb retromer‐mediated sorting of GLUT 1 and CI ‐ MPR HeLa, SNX5/6 double KO, and TBC1D5 KO cells were fixed in PFA and stained for CI‐MPR (red) and the trans ‐Golgi network marker TGN46. Note that knockout of SNX5/6 results in dispersal of CI‐MPR from the TGN. HeLa, VPS29, and TBC1D5 KO cells were fixed in PFA and stained for GLUT1 (green) and LAMP2 (red). Co‐localization between GLUT1 and LAMP2 was quantified over ten images for each condition. HeLa cells were transfected with a pool of three distinct CRISPR/Cas9 plasmids targeting the RAB7a gene at three sites together with a puromycin resistance plasmid. Following puromycin selection and 5 days of incubation, the RAB7a KO cells were transduced with lentiviruses expressing the indicated GFP‐RAB7 proteins. A Western blot for RAB7 confirms high knockout efficiency and demonstrates that the GFP‐RAB7 variants are expressed at low endogenous levels. Parental HeLa cells and clonal VPS29 and VPS35 KO cells transduced with GFP‐LC3b were starved in EBSS for 4 h without (EBSS) or with addition of bafilomycin A1 to the EBSS (EBSS + Bafi) to assess autophagic flux. Autophagic flux was calculated from the increase in lipidated GFP‐LC3 in the EBSS + bafilomycin samples compared to the EBSS‐only sample. The quantification was done across four independent experiments. Data information: All scale bars = 10 μm, all error bars = SD, and * P

    Article Snippet: GFP‐RAB7 was then isolated with GFP‐trap beads (Chromotek), and the IPs were combined during the final washing steps.

    Techniques: Staining, Marker, Knock-Out, Transfection, CRISPR, Plasmid Preparation, Selection, Incubation, Transduction, Expressing, Western Blot

    NCDN colocalises with SNRPN and SMN in the cytoplasm, but not the nucleus, and is expressed in motor neurons in mouse spinal cord. (A) NCDN–GFP and mCherry–SNRPN colocalise in vesicle-like structures (chevron arrowheads) in neurites of SH-SY5Y cells constitutively expressing mCherry–SNRPN, and transiently expressing NCDN-GFP (left hand panels). White areas in the merged image show areas of colocalisation. Colocalisation images (bottom row) were generated in Volocity, using automatic thresholds on undeconvolved z-sections before excluding values below 0.05 (see Materials and Methods). No colocalisation is seen in the same cell line transiently expressing YFP alone (right hand panel). Triangular arrowheads show structures containing mCherry–SNRPN but not YFP. (B) mCherry–SMN and NCDN–GFP colocalise in vesicles (chevron arrowheads) in the cytoplasm of co-transfected SH-SY5Y cells (left hand panels). White areas in the merged image show areas of colocalisation. Colocalisation images (bottom row) were generated as above. No colocalisation is observed between mCherry-SMN and YFP (triangular arrowheads, right hand panels). (C) NCDN forms nuclear foci (arrows) in the nuclei of a small proportion of SH-SY5Y cells (≤2%, two independent experiments, n =100 cells per experiment). These do not colocalise with nuclear foci stained with coilin (arrowheads, left hand panels) or SMN (arrowheads, right hand panels). (D) NCDN (green) is expressed throughout the spinal cord, with increased expression in motor neurons (arrows), as identified with anti-ChAT antibody (magenta). (E) Higher magnification imaging confirms the presence of NCDN in ChAT-positive motor neurons (single deconvolved z-section). Scale bars: 7 µm (A,B); 500 µm (C,D); 10 µm (E).

    Journal: Journal of Cell Science

    Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology

    doi: 10.1242/jcs.211482

    Figure Lengend Snippet: NCDN colocalises with SNRPN and SMN in the cytoplasm, but not the nucleus, and is expressed in motor neurons in mouse spinal cord. (A) NCDN–GFP and mCherry–SNRPN colocalise in vesicle-like structures (chevron arrowheads) in neurites of SH-SY5Y cells constitutively expressing mCherry–SNRPN, and transiently expressing NCDN-GFP (left hand panels). White areas in the merged image show areas of colocalisation. Colocalisation images (bottom row) were generated in Volocity, using automatic thresholds on undeconvolved z-sections before excluding values below 0.05 (see Materials and Methods). No colocalisation is seen in the same cell line transiently expressing YFP alone (right hand panel). Triangular arrowheads show structures containing mCherry–SNRPN but not YFP. (B) mCherry–SMN and NCDN–GFP colocalise in vesicles (chevron arrowheads) in the cytoplasm of co-transfected SH-SY5Y cells (left hand panels). White areas in the merged image show areas of colocalisation. Colocalisation images (bottom row) were generated as above. No colocalisation is observed between mCherry-SMN and YFP (triangular arrowheads, right hand panels). (C) NCDN forms nuclear foci (arrows) in the nuclei of a small proportion of SH-SY5Y cells (≤2%, two independent experiments, n =100 cells per experiment). These do not colocalise with nuclear foci stained with coilin (arrowheads, left hand panels) or SMN (arrowheads, right hand panels). (D) NCDN (green) is expressed throughout the spinal cord, with increased expression in motor neurons (arrows), as identified with anti-ChAT antibody (magenta). (E) Higher magnification imaging confirms the presence of NCDN in ChAT-positive motor neurons (single deconvolved z-section). Scale bars: 7 µm (A,B); 500 µm (C,D); 10 µm (E).

    Article Snippet: To further investigate the interaction between SMN and NCDN observed in B, a reciprocal experiment was performed using GFP-TRAP to affinity purify GFP–SMN from an SH-SY5Y cell line constitutively expressing GFP–SMN ( ).

    Techniques: Expressing, Generated, Transfection, Staining, Imaging

    SNRPN exhibits similar behaviour to SNRPB in SH-SY5Y cells. (A) SH-SY5Y cells transiently expressing YFP–SNRPN and fixed after 24, 48 and 72 h show variations in distribution of the YFP–SNRPN with time. Immunostaining with Y12 (detecting Sm proteins, red on overlay) and anti-coilin (white on overlay) shows splicing speckles (chevron arrowheads) and cajal bodies (CBs, triangular arrowheads) respectively. Images are deconvolved z -stacks with 0.2 µm spacing. (B) After transient expression, SNRPN initially localises diffusely in the cytoplasm, before localising to speckles at the 48 and 72 h time-points. Results are mean±s.d. from three independent experiments, n =100 cells per experiment. (C) Western blot analysis of snRNPs immunoprecipitated using TMG beads (against the characteristic tri-methyl guanosine Cap of snRNAs, left hand lane) confirms that both YFP–SNRPN (detected with anti-YFP, top row) and mCherry–SNRPN (detected with anti-mCherry, bottom row) are incorporated into snRNPs. (D) mCherry–SNRPN cytoplasmic structures are mobile and stain with the lipophilic dye BODIPY 493. Chevron arrowheads identify mCherry–SNRPN structures stained with BODIPY 493; triangular arrowheads identify BODIPY 493-stained vesicles not containing mCherry–SNRPN. mCherry alone does not accumulate in BODIPY 493-stained vesicles. Cells were imaged approximately every 4 s for 9 min. Images are single deconvolved z -sections. (E) mCherry–SNRPN and GFP–SMN colocalise in cytoplasmic foci in SH-SY5Y cells (chevron arrowheads in left hand panels), whereas YFP alone shows no accumulation in mCherry-SNRPN foci (triangular arrowheads in right hand panels). White signal on the overlay indicates areas of colocalisation. Images are single deconvolved z -sections. (F) Comparison of the percentage of mCherry–SNRPN vesicles per cell colocalising with GFP–SMN to those showing co-incidental overlap with YFP alone confirms the colocalisation. Results are mean±s.d., n =5 ( P

    Journal: Journal of Cell Science

    Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology

    doi: 10.1242/jcs.211482

    Figure Lengend Snippet: SNRPN exhibits similar behaviour to SNRPB in SH-SY5Y cells. (A) SH-SY5Y cells transiently expressing YFP–SNRPN and fixed after 24, 48 and 72 h show variations in distribution of the YFP–SNRPN with time. Immunostaining with Y12 (detecting Sm proteins, red on overlay) and anti-coilin (white on overlay) shows splicing speckles (chevron arrowheads) and cajal bodies (CBs, triangular arrowheads) respectively. Images are deconvolved z -stacks with 0.2 µm spacing. (B) After transient expression, SNRPN initially localises diffusely in the cytoplasm, before localising to speckles at the 48 and 72 h time-points. Results are mean±s.d. from three independent experiments, n =100 cells per experiment. (C) Western blot analysis of snRNPs immunoprecipitated using TMG beads (against the characteristic tri-methyl guanosine Cap of snRNAs, left hand lane) confirms that both YFP–SNRPN (detected with anti-YFP, top row) and mCherry–SNRPN (detected with anti-mCherry, bottom row) are incorporated into snRNPs. (D) mCherry–SNRPN cytoplasmic structures are mobile and stain with the lipophilic dye BODIPY 493. Chevron arrowheads identify mCherry–SNRPN structures stained with BODIPY 493; triangular arrowheads identify BODIPY 493-stained vesicles not containing mCherry–SNRPN. mCherry alone does not accumulate in BODIPY 493-stained vesicles. Cells were imaged approximately every 4 s for 9 min. Images are single deconvolved z -sections. (E) mCherry–SNRPN and GFP–SMN colocalise in cytoplasmic foci in SH-SY5Y cells (chevron arrowheads in left hand panels), whereas YFP alone shows no accumulation in mCherry-SNRPN foci (triangular arrowheads in right hand panels). White signal on the overlay indicates areas of colocalisation. Images are single deconvolved z -sections. (F) Comparison of the percentage of mCherry–SNRPN vesicles per cell colocalising with GFP–SMN to those showing co-incidental overlap with YFP alone confirms the colocalisation. Results are mean±s.d., n =5 ( P

    Article Snippet: To further investigate the interaction between SMN and NCDN observed in B, a reciprocal experiment was performed using GFP-TRAP to affinity purify GFP–SMN from an SH-SY5Y cell line constitutively expressing GFP–SMN ( ).

    Techniques: Expressing, Immunostaining, Western Blot, Immunoprecipitation, Staining

    Reduction of endogenous SMN causes a reduction in cytoplasmic NCDN foci in SH-SY5Y cells. (A) SH-SY5Y cells were transfected with plasmids to express shRNAs targeting SMN (shSMN), Cyclophilin B (shCyclophilin) or with the empty pSuper GFP vector (data not shown), fixed after 72 h, and immunostained for endogenous NCDN and SMN allowing detection of NCDN foci within the cytoplasm (chevron arrowheads), as well as SMN-positive nuclear gems (triangular arrowheads). Images are single deconvolved z-sections. Scales bars: 7 µm. (B) The depletion of SMN results in a reduction in the number of NCDN foci present in the cytoplasm to 15.3 (±7.2) (mean±s.d.) from 20.6 (±12.0) and 19.5 (±7.6) compared to cells transfected with either shCyclophilin B or the empty pSuper GFP vector, respectively (ANOVA, P

    Journal: Journal of Cell Science

    Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology

    doi: 10.1242/jcs.211482

    Figure Lengend Snippet: Reduction of endogenous SMN causes a reduction in cytoplasmic NCDN foci in SH-SY5Y cells. (A) SH-SY5Y cells were transfected with plasmids to express shRNAs targeting SMN (shSMN), Cyclophilin B (shCyclophilin) or with the empty pSuper GFP vector (data not shown), fixed after 72 h, and immunostained for endogenous NCDN and SMN allowing detection of NCDN foci within the cytoplasm (chevron arrowheads), as well as SMN-positive nuclear gems (triangular arrowheads). Images are single deconvolved z-sections. Scales bars: 7 µm. (B) The depletion of SMN results in a reduction in the number of NCDN foci present in the cytoplasm to 15.3 (±7.2) (mean±s.d.) from 20.6 (±12.0) and 19.5 (±7.6) compared to cells transfected with either shCyclophilin B or the empty pSuper GFP vector, respectively (ANOVA, P

    Article Snippet: To further investigate the interaction between SMN and NCDN observed in B, a reciprocal experiment was performed using GFP-TRAP to affinity purify GFP–SMN from an SH-SY5Y cell line constitutively expressing GFP–SMN ( ).

    Techniques: Transfection, Plasmid Preparation

    Reduction of endogenous NCDN through siRNA increases localisation of SMN to nuclear foci. (A) Transfection of SH-SY5Y cells constitutively expressing GFP–SMN (green) with siRNAs shows an increase in the number of SMN-positive nuclear foci (arrowheads) in cells transfected with siRNAs against NCDN (siNCDN, pooled represents all four siRNA molecules together) or SNRPB (siSNRPB), and a decrease in the number of SMN-positive nuclear foci in cells transfected with siRNAs against SMN (siSMN) in comparison to cells transfected with non-targeting siRNAs (siControl) or siRNAs against lamin A/C (siLamin A/C) as a ‘targeting’ control. Transfection of SH-SY5Y cells with a plasmid to express GFP–SMNΔ7 also results in increased numbers of SMN-positive nuclear foci. Cell nuclei are stained with Hoechst 33342 (grey on images). Transfection efficiency with siRNAs was greater than 90%, measured by transfection with siGlo Cyclophillin B (data not shown). Scale bars: 7 µm. Images are deconvolved z -stacks taken with 0.2 µm spacing. (B) Quantification of numbers of SMN-positive nuclear foci per nucleus showing that there is a significant increase following reduction of NCDN [10.2 (±4.1) with siNCDN 18, 10.4 (±5.0) with siNCDN 19, 9.9 (±4.1) with siNCDN 20, 9.5 (±3.6) with siNCDN 21, and 10.7 (±4.6) with siNCDN pooled] compared to 4.4 (±2.5) in cells treated with non-targeting siRNA (siControl) and 4.2 (±2.3) in cells treated with siRNAs targeting lamin A/C (siLaminA/C). Reduction of SNRPB also shows an increase in numbers of SMN-positive nuclear foci [to 16.7 (±6.8) with siSNRPB], while reduction of SMN leads to a decrease in numbers of SMN-positive nuclear foci [to 0.7 (±1.4) with siSMN]. Expression of GFP–SMNΔ7 results in an increase of numbers of SMN-positive nuclear foci to 18.2 (±5.3). All values are mean±s.d. The difference between each siNCDN and controls is statistically significant (AVOVA; P

    Journal: Journal of Cell Science

    Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology

    doi: 10.1242/jcs.211482

    Figure Lengend Snippet: Reduction of endogenous NCDN through siRNA increases localisation of SMN to nuclear foci. (A) Transfection of SH-SY5Y cells constitutively expressing GFP–SMN (green) with siRNAs shows an increase in the number of SMN-positive nuclear foci (arrowheads) in cells transfected with siRNAs against NCDN (siNCDN, pooled represents all four siRNA molecules together) or SNRPB (siSNRPB), and a decrease in the number of SMN-positive nuclear foci in cells transfected with siRNAs against SMN (siSMN) in comparison to cells transfected with non-targeting siRNAs (siControl) or siRNAs against lamin A/C (siLamin A/C) as a ‘targeting’ control. Transfection of SH-SY5Y cells with a plasmid to express GFP–SMNΔ7 also results in increased numbers of SMN-positive nuclear foci. Cell nuclei are stained with Hoechst 33342 (grey on images). Transfection efficiency with siRNAs was greater than 90%, measured by transfection with siGlo Cyclophillin B (data not shown). Scale bars: 7 µm. Images are deconvolved z -stacks taken with 0.2 µm spacing. (B) Quantification of numbers of SMN-positive nuclear foci per nucleus showing that there is a significant increase following reduction of NCDN [10.2 (±4.1) with siNCDN 18, 10.4 (±5.0) with siNCDN 19, 9.9 (±4.1) with siNCDN 20, 9.5 (±3.6) with siNCDN 21, and 10.7 (±4.6) with siNCDN pooled] compared to 4.4 (±2.5) in cells treated with non-targeting siRNA (siControl) and 4.2 (±2.3) in cells treated with siRNAs targeting lamin A/C (siLaminA/C). Reduction of SNRPB also shows an increase in numbers of SMN-positive nuclear foci [to 16.7 (±6.8) with siSNRPB], while reduction of SMN leads to a decrease in numbers of SMN-positive nuclear foci [to 0.7 (±1.4) with siSMN]. Expression of GFP–SMNΔ7 results in an increase of numbers of SMN-positive nuclear foci to 18.2 (±5.3). All values are mean±s.d. The difference between each siNCDN and controls is statistically significant (AVOVA; P

    Article Snippet: To further investigate the interaction between SMN and NCDN observed in B, a reciprocal experiment was performed using GFP-TRAP to affinity purify GFP–SMN from an SH-SY5Y cell line constitutively expressing GFP–SMN ( ).

    Techniques: Transfection, Expressing, Plasmid Preparation, Staining

    Detergent-free fractionation of SH-SY5Y cells reveals that SMN, coatomer proteins, NCDN, SNRPB and SNRPN are all enriched in the 100,000 g vesicle pellet. (A) Immunoblotting of equal protein amounts from fractionated SH-SY5Y cells reveals that SMN (top row) is highly enriched in the 100,000 g (RCF) pellet (small membrane-bound structures), with smaller amounts seen in the 16,100 g pellet (larger membrane-bound structures) and the nuclear pellet. The coatomer protein, γCOP (also known as COPG1; second row) is also enriched in the 100,000 g pellet as well as the 16,100 g pellet. Antibodies against histone H3 and tubulin confirm minimal nuclear contamination in cytoplasmic fractions, and minimal cytoplasmic contamination in the nuclear pellet, respectively. (B) Quantification of immunoblot analysis confirms that SMN is highly enriched in the 100,000 g pellet, with enrichment of γCOP also seen. Histone H3 and tubulin are highly enriched in the nucleus and cytoplasm, respectively. Quantification (mean±s.d.) of tubulin and histone H3 band density was from seven immunoblots, with values from SMN and γCOP from five and four immunoblots, respectively. (C) Immunoblotting of equal protein amounts from fractionated SH-SY5Y cells constitutively expressing NCDN–GFP, YFP–SNRPB, YFP–SNRPN or YFP alone (all detected with anti-GFP antibody) reveals that NCDN–GFP is enriched in the 100,000 g pellet, with smaller amounts seen in the 16,100 g pellet and the cytosolic supernatant. YFP–SNRPB and YFP–SNRPN are both also found in the 100,000 g pellet, in addition to the nuclear pellet and cytosolic supernatant. YFP alone is found almost exclusively in the cytosolic supernatant, with none detected in the 100,000 g or 16,100 g pellets. (D) Quantification of the band densities for the immunoblot shown in C confirms the presence of NCDN–GFP, YFP–SNRPB and YFP–SNRPN in the 100,000 g pellet, together with the restriction of YFP alone to the residual cytosolic supernatant.

    Journal: Journal of Cell Science

    Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology

    doi: 10.1242/jcs.211482

    Figure Lengend Snippet: Detergent-free fractionation of SH-SY5Y cells reveals that SMN, coatomer proteins, NCDN, SNRPB and SNRPN are all enriched in the 100,000 g vesicle pellet. (A) Immunoblotting of equal protein amounts from fractionated SH-SY5Y cells reveals that SMN (top row) is highly enriched in the 100,000 g (RCF) pellet (small membrane-bound structures), with smaller amounts seen in the 16,100 g pellet (larger membrane-bound structures) and the nuclear pellet. The coatomer protein, γCOP (also known as COPG1; second row) is also enriched in the 100,000 g pellet as well as the 16,100 g pellet. Antibodies against histone H3 and tubulin confirm minimal nuclear contamination in cytoplasmic fractions, and minimal cytoplasmic contamination in the nuclear pellet, respectively. (B) Quantification of immunoblot analysis confirms that SMN is highly enriched in the 100,000 g pellet, with enrichment of γCOP also seen. Histone H3 and tubulin are highly enriched in the nucleus and cytoplasm, respectively. Quantification (mean±s.d.) of tubulin and histone H3 band density was from seven immunoblots, with values from SMN and γCOP from five and four immunoblots, respectively. (C) Immunoblotting of equal protein amounts from fractionated SH-SY5Y cells constitutively expressing NCDN–GFP, YFP–SNRPB, YFP–SNRPN or YFP alone (all detected with anti-GFP antibody) reveals that NCDN–GFP is enriched in the 100,000 g pellet, with smaller amounts seen in the 16,100 g pellet and the cytosolic supernatant. YFP–SNRPB and YFP–SNRPN are both also found in the 100,000 g pellet, in addition to the nuclear pellet and cytosolic supernatant. YFP alone is found almost exclusively in the cytosolic supernatant, with none detected in the 100,000 g or 16,100 g pellets. (D) Quantification of the band densities for the immunoblot shown in C confirms the presence of NCDN–GFP, YFP–SNRPB and YFP–SNRPN in the 100,000 g pellet, together with the restriction of YFP alone to the residual cytosolic supernatant.

    Article Snippet: To further investigate the interaction between SMN and NCDN observed in B, a reciprocal experiment was performed using GFP-TRAP to affinity purify GFP–SMN from an SH-SY5Y cell line constitutively expressing GFP–SMN ( ).

    Techniques: Fractionation, Western Blot, Expressing

    NCDN does not co-purify with snRNPs, while NCDN and SMN interact with Rab5 and colocalise with a subset of Rab5 vesicles within neurites of SH-SY5Y cells. (A) Incubation of whole-cell lysate from an SH-SY5Y cell line constitutively expressing NCDN–GFP with agarose beads conjugated to antibodies against the tri-methyl guanosine cap (Me3Gppp) of snRNAs (TMG beads) affinity purifies snRNPs as evidenced by the enrichment of the core snRNP protein SNRPN (detected with anti-SNRPN antibody, bottom row). The enriched snRNP fraction also contains SMN, which is essential for snRNP assembly. NCDN–GFP, however, does not co-enrich with snRNPs. Also shown is the core structure of mature snRNPs consisting of the heptameric Sm protein ring bound at the Sm-binding site of snRNA, as well as the characteristic tri-methyl guanosine Cap of snRNAs (Me 3 Gppp) at the 5′ end. (B) Affinity isolation of mRFP–Rab5 using RFP-Trap from cells co-transfected with plasmids to express mRFP–Rab5 together with NCDN–GFP, GFP–SMN or YFP alone co-enriches both NCDN–GFP (top row, detected with anti-GFP antibody, band is present in RFP-Trap lane but not Sepharose beads lane) and SMN-GFP (second row, detected with anti-GFP antibody, band is present in RFP-Trap lane but not Sepharose beads lane), but not YFP (third row, no band detected in RFP-Trap lane). Endogenous SMN (fourth row, detected with mouse anti-SMN) co-enriches with mRFP–Rab5 in all three samples. Detection of mRFP–Rab5 (bottom row, detected with anti-RFP antibody) confirms substantial enrichment of mRFP–Rab5 in all three samples. (C) Both GFP–SMN and NCDN–GFP partially colocalise with mRFP–Rab5 in a subset of mRFP–Rab5-containing vesicles in co-transfected SH-SY5Y cells (white signal in overlaid images, top row; yellow signal in colocalisation images, bottom row). (D) Enlargement of the boxed areas in C confirms that the colocalisation between SMN or NCDN and Rab5 occurs in punctate structures. Arrowheads identify areas of colocalisation. Colocalisation images were generated by Volocity using automatic thresholds on non-deconvolved z -sections before excluding values below 0.05. Images (excluding the colocalisation images) are single deconvolved z -sections. Scale bars: 7 µm.

    Journal: Journal of Cell Science

    Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology

    doi: 10.1242/jcs.211482

    Figure Lengend Snippet: NCDN does not co-purify with snRNPs, while NCDN and SMN interact with Rab5 and colocalise with a subset of Rab5 vesicles within neurites of SH-SY5Y cells. (A) Incubation of whole-cell lysate from an SH-SY5Y cell line constitutively expressing NCDN–GFP with agarose beads conjugated to antibodies against the tri-methyl guanosine cap (Me3Gppp) of snRNAs (TMG beads) affinity purifies snRNPs as evidenced by the enrichment of the core snRNP protein SNRPN (detected with anti-SNRPN antibody, bottom row). The enriched snRNP fraction also contains SMN, which is essential for snRNP assembly. NCDN–GFP, however, does not co-enrich with snRNPs. Also shown is the core structure of mature snRNPs consisting of the heptameric Sm protein ring bound at the Sm-binding site of snRNA, as well as the characteristic tri-methyl guanosine Cap of snRNAs (Me 3 Gppp) at the 5′ end. (B) Affinity isolation of mRFP–Rab5 using RFP-Trap from cells co-transfected with plasmids to express mRFP–Rab5 together with NCDN–GFP, GFP–SMN or YFP alone co-enriches both NCDN–GFP (top row, detected with anti-GFP antibody, band is present in RFP-Trap lane but not Sepharose beads lane) and SMN-GFP (second row, detected with anti-GFP antibody, band is present in RFP-Trap lane but not Sepharose beads lane), but not YFP (third row, no band detected in RFP-Trap lane). Endogenous SMN (fourth row, detected with mouse anti-SMN) co-enriches with mRFP–Rab5 in all three samples. Detection of mRFP–Rab5 (bottom row, detected with anti-RFP antibody) confirms substantial enrichment of mRFP–Rab5 in all three samples. (C) Both GFP–SMN and NCDN–GFP partially colocalise with mRFP–Rab5 in a subset of mRFP–Rab5-containing vesicles in co-transfected SH-SY5Y cells (white signal in overlaid images, top row; yellow signal in colocalisation images, bottom row). (D) Enlargement of the boxed areas in C confirms that the colocalisation between SMN or NCDN and Rab5 occurs in punctate structures. Arrowheads identify areas of colocalisation. Colocalisation images were generated by Volocity using automatic thresholds on non-deconvolved z -sections before excluding values below 0.05. Images (excluding the colocalisation images) are single deconvolved z -sections. Scale bars: 7 µm.

    Article Snippet: To further investigate the interaction between SMN and NCDN observed in B, a reciprocal experiment was performed using GFP-TRAP to affinity purify GFP–SMN from an SH-SY5Y cell line constitutively expressing GFP–SMN ( ).

    Techniques: Incubation, Expressing, Binding Assay, Isolation, Transfection, Generated

    NCDN interacts with SNRPN, SNRPB and SMN in cell lines and in mice . (A) Affinity isolation of NCDN–GFP using GFP-Trap, detected with anti-GFP antibody (top row) co-enriches mCherry–SNRPB, detected with anti-mCherry (bottom row) in transiently co-transfected SH-SY5Y cells. (B) In an SH-SY5Y cell line constitutively expressing NCDN–GFP, affinity isolation of NCDN–GFP, detected with anti-GFP antibody (top row) co-enriches SMN, SNRPB, SNRPN and the coatomer protein βCOP, all detected with antibodies against the endogenous proteins (as labelled). (C) In an SH-SY5Y cell line constitutively expressing YFP, affinity isolation of YFP, detected with anti-GFP antibody (top row) does not co-enrich SMN, SNRPB or βCOP, all detected with antibodies to the endogenous proteins (as labelled). (D) In an SH-SY5Y cell line constitutively expressing GFP–SMN, affinity isolation of GFP–SMN, detected with anti-GFP antibody (top row) co-enriches endogenous NCDN, detected with anti-NCDN antibody (middle row). Endogenous SMN, detected with anti-SMN (bottom row) is also co-enriched. (E) Immunoprecipitation (IP) of endogenous SMN co-enriches endogenous NCDN in SH-SY5Y cells. (F) Immunoprecipitation of endogenous SMN from murine P8 brain lysate co-enriches NCDN.

    Journal: Journal of Cell Science

    Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology

    doi: 10.1242/jcs.211482

    Figure Lengend Snippet: NCDN interacts with SNRPN, SNRPB and SMN in cell lines and in mice . (A) Affinity isolation of NCDN–GFP using GFP-Trap, detected with anti-GFP antibody (top row) co-enriches mCherry–SNRPB, detected with anti-mCherry (bottom row) in transiently co-transfected SH-SY5Y cells. (B) In an SH-SY5Y cell line constitutively expressing NCDN–GFP, affinity isolation of NCDN–GFP, detected with anti-GFP antibody (top row) co-enriches SMN, SNRPB, SNRPN and the coatomer protein βCOP, all detected with antibodies against the endogenous proteins (as labelled). (C) In an SH-SY5Y cell line constitutively expressing YFP, affinity isolation of YFP, detected with anti-GFP antibody (top row) does not co-enrich SMN, SNRPB or βCOP, all detected with antibodies to the endogenous proteins (as labelled). (D) In an SH-SY5Y cell line constitutively expressing GFP–SMN, affinity isolation of GFP–SMN, detected with anti-GFP antibody (top row) co-enriches endogenous NCDN, detected with anti-NCDN antibody (middle row). Endogenous SMN, detected with anti-SMN (bottom row) is also co-enriched. (E) Immunoprecipitation (IP) of endogenous SMN co-enriches endogenous NCDN in SH-SY5Y cells. (F) Immunoprecipitation of endogenous SMN from murine P8 brain lysate co-enriches NCDN.

    Article Snippet: To further investigate the interaction between SMN and NCDN observed in B, a reciprocal experiment was performed using GFP-TRAP to affinity purify GFP–SMN from an SH-SY5Y cell line constitutively expressing GFP–SMN ( ).

    Techniques: Mouse Assay, Isolation, Transfection, Expressing, Immunoprecipitation

    The interactomes of SNRPB and SNRPN are similar, but there are differences at the level of individual proteins. (A) Immunoblot analysis confirms efficient affinity purification of YFP–SNRPN, YFP–SNRPB and YFP. 10% of the affinity purified material (left hand lane in each panel) was compared to 80 µg of precleared lysate (Input) and unbound material using anti-GFP antibody. GFP-Trap effectively immunoprecipitated all three proteins. (B) After processing the MS data and sorting identified proteins into groups based on Gene Ontology annotations, the interactomes of SNRPN and SNRPB are very similar. (C) A comparison between the amino acid sequences of SNRPN and SNRPB reveals their similarity. Differences in amino acid sequence are in red. Sequences are from Uniprot [entries P63162 (SNRPN) and P14678-2 (SNRPB)]. (D) NCDN was identified in the interactome of YFP–SNRPN, with five unique peptide hits encompassing 9% sequence coverage. Each Ion score (Mascot Ion Score) was above the threshold for peptide identity (Mascot Identity Score), with two out of the five identified peptides having a score of above double the threshold score.

    Journal: Journal of Cell Science

    Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology

    doi: 10.1242/jcs.211482

    Figure Lengend Snippet: The interactomes of SNRPB and SNRPN are similar, but there are differences at the level of individual proteins. (A) Immunoblot analysis confirms efficient affinity purification of YFP–SNRPN, YFP–SNRPB and YFP. 10% of the affinity purified material (left hand lane in each panel) was compared to 80 µg of precleared lysate (Input) and unbound material using anti-GFP antibody. GFP-Trap effectively immunoprecipitated all three proteins. (B) After processing the MS data and sorting identified proteins into groups based on Gene Ontology annotations, the interactomes of SNRPN and SNRPB are very similar. (C) A comparison between the amino acid sequences of SNRPN and SNRPB reveals their similarity. Differences in amino acid sequence are in red. Sequences are from Uniprot [entries P63162 (SNRPN) and P14678-2 (SNRPB)]. (D) NCDN was identified in the interactome of YFP–SNRPN, with five unique peptide hits encompassing 9% sequence coverage. Each Ion score (Mascot Ion Score) was above the threshold for peptide identity (Mascot Identity Score), with two out of the five identified peptides having a score of above double the threshold score.

    Article Snippet: To further investigate the interaction between SMN and NCDN observed in B, a reciprocal experiment was performed using GFP-TRAP to affinity purify GFP–SMN from an SH-SY5Y cell line constitutively expressing GFP–SMN ( ).

    Techniques: Affinity Purification, Immunoprecipitation, Mass Spectrometry, Sequencing

    NLRP1 is required for HRV-induced inflammasome assembly and IL-18 secretion in primary human bronchial epithelial cells. A. HRV16 infection causes NLRP1 cleavage. HeLa-Ohio cells stably expressing NLRP1-HA and a vector control were infected with HRV16 at MOI=1 in duplicates. Rupintrivir (10 nM) was added at the time of infection and cell pellets were harvested 24 hours after inoculation. B. The effect of HRV16 infection on ASC-GFP speck formation in HeLa-Ohio-ASC-GFP cells expressing wild-type NLRP1 or NLRP1 Q130A . ***, p

    Journal: bioRxiv

    Article Title: Direct cleavage of human NLRP1 by enteroviral 3C protease triggers inflammasome activation in airway epithelium

    doi: 10.1101/2020.10.14.325076

    Figure Lengend Snippet: NLRP1 is required for HRV-induced inflammasome assembly and IL-18 secretion in primary human bronchial epithelial cells. A. HRV16 infection causes NLRP1 cleavage. HeLa-Ohio cells stably expressing NLRP1-HA and a vector control were infected with HRV16 at MOI=1 in duplicates. Rupintrivir (10 nM) was added at the time of infection and cell pellets were harvested 24 hours after inoculation. B. The effect of HRV16 infection on ASC-GFP speck formation in HeLa-Ohio-ASC-GFP cells expressing wild-type NLRP1 or NLRP1 Q130A . ***, p

    Article Snippet: Antibodies and cytokine analysisThe following antibodies were used in this study: c-Myc (Santa Cruz Biotechnology #sc-40), HA tag (Santa Cruz Biotechnology, #sc-805), GAPDH (Santa Cruz Biotechnology, #sc-47724), ASC (Apipogen, #AL-177), CASP1 (Santa Cruz Biotechnology, #sc-622), IL1B (R & D systems, #AF-201), FLAG (SigmaAldrich, #F3165), GFP (Abcam, #ab290), NLRP1 (R & D systems, #AF6788), IL18 (R & D systems, MAB9124) and VP2 (QED Bioscience, #18758).

    Techniques: Infection, Stable Transfection, Expressing, Plasmid Preparation

    A. HRV16 causes ASC-GFP oligomerization in a cleavage-dependent manner. HeLa-Ohio-ASC-GFP reporter cells were stably transduced with wild-type NLRP1 or the cleavage-resistant NLRP1 Q130A mutant and infected with HRV16 (MOI=1) as in a. ASC-GFP oligomers were crosslinked with 1% DSS and solubilized with 1% SDS. Note that level of overall ASC-GFP was reduced in HRV16-infected cells. B. The effect of HRV16 infection and Talabostat on HeLa-Ohio-ASC-GFP cells expressing NLRP1. Yellow arrows, ASC-GFP specks. C. Morphology of cell death in HRV16-infected NHBEs. Yellow arrows, characteristic pyroptotic cell death (thistle-like, elongated cell bodies with membrane ‘balloons’). Blue arrows, late-stage apoptosis (membrane blebs with shrunken cell bodies). D. Functional validation of CRISPR/Cas9 knockout NHBEs. Cell death was visualized by propidium iodide staining.

    Journal: bioRxiv

    Article Title: Direct cleavage of human NLRP1 by enteroviral 3C protease triggers inflammasome activation in airway epithelium

    doi: 10.1101/2020.10.14.325076

    Figure Lengend Snippet: A. HRV16 causes ASC-GFP oligomerization in a cleavage-dependent manner. HeLa-Ohio-ASC-GFP reporter cells were stably transduced with wild-type NLRP1 or the cleavage-resistant NLRP1 Q130A mutant and infected with HRV16 (MOI=1) as in a. ASC-GFP oligomers were crosslinked with 1% DSS and solubilized with 1% SDS. Note that level of overall ASC-GFP was reduced in HRV16-infected cells. B. The effect of HRV16 infection and Talabostat on HeLa-Ohio-ASC-GFP cells expressing NLRP1. Yellow arrows, ASC-GFP specks. C. Morphology of cell death in HRV16-infected NHBEs. Yellow arrows, characteristic pyroptotic cell death (thistle-like, elongated cell bodies with membrane ‘balloons’). Blue arrows, late-stage apoptosis (membrane blebs with shrunken cell bodies). D. Functional validation of CRISPR/Cas9 knockout NHBEs. Cell death was visualized by propidium iodide staining.

    Article Snippet: Antibodies and cytokine analysisThe following antibodies were used in this study: c-Myc (Santa Cruz Biotechnology #sc-40), HA tag (Santa Cruz Biotechnology, #sc-805), GAPDH (Santa Cruz Biotechnology, #sc-47724), ASC (Apipogen, #AL-177), CASP1 (Santa Cruz Biotechnology, #sc-622), IL1B (R & D systems, #AF-201), FLAG (SigmaAldrich, #F3165), GFP (Abcam, #ab290), NLRP1 (R & D systems, #AF6788), IL18 (R & D systems, MAB9124) and VP2 (QED Bioscience, #18758).

    Techniques: Stable Transfection, Transduction, Mutagenesis, Infection, Expressing, Functional Assay, CRISPR, Knock-Out, Staining

    3CPros activate NLRP1 by direct cleavage at a single site. A. HRV14-3Cpro cleaves NLRP1 close to its N-terminus. Top panel: the antibodies used to detect the NLRP1 auto-proteolytic fragments. The epitope of the N-terminal NLRP1 antibody is between NLRP1 a.a. 130 and a.a. 230. Bottom panel: 293T cells were transfected with C-terminally HA-tagged NLRP1 and Myc-tagged HRV14-3Pro. Full length NLRP1 and its cleavage products were B. visualized with the N-terminal fragment-specific NLRP1 antibody and an antibody against the C-terminal HA tag. Red arrows indicate the proposed proteolytic relationship between the observed NLRP1 fragments. Note that the presence of catalytically active 3Cpro decreased the expression all transfected plasmids (see also Fig. 2b, d and fig. S1e ). C. HRV14-3Cpro cleaves NLRP1 F1212A at a single site. 293T cells were transfected with FLAG-tagged NLRP1 F1212A with increasing amounts of HRV14-3Cpro. Cell lysates were harvested 48 hours post transfection. D. Recombinant HRV14-3Cpro cleaves human NLRP1. Cell-free lysate from NLRP1-HA-transfected 293T cells were incubated with recombinant HRV14-3Cpro at 33°C for 90 mins and analyzed by SDS-PAGE. E. Mapping of the 3Cpro cleavage site. Top: NLRP1 linker region immediately after the PYRIN domain (PYD). Glutamine (Q) residues are highlighted in red. Bottom: 293T cells were co-transfected with the indicated NLRP1 Q > A mutants and HRV14-3Cpro. Total cell lysates were harvested 48 hours post transfection. F. 3Cpros cannot activate the cleavage-resistant NLRP1 Q130A mutant. 293T-ASC-GFP cells were fixed 24 hours post transfection. The number of cells with ASC-GFP specks were visually scored with wide-field epifluorescence microscopy at 20x maganification. *, p

    Journal: bioRxiv

    Article Title: Direct cleavage of human NLRP1 by enteroviral 3C protease triggers inflammasome activation in airway epithelium

    doi: 10.1101/2020.10.14.325076

    Figure Lengend Snippet: 3CPros activate NLRP1 by direct cleavage at a single site. A. HRV14-3Cpro cleaves NLRP1 close to its N-terminus. Top panel: the antibodies used to detect the NLRP1 auto-proteolytic fragments. The epitope of the N-terminal NLRP1 antibody is between NLRP1 a.a. 130 and a.a. 230. Bottom panel: 293T cells were transfected with C-terminally HA-tagged NLRP1 and Myc-tagged HRV14-3Pro. Full length NLRP1 and its cleavage products were B. visualized with the N-terminal fragment-specific NLRP1 antibody and an antibody against the C-terminal HA tag. Red arrows indicate the proposed proteolytic relationship between the observed NLRP1 fragments. Note that the presence of catalytically active 3Cpro decreased the expression all transfected plasmids (see also Fig. 2b, d and fig. S1e ). C. HRV14-3Cpro cleaves NLRP1 F1212A at a single site. 293T cells were transfected with FLAG-tagged NLRP1 F1212A with increasing amounts of HRV14-3Cpro. Cell lysates were harvested 48 hours post transfection. D. Recombinant HRV14-3Cpro cleaves human NLRP1. Cell-free lysate from NLRP1-HA-transfected 293T cells were incubated with recombinant HRV14-3Cpro at 33°C for 90 mins and analyzed by SDS-PAGE. E. Mapping of the 3Cpro cleavage site. Top: NLRP1 linker region immediately after the PYRIN domain (PYD). Glutamine (Q) residues are highlighted in red. Bottom: 293T cells were co-transfected with the indicated NLRP1 Q > A mutants and HRV14-3Cpro. Total cell lysates were harvested 48 hours post transfection. F. 3Cpros cannot activate the cleavage-resistant NLRP1 Q130A mutant. 293T-ASC-GFP cells were fixed 24 hours post transfection. The number of cells with ASC-GFP specks were visually scored with wide-field epifluorescence microscopy at 20x maganification. *, p

    Article Snippet: Antibodies and cytokine analysisThe following antibodies were used in this study: c-Myc (Santa Cruz Biotechnology #sc-40), HA tag (Santa Cruz Biotechnology, #sc-805), GAPDH (Santa Cruz Biotechnology, #sc-47724), ASC (Apipogen, #AL-177), CASP1 (Santa Cruz Biotechnology, #sc-622), IL1B (R & D systems, #AF-201), FLAG (SigmaAldrich, #F3165), GFP (Abcam, #ab290), NLRP1 (R & D systems, #AF6788), IL18 (R & D systems, MAB9124) and VP2 (QED Bioscience, #18758).

    Techniques: Transfection, Expressing, Recombinant, Incubation, SDS Page, Mutagenesis, Epifluorescence Microscopy

    A. Polio- and EV71-3Cpros do not significantly induce ASC-GFP speck formation in 293T-ASC-GFP cells expressing NLRP1. B. Expression levels of viral proteases in 293T-ASC-GFP cells. All indicated viral proteases were tagged with Myc at the N-terminus. Cell pellets were harvested 48 hours post plasmid transfection. C. Validation of pooled CRISPR/Cas9 PYCARD/ASC knockout in immortalized keratinocytes. The cell lines that were used for downstream analyses are highlighted in red. D. Validation of pooled CRISPR/Cas9 CASP1 knockout in immortalized keratinocytes. E. NLRP1 Q130A is resistant to cleavage by multiple 3Cpros. 293T-ASC-GFP cells were transfected as in Fig. 2d . Cell pellets were harvested 48 hours post transfection. Asterisk, endogenous c-myc in 293T cells (unrelated to myc-3Cpros). F. NLRP1 WT -HA and NLRP1 Q130A -HA were expressed to similar levels in NLRP1 KO immortalized keratinocytes.

    Journal: bioRxiv

    Article Title: Direct cleavage of human NLRP1 by enteroviral 3C protease triggers inflammasome activation in airway epithelium

    doi: 10.1101/2020.10.14.325076

    Figure Lengend Snippet: A. Polio- and EV71-3Cpros do not significantly induce ASC-GFP speck formation in 293T-ASC-GFP cells expressing NLRP1. B. Expression levels of viral proteases in 293T-ASC-GFP cells. All indicated viral proteases were tagged with Myc at the N-terminus. Cell pellets were harvested 48 hours post plasmid transfection. C. Validation of pooled CRISPR/Cas9 PYCARD/ASC knockout in immortalized keratinocytes. The cell lines that were used for downstream analyses are highlighted in red. D. Validation of pooled CRISPR/Cas9 CASP1 knockout in immortalized keratinocytes. E. NLRP1 Q130A is resistant to cleavage by multiple 3Cpros. 293T-ASC-GFP cells were transfected as in Fig. 2d . Cell pellets were harvested 48 hours post transfection. Asterisk, endogenous c-myc in 293T cells (unrelated to myc-3Cpros). F. NLRP1 WT -HA and NLRP1 Q130A -HA were expressed to similar levels in NLRP1 KO immortalized keratinocytes.

    Article Snippet: Antibodies and cytokine analysisThe following antibodies were used in this study: c-Myc (Santa Cruz Biotechnology #sc-40), HA tag (Santa Cruz Biotechnology, #sc-805), GAPDH (Santa Cruz Biotechnology, #sc-47724), ASC (Apipogen, #AL-177), CASP1 (Santa Cruz Biotechnology, #sc-622), IL1B (R & D systems, #AF-201), FLAG (SigmaAldrich, #F3165), GFP (Abcam, #ab290), NLRP1 (R & D systems, #AF6788), IL18 (R & D systems, MAB9124) and VP2 (QED Bioscience, #18758).

    Techniques: Expressing, Plasmid Preparation, Transfection, CRISPR, Knock-Out

    3C proteases activate the human NLRP1 inflammasome A. Domain structures of human NLRP1 and rodent Nlrp1 homologues. B. Percentage of 293T-ASC-GFP cells with ASC-GFP specks after over-expression of vector control or NLRP1 with various viral proteases. Cells were fixed 24 hours after co-transfection of the indicated plasmids and > 100 cells were scored for ASC-GFP speck formation. n=3 biological replicates. ****: p

    Journal: bioRxiv

    Article Title: Direct cleavage of human NLRP1 by enteroviral 3C protease triggers inflammasome activation in airway epithelium

    doi: 10.1101/2020.10.14.325076

    Figure Lengend Snippet: 3C proteases activate the human NLRP1 inflammasome A. Domain structures of human NLRP1 and rodent Nlrp1 homologues. B. Percentage of 293T-ASC-GFP cells with ASC-GFP specks after over-expression of vector control or NLRP1 with various viral proteases. Cells were fixed 24 hours after co-transfection of the indicated plasmids and > 100 cells were scored for ASC-GFP speck formation. n=3 biological replicates. ****: p

    Article Snippet: Antibodies and cytokine analysisThe following antibodies were used in this study: c-Myc (Santa Cruz Biotechnology #sc-40), HA tag (Santa Cruz Biotechnology, #sc-805), GAPDH (Santa Cruz Biotechnology, #sc-47724), ASC (Apipogen, #AL-177), CASP1 (Santa Cruz Biotechnology, #sc-622), IL1B (R & D systems, #AF-201), FLAG (SigmaAldrich, #F3165), GFP (Abcam, #ab290), NLRP1 (R & D systems, #AF6788), IL18 (R & D systems, MAB9124) and VP2 (QED Bioscience, #18758).

    Techniques: Over Expression, Plasmid Preparation, Cotransfection

    Expression of the SIBR cassette from an intron. ( A ) Schematic diagrams of SIBR vectors: US2-SIBR ΔpA vector contains the SIBR cassette in the second exon of human ubC gene, under control of the human ubC promoter, but without a polyA signal. There is no coding region present in either the CS2+SIBR ΔpA or US2-SIBR ΔpA vector. UI2 vectors also use the human ubC promoter but contain the SIBR cassette in the first intron of the ubC gene, with the GFP or puromycin-resistance proteins expressed from the second exon. SD and SA indicate splice donor and splice acceptor, respectively. Exon and intron sizes not to scale. ( B ) Different SIBR vector designs expressing the ND1-1888 miRNA against NeuroD1 were cotransfected with the NeuroD1 3′-UTR luciferase reporter (see Figure 2 ). All four designs with ND1-1888 showed similar levels of inhibition of the reporter. Control SIBR vectors used the same designs but expressed an unrelated miRNA directed against the mouse POSH mRNA. Standard errors are indicated. ( C ) Comparable GFP fluorescence was detected 24 h after transfection with GFP expressed from ubC-based expression vectors, whether or not a functional SIBR cassette was present in the ubC intron.

    Journal: Nucleic Acids Research

    Article Title: Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

    doi: 10.1093/nar/gkl143

    Figure Lengend Snippet: Expression of the SIBR cassette from an intron. ( A ) Schematic diagrams of SIBR vectors: US2-SIBR ΔpA vector contains the SIBR cassette in the second exon of human ubC gene, under control of the human ubC promoter, but without a polyA signal. There is no coding region present in either the CS2+SIBR ΔpA or US2-SIBR ΔpA vector. UI2 vectors also use the human ubC promoter but contain the SIBR cassette in the first intron of the ubC gene, with the GFP or puromycin-resistance proteins expressed from the second exon. SD and SA indicate splice donor and splice acceptor, respectively. Exon and intron sizes not to scale. ( B ) Different SIBR vector designs expressing the ND1-1888 miRNA against NeuroD1 were cotransfected with the NeuroD1 3′-UTR luciferase reporter (see Figure 2 ). All four designs with ND1-1888 showed similar levels of inhibition of the reporter. Control SIBR vectors used the same designs but expressed an unrelated miRNA directed against the mouse POSH mRNA. Standard errors are indicated. ( C ) Comparable GFP fluorescence was detected 24 h after transfection with GFP expressed from ubC-based expression vectors, whether or not a functional SIBR cassette was present in the ubC intron.

    Article Snippet: GFP fluorescence in cells transfected with ubC-driven GFP vectors was photographed in cells fixed 45–48 h after transfection, using a Zeiss Axiovert S100 inverted fluorescence microscope with a DAGE 330 video camera and ImageJ software version 1.34.

    Techniques: Expressing, Plasmid Preparation, Luciferase, Inhibition, Fluorescence, Transfection, Functional Assay

    Knock-down of endogenous genes using UI2 SIBR vectors. ( A ) qRT–PCR measurements of mRNA levels in P19 cells transiently cotransfected with an expression vector for Ngn2 (to activate endogenous NeuroD1 expression), and various UI2-puro-SIBR vectors. The level of NeuroD1 mRNA was reduced by miRNAs directed against the NeuroD1 3′-UTR (see Figure 2 ) or coding region (ND1-380), but not by a control miRNA directed against Luciferase. GAPDH and HPRT mRNA levels were not reduced. ( B ) UI2-GFP-SIBR vectors expressing miRNAs that target the HP1γ or RASSF1 mRNAs reduced the levels of the corresponding endogenous mRNA in transfected P19 cells, but not the HPRT mRNA. ( C ) UI2-puro-SIBR vectors expressing miRNAs directed against GAPDH reduce endogenous GAPDH mRNA levels. ( D ) Expression of the GAPDH-240 miRNA leads to cleavage of the endogenous GAPDH mRNA at the expected site (see text). In A–D, transfected cells were selected with puromycin to remove untransfected cells. UI2-GFP-SIBR vectors in B were cotransfected with US2-puro to permit selection.

    Journal: Nucleic Acids Research

    Article Title: Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

    doi: 10.1093/nar/gkl143

    Figure Lengend Snippet: Knock-down of endogenous genes using UI2 SIBR vectors. ( A ) qRT–PCR measurements of mRNA levels in P19 cells transiently cotransfected with an expression vector for Ngn2 (to activate endogenous NeuroD1 expression), and various UI2-puro-SIBR vectors. The level of NeuroD1 mRNA was reduced by miRNAs directed against the NeuroD1 3′-UTR (see Figure 2 ) or coding region (ND1-380), but not by a control miRNA directed against Luciferase. GAPDH and HPRT mRNA levels were not reduced. ( B ) UI2-GFP-SIBR vectors expressing miRNAs that target the HP1γ or RASSF1 mRNAs reduced the levels of the corresponding endogenous mRNA in transfected P19 cells, but not the HPRT mRNA. ( C ) UI2-puro-SIBR vectors expressing miRNAs directed against GAPDH reduce endogenous GAPDH mRNA levels. ( D ) Expression of the GAPDH-240 miRNA leads to cleavage of the endogenous GAPDH mRNA at the expected site (see text). In A–D, transfected cells were selected with puromycin to remove untransfected cells. UI2-GFP-SIBR vectors in B were cotransfected with US2-puro to permit selection.

    Article Snippet: GFP fluorescence in cells transfected with ubC-driven GFP vectors was photographed in cells fixed 45–48 h after transfection, using a Zeiss Axiovert S100 inverted fluorescence microscope with a DAGE 330 video camera and ImageJ software version 1.34.

    Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation, Luciferase, Transfection, Selection

    Increased inhibition by multiple SIBR cassettes expressed from a single intron vector, and by use of a two intron vector. ( A ) Schematic representation of UI2-GFP-SIBR vectors expressing one to eight tandem copies of the same synthetic miRNA against luciferase. ( B ) P19 Cells transfected with a fixed amount of target luciferase reporter and a fixed total amount of DNA show dose dependent inhibition of luciferase. At three different DNA concentrations, an increased number of copies of the luc-1601 SIBR cassette in the UI2-GFP vector provided better inhibition. The UI2-GFP-SIBR POSH-2852 control vector expresses a functional synthetic miRNA directed against the mouse POSH gene. A vector with eight copies of the POSH miRNA does not inhibit luciferase. Total DNA amount was kept constant by replacing the SIBR vector with the US2-MT vector, which does not express a miRNA. Standard errors are indicated. ( C ) Schematics of the two intron UI4-GFP-SIBR vectors. UI4-GFP vectors contain the SIBR cassettes in rabbit globin intron, inserted between the exon 2 and GFP in exon 3. Exons 1 and 2 are noncoding. SD and SA indicate splice donor and splice acceptor, respectively. Approximate intron size is indicated. ( D ) Cotransfection reporter assay comparing the inhibition by UI4-GFP-SIBR vectors to UI2-GFP-SIBR vectors expressing one or two miRNAs against luciferase. At three different plasmid concentrations, the UI4-GFP-SIBR vectors showed increased inhibition of luciferase relative to the UI2-GFP-SIBR vectors. Standard errors are indicated.

    Journal: Nucleic Acids Research

    Article Title: Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

    doi: 10.1093/nar/gkl143

    Figure Lengend Snippet: Increased inhibition by multiple SIBR cassettes expressed from a single intron vector, and by use of a two intron vector. ( A ) Schematic representation of UI2-GFP-SIBR vectors expressing one to eight tandem copies of the same synthetic miRNA against luciferase. ( B ) P19 Cells transfected with a fixed amount of target luciferase reporter and a fixed total amount of DNA show dose dependent inhibition of luciferase. At three different DNA concentrations, an increased number of copies of the luc-1601 SIBR cassette in the UI2-GFP vector provided better inhibition. The UI2-GFP-SIBR POSH-2852 control vector expresses a functional synthetic miRNA directed against the mouse POSH gene. A vector with eight copies of the POSH miRNA does not inhibit luciferase. Total DNA amount was kept constant by replacing the SIBR vector with the US2-MT vector, which does not express a miRNA. Standard errors are indicated. ( C ) Schematics of the two intron UI4-GFP-SIBR vectors. UI4-GFP vectors contain the SIBR cassettes in rabbit globin intron, inserted between the exon 2 and GFP in exon 3. Exons 1 and 2 are noncoding. SD and SA indicate splice donor and splice acceptor, respectively. Approximate intron size is indicated. ( D ) Cotransfection reporter assay comparing the inhibition by UI4-GFP-SIBR vectors to UI2-GFP-SIBR vectors expressing one or two miRNAs against luciferase. At three different plasmid concentrations, the UI4-GFP-SIBR vectors showed increased inhibition of luciferase relative to the UI2-GFP-SIBR vectors. Standard errors are indicated.

    Article Snippet: GFP fluorescence in cells transfected with ubC-driven GFP vectors was photographed in cells fixed 45–48 h after transfection, using a Zeiss Axiovert S100 inverted fluorescence microscope with a DAGE 330 video camera and ImageJ software version 1.34.

    Techniques: Inhibition, Plasmid Preparation, Expressing, Luciferase, Transfection, Functional Assay, Cotransfection, Reporter Assay

    Comparable inhibition by SIBR vectors and a U6 shRNA vector. ( A ) A reporter assay comparing inhibition of a luciferase reporter containing 3′-UTR of mouse tubulin β3 (luc-Tubb3-UTR) cotransfected with either the UI2-GFP-SIBR Tubb3-1549 vector or the U6-Tubb3HP2 vector. At three different plasmid concentrations, both vectors showed comparable levels of inhibition. Standard errors are indicated. ( B ) Reduction of endogenous mouse tubulin β3 protein at a single cell level demonstrated by immunocytochemistry. U6 or UI2-GFP-SIBR vectors were cotransfected into P19 cells together with the US2-Ngn2 vector to induce neuronal differentiation and β3 tubulin expression. U6 transfections included the US2-GFP vector to label transfected cells. P19 cells transfected with a U6 shRNA vector or either of two UI2-GFP-SIBR vectors (green) directed against β3 tubulin showed substantially reduced tubulin β3 protein (red) by indirect immunfluorescence, relative to cells transfected with control vectors.

    Journal: Nucleic Acids Research

    Article Title: Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

    doi: 10.1093/nar/gkl143

    Figure Lengend Snippet: Comparable inhibition by SIBR vectors and a U6 shRNA vector. ( A ) A reporter assay comparing inhibition of a luciferase reporter containing 3′-UTR of mouse tubulin β3 (luc-Tubb3-UTR) cotransfected with either the UI2-GFP-SIBR Tubb3-1549 vector or the U6-Tubb3HP2 vector. At three different plasmid concentrations, both vectors showed comparable levels of inhibition. Standard errors are indicated. ( B ) Reduction of endogenous mouse tubulin β3 protein at a single cell level demonstrated by immunocytochemistry. U6 or UI2-GFP-SIBR vectors were cotransfected into P19 cells together with the US2-Ngn2 vector to induce neuronal differentiation and β3 tubulin expression. U6 transfections included the US2-GFP vector to label transfected cells. P19 cells transfected with a U6 shRNA vector or either of two UI2-GFP-SIBR vectors (green) directed against β3 tubulin showed substantially reduced tubulin β3 protein (red) by indirect immunfluorescence, relative to cells transfected with control vectors.

    Article Snippet: GFP fluorescence in cells transfected with ubC-driven GFP vectors was photographed in cells fixed 45–48 h after transfection, using a Zeiss Axiovert S100 inverted fluorescence microscope with a DAGE 330 video camera and ImageJ software version 1.34.

    Techniques: Inhibition, shRNA, Plasmid Preparation, Reporter Assay, Luciferase, Immunocytochemistry, Expressing, Transfection

    Knock-down of two endogenous genes using a single UI2 SIBR vector. ( A ) Schematic of the UI2-GFP/puro-SIBR vectors showing unique restriction sites flanking the SIBR cassette. Indicated in bold are restriction enzyme sites used for multiplexing SIBR cassettes. ( B ) Using appropriate restriction enzymes, it is possible to create vectors with tandem SIBR cassettes rapidly. ( C ) Schematic of vectors with miRNAs directed against the B-Raf and/or c-Raf kinases, including a vector with tandem B-Raf and c-Raf SIBR cassettes. Schematics in A-C are not to scale. ( D ) Western blot showing reduced levels of either B-Raf or c-Raf protein in cells transfected with SIBR vectors expressing a miRNA against the corresponding mRNA, but not in cells transfected with a control vector expressing a miRNA directed against luciferase. The vector expressing two miRNAs targeting the B-Raf and c-Raf mRNAs reduced the levels of both Raf proteins, but not the ERK kinase.

    Journal: Nucleic Acids Research

    Article Title: Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

    doi: 10.1093/nar/gkl143

    Figure Lengend Snippet: Knock-down of two endogenous genes using a single UI2 SIBR vector. ( A ) Schematic of the UI2-GFP/puro-SIBR vectors showing unique restriction sites flanking the SIBR cassette. Indicated in bold are restriction enzyme sites used for multiplexing SIBR cassettes. ( B ) Using appropriate restriction enzymes, it is possible to create vectors with tandem SIBR cassettes rapidly. ( C ) Schematic of vectors with miRNAs directed against the B-Raf and/or c-Raf kinases, including a vector with tandem B-Raf and c-Raf SIBR cassettes. Schematics in A-C are not to scale. ( D ) Western blot showing reduced levels of either B-Raf or c-Raf protein in cells transfected with SIBR vectors expressing a miRNA against the corresponding mRNA, but not in cells transfected with a control vector expressing a miRNA directed against luciferase. The vector expressing two miRNAs targeting the B-Raf and c-Raf mRNAs reduced the levels of both Raf proteins, but not the ERK kinase.

    Article Snippet: GFP fluorescence in cells transfected with ubC-driven GFP vectors was photographed in cells fixed 45–48 h after transfection, using a Zeiss Axiovert S100 inverted fluorescence microscope with a DAGE 330 video camera and ImageJ software version 1.34.

    Techniques: Plasmid Preparation, Multiplexing, Western Blot, Transfection, Expressing, Luciferase

    Reduction of endogenous HP1γ protein in single cells identified by coexpressed GFP. P19 cells transfected with the UI2-GFP-SIBR HP1γ-664 vector express GFP (green) and show reduced HP1γ by indirect immunfluorescence (red) when compared with untransfected cells (no GFP). The HP1γ signal in cells transfected with the UI2-GFP-luc1601 control vector remains unchanged.

    Journal: Nucleic Acids Research

    Article Title: Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155

    doi: 10.1093/nar/gkl143

    Figure Lengend Snippet: Reduction of endogenous HP1γ protein in single cells identified by coexpressed GFP. P19 cells transfected with the UI2-GFP-SIBR HP1γ-664 vector express GFP (green) and show reduced HP1γ by indirect immunfluorescence (red) when compared with untransfected cells (no GFP). The HP1γ signal in cells transfected with the UI2-GFP-luc1601 control vector remains unchanged.

    Article Snippet: GFP fluorescence in cells transfected with ubC-driven GFP vectors was photographed in cells fixed 45–48 h after transfection, using a Zeiss Axiovert S100 inverted fluorescence microscope with a DAGE 330 video camera and ImageJ software version 1.34.

    Techniques: Transfection, Plasmid Preparation