Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A-B Confocal images from a time-lapse of a tip cell from a transgenic Tg(kdrl:mCherry-CAAX) S916 embryo shown in magenta. B-B’’’ Inverted contrast of mCherry-CAAX from A shows CAAX dots which elongate along the apical membrane. Pink pseudo-color indicates the vascular lumen. C-E Confocal images of the tip cell of a double transgenic Tg(fli:eGFP-Rab7a) ubs48 ; Tg(kdrl:mCherry-CAAX) S916 embryo at 32hpf . D Inverted contrast image of the EGFP-Rab7 channel. E Inverted contrast image showing the membrane marker mCherry-CAAX in ECs. Arrows point to co-localisation of Rab7 and CAAX at dots. F Distribution of Pearson Correlation Coefficient (PCC) of EGFP-Rab7a ROIs in correlation to mCherry-CAAX, showing a correlation between EGFP and mCherry signal (n=8 tip cells, N>3 different embryos). F A tip cell from a transgenic Tg (kdrl:mCherry-CAAX) S916 embryo at 30hpf, transiently expressing fli:EGFP-Rab7a . F’-F’’’ Stills from the ROI from D showing EGFP-Rab7 and mCherry-CAAX dots that move along the expanding apical membrane. G-G’’’ EGFP-Rab7a signal alone. H-H’’’ mCherry-CAAX signal alone. Arrows point to the EGFP-Rab7a and mCherry-CAAX dots.

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Transgenic Assay, Marker, Expressing

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A-B Confocal images of a tip cell from a transgenic TgBAC(Lamp2-RFP) pd1117 embryo at 30hpf, transiently expressing fli:GFP-Rab7a . A Z-projection of the tip cell. A’ Single z-slice of the ROI in A. Two vesicles are depicted with signal positive for GFP-Rab7a and Lamp2-RFP. The largest vesicle shows a reduced signal in the centre for both GFP-Rab7a and Lamp2-RFP, resembling late endosomal/lysosomal structures. B-B’ EGFP-Rab7a signal alone in endothelial cells. C-C’ Lamp2-RFP signal alone. D Schematic representation of the blood vessel (tip EC) undergoing transcellular lumen formation. Rab7, Lamp2 and CAAX co-localise at dots which migrate along the expanding apical membrane. D Tip cell of a double transgenic Tg(kdrl:GFP-CAAX) s916 ; TgBAC(Lamp2-RFP) pd1117 embryo at 34hpf. D’-D’’’ Timelapse from the ROI in E showing Lamp2-RFP dot-like structure elongating along the apical, invaginating membrane. E-E’’’ Lamp2 signal alone. F-F’’’ mCherry-CAAX signal alone. Arrows point to the EGFP-Rab7 and mCherry-CAAX dot-like structures

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Transgenic Assay, Expressing

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A All three rab7 protein sequences and their predicted mutant sequence. Yellow boxes indicate an important prenylation site responsible for mediating post-translational prenylation of the C-terminal XCXC motif and membrane insertion, and the switch domains important for effector binding after GTP-activation and the hypervariable region responsible for proper membrane recognition. Green letters represent aa that are identical in all three Rab7 isoforms, while black letters represent different aa in Rab7 isoforms. In mutant protein sequences, blue letters are aa encoded out of frame and STOP marks the premature terminating codon. B-C Individual value scatter plots of relative protein expression of Rab7ba and the total Rab7 amount. Levels were measured in two different pooled samples of wild-type, rab7a mat-zyg, rab7ba mat-zyg and rab7bb mat-zyg homozygous embryos. Values were then normalized to total amount of protein measured per sample and to the amount of wild-type sample (n= 2 pools of 20 embryos). D-D’’ Sequencing results of PCR products of the respective rab7 loci from 3 months old homozygous fish. E Brightfield images of wild-type, rab7a, rab7ba and rab7bb homozygous mutant embryos at 30 hpf and 48 hpf. F Images of wild-type, rab7a, rab7ba and rab7bb adult fish at 5 months. G Percentage of rab7a, rab7ba and rab7bb mutations found in adult fish from a heterozygous incross of the respective mutant ( rab7a : N= 3 independent experiments, n= 100 fish; rab7b a: N= 3, n= 100; rab7bb N= 2, n= 70).

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Mutagenesis, Sequencing, Binding Assay, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A Phylogenetic tree constructed from the protein sequence derived from amphioxus (outgroup), human, mouse and zebrafish rab7 genes, scale: 0.9 amino acid substitutions. B Phylogenetic tree constructed from the protein sequences of rab7a, rab7b and zgc:100918 genes from zebrafish, other cyprinid family members and amphioxus (outgroup), scale: 0.3 amino acid substitutions. C Representation of the chromosomal region around the genes rab7b on chromosome 8 and zgc:100918 on chromosome 10. Indicated are the genes that are already annotated copies of each other. D Analysis of the expression of rab7 genes in endothelial and non-endothelial cells from zebrafish transcriptomics data (Lawson, Li et al. 2020). E Heatmap of expression of rab7 genes, a maternally contributed gene ( smarca2 ) and an endothelial-specific gene ( cdh5 ) during zebrafish development, based on data from an EMBL expression atlas (Papatheodorou et al. 2020 see Material and Methods). F Schematic representation of the gene structure of rab7 genes in zebrafish. 5’ and 3’ UTRs and ATG (start codon) are highlighted in dark blue, alternating coding exons are represented in light green and light blue. Sequence of the exons 2 of rab7a, rab7ba and rab7bb . Below each gene sequence appears the respective sequence of mutant alleles ubs51 , ubs52 and ubs53 . Deleted base pairs are underlined at the wild-type sequence, inserted base pairs are represented in light green and deletions are shown in red in mutant alleles. Red asterisk shows premature stop codon in the sequence of exon2.

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Construct, Sequencing, Derivative Assay, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A All three rab7 amino acid sequences and their predicted mutant sequences. Peptides used for the Mass Spectrometry experiments are shown on top of each sequence. Peptides that recognise all three isoforms are shown in blue (PanRab7), isoform-specific peptides are shown in magenta and the common peptide for Rab7a and Rab7ba is shown in green. B Graph showing the contribution in % of the individual Rab7 isoforms to total Rab7 protein in wild-type (n= 2 pools of 20 embryos). C and D Individual value scatter plots of relative protein expression of the three different Rab7 isoforms. Levels were measured in two different pooled samples of wild-type, rab7a mat-zyg, rab7ba mat-zyg and rab7bb mat-zyg homozygous embryos. Values were then normalized to total amount of protein measured per sample and to the amount of wild-type sample (n= 2 pools of 20 embryos). G-J Bright field images of wild-type or maternal-zygotic rab7a; rab7bb double homozygous mutant embryos at 1-cell stage and 8-cell stage. K Bar graph showing the percentage of embryos with enlarged yolk granules in different rab7 mutant crosses (n= 289-515 embryos, N=3 different single crosses per condition). L Survival plot of clutches from different mutant crosses. Percentage of surviving embryos from wild-type and rab7a, rab7ba and rab7bb homozygous incrosses, as well as incrosses from rab7a homozygous , rab7bb heterozygous adults and rab7a; rab7bb double homozygous parents (n=499-1438 embryos N= 2-7 crosses per condition.). M Triple mutant survival from a cross of a rab7a -/- ; rab7ba -/- ; rab7bb +/- mother and a rab7a -/- ; rab7ba +/- ; rab7bb -/- father (n=16 embryos at 24hpf, 34 adults after 3 months).

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Mutagenesis, Mass Spectrometry, Sequencing, Expressing

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A-E Confocal still pictures from time-lapse movies from transgenic Tg(kdrl:EGFP-CAAX) s916 embryos at 34-44 hpf showing blood vessel lumenization in wild-type (A), maternal zygotic homozygous mutant for rab7a (B) , rab7ba (C) , maternal-zygotic double homozygous mutant for rab7a; rab7ba (D) and rab7a;rab7bb (E) . Black arrowheads show invaginating luminal front. The final image represents the fully lumenized state of the blood vessels around 44 hpf. F Schematic representation of how lumen diameter was measured. The diameter of the vessel was measured perpendicular to vessel axis at 3 positions. The membrane marker Tg(kdrl:mcherry-CAAX) s916 was used as reference to how far the lumen expanded. G Violin plot showing lumen diameter in rab7 mutants. Median is indicated by thick black line (wild-type: N= 8 fish, n= 24 blood vessels; rab7a : N= 4, n= 10 (maternal-zygotic homozygous); rab7ba : N= 8, n= 22 (maternal-zygotic); rab7a; rab7ba : N= 10, n= 31 (maternal-zygotic); rab7a; rab7bb : N=7, n= 19 (maternal-zygotic).

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Transgenic Assay, Mutagenesis, Marker

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A-D Stills from high resolution confocal imaging of lumen fusion in the DLAV (anterior to the left) of double transgenic Tg(kdrl:mcherry-CAAX) s916 ; Tg(fli:Pecam1-EGFP) ncv27 wild-type ( A ), maternal-zygotic homozygous rab7a double ( B ), maternal-zygotic homozygous rab7ba ( C ) and maternal-zygotic double homozygous rab7a ubs51 ; rab7ba us521 ( D ) embryos. A’-D’ Isolated mcherry-CAAX signal labelling the apical membrane of ROIs from A-D. Arrows indicate the invaginating luminal front.

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Imaging, Transgenic Assay, Isolation

Journal: bioRxiv

Article Title: Genetic analysis of rab7 mutants in zebrafish

doi: 10.1101/2023.03.09.531857

Figure Lengend Snippet: A-B ’ Representation of measurement of vesicle size. Single z-stack of a non-lumenized tip-cell is chosen using the endothelial marker kdrl:EGFP-CAAX . B’ In a zoom-in window of the ROI in B, every Lamp2-RFP positive signal is measured (using as upper cut off 2 pixels). G Violin plots of measured vesicle sizes in rab7 mutants (wild-type N= 5, n= 283, rab7a;rab7ba heterozygous N= 4, n= 347, rab7a homozygous rab7ba heterozygous N= 5, n=356, rab7a heterozygous; rab7ba homozygous N= 7, n= 539, rab7a;rab7ba homozygous N= 6, n= 260, p***<0.001, red line indicates the median) and in rab7 morphants (standard-MO N= 8, n= 930, rab7a -MO N= 6, n= 129, rab7bb -MO N= 6, n=259, p****<0.0001, red line indicates the median). D-J Confocal images expressing an endothelial marker and the late endosome marker Lamp2 (double transgenic embryos Tg(kdrl:EGFP-CAAX);TgBAC(Lamp2-RFP) pd1117 or Tg(fli:Pecam1-EGFP) ncv27 ;TgBAC(Lamp2-RFP) pd1117 . D’-J’ Zoom-in areas indicated in D-J showing isolated Lamp2-RFP signal. D-F Images from wild-type or rab7 mutant embryos. G-J Images from embryos injected with a standard morpholino or rab7 morpholinos.

Article Snippet: For generation of the fli1: EGFP-rab7a vector, a pDestTol2CG2-heart-gfp with the independent marker cmlc2:EGFP, a fli1 P-5’entry clone (Addgene, Lawson Lab), an EGFP p-middle entry (Addgene, Kwan, Chien lab) and rab7a p-3’entry clone ( ) were used.

Techniques: Marker, Expressing, Transgenic Assay, Isolation, Mutagenesis, Injection

Journal: Nature communications

Article Title: Negative regulation of EGFR signalling by the human folliculin tumour suppressor protein.

doi: 10.1038/ncomms15866

Figure Lengend Snippet: Figure 4 | Expression of Rab7A phenocopies the effect of FLCN on ligand-dependent EGFR activation. (a) FLCN-deficient FTC-133 cells (FLCN / ) and wild-type FLCN-replete cells (FLCN WT) were stably infected with viruses expressing a vector, wild-type Rab7A or the constitutively active (Q67L) or dominant-negative (T22N) Rab7A mutants (indicated as vector only (VO), WT, CA and DN). After 2.5 h of starvation, the cells were stimulated with 20 ng ml 1 EGF for 3 h and protein lysates were immunoblotted as indicated. A representative western blot demonstrating decreased pEGFR, pERK and pS6 signalling in FLCN / cells expressing WT or CA Rab7A. (b) Quantification of western blot signal intensity from two independent experiments, presented as the as the mean±s.d. The densitometry of the bands was determined with BioRad Image Lab Software, normalized to the loading control (actin), and expressed relative to the FLCN replete cells (FLCN WT) infected with the vector only.

Article Snippet: The HA-eGFP-Rab7A WT and the HA-eGFP-Rab7A Q67L mutant were cloned into the pCDNA3 backbone (previously described57) and obtained from Addgene (plasmid 28047 and plasmid 28049).

Techniques: Expressing, Activation Assay, Stable Transfection, Infection, Plasmid Preparation, Dominant Negative Mutation, Western Blot, Software, Control

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Relative numbers Dnm2-EndoB1 PLA spots increase, while Dnm2-EndoA1 PLA spots marginally decrease when autophagy is induced. B PLA spots for endogenous Dnm2 and EndoB1 (red) colocalize with EGFP-LC3 (green) when mitophagy is induced (4 hr CCCP). C A larger fraction of Dnm2-EndoB1 PLA spots colocalize with GFP-tagged LC3, Atg2 and Fip200 when autophagy is induced with rapamycin or CCCP, suggesting that Dnm2 is connected with autophagy. The fraction that colocalizes with GFP-tagged Lamp1 decreases and remains low for Stx17 and Rab7. For the histograms, 50 cells per experiment, SEM, n=3, unpaired Student’s t-test. Scale bar is 10 μm.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques:

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Effects of CCCP on mitophagy in WT, Dnm2 -/-, EndoB1 -/- and DKO cells observed with immunofluorescence microscopy using Tom20 (red) and Hsp60 (green) antibodies. At 24 hr, most mitochondria are degraded, except in EndoB1 -/- cells, where mitophagy is inhibited. Scale bar is 10 μm. B Western blots showing the effects of CCCP on Hsp60 levels in the mutant cells. The bottom panel shows average intensities of Hsp60 bands in four independent experiments, relative to tubulin levels and normalized at 100% for 0hr time point. C Super-resolution images (SIM) show the formation of LC3-membranes (green) encapsulating mitochondria (red) in cells treated with CCCP. These structures are observed in WT, Dnm2 -/- and DKO cells, but not in EndoB1 -/- cells. Cells were treated for 6 hr with 20 μM CCCP. Mitochondria were detected with mitoDsRed and autophagic membranes were detected with EGFP-LC3. Scale bar is 10 μm for top panels and 1 μm for the enlargements. D Western blots showing the effects of CCCP and Bafilomycin A treatments on LC3 lipidation in wildtype, Dnm2 -/-, EndoB1 -/- and DKO cells. E Effects of Dnm2 and EndoB1 deletions on colocalization of RFP-LC3 and GFP-Rab7. Autophagy was induced for 1 hr with Rapamycin, Scale bar is 10 μm. F Reduced levels of LC3 and Rab7 colocalization expressed as Mander’s coefficient for untreated cells and for cells treated with Rapamycin, Bafilomycin or both. There were 50 cells per experiment, SEM, n=3, unpaired Student’s t-test.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Immunofluorescence, Microscopy, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Time lapse images of GFP-Atg9, Dnm2-RFP and BFP-LC3. Images were taken at 350 msec intervals. An arrow in the middle panel points to a potential scission event. The bar in the first panel shows the line used for the intensity tracings shown in the bottom panels. Scale bar is 1 μm. B Mean frequencies of Dnm2-RFP spots transiently wedged between GFP-Atg9 and BFP-LC3 spots in untreated cells and cells treated with Dynole 34-2 or Ryngo 1-23 (n = 20, 17 and 22, SEM, unpaired T test). The frequencies were low because only a small part of a cell was observed for a limited time (50 frames), but their transient nature and the opposing effects of a dynamin inhibiter and an activator suggest a functional connection with Atg9 and LC3 fission and fusion events. C RFP-Atg9 colocalizes with EGFP-LC3 in Dnm2 -/- cells treated with CCCP, but not in wild type cells. Scale bar is 10 μm for whole cells and 3 μm for enlarged portions. D Histogram showing increased colocalization of RFP-ATG9 and GFP-LC3 in Dnm2 -/- cells, dynasore-treated WT cells and EndoB1 -/- cells after incubation with rapamycin or CCCP. E The number of Atg9 vesicles (RFP spots without GFP label) decreases when Dnm2 -/- cells, dynasore-treated WT cells and EndoB1 -/- cells are incubated with rapamycin or CCCP. For panels D and E, 50 cells were counted per experiment, n= 3, SEM, unpaired students’ t-test. F Images of autophagosome containing fractions from WT and Dnm2 -/- cells transfected with EGFP-Atg9 and RFP-LC3 followed by 4hr treatments with CCCP and Bafilomycin to induce mitophagy and to accumulate autophagosomes. Autophagosomes were enriched by differential centrifugation. Enlargements show individual spots with line tracings to plot fluorescence intensities. Scale bar is 10 μm in the overview images and 2 μm in the enlargements. The x-axis in the fluorescence intensity plots show numbers of pixels (24 pixel/μm) and the bars show 500 nm. G Scatter plot of peak fluorescence intensities for individual spots, as shown in panel F (arbitrary units). H Histogram of the peak fluorescence intensities of Atg9 in LC3 spots relative to the peak intensities of Atg9 in spots without LC3. Bar is SE, with Student’s t-test for 3 independent experiments. 30 spots for each condition were measured in each experiment. I Subcellular fractionation of homogenates from WT and Dnm -/- MEF cells, which were treated for 4 hr with Rapamycin. Membranous organelles were separated with a 1-22% Ficoll density gradient and analyzed with Western blots. The bottom fraction (#20) contains mitochondria and other dense organelles such as mitophagosomes, autophagolysosomes or lysosomes. Atg9 is normally present in light fractions (fraction #6), but partially shifts to an intermediate density (fraction #14) in Dnm2 -/- cells, along with some p62, LC3-II and Rab7a. J Delayed dissociation of Atg9 and p62 in Dnm2 -/- cells. A buildup of Atg9 and p62 was induced for 16h with the Vps34 inhibitor Vps34-IN1. This was followed by recovery through washout of the inhibitor for the indicated lengths of time. Association and dissociation of Atg9 and p62 was monitored by immunofluorescence microscopy of endogenous proteins in WT and Dnm2 -/- HeLa cells.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Functional Assay, Incubation, Transfection, Centrifugation, Fluorescence, Fractionation, Western Blot, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Immunofluorescence of endogenous Dnm2 (green) and EndoB1 (red) is shown without or with 2 hr rapamycin. B Mander’s coefficients for colocalization shown for untreated cells and after 2 or 4 hr with rapamycin or CCCP (50 cells were counted per experiment, SE for 3 independent experiments, unpaired Student’s t-test). C, D Similar experiments with transfected cells expressing Dnm2-GFP and EndoB1-mRuby2. E Colocalization of Dnm2 and EndoB1 tested with Proximity Ligation Assay (PLA) shown with or without 2 hr Antimycin A treatment. F Average numbers of PLA dots/cell shown for Dnm2 and EndoB1 colocalization in untreated cells, cells treated with staurosporine (STS) or actinomycin D to induce apoptosis, or with CCCP, Antimycin A, rapamycin, starvation and ionomycin to induce autophagy. Numbers of dots were counted in 50 cells/experiment (SEM, n=3, unpaired student’s t-test). Scale bar is 10 μm for whole cells and 5 μm for enlarged portions.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Immunofluorescence, Transfection, Expressing, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Scatterplots of immunofluorescence images of cells treated for 2h with or without rapamycin to induce autophagy. B Fraction of spots in the colocalizing quadrants of the scatterplots, shown before and after 2 or 4 hr treatment with rapamycin or CCCP. (SE and unpaired Student’s t-test, n= 3). C, D As in panels A and B, but with transiently expressed GFP- and RFP-tagged proteins. E Western blots showing derivation of Dnm2, EndoB1 and DKO cells. Dnm2 and EndoB1 -/- cells were generated by CRISPR/Cas9 in MEFs. DKO cells were generated by additional knockout of EndoB1 in Dnm2 -/- cells. Clone A4 was chosen for further studies. F PLA signals for Dnm2 and EndoB1 interactions were absent from cells with mutations in one or both of these proteins. G As negative control for PLA, cells were incubated with Fis1 and Hsp60 antibodies, which detect proteins that are on the outside or inside of mitochondria and are therefore too far apart for generating PLA signals. As positive control, cells were incubated with antibodies for Drp1 and Mff, which detect well-characterized binding partners. Scale bar is 10 μm. H Western blots of conditional dynamin knockout cells. Cre-recombinase was induced with tamoxifen at the indicated concentrations for 24 hr and cells were grown for an additional 4 days with fresh medium before western blot analysis of Dnm1 and Dnm2 expression. Although Dnm1 is not normally expressed in these cells and Dnm3 is undetectable with currently available antibodies , all three genes were floxed with 3 μM tamoxifen to ensure complete lack of dynamins.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Immunofluorescence, Western Blot, Generated, CRISPR, Knock-Out, Negative Control, Incubation, Positive Control, Binding Assay, Expressing

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Bax translocation to mitochondria is delayed when apoptosis is induced in Dnm2 -/- cells, but not in EndoB1 -/- cells. B, C Similar effects were observed with cytochrome c release from mitochondria and with the appearance of pyknotic nuclei, which are two other indicators of apoptosis. D Bax translocation to mitochondria is delayed in cells lacking all three dynamins. Triple knockout cells (TKO) were generated by treating conditional cells (labeled as WT) with tamoxifen to induce Cre recombinase. 50 cells per experiment, SEM, n=3, unpaired Student’s t-test.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Translocation Assay, Triple Knockout, Generated, Labeling

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Immunofluorescence of cells stained with antibodies for Tom20 (red) and Hsp60 (green) show no effects of Dnm2 or EndoB1 mutations on mitochondrial fission. The top row has images of untreated cells, the middle row has cells treated for 30 min with CCCP and the bottom row has cells treated for 30 min with valinomycin to induce mitochondrial fission. B Images of Drp1 -/- cells treated without or with CCCP. C Histogram showing that mutations in Dnm2 and EndoB1 do not prevent CCCP or valinomycin induced mitochondrial fission. D Images of cells before and after treatment with tamoxifen to induce knockout of all three dynamin genes. Cells were then treated with CCCP or valinomycin to induce mitochondrial fission. E Histogram showing that mutations in all three dynamin genes do not prevent CCCP or valinomycin induced mitochondrial fission. 50 cells/experiment, SEM, n=3, unpaired student’s t-test. Scale bar is 10 μm.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Immunofluorescence, Staining, Knock-Out

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Relative numbers Dnm2-EndoB1 PLA spots increase, while Dnm2-EndoA1 PLA spots marginally decrease when autophagy is induced. B PLA spots for endogenous Dnm2 and EndoB1 (red) colocalize with EGFP-LC3 (green) when mitophagy is induced (4 hr CCCP). C A larger fraction of Dnm2-EndoB1 PLA spots colocalize with GFP-tagged LC3, Atg2 and Fip200 when autophagy is induced with rapamycin or CCCP, suggesting that Dnm2 is connected with autophagy. The fraction that colocalizes with GFP-tagged Lamp1 decreases and remains low for Stx17 and Rab7. For the histograms, 50 cells per experiment, SEM, n=3, unpaired Student’s t-test. Scale bar is 10 μm.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques:

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Overexpressed GFP-Dnm2 spontaneously colocalizes with RFP-LC3 in Dnm2 -/- cells, but not in WT cells. This colocalization is disrupted by the PIKfyve inhibitor YM201636. Enlarged portions of the cells (2X) are shown below each image with merged, Dnm2 (green) and LC3 (red) channels. B Colocalization of GFP fused to the PH domain of PLC delta with RFP-LC3 is also observed in Dnm2 -/- cells. C Histograms show increased colocalization of the GFP-tagged PH domain of PLC delta with RFP-LC3 in Dnm2 -/- cells. For the histograms, 50 cells per experiment, SEM, n=3, unpaired Student’s t-test. Scale bar is 10 μm. D Example of Dnm2 and EndoB1 -/- effects on CCCP induced LC3 lipidation. There is an accumulation of LC3-II in EndoB1 -/- cells but not in Dnm2 -/- cells. E Starvation induced autophagy is not affected by mutations in Dnm2 or EndoB1 as shown with p62 turnover and LC3 lipidation.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques:

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Effects of CCCP on mitophagy in WT, Dnm2 -/-, EndoB1 -/- and DKO cells observed with immunofluorescence microscopy using Tom20 (red) and Hsp60 (green) antibodies. At 24 hr, most mitochondria are degraded, except in EndoB1 -/- cells, where mitophagy is inhibited. Scale bar is 10 μm. B Western blots showing the effects of CCCP on Hsp60 levels in the mutant cells. The bottom panel shows average intensities of Hsp60 bands in four independent experiments, relative to tubulin levels and normalized at 100% for 0hr time point. C Super-resolution images (SIM) show the formation of LC3-membranes (green) encapsulating mitochondria (red) in cells treated with CCCP. These structures are observed in WT, Dnm2 -/- and DKO cells, but not in EndoB1 -/- cells. Cells were treated for 6 hr with 20 μM CCCP. Mitochondria were detected with mitoDsRed and autophagic membranes were detected with EGFP-LC3. Scale bar is 10 μm for top panels and 1 μm for the enlargements. D Western blots showing the effects of CCCP and Bafilomycin A treatments on LC3 lipidation in wildtype, Dnm2 -/-, EndoB1 -/- and DKO cells. E Effects of Dnm2 and EndoB1 deletions on colocalization of RFP-LC3 and GFP-Rab7. Autophagy was induced for 1 hr with Rapamycin, Scale bar is 10 μm. F Reduced levels of LC3 and Rab7 colocalization expressed as Mander’s coefficient for untreated cells and for cells treated with Rapamycin, Bafilomycin or both. There were 50 cells per experiment, SEM, n=3, unpaired Student’s t-test.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Immunofluorescence, Microscopy, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Time lapse images of GFP-Atg9, Dnm2-RFP and BFP-LC3. Images were taken at 350 msec intervals. An arrow in the middle panel points to a potential scission event. The bar in the first panel shows the line used for the intensity tracings shown in the bottom panels. Scale bar is 1 μm. B Mean frequencies of Dnm2-RFP spots transiently wedged between GFP-Atg9 and BFP-LC3 spots in untreated cells and cells treated with Dynole 34-2 or Ryngo 1-23 (n = 20, 17 and 22, SEM, unpaired T test). The frequencies were low because only a small part of a cell was observed for a limited time (50 frames), but their transient nature and the opposing effects of a dynamin inhibiter and an activator suggest a functional connection with Atg9 and LC3 fission and fusion events. C RFP-Atg9 colocalizes with EGFP-LC3 in Dnm2 -/- cells treated with CCCP, but not in wild type cells. Scale bar is 10 μm for whole cells and 3 μm for enlarged portions. D Histogram showing increased colocalization of RFP-ATG9 and GFP-LC3 in Dnm2 -/- cells, dynasore-treated WT cells and EndoB1 -/- cells after incubation with rapamycin or CCCP. E The number of Atg9 vesicles (RFP spots without GFP label) decreases when Dnm2 -/- cells, dynasore-treated WT cells and EndoB1 -/- cells are incubated with rapamycin or CCCP. For panels D and E, 50 cells were counted per experiment, n= 3, SEM, unpaired students’ t-test. F Images of autophagosome containing fractions from WT and Dnm2 -/- cells transfected with EGFP-Atg9 and RFP-LC3 followed by 4hr treatments with CCCP and Bafilomycin to induce mitophagy and to accumulate autophagosomes. Autophagosomes were enriched by differential centrifugation. Enlargements show individual spots with line tracings to plot fluorescence intensities. Scale bar is 10 μm in the overview images and 2 μm in the enlargements. The x-axis in the fluorescence intensity plots show numbers of pixels (24 pixel/μm) and the bars show 500 nm. G Scatter plot of peak fluorescence intensities for individual spots, as shown in panel F (arbitrary units). H Histogram of the peak fluorescence intensities of Atg9 in LC3 spots relative to the peak intensities of Atg9 in spots without LC3. Bar is SE, with Student’s t-test for 3 independent experiments. 30 spots for each condition were measured in each experiment. I Subcellular fractionation of homogenates from WT and Dnm -/- MEF cells, which were treated for 4 hr with Rapamycin. Membranous organelles were separated with a 1-22% Ficoll density gradient and analyzed with Western blots. The bottom fraction (#20) contains mitochondria and other dense organelles such as mitophagosomes, autophagolysosomes or lysosomes. Atg9 is normally present in light fractions (fraction #6), but partially shifts to an intermediate density (fraction #14) in Dnm2 -/- cells, along with some p62, LC3-II and Rab7a. J Delayed dissociation of Atg9 and p62 in Dnm2 -/- cells. A buildup of Atg9 and p62 was induced for 16h with the Vps34 inhibitor Vps34-IN1. This was followed by recovery through washout of the inhibitor for the indicated lengths of time. Association and dissociation of Atg9 and p62 was monitored by immunofluorescence microscopy of endogenous proteins in WT and Dnm2 -/- HeLa cells.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Functional Assay, Incubation, Transfection, Centrifugation, Fluorescence, Fractionation, Western Blot, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Example of a Dnm2 spot transiently wedged between Atg9 and LC3 containing organelles. Time lapse images of GFP-Atg9, RFP-Dnm2 and BFP-LC3 were taken at 350 msec intervals. An arrow in the middle panel points to a potential scission event. The bar in the first panel shows the line used for the intensity tracings shown in the bottom panels. Scale bar is 1 μm. B, C Effects of Dnm2 on Atg9 retrieval in live cells. Images of BFP-LC3 (shown in red) and GFP-Atg9 in live WT and Dnm2 -/- cells, untreated or after 5 hr with Bafilomycin. Scale bar is 5 μm. Histograms showing increased colocalization of BFP-LC3 and GFP-Atg9 in Dnm2 -/- cells with or without Bafilomycin treatments. 50 cells per condition in each experiment, n=3, SE and unpaired student’s T test. D Western blot showing depletion of Dnm2 after transfection of WT cells with siRNA oligonucleotides. E RFP-Atg9 colocalizes with EGFP-LC3 after 4h treatment with CCCP in Dnm2 siRNA cells, but not in mock-transfected cells. F Histogram showing increased colocalization of RFP-ATG9 and GFP-LC3 in CCCP-treated Dnm2 siRNA cells. Scale bar is 10 μm. G Western blot showing depletion of Dnm2 in a CRISPR/Cas9 generated clone HeLa cells. Clone G8 was chosen for further analysis. H RFP-Atg9 colocalizes with EGFP-LC3 in Dnm2 -/- HeLa cells treated with CCCP, but not in wild type cells. Scale bar is 10 μm for whole cells and 3 μm for enlarged portions. I Histogram showing increased colocalization of RFP-ATG9 and GFP-LC3 in Dnm2 -/- cells after incubation with rapamycin or CCCP. J Image of autophagosome containing fraction from Dnm2 -/- cells transfected with EGFP-Atg9 and RFP-LC3 followed by 4hr treatments with CCCP and Bafilomycin to induce mitophagy and to accumulate autophagosomes. Autophagosomes were enriched by differential centrifugation. Scale bar is 10 μm. K Enlargements show an individual spot with the line used to plot intensities. Scale bar is 2 μm. The x-axis in the fluorescence intensity plot shows numbers of pixels (24 pixel/μm) and the bar shows 500 nm. L Line tracings show fluorescence intensities

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Western Blot, Transfection, CRISPR, Generated, Incubation, Centrifugation, Fluorescence

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Tracking autophagy with RFP-GFP-LC3 marker shows transfer of LC3 to autophagolysomes, visible as red spots due to acidic quenching of GFP. B Numbers of phagophores or autophagosomes per cells, detected as yellow spots are increased in Dnm2 -/- cells. C Numbers of autophagolysosomes, detected as red spots, are dramatically increased in Dnm2 -/- cells treated with CCCP. D Colocalization of RFP-LC3 with GFP-Lamp1 is similarly increased in Dnm2 -/- cells. E, F Colocalization of GFP-VAMP7 with RFP-LC3 is increased in Dnm2 -/- cells and increases more after CCCP treatment. G, H Colocalization of GFP-Fip200 with RFP-LC3 is increased in Dnm2 -/- cells and increases more after CCCP treatment. I, J There is also increased colocalization of RFP-Atg9 and GFP-LAMP1 after treatment with CCCP. Cells were incubated with pepstatin and E64d to prevent Atg9 digestion in lysosomes. For panels B, C, F, H and J, 50 cells were counted per experiment, n= 3, unpaired students’ t-test. Scale bar is 10 μm for whole cells and 5 μm for enlarged portions. K Quantification of Atg9 protein levels in wildtype and Dnm2 -/- cells. Levels decrease in Dnm2 -/- cells after treatment with CCCP, but this decrease is partially prevented by Bafilomycin A, suggesting that Atg9 is degraded by autophagy in Dnm2 -/- cells. Band intensities were determined with Licor software, n = 4, unpaired Students’ t-test. L Genetic representation of the proposed roles for Dnm2 and EndoB1 during autophagy. The initial stages of phagophore formation during which EndoB1, as part of the Vps34 complex, promotes the role of Dnm2 in the retrieval of Atg9 through fission from phagophores (1a). At this stage, Dnm2 also plays an inhibitory role, preventing fusion to late endosomes or lysosomes (1b), so that further maturation of the phagophore only occurs when growth of the phagophore is complete. M A later stage during which EndoB1 switches from a positive to a negative function, suppressing the inhibitory effects of Dnm2 on fusion to late endosomes and lysosomes (2).

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Marker, Incubation, Software

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: Atg9 vesicles derived from other compartments, such as the Golgi and early endosomes collect near the site of autophagy, where they undergo homotypic fusion assisted by Fip200. These larger Atg9 containing structures can then fuse to the growing phagophore. After depositing membrane, Atg9 is retrieved by Dnm2 and EndoB1 mediated scission and returned to the originating organelles.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Immunofluorescence of endogenous Dnm2 (green) and EndoB1 (red) is shown without or with 2 hr rapamycin. B Mander’s coefficients for colocalization shown for untreated cells and after 2 or 4 hr with rapamycin or CCCP (50 cells were counted per experiment, SE for 3 independent experiments, unpaired Student’s t-test). C, D Similar experiments with transfected cells expressing Dnm2-GFP and EndoB1-mRuby2. E Colocalization of Dnm2 and EndoB1 tested with Proximity Ligation Assay (PLA) shown with or without 2 hr Antimycin A treatment. F Average numbers of PLA dots/cell shown for Dnm2 and EndoB1 colocalization in untreated cells, cells treated with staurosporine (STS) or actinomycin D to induce apoptosis, or with CCCP, Antimycin A, rapamycin, starvation and ionomycin to induce autophagy. Numbers of dots were counted in 50 cells/experiment (SEM, n=3, unpaired student’s t-test). Scale bar is 10 μm for whole cells and 5 μm for enlarged portions.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Immunofluorescence, Transfection, Expressing, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Scatterplots of immunofluorescence images of cells treated for 2h with or without rapamycin to induce autophagy. B Fraction of spots in the colocalizing quadrants of the scatterplots, shown before and after 2 or 4 hr treatment with rapamycin or CCCP. (SE and unpaired Student’s t-test, n= 3). C, D As in panels A and B, but with transiently expressed GFP- and RFP-tagged proteins. E Western blots showing derivation of Dnm2, EndoB1 and DKO cells. Dnm2 and EndoB1 -/- cells were generated by CRISPR/Cas9 in MEFs. DKO cells were generated by additional knockout of EndoB1 in Dnm2 -/- cells. Clone A4 was chosen for further studies. F PLA signals for Dnm2 and EndoB1 interactions were absent from cells with mutations in one or both of these proteins. G As negative control for PLA, cells were incubated with Fis1 and Hsp60 antibodies, which detect proteins that are on the outside or inside of mitochondria and are therefore too far apart for generating PLA signals. As positive control, cells were incubated with antibodies for Drp1 and Mff, which detect well-characterized binding partners. Scale bar is 10 μm. H Western blots of conditional dynamin knockout cells. Cre-recombinase was induced with tamoxifen at the indicated concentrations for 24 hr and cells were grown for an additional 4 days with fresh medium before western blot analysis of Dnm1 and Dnm2 expression. Although Dnm1 is not normally expressed in these cells and Dnm3 is undetectable with currently available antibodies , all three genes were floxed with 3 μM tamoxifen to ensure complete lack of dynamins.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Immunofluorescence, Western Blot, Generated, CRISPR, Knock-Out, Negative Control, Incubation, Positive Control, Binding Assay, Expressing

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Bax translocation to mitochondria is delayed when apoptosis is induced in Dnm2 -/- cells, but not in EndoB1 -/- cells. B, C Similar effects were observed with cytochrome c release from mitochondria and with the appearance of pyknotic nuclei, which are two other indicators of apoptosis. D Bax translocation to mitochondria is delayed in cells lacking all three dynamins. Triple knockout cells (TKO) were generated by treating conditional cells (labeled as WT) with tamoxifen to induce Cre recombinase. 50 cells per experiment, SEM, n=3, unpaired Student’s t-test.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Translocation Assay, Triple Knockout, Generated, Labeling

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Immunofluorescence of cells stained with antibodies for Tom20 (red) and Hsp60 (green) show no effects of Dnm2 or EndoB1 mutations on mitochondrial fission. The top row has images of untreated cells, the middle row has cells treated for 30 min with CCCP and the bottom row has cells treated for 30 min with valinomycin to induce mitochondrial fission. B Images of Drp1 -/- cells treated without or with CCCP. C Histogram showing that mutations in Dnm2 and EndoB1 do not prevent CCCP or valinomycin induced mitochondrial fission. D Images of cells before and after treatment with tamoxifen to induce knockout of all three dynamin genes. Cells were then treated with CCCP or valinomycin to induce mitochondrial fission. E Histogram showing that mutations in all three dynamin genes do not prevent CCCP or valinomycin induced mitochondrial fission. 50 cells/experiment, SEM, n=3, unpaired student’s t-test. Scale bar is 10 μm.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Immunofluorescence, Staining, Knock-Out

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Relative numbers Dnm2-EndoB1 PLA spots increase, while Dnm2-EndoA1 PLA spots marginally decrease when autophagy is induced. B PLA spots for endogenous Dnm2 and EndoB1 (red) colocalize with EGFP-LC3 (green) when mitophagy is induced (4 hr CCCP). C A larger fraction of Dnm2-EndoB1 PLA spots colocalize with GFP-tagged LC3, Atg2 and Fip200 when autophagy is induced with rapamycin or CCCP, suggesting that Dnm2 is connected with autophagy. The fraction that colocalizes with GFP-tagged Lamp1 decreases and remains low for Stx17 and Rab7. For the histograms, 50 cells per experiment, SEM, n=3, unpaired Student’s t-test. Scale bar is 10 μm.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques:

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Overexpressed GFP-Dnm2 spontaneously colocalizes with RFP-LC3 in Dnm2 -/- cells, but not in WT cells. This colocalization is disrupted by the PIKfyve inhibitor YM201636. Enlarged portions of the cells (2X) are shown below each image with merged, Dnm2 (green) and LC3 (red) channels. B Colocalization of GFP fused to the PH domain of PLC delta with RFP-LC3 is also observed in Dnm2 -/- cells. C Histograms show increased colocalization of the GFP-tagged PH domain of PLC delta with RFP-LC3 in Dnm2 -/- cells. For the histograms, 50 cells per experiment, SEM, n=3, unpaired Student’s t-test. Scale bar is 10 μm. D Example of Dnm2 and EndoB1 -/- effects on CCCP induced LC3 lipidation. There is an accumulation of LC3-II in EndoB1 -/- cells but not in Dnm2 -/- cells. E Starvation induced autophagy is not affected by mutations in Dnm2 or EndoB1 as shown with p62 turnover and LC3 lipidation.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques:

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Effects of CCCP on mitophagy in WT, Dnm2 -/-, EndoB1 -/- and DKO cells observed with immunofluorescence microscopy using Tom20 (red) and Hsp60 (green) antibodies. At 24 hr, most mitochondria are degraded, except in EndoB1 -/- cells, where mitophagy is inhibited. Scale bar is 10 μm. B Western blots showing the effects of CCCP on Hsp60 levels in the mutant cells. The bottom panel shows average intensities of Hsp60 bands in four independent experiments, relative to tubulin levels and normalized at 100% for 0hr time point. C Super-resolution images (SIM) show the formation of LC3-membranes (green) encapsulating mitochondria (red) in cells treated with CCCP. These structures are observed in WT, Dnm2 -/- and DKO cells, but not in EndoB1 -/- cells. Cells were treated for 6 hr with 20 μM CCCP. Mitochondria were detected with mitoDsRed and autophagic membranes were detected with EGFP-LC3. Scale bar is 10 μm for top panels and 1 μm for the enlargements. D Western blots showing the effects of CCCP and Bafilomycin A treatments on LC3 lipidation in wildtype, Dnm2 -/-, EndoB1 -/- and DKO cells. E Effects of Dnm2 and EndoB1 deletions on colocalization of RFP-LC3 and GFP-Rab7. Autophagy was induced for 1 hr with Rapamycin, Scale bar is 10 μm. F Reduced levels of LC3 and Rab7 colocalization expressed as Mander’s coefficient for untreated cells and for cells treated with Rapamycin, Bafilomycin or both. There were 50 cells per experiment, SEM, n=3, unpaired Student’s t-test.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Immunofluorescence, Microscopy, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Time lapse images of GFP-Atg9, Dnm2-RFP and BFP-LC3. Images were taken at 350 msec intervals. An arrow in the middle panel points to a potential scission event. The bar in the first panel shows the line used for the intensity tracings shown in the bottom panels. Scale bar is 1 μm. B Mean frequencies of Dnm2-RFP spots transiently wedged between GFP-Atg9 and BFP-LC3 spots in untreated cells and cells treated with Dynole 34-2 or Ryngo 1-23 (n = 20, 17 and 22, SEM, unpaired T test). The frequencies were low because only a small part of a cell was observed for a limited time (50 frames), but their transient nature and the opposing effects of a dynamin inhibiter and an activator suggest a functional connection with Atg9 and LC3 fission and fusion events. C RFP-Atg9 colocalizes with EGFP-LC3 in Dnm2 -/- cells treated with CCCP, but not in wild type cells. Scale bar is 10 μm for whole cells and 3 μm for enlarged portions. D Histogram showing increased colocalization of RFP-ATG9 and GFP-LC3 in Dnm2 -/- cells, dynasore-treated WT cells and EndoB1 -/- cells after incubation with rapamycin or CCCP. E The number of Atg9 vesicles (RFP spots without GFP label) decreases when Dnm2 -/- cells, dynasore-treated WT cells and EndoB1 -/- cells are incubated with rapamycin or CCCP. For panels D and E, 50 cells were counted per experiment, n= 3, SEM, unpaired students’ t-test. F Images of autophagosome containing fractions from WT and Dnm2 -/- cells transfected with EGFP-Atg9 and RFP-LC3 followed by 4hr treatments with CCCP and Bafilomycin to induce mitophagy and to accumulate autophagosomes. Autophagosomes were enriched by differential centrifugation. Enlargements show individual spots with line tracings to plot fluorescence intensities. Scale bar is 10 μm in the overview images and 2 μm in the enlargements. The x-axis in the fluorescence intensity plots show numbers of pixels (24 pixel/μm) and the bars show 500 nm. G Scatter plot of peak fluorescence intensities for individual spots, as shown in panel F (arbitrary units). H Histogram of the peak fluorescence intensities of Atg9 in LC3 spots relative to the peak intensities of Atg9 in spots without LC3. Bar is SE, with Student’s t-test for 3 independent experiments. 30 spots for each condition were measured in each experiment. I Subcellular fractionation of homogenates from WT and Dnm -/- MEF cells, which were treated for 4 hr with Rapamycin. Membranous organelles were separated with a 1-22% Ficoll density gradient and analyzed with Western blots. The bottom fraction (#20) contains mitochondria and other dense organelles such as mitophagosomes, autophagolysosomes or lysosomes. Atg9 is normally present in light fractions (fraction #6), but partially shifts to an intermediate density (fraction #14) in Dnm2 -/- cells, along with some p62, LC3-II and Rab7a. J Delayed dissociation of Atg9 and p62 in Dnm2 -/- cells. A buildup of Atg9 and p62 was induced for 16h with the Vps34 inhibitor Vps34-IN1. This was followed by recovery through washout of the inhibitor for the indicated lengths of time. Association and dissociation of Atg9 and p62 was monitored by immunofluorescence microscopy of endogenous proteins in WT and Dnm2 -/- HeLa cells.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Functional Assay, Incubation, Transfection, Centrifugation, Fluorescence, Fractionation, Western Blot, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Example of a Dnm2 spot transiently wedged between Atg9 and LC3 containing organelles. Time lapse images of GFP-Atg9, RFP-Dnm2 and BFP-LC3 were taken at 350 msec intervals. An arrow in the middle panel points to a potential scission event. The bar in the first panel shows the line used for the intensity tracings shown in the bottom panels. Scale bar is 1 μm. B, C Effects of Dnm2 on Atg9 retrieval in live cells. Images of BFP-LC3 (shown in red) and GFP-Atg9 in live WT and Dnm2 -/- cells, untreated or after 5 hr with Bafilomycin. Scale bar is 5 μm. Histograms showing increased colocalization of BFP-LC3 and GFP-Atg9 in Dnm2 -/- cells with or without Bafilomycin treatments. 50 cells per condition in each experiment, n=3, SE and unpaired student’s T test. D Western blot showing depletion of Dnm2 after transfection of WT cells with siRNA oligonucleotides. E RFP-Atg9 colocalizes with EGFP-LC3 after 4h treatment with CCCP in Dnm2 siRNA cells, but not in mock-transfected cells. F Histogram showing increased colocalization of RFP-ATG9 and GFP-LC3 in CCCP-treated Dnm2 siRNA cells. Scale bar is 10 μm. G Western blot showing depletion of Dnm2 in a CRISPR/Cas9 generated clone HeLa cells. Clone G8 was chosen for further analysis. H RFP-Atg9 colocalizes with EGFP-LC3 in Dnm2 -/- HeLa cells treated with CCCP, but not in wild type cells. Scale bar is 10 μm for whole cells and 3 μm for enlarged portions. I Histogram showing increased colocalization of RFP-ATG9 and GFP-LC3 in Dnm2 -/- cells after incubation with rapamycin or CCCP. J Image of autophagosome containing fraction from Dnm2 -/- cells transfected with EGFP-Atg9 and RFP-LC3 followed by 4hr treatments with CCCP and Bafilomycin to induce mitophagy and to accumulate autophagosomes. Autophagosomes were enriched by differential centrifugation. Scale bar is 10 μm. K Enlargements show an individual spot with the line used to plot intensities. Scale bar is 2 μm. The x-axis in the fluorescence intensity plot shows numbers of pixels (24 pixel/μm) and the bar shows 500 nm. L Line tracings show fluorescence intensities

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Western Blot, Transfection, CRISPR, Generated, Incubation, Centrifugation, Fluorescence

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: A Tracking autophagy with RFP-GFP-LC3 marker shows transfer of LC3 to autophagolysomes, visible as red spots due to acidic quenching of GFP. B Numbers of phagophores or autophagosomes per cells, detected as yellow spots are increased in Dnm2 -/- cells. C Numbers of autophagolysosomes, detected as red spots, are dramatically increased in Dnm2 -/- cells treated with CCCP. D Colocalization of RFP-LC3 with GFP-Lamp1 is similarly increased in Dnm2 -/- cells. E, F Colocalization of GFP-VAMP7 with RFP-LC3 is increased in Dnm2 -/- cells and increases more after CCCP treatment. G, H Colocalization of GFP-Fip200 with RFP-LC3 is increased in Dnm2 -/- cells and increases more after CCCP treatment. I, J There is also increased colocalization of RFP-Atg9 and GFP-LAMP1 after treatment with CCCP. Cells were incubated with pepstatin and E64d to prevent Atg9 digestion in lysosomes. For panels B, C, F, H and J, 50 cells were counted per experiment, n= 3, unpaired students’ t-test. Scale bar is 10 μm for whole cells and 5 μm for enlarged portions. K Quantification of Atg9 protein levels in wildtype and Dnm2 -/- cells. Levels decrease in Dnm2 -/- cells after treatment with CCCP, but this decrease is partially prevented by Bafilomycin A, suggesting that Atg9 is degraded by autophagy in Dnm2 -/- cells. Band intensities were determined with Licor software, n = 4, unpaired Students’ t-test. L Genetic representation of the proposed roles for Dnm2 and EndoB1 during autophagy. The initial stages of phagophore formation during which EndoB1, as part of the Vps34 complex, promotes the role of Dnm2 in the retrieval of Atg9 through fission from phagophores (1a). At this stage, Dnm2 also plays an inhibitory role, preventing fusion to late endosomes or lysosomes (1b), so that further maturation of the phagophore only occurs when growth of the phagophore is complete. M A later stage during which EndoB1 switches from a positive to a negative function, suppressing the inhibitory effects of Dnm2 on fusion to late endosomes and lysosomes (2).

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Marker, Incubation, Software

Journal: bioRxiv

Article Title: Dynamin-2 controls phagophore maturation

doi: 10.1101/241901

Figure Lengend Snippet: Atg9 vesicles derived from other compartments, such as the Golgi and early endosomes collect near the site of autophagy, where they undergo homotypic fusion assisted by Fip200. These larger Atg9 containing structures can then fuse to the growing phagophore. After depositing membrane, Atg9 is retrieved by Dnm2 and EndoB1 mediated scission and returned to the originating organelles.

Article Snippet: Addgene provided pcDNA3-mRuby2 (#40260), GFP-C1-PLCdelta-PH (#21179), Dnm2-EGFP (#34686), Dnm2-mCherryN1 (#27689), ptfLC3 (#21074), pMXs-puro-RFP-ATG9A (#60609), pEGFP-LC3 (# 24920), GFP-Rab7A (#61803), mTaqBFP2-ER-5 (#55294), pcDNA3-mRuby2 (#40260), GFP-Atg2 (#36456), GFP-Fip200 (#38192), GFP-VAMP7 (#42316) and pmRFP-LC3 (#21073).

Techniques: Derivative Assay