Journal: EMBO Reports

Article Title: A novel bacterial effector protein mediates ER-LD membrane contacts to regulate host lipid droplets

doi: 10.1038/s44319-024-00266-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-VAPA , Proteintech , 15275-1-AP.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Modification, Cloning, Staining, Magnetic Beads, Protease Inhibitor, Software, Microscopy, Western Blot

Journal: eLife

Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

doi: 10.7554/eLife.85962

Figure Lengend Snippet: ( A ) Left panel: Representative immunoblots showing the levels of VAPA and Tubulin in Control and VAPA KO cells. Tubulin expression level was used as loading control. Right panel: quantification of relative VAPA density in Control and VAPA KO cells normalized to Tubulin levels (mean ± SEM from three independent experiments). Data were analysed using a single-sample Student t-test. ( B ) Confocal images of Control and VAPA KO cells immunostained for VAPA. Scale bar 20 µm. ( C ) Confocal images of VAPA KO leader cells expressing wild-type mCherry-VAPA (VAPA KO +WT) or mCherry-VAPA KD/MD (VAPA KO +KDMD). Scale bar 10 µm. ( D ) Phase contrast images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells migrating collectively 48 hr after space release. For each condition, the trajectory of a leader cell over 20 hr is shown in green. Scale bar: 100 µm. ( E ) Analysis of relative colonized surface within the last 20 h by Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells (mean ± SEM from three independent experiments). Data were analysed using a One-way Anova paired test. ( F, G ) Analysis of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cell velocity ( F ) n=40, 40, 3,9 and 39 cells respectively from three independent experiments and directionality coefficient ( G ) n=34, 36, 32, and 26 cells respectively from three independent experiments. Data were analysed using a One-way Anova Kruskal-Wallis test. ( H ) Phase contrast images of a Control and VAPA KO individual cell displacing on fibronectin-coated glass during 5 minutes. The cell trajectory is shown in blue. Scale bar: 20 µm. ( I, J ) Analysis of cell velocity ( I ) and directionality coefficient ( J ) of Control and VAPA KO individual cells displacing on fibronectin-coated glass during at least 3 h (mean ± SEM; Control: n=64 cells; VAPA KO: n=33 cells from two independent experiments). data were analysed using non parametric Mann-Whitney t-test. ( K ) Epifluorescence images of Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD cells after 1 hour spreading on fibronectin-coated glass and stained as indicated. Scale bar: 50 µm. ( L ) Analysis of cells area after 1 hour spreading on fibronectin-coated glass (mean ± SEM; n=117, 134, 113, and 128 cells respectively from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 1—source data 1. Table containing the raw data used for the quantifications in . Figure 1—source data 2. Folder containing the 2 original files of the full raw unedited blots for VAPA and Tubulin presented in figure with the uncropped annotated blots.

Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

Techniques: Western Blot, Control, Expressing, MANN-WHITNEY, Staining

Journal: eLife

Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

doi: 10.7554/eLife.85962

Figure Lengend Snippet: ( A ) Left panel: representative immunoblots showing the levels of VAPB and Tubulin in Control and VAPA KO cells. Tubulin level was used as loading control. Right panel: quantification of relative VAPB density in Control and VAPA KO cells normalized to Tubulin levels (mean ± SEM from three independent experiments). Data were analysed using a single-sample Student t-test (ns: non significant). ( B ) Confocal images of Control and VAPA KO leader cells immunostained for VAPB. Scale bar: 20 µm (5 µm in insets). Figure 1—figure supplement 1—source data 1. Table containing the raw data used for the quantifications . Figure 1—figure supplement 1—source data 2. Folder containing the 2 original files of the full raw unedited blots for VAPB and Tubulin presented in and a figure with the uncropped annotated blots.

Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

Techniques: Western Blot, Control

Journal: eLife

Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

doi: 10.7554/eLife.85962

Figure Lengend Snippet: ( A ) Confocal images with zoom boxes of central (green squares) and peripheral (blue squares) paxillin-labeled focal adhesions from Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells in migrating monolayers 24 -48hr after insert removal. Scale bar: 20 µm (2 µm in insets). ( B, C ) Analysis of central ( B ) and peripheral ( C ) focal adhesion area quantified from images in A, in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (Control: n=2968 central and 353 peripheral focal adhesions from 20 cells, VAPA KO: n=4329 central and 293 peripheral focal adhesions from 22 cells, VAPA KO +WT: n=3818 central and 336 peripheral focal adhesions from 24 cells, VAPA KO +KDMD: n=2801 central and 297 peripheral focal adhesions from 19 cells, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test.( D ) Confocal images of actin cytoskeleton network in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells stained for cortactin and F-actin. Green arrows point to F-Actin transversal arcs. Plot profiles of the cortactin signal along the leading edge are shown on the right. Pink lines highlight the cortactin-rich protrusive subdomains. Scale bar: 20 µm.( E ) Differential Interference Contrast (DIC) images (left) and kymographs (right) along the yellow lines of Control and VAPA KO leader cells from a 30 min movie at 1 frame every 3 seconds, showing protrusion and retraction phases of the leading edge. Scale bar: 20 µm (left) and 5 µm (right). ( F, G ) Analysis of proportion of the leading edge enriched with cortactin ( F ) and mean size of cortactin-enriched domains at the leading edge normalized to the length of the leading edge ( G ) quantified from images in D (mean ± SEM; n=29 cells for each cell line, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. ( H ) Quantification of protrusion phases frequency per 30 min quantified from the kymographs in E, in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (mean ± SEM; n=16, 21, 17 and 12 cells respectively, from three independent experiments). Data were analysed using a non parametric Mann-Whitney t-test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 2—source data 1. Table containing the raw data used for the quantifications .

Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

Techniques: Labeling, Control, Staining, MANN-WHITNEY

Journal: eLife

Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

doi: 10.7554/eLife.85962

Figure Lengend Snippet: ( A, B ) Confocal images and zoom boxes of PI(4)P distribution in Control and VAPA KO leader cells expressing GFP-PH-OSBP and immunostained for TGN46 ( A ) or EEA1 ( B ). Scale Bar: 10 µm. ( C ) Analysis of Golgi(TGN46)/cytosol (left panel) and Early endosomes(EEA1)/Cytosol (right panel) ratio of GFP-PH-OSBP signal, quantified from images in A and B, in Control and VAPA KO leader cells (Left panel: n=24–25 cells, Right panel: n=13 cells; from three independent experiments). ( D, H ) Top: Sum projection of confocal images of PI(4)P ( D ) and PI(4,5)P2 ( H ) distribution in Control and VAPA KO leader cells expressing mCherry-P4M SidM or RFP-PH-PLCδ1 respectively, represented as a color-coded heat map. Scale bar: 10 µm. Bottom: Plot profiles of normalized grey levels along the pink lines. ( E, I ) Analysis of PM/Cytosol ratio of mCherry-P4M SidM ( E ) and RFP-PH-PLCδ1 ( I ), quantified from plot profiles represented in D and H respectively, in protrusive domains at the leading edge of Control and VAPA KO leader cells (E: n=14–16 cells, I: n=26–27 cells; from three independent experiments). ( F ) XZ view of confocal images of Control and VAPA KO cells immunostained for PI(4,5)P2. Scale Bar: 5 µm. ( G ) Analysis of PI(4,5)P2 peak intensity, quantified from images in F, in Control and VAPA KO leader cells (Control: n=58 cells; VAPA KO: n=53 cells, from three independent experiments). All data were analysed using non parametric Mann-Whitney t-test (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 3—source data 1. Table containing the raw data used for the quantifications .

Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

Techniques: Control, Expressing, MANN-WHITNEY

Journal: eLife

Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

doi: 10.7554/eLife.85962

Figure Lengend Snippet: ( A ) TIRF microscopy images of GFP-MAPPER foci distribution along the ER in Control and VAPA KO leader cells expressing GFP-MAPPER and RFP-KDEL. Scale bar: 5 µm. ( B ) Sequential TIRF microscopy images of ER and GFP-MAPPER foci accumulation at the front of a Control leader cell expressing GFP-MAPPER and RFP-KDEL. Scale bar: 1 µm. ( C ) Confocal images of GFP-MAPPER foci distribution along Cherry-VAPA containing ER in Control cells transiently expressing Cherry-VAPA. Scale bar: 1 µm. ( D ) Transmission Electron Microscopy images of transversal cuts of a Control leader cell, showing the leading edge. Arrows point to ER-PM contact sites at the bottom of the cell. Scale bar: 1 µm (top) and 100 nm in insets 1 and 2. ( E ) Sequential TIRF microscopy images of GFP-MAPPER foci at the front of Control and VAPA KO leader cells expressing GFP-MAPPER. Individual GFP-MAPPER foci at each time point are pictured in the frames below images. Scale bar: 1 µm. ( F, G ). Analysis of the lifetime ( F ) and the speed ( G ) of ventral GFP-MAPPER foci, quantified from images in E, in Control and VAPA KO leader cells (Control: n=92 and 52 foci from 8 cells; VAPA KO: n=77 and 58 foci from 6 cells, from four independent experiments). All data were analysed using non parametric Mann-Whitney t-test (***p-values <0.001). Figure 4—source data 1. Table containing the raw data used for the quantifications .

Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

Techniques: Microscopy, Control, Expressing, Transmission Assay, Electron Microscopy, MANN-WHITNEY

Journal: eLife

Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

doi: 10.7554/eLife.85962

Figure Lengend Snippet: ( A ) Confocal images of GFP-MAPPER foci distribution along anti-VAPA-stained ER in Control cells. On the right: plot profiles of normalized gray values along the two lines depicted in yellow. Scale bar: 1 µm. ( B ) Sequential spinning disk images of dorsal (magenta) and ventral (green) GFP-MAPPER foci at the front of a Control leader cell expressing GFP-MAPPER. Top and bottom plane images were color-coded in magenta and green respectively. Circles highlight single GFP-MAPPER foci at the dorsal (blue) and ventral (red) sides. Scale bar: 3 µm. ( C ) Representative Transmission Electron Microscopy images of a Control and a VAPA KO cell showing ER-PM contact sites. Scale bar: 0.3 µm. The ER compartment and the PM are pictured in the right framebox. ( D, E ). Analysis of the ER portion in contact with the PM ( D ) and the percentage of the PM in contact with the ER ( E ), quantified from images in A, in Control and VAPA KO leader cells (mean ± SEM; Control: n=25 cells: VAPA KO: n=14 cells, from three independent experiments). All data were analysed using non parametric Mann-Whitney t-test (ns: non significant). Figure 4—figure supplement 1—source data 1. Table containing the raw data used for the quantifications and 1E.

Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

Techniques: Staining, Control, Expressing, Transmission Assay, Electron Microscopy, MANN-WHITNEY

Journal: eLife

Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

doi: 10.7554/eLife.85962

Figure Lengend Snippet: ( A ) Sequential TIRF microscopy images of focal adhesions in Control and VAPA KO leader cells expressing mCherry-Vinculin. Scale Bar: 1 µm. ( B, C ) Analysis of assembly rate ( B ) and disassembly rate ( C ) of focal adhesions (FA), quantified from time-lapse images in A, in Control and VAPA KO leader cells (whisker plots with 10–90 percentile; Control: n=217 FA from 9 cells; VAPA KO: n=342 FA from 8 cells, from three independent experiments). Data were analysed using non parametric Mann-Whitney t-test. ( D ) Distribution of focal adhesion life times in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (Control: n=98 FA from 10 cells; VAPA KO: n=120 FA from 12 cells, VAPA KO +WT: n=79 FA from 8 cells, VAPA KO +KDMD: n=90 FA from 9 cells, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. ( E ) Confocal images of focal adhesions after nocodazole treatment and wash-out in migrating Control and VAPA KO leader cells immunostained for paxillin. Scale bar: 10 µm (2 µm in insets). ( F ) Analysis of relative focal adhesions (FA) size, quantified from images in E, in Control and VAPA KO leader cells after 0 min, 15 min, 30 min, and 60 min after nocodazole wash-out (FA from 22 to 25 cells were analysed, from 3 independent experiments. Control T0min: n=6053 FA; Control T15 min: n=5146 FA; Control T30 min: n=5543 FA; Control T60 min: n=3913 FA; VAPA KO T0 min: n=4481 FA, VAPA KO T15 min: n=3878 FA, VAPA KO T30 min: n=4165 FA; T60 min: n=4889 FA). Data were analysed using non parametric Mann-Whitney t-test. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 5—source data 1. Table containing the raw data used for the quantifications .

Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

Techniques: Microscopy, Control, Expressing, Whisker Assay, MANN-WHITNEY

Journal: eLife

Article Title: The ER tether VAPA is required for proper cell motility and anchors ER-PM contact sites to focal adhesions

doi: 10.7554/eLife.85962

Figure Lengend Snippet: ( A ) TIRF microscopy images of GFP-MAPPER and focal adhesions in Control and VAPA KO leader cells expressing GFP-MAPPER and mCherry-Vinculin. Scale bars: 10 µm (1 µm in insets). ( B ) Analysis of the percentage of central (top) or peripheral (bottom) focal adhesions in contact with GFP-MAPPER foci in Control, VAPA KO, VAPA KO +WT and VAPA KO +KDMD leader cells (n=10–12 cells, from three independent experiments). Data were analysed using a One-way Anova Kruskal-Wallis test. ( C ) Sequential TIRF microscopy images of GFP-MAPPER foci and focal adhesions before and after its disassembly in a Control and 2 representative VAPA KO leader cells expressing GFP-MAPPER and mCherry-Vinculin. Scale bar: 1 µm. ( D ) Time course of GFP-MAPPER (green) and mCherry-Vinculin (magenta) signals during the lifetime of focal adhesions in the yellow ROI depicted in C. The signals were smoothed, readjusted to the minimal value and expressed as % of the maximal value. ( E ) Histogram representing the repartition of first anchoring time of GFP-MAPPER foci relative to focal adhesion disassembly in Control and VAPA KO leader cells, quantified from images in D (n=21 focal adhesions from 7 cells from three independent experiments). ( F ) Analysis of duration of the first anchoring of GFP-MAPPER foci to focal adhesions in Control and VAPA KO leader cells, from images in D (n=21 focal adhesions from 7 cells from three independent experiments). Data were analysed using a non parametric Mann-Whitney t-test. ( G ) TIRF microscopy images of iRFP-Vinculin, GFP-MAPPER and Cherry-VAPA WT in a VAPA KO leader cell. Scale bar: 2 µm. ( H ) Plot profiles of normalized grey levels along the two lines depicted in G. (ns: non significant, ***p-values <0.001, **p-values <0.01, *p-values <0.05). Figure 6—source data 1. Table containing the raw data used for the quantifications .

Article Snippet: The following primary antibodies were used: mouse anti-EEA1 monoclonal antibody (BD transduction), rabbit anti-VAPB polyclonal antibody (Atlas antibody), mouse anti-VAPA monoclonal antibody and rabbit anti-VAPA polyclonal antibody for the immunoblot (Sigma), rabbit anti-TGN46 polyclonal antibody (Abcam), mouse anti-paxilline monoclonal antibody (Merck), mouse anti-cortactin monoclonal antibody (Merck), mouse anti-tubulin monoclonal antibody (GeneTex), mouse anti-PI(4,5)P2 monoclonal IgM antibody (Echelon).

Techniques: Microscopy, Control, Expressing, MANN-WHITNEY