Journal: Life Science Alliance

Article Title: MERLIN: a novel BRET-based proximity biosensor for studying mitochondria–ER contact sites

doi: 10.26508/lsa.201900600

Figure Lengend Snippet: Top: representative images of cells expressing different-size MERLIN in the absence ( left ) and presence ( right ) of staurosporine (STS) in HCT116 cells. Bottom: quantification of cell viability upon MERLIN expression by measuring Smac-mCherry release in the presence/absence of STS and relativized to mVenus transfected cells.

Article Snippet: In the cell viability experiments, the cells were grown as described above and transfected with Smac-mCherry and MERLIN (Smac/donor/acceptor in a 2:1:3 ratio).

Techniques: Expressing, Transfection

Journal: Life Science Alliance

Article Title: MERLIN: a novel BRET-based proximity biosensor for studying mitochondria–ER contact sites

doi: 10.26508/lsa.201900600

Figure Lengend Snippet: (A, B) Scheme and saturation curve for MERLIN based on the 12-nm linker with (A) the donor targeted to the ER and the acceptor targeted to mitochondria and (B) the donor targeted to mitochondria and the acceptor targeted to the ER. (C) Maximum BRET signals for the different linker lengths and organelle localizations of the MERLIN components. (D) BRET signal for the negative controls sCal-L1-RLuc (3-nm donor) and mVen-ER5 (luminal ER protein) or mVen-H2B6 (nucleus). (E) BRET signal of the positive control mVen-L1-RLuc compared with the 3- and 6-nm linker lengths.

Article Snippet: In the cell viability experiments, the cells were grown as described above and transfected with Smac-mCherry and MERLIN (Smac/donor/acceptor in a 2:1:3 ratio).

Techniques: Positive Control

Journal: Life Science Alliance

Article Title: MERLIN: a novel BRET-based proximity biosensor for studying mitochondria–ER contact sites

doi: 10.26508/lsa.201900600

Figure Lengend Snippet: (A) Synthetic tether alone is not capable of energy transfer to Scal-mVenus in the presence of coelenterazine. Break from 10 5 to 10 6 a.u. (B) Transfection with increasing amounts of DNA do not affect the % of transfected cells but increases linearly the mVenus fluorescence signal per cell. (C) Validation of MERLIN with an alternative method to quantify MERCs. Quantification of MERCs by EM in HCT116 cells and MERLIN-containing HCT116 cells untreated and in presence of tunicamycin or starvation. (D, E, F) Localization of the donor and acceptor to the mitochondria and ER, respectively. Scale bar 10 μm. Stable expression of MERLIN does not affect viability nor sensitivity to apoptosis induced by treatment with 1 μM STS for 4 h, raw data and quantification, respectively. Scale bar 5 μm. (G) Left: starvation, hypoxia, or treatment with bortezomib, Taxol, or tunicamycin does not affect luciferase activity. Right: hypoxia effects in HCT116 wt cells measured by BODIPY. Scale bar 100 μm. (H) coelenterazine H concentration does not affect the BRET ratio.

Article Snippet: In the cell viability experiments, the cells were grown as described above and transfected with Smac-mCherry and MERLIN (Smac/donor/acceptor in a 2:1:3 ratio).

Techniques: Transfection, Fluorescence, Biomarker Discovery, Expressing, Luciferase, Activity Assay, Concentration Assay

Journal: Life Science Alliance

Article Title: MERLIN: a novel BRET-based proximity biosensor for studying mitochondria–ER contact sites

doi: 10.26508/lsa.201900600

Figure Lengend Snippet: (A, B, C, D) PDZD8 modulates ER–mitochondria distance. (A, B, C) Representative Western blot of the PDZD8 levels when transiently transfected and (B) upon silencing with siRNA_PDZD8 in HCT116 cells, whose quantification is shown in (C) (n = 3). (D) BRET signal in cells co-expressing Rluc-L1-B33C and Scal-L1-mVenus biosensor combination, in the presence of overexpressed PDZD8, the synthetic tether mTagBFP2 and PDZD8 knockdown in HCT116 cells. (** P < 0.025, *** P ≤ 0.001). t test, data are expressed as mean ± SD. (E, F) The BRET signal of MERLIN is increased in apoptotic cells. (E) Confocal images of Cos1 cells transfected with sCal-L1-mVen (green) and RLuc-L1-B33C (magenta) under healthy condition and upon apoptosis induction with 1 μM STS at different times. Scale bar 10 μM. (F) Scheme and graph showing the change of the BRET signal in apoptotic cells over time for the 12-nm linker MERLIN. Black lines represent four individual measurements and the grey line the control measurement without induction of apoptosis. Apoptosis was induced at time point 0 h by addition of 1 μM STS. (N = 4). (G) MERLIN detects a NAC-induced decrease in MERCs (** P < 0.025) t test, data are expressed as mean ± SD.

Article Snippet: In the cell viability experiments, the cells were grown as described above and transfected with Smac-mCherry and MERLIN (Smac/donor/acceptor in a 2:1:3 ratio).

Techniques: Western Blot, Transfection, Expressing, Knockdown, Control

Journal: Life Science Alliance

Article Title: MERLIN: a novel BRET-based proximity biosensor for studying mitochondria–ER contact sites

doi: 10.26508/lsa.201900600

Figure Lengend Snippet: (A) Measurement of BRET signal of MERLIN as a function of time in HCT116 cells exposed to stress: starvation (green), bortezomib (purple), Taxol (orange), staurosporine (dark blue), tunicamycin (cyan), and hypoxia (grey). Control shown in black. BRET was quantified before treatment (−4 h), after 4 h of stress (0 h) and upon recovery at 4 and 16 h. (B) Localization of the donor and acceptor to the mitochondria and ER, respectively in neuroprogenitor cells. Scale bar 5 μm. (C) Representative image of a differentiated dopaminergic (top) and embryonic mice primary neurons (bottom). Scale bar 100 and 20 μm, respectively. (D) Quantification of BRET signal in neuroprogenitor cell (magenta) and dopaminergic neurons (grey) in the presence of absence of PDZD8. (** P < 0.025 and *** P < 0.001). T test, data are expressed as mean ± SD.

Article Snippet: In the cell viability experiments, the cells were grown as described above and transfected with Smac-mCherry and MERLIN (Smac/donor/acceptor in a 2:1:3 ratio).

Techniques: Control

Journal: Life Science Alliance

Article Title: MERLIN: a novel BRET-based proximity biosensor for studying mitochondria–ER contact sites

doi: 10.26508/lsa.201900600

Figure Lengend Snippet: (A) Upper plane shows a representative Cos1 cell transfected with mCer-L1-B33C (blue) and sCal-L1-mVen (yellow). Scale bar 25 μM. The area in the white rectangle was used for FLIM-FRET measurement. Lower plane shows the zoom in in this area. Scale bar 5 μM. (B) The fluorescence lifetime is shown for the donor fluorophore with the 6-nm linker MERLIN, the negative and the positive control as well as the donor only control. Graph shows three biological replicates with n = 10, Error bars SD.

Article Snippet: In the cell viability experiments, the cells were grown as described above and transfected with Smac-mCherry and MERLIN (Smac/donor/acceptor in a 2:1:3 ratio).

Techniques: Transfection, Fluorescence, Positive Control, Control

Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: (A) Western blots of siCTL–, siRTN4–, and siCLIMP-63–transfected HT-1080 cells were probed with anti-RTN4, anti-CLIMP-63, and anti-β-actin as a loading control. (B) Representative images of ER tubules in HT-1080 cells transfected with ERmoxGFP and siCTL, siRTN4, or siCLIMP-63. Arrowheads indicate the tubules with increased blob length. Scale bar, 2 μm. (C) The quantification of maxima length, variation of maxima length (SD), and maxima-minima intensity differentials of ER tubules in HT-1080 cells transfected with siRTN4, siCLIMP-63, or siCTL. Bar graphs show mean ± SEM and scatter dot plots median with interquartile range. Significance assessed by Student t test from three independent experiments (20–40 line scans/each repeat). ** P < 0.01; *** P < 0.001. Numerical values that underlie the graphs and plots are shown in . CLIMP-63, cytoskeleton-linking membrane protein 63; ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; RTN4, reticulon4; SD, standard deviation; siCLIMP-63, siRNA to CLIMP-63; siCTL, siControl; siRTN4, siRNA to RTN4.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Western Blot, Transfection, Standard Deviation

Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: (A) STED images of ERmoxGFP in HT-1080 cells transfected with ERmoxGFP or cotransfected with mCherry-CLIMP-63 (CLIMP-63), mCherry-RTN4a (RTN4a), or mCherry-ATL1 (ATL1). Peripheral ER regions (white boxes) are shown as zooms; line scans of selected tubules in these regions (yellow boxes) are shown with ERmoxGFP in green and ER-shaping proteins in red. Scale bar, 5 μm; zooms, 2 μm. (B) Peripheral ER tubule maxima length, variation of maxima length (SD), and maxima-to-minima intensity differential are shown for cells transfected with ERmoxGFP alone (CTL) or cotransfected with mCherry-CLIMP-63 (CLIMP-63), mCherry-RTN4a (RTN4a), or mCherry-ATL1 (ATL1). Significance assessed by one-way ANOVA from three independent experiments (40 line scans/each repeat). Bar graphs show mean ± SEM and scatter dot plots median with interquartile range. * P < 0.05; ** P < 0.01; *** P < 0.001. Numerical values that underlie the graphs and plots are shown in . (C) Based on line scan analysis of peripheral ER tubules of HT-1080 cells cotransfected with mCherry-CLIMP-63 (CLIMP-63), mCherry-RTN4a (RTN4a), or mCherry-ATL1 (ATL1), percent localization of CLIMP-63, RTN4a, and ATL1 puncta to minima or maxima of lumenal ERmoxGFP-labeled tubules was quantified. Significance assessed by one-way ANOVA from four independent experiments (40 line scans/each repeat). Bar graphs show mean ± SEM. * P < 0.05; *** P < 0.001. Numerical values that underlie the graphs are shown in . ATL, atlastin; CLIMP-63, cytoskeleton-linking membrane protein 63; CTL, control ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; RTN4a, reticulon4a; SD, standard deviation; STED, stimulated emission depletion.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Transfection, Labeling, Standard Deviation

Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: (A) Representative merged images of single peripheral ER tubules expressing ERmoxGFP or Sec61βGFP labeled for calnexin or derlin-1. The dashed line indicates the site of line scan analysis along tubule. Fluorescence intensities of ER reporter (green) and protein (red) from line scans are presented as graphs. Scale bar, 0.5 μm. (B) Based on line scan analysis of peripheral ER tubules, percent localization of calnexin and derlin-1 puncta to ERmoxGFP or Sec61βGFP maxima and minima was quantified. Values plotted are mean ± SEM from three independent experiments (40 tubules per repeat) with one-way ANOVA for significance. *** P < 0.001. Numerical values that underlie the graphs are shown in . (C) Based on line scan analysis of peripheral ER tubules, percent localization of calnexin and derlin-1 puncta to ERmoxGFP maxima and minima was quantified in cells transfected with siCTL, siCLIMP-63, or siRTN4. Significance was assessed by χ 2 test from three independent experiments (20–40 tubules per repeat). *** P < 0.001. Numerical values that underlie the graphs are shown in . (D) Based on line scan analysis of peripheral ER tubules, percent localization of calnexin puncta to ERmoxGFP maxima and minima was quantified in HT-1080 cells cotransfected with mCherry-CLIMP-63, mCherry-RTN4a, or mCherry-ATL1 compared with CTL. Significance assessed by χ 2 test from three independent experiments (40 tubules per repeat). *** P < 0.001. Numerical values that underlie the graphs are shown in . ATL, atlastin; CLIMP-63, cytoskeleton-linking membrane protein 63; CTL, control; ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; RTN4a, reticulon4a; siCLIMP-63, siRNA to CLIMP-63; siCTL, siControl; siRTN4, siRNA to RTN4.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Expressing, Labeling, Fluorescence, Transfection

Journal: PLoS Biology

Article Title: Reticulon and CLIMP-63 regulate nanodomain organization of peripheral ER tubules

doi: 10.1371/journal.pbio.3000355

Figure Lengend Snippet: STED live cell imaging (40 ms/frame over 4 seconds) of isolated ROIs of peripheral ERmoxGFP-labeled tubules (a) was performed for COS-7 cells cotransfected with mCherry-CLIMP-63 or mCherry-RTN4a (A) or transfected with siCLIMP-63, siRTN4, or siCTL (B). Kymograms show the distribution of ERmoxGFP at specific sites along ER tubules over time (b). From plots of normalized average intensity over time (c), we determined the CoV along the tubule length as a measure of localized distribution of ERmoxGFP to distinct domains along peripheral ER tubules (d). Scatter dot plots show median with interquartile range from three independent experiments (20–50 tubules per condition) with one-way ANOVA for significance. * P < 0.05; *** P < 0.001. Numerical values that underlie the plots are shown in . CLIMP-63, cytoskeleton-linking membrane protein 63; CoV, coefficient of variation; CTL, control; ER, endoplasmic reticulum; ERmoxGFP, ER monomeric oxidizing environment-optimized green fluorescent protein; ns, not significant; ROI, region of interest; RTN4a, reticulon4a; siCLIMP-63, siRNA to CLIMP-63; siCTL, siControl; siRNA, small interfering RNA; siRTN4, siRNA to RTN4; STED, stimulated emission depletion.

Article Snippet: ERmoxGFP was a gift from Dr. Erik Snapp (Albert Einstein College of Medicine, presently at Howard Hughes Medical Institute Janelia Research Campus, VA) (Addgene plasmid #68072), mCherry-CLIMP-63 and Sec61βGFP from Dr. Gia Voeltz (University of Colorado, Boulder, CO), Sec61βmRFP from Dr. Patrick Lajoie (University of Western Ontario, London, ON, Canada), and mCherry-RTN4a and mCherry-ATL1 from Dr. Tom Rapoport (Harvard University, MA) (Addgene plasmid #86683 and #86678, respectively).

Techniques: Live Cell Imaging, Isolation, Labeling, Transfection, Small Interfering RNA

Journal: Research Square

Article Title: “MrgprA3 neurons selectively control myeloid-derived cytokines for IL-17 dependent cutaneous immunity”

doi: 10.21203/rs.3.rs-3644984/v1

Figure Lengend Snippet: ( A-D ), UMAP plots illustrating cytokine and chemokine gene expression among myeloid cell clusters generated by single cell RNA-sequencing of skin-resident CD11c+ MHC-II+ populations sort-purified from unmanipulated control or CD11c-IL-33KO mice. ( E-H ), Violin plots of cytokine and chemokine gene expression in specific myeloid APC clusters from control or CD11c-IL-33KO skin. ( I ), Cytokine-expressing cells from control or CD11c-IL-33KO BMDMs treated with increasing concentrations of LPS. ( J ), LPS-induced cytokine expression of BMDMs generated from control or CMV-IL-33KO mice. ( K ), Experimental approach to transfect control or CMV-IL-33KO BMDMs with empty mCherry plasmid or IL-33-mCherry plasmid followed by quantification of LPS-induced cytokines 2 days post-transfection. ( L,M ), Representative counterplots of pro-IL-1β staining and quantification of pro-IL-1β+ and TNFα+ cells in control or CMV-IL-33KO BMDMs that were transfected with empty or IL-33-containing plasmid. P values were determined by two-tailed Student’s t-tests or One-way ANOVA with post hoc correction. *P<0.05, **P<0.01, ***P<0.001. I-M, Representative of 2–3 independent experiments, each with ≥4 biological replicates.

Article Snippet: As described in the literature , BMDMs were transfected with 3μg of empty mCherry- or IL-33-mCherry plasmid generated by VectorBuilder.

Techniques: Gene Expression, Generated, RNA Sequencing, Purification, Control, Expressing, Plasmid Preparation, Transfection, Staining, Two Tailed Test

Journal: Research Square

Article Title: “MrgprA3 neurons selectively control myeloid-derived cytokines for IL-17 dependent cutaneous immunity”

doi: 10.21203/rs.3.rs-3644984/v1

Figure Lengend Snippet:

Article Snippet: As described in the literature , BMDMs were transfected with 3μg of empty mCherry- or IL-33-mCherry plasmid generated by VectorBuilder.

Techniques: Purification, Recombinant, Control, Activation Assay, Staining, Reverse Transcription, SYBR Green Assay, Lysis, Protease Inhibitor, Electron Microscopy, Injection, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Software

Journal: eLife

Article Title: An open cortico-basal ganglia loop allows limbic control over motor output via the nigrothalamic pathway

doi: 10.7554/elife.49995

Figure Lengend Snippet: Figure 1. Trans-synaptic wild-type rabies tracing reveals both closed and open cortico-basal ganglia loops. (A) The strategy to label striatal neurons connecting to the cerebral cortex by Wt-RABV trans-synaptic retrograde tracing, and CTb-based non-trans-synaptic anterograde tracing for mapping cortico-striatal terminals. (B) Example image of Wt-RABV/CTb injection into M1 (left). After 66–70 hr of survival time, Wt-RABV was transfected up to 3rd- order neurons, which were found in various striatal subregions (right). Scale bars, 1 mm (left), 500 mm (right). (C) 3D-reconstruction of Wt-RABV+ striatal neurons from the M1 injection case shown in (B). The two different angles emphasize the presence of Wt-RABV+ neurons throughout all of the striatum (VS, DMS, DLS, and TS). (D) Schema of Wt-RABV/CTb injection in M1. (E) Anterogradely labeled CTb+ cortico-striatal terminals (green) and retrogradely labeled Wt-RABV+ striatal neurons (purple) from the M1 injection case shown in (B). (F) Density map showing the distribution of Wt-RABV+ neurons throughout the striatum from M1 injection. Black contours indicate approximate areas receiving cortico-striatal inputs from M1. Color maps indicate the intensity of Wt-RABV+ labeling. (G–I) The same analyses for Wt-RABV/CTb injection in M2. (J–L) The same analyses for Wt-RABV/CTb injection in mPFC. (M) Normalized distribution of Wt-RABV+ neurons across five striatal regions (VMS, VLS, DMS, DLS, TS) showing differences Figure 1 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.49995 15 of 29 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Strain, strain background (recombinant rabies virus) EnvA.dGRabies.eGFP Salk Vector Core RRID: SCR_014847 Strain, strain background (adeno-associated virus) AAV9.FLEX.tdTomato University of Penn Viral Vector Core RRID: SCR_015406 Strain, strain background (adeno-associated virus) AAV9.FLEX.eGFP University of Penn Viral Vector Core RRID: SCR_015406 Strain, strain background (adeno-associated virus) AAV9.CAG.tdTomato UNC Viral Vector Core RRID: SCR_002448 Strain, strain background (adeno-associated virus) AAV9.hsyn.ChR2.eGFP University of Penn Viral Vector Core RRID: SCR_015406 Strain, strain background (adeno-associated virus) AAV5-EF1a-DIOhChR2(H134R)-mCherry University of Penn Viral Vector Core Antibody anti-wild type rabies phosphoprotein mouse monoclonal antibody commercially unavailable (Raux et al., 1997) (1:5000) Antibody anti-cholera toxin b-subunit goat polyclonal antibody List Biological Laboratories Cat.# 704 (1:15000) Antibody anti-tyrosine hydroxylase (TH) mouse monoclonal antibody Millipore Cat.# MAB318 (1:1000) Antibody anti-NeuN rabbit polyclonal antibody Abcam Cat.# ab104225 (1:1000) Antibody anti-GFP chicken polyclonal antibody Novus Biologicals Cat.# NB100-1614 (1:1000) Antibody anti-substance P mouse monoclonal antibody Abcam Cat.# ab14184 (1:1000) Antibody anti-mouse IgG horseradish peroxydase (HRP) (host: rabbit, polyclonal) DAKO Cat.# P260 (1:200) Antibody anti-goat IgG horseradish peroxydase (HRP) (host: rabbit, polyclonal) DAKO Cat.# P044901-2 (1:200) Antibody anti-mouse Alexa Fluor 488 (host: donkey, polyclonal) Jackson ImmunoResearch Laboratories Cat.# 715-545-151 (1:250) Antibody anti-mouse Cy3 (host: donkey, polyclonal) Jackson ImmunoReseach Laboratories Cat.# 715-165-151 (1:250) Antibody anti-mouse Cy5 (host: donkey, polyclonal) Jackson ImmunoReseach Laboratories Cat.# 715-175-151 (1:250) Continued on next page Aoki et al. eLife 2019;8:e49995.

Techniques: Retrograde Tracing, Anterograde Tracing, Injection, Transfection, Labeling