Journal: bioRxiv

Article Title: Apoptotic Extracellular Vesicles (ApoEVs) Safeguard Liver Homeostasis and Regeneration via Assembling an ApoEV-Golgi Organelle

doi: 10.1101/2021.02.24.432630

Figure Lengend Snippet: (A) Representative immunofluorescent images show apoEV (labeled with CellMask™ Deep Red, red) surface expression of vesicle-localized-soluble N -ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs, green) VAMP3, V VAMP4 and Golgi SNARE of 15 kDa (GS15). Flow cytometric analysis was performed to determine percentages of positively stained apoEVs. Scale bars = 25 μm. (B) Western blot analysis show expression of VAMPs in apoEVs compared to their source MSCs. (C) Western blot analysis show expression of VAMPs in apoEVs. Source MSCs were transfected with small interferon RNAs (siRNAs) as a negative control (siCtrl) for VAMP3 (si Vamp3 ), VAMP4 (si Vamp4 ), and both VAMP3 and VAMP4 (si Vamp3/4 ). Flow cytometric analysis was performed to determine percentages of positively stained apoEVs after knockdown of VAMPs. (D) Representative images show cultured Fas mutant ( Fas mut ) tetraploid hepatocytes (4N-HCs) with 130 kDa Golgi matrix protein (GM130) immunofluorescent staining (green), PKH26-labeled apoEVs (red), and DAPI counterstain (blue). White dashed lines depict cell borders of cultured HCs. Imaging analysis was performed to quantify apoEV uptake and percentages of Golgi-contacted apoEVs. Scale bars = 10 μm. N = 4 per group. (E) Representative fluorescent images show cultured Fas mut 4N-HCs with ac-α-tubulin immunostaining (white), N -acetylgalactosaminyltransferase 2 (GALNT2)-GFP-labeled Golgi (green), PKH26-labeled apoEVs (red), and DAPI counterstain (blue). Imaging analysis was performed to quantify HC percentages with Golgi fragmentation and acetylated microtubules (MTs) fluorescence intensity. Scale bars = 5 μm. N = 4 per group. (F) Western blot analysis showed human VAMP3 (hVAMP3) expression in isolated HC Golgi. ApoEVs were derived from human MSCs (hApoEVs). GM130 was used as an internal control. (G) Western blot analysis showed VAMP3 expression in apoEVs without or with treatment by 250 ng/ml Tetanus toxin (TeNT). (H) Representative Golgin84 immunofluorescent staining (green) images of liver Golgi counterstained with DAPI (blue). WT, wild type. Imaging analyses were performed to quantify HC percentages with Golgi fragmentation. Scale bars = 5 μm. N = 4 per group. (I) Golgi apparatus was isolated from the liver and Golgi protein mass was determined using the BCA method. N = 4 per group. (J) Representative liver fluorescent images showed HCs with different nuclei (blue, DAPI for DNA) and their cell borders (green, phalloidin for F-actin). # indicates binucleated HCs. Scale bars = 25 μm. N = 4 per group. (K) After PI staining, percentages of binucleated HCs were quantified. Diploid HCs (2N-HCs) were analyzed using flow cytometry. N = 4 per group. (L) Hematoxylin and eosin (H&E) staining of liver tissues in periportal vein (PV) areas. Hepatic injury scores were examined based on pathological parameters. Scale bars = 50 μm. N = 4 per group. (M) Serum alanine aminotransferase (ALT) levels were determined. N = 4 per group. (N) Graphical summary illustrating that apoEVs use VAMP3 to assemble with Golgi for HC and liver regulation. Data represent mean ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; NS, not significant, P > 0.05. Statistical analyses were performed by Student’s t test for two-group analysis or one-way analysis of variance followed by the Newman-Keuls post hoc tests for multiple group comparisons.

Article Snippet: For IF staining of surface markers of apoEVs, after isolation, apoEVs were stained with rabbit anti-human/mouse VAMP3 primary antibody (13640, Cell Signaling Technology, USA), rabbit anti-mouse VAMP4 primary antibody (PA1-768, Invitrogen, USA), mouse anti-mouse GS15 primary antibody (610960, BD Transduction Laboratories, UK), rabbit anti-mouse C1q primary antibody (ab71940, Abcam, UK), or mouse anti-mouse TSP1 primary antibody (sc-59887, Santa Cruz Biotechnology, USA) at 4°C for 1 h at a concentration of 1:100 in PBS.

Techniques: Labeling, Expressing, Staining, Western Blot, Transfection, Negative Control, Cell Culture, Mutagenesis, Imaging, Immunostaining, Fluorescence, Isolation, Derivative Assay, Flow Cytometry, Standard Deviation

Journal: Cell reports

Article Title: ELKS1 Captures Rab6-Marked Vesicular Cargo in Presynaptic Nerve Terminals

doi: 10.1016/j.celrep.2020.107712

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: rabbit anti-VAMP4 , Sysy , RRID:AB_887816.

Techniques: Recombinant, Software

Journal: Cell reports

Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

doi: 10.1016/j.celrep.2021.109678

Figure Lengend Snippet: Figure 1. VAMP2 and VAMP4 are markers of recycling endosome exocytosis in the soma and dendrites of hippocampal neurons (A–C) Images (top) and kymographs (bottom) of neurons (14 DIV) transfected with TfR-SEP (A), VAMP2-SEP (B), or VAMP4-SEP (C). Exocytosis events (sudden appearance of a bright cluster) are marked with green arrowheads. In (A), dim stable spots represent clathrin coated endocytic zones. Scale bar, 2 mm. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

Techniques: Transfection

Journal: Cell reports

Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

doi: 10.1016/j.celrep.2021.109678

Figure Lengend Snippet: Figure 2. Downregulation of VAMP4, but not VAMP2, impairs RE exocytosis and recycling to the plasma membrane (A) Frequency of exocytosis events in neurons transfected with TfR-SEP and TeNT-LC E234Q (n = 6) or TeNT-LC (n = 10). (B) Images of neurons co-transfected with VAMP2-SEP and TeNT-LC E234Q or TeNT-LC. VAMP2-SEP is enriched in the axon (cyan arrows) in the first case but not the second case. Scale bar, 10 mm. (C) Immunofluorescence images of endogenous VAMP4 in cells expressing GFP and a combination of shRNA targeted against VAMP4 for four days. In cells expressing GFP and the shRNA (cyan arrows), the labeling is strongly decreased compared to untransfected cells or cells expressing scramble (scr) shRNA. In cells co-expressing TfR, VAMP4-HA, and KD1, the VAMP4 staining is strong. Scale bar, 10 mm. Bottom, quantification of VAMP4 staining in the area delimited by the GFP mask (soma and dendrites). The staining is decreased by 50% in all KD conditions. The number of cells is indicated above the bars for all conditions. Comparison with scr with one-way ANOVA; *p < 0.05 and ***p < 0.001. (D) Frequency of exocytosis events recorded in cells expressing TfR-SEP and shRNAs targeted to VAMP4: scr (33 cells; 3 cells have frequencies of 0.132, 0.157, and 0.119 events.mm2.min1 and are represented above the axis limit), KD1 (23 cells), KD2 (10 cells), KD1+2 (18 cells), cells expressing VAMP4-HA (8 cells), and KD1+VAMP4-HA (12 cells). *p < 0.05 one-way ANOVA. (E) Images of neurons expressing scr or KD1 shRNAs in GFP vectors, labeled with A568-Tf (50 mg/ml) for 5 min and chased with unlabeled transferrin (2 mg/ml) at 37C for the indicated times. Scale bar, 10 mm. (F) Quantification of the Alexa568 fluorescence in the GFP mask from the pulse-chase experiments described in (E). 70 to 88 cells per condition from 4 inde- pendent experiments. Error bars represent SEM; **p < 0.01. (G) Estimation of TfR-SEP surface fraction. Top, cartoons showing the fraction of fluorescent TfR-SEP. At pH 7.4, surface receptors are fluorescent, but not at pH 5.5. Receptors in acidic intracellular organelles are not fluorescent, but become fluorescent with NH4Cl. Bottom left, images of a dendrite bathed successively in solutions at pH 7.4 (images 1, 3, and 5), pH 5.5 (image 2), and pH 7.4 containing NH4Cl (image 4). For image 4, the contrast is 23 lower than in the other images. Bottom right, quantification of the TfR-SEP surface fraction for neurons transfected with scr (n = 27) and KD1 (n = 26). See STAR Methods for calculation. ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

Techniques: Clinical Proteomics, Membrane, Transfection, Expressing, shRNA, Labeling, Staining, Comparison, Pulse Chase

Journal: Cell reports

Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

doi: 10.1016/j.celrep.2021.109678

Figure Lengend Snippet: Figure 3. TfR-SEP and VAMP4-SEP exocytosis increase after chemical LTP (A) Images of a neuron transfected with TfR-SEP before and 15 min after induction of cLTP. Cyan crosses show the location of detected exocytosis events. Scale bar, 5 mm. (B) Normalized exocytosis frequency of neurons transfected with TfR-SEP at times relative to cLTP induction (n = 16, control [ctrl]). The light blue area denotes the time of incubation with cLTP inducing medium. The increase in frequency is significant 15 min after induction (Dunnett’s multiple comparison test, p = 0.003). In the presence of APV (100 mM), the frequency does not increase (n = 12). (C) Exocytosis frequencies before and 15 min after LTP induction. Paired t test p = 0.0008 (ctrl) and p = 0.14 (APV). (D) Normalized change in fluorescence intensity of TfR-SEP before and after cLTP induction. The increase is significant after 10 min or more (Dunnett’s multiple comparison). (E) Changes in TfR-SEP fluorescence 20 min after cLTP induction, in ctrl (carmin dots) or with APV (gray dots). Paired t test p = 0.0002 (ctrl) and p = 0.89 (APV). (F–J) Same as (A)–(E) for neurons transfected with VAMP2-SEP (n = 9) and with APV (n = 7). The increase in frequency is significant 10 min or more after induction (p = 0.002). (K–O) Same as (A)–(E) for neurons transfected with VAMP4-SEP (n = 15) and with APV (n = 15). The increase in frequency is significant 15 min or more after induction (p = 0.0082).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

Techniques: Transfection, Control, Incubation, Comparison

Journal: Cell reports

Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

doi: 10.1016/j.celrep.2021.109678

Figure Lengend Snippet: Figure 4. Effect of TeNT-LC and VAMP4 KD on TfR-SEP exocytosis after cLTP (A) Exocytosis frequencies before and after LTP induction in neurons expressing TfR-SEP and either TeNT-LC E234Q (n = 13) or TeNT-LC (n = 13). In both conditions the increase in frequency is significant. (B) Images of dendrites before and after induction of cLTP. Scale bar, 5 mm. (C) TfR-SEP fluorescence in dendrites of neurons before and after cLTP induction. (D–F) Same as (A)–(C) for neurons expressing TfR-SEP and either scr (n = 10) or VAMP4 KD1 (n = 8) shRNA.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

Techniques: Expressing, shRNA

Journal: Cell reports

Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

doi: 10.1016/j.celrep.2021.109678

Figure Lengend Snippet: Figure 6. Effect of post-synaptic VAMP4 KD and TeNT on glutamatergic synaptic transmission (A) Confocal image of an organotypic hippocampal slice culture infected with scr-mScarlet lentivirus at 1 DIV and fixed at 9 DIV. Many pyramidal neurons in CA1 are brightly fluorescent. (B) DIC image of two pyramidal neurons recorded simultaneously with patch pipettes (asterisks). Epifluorescent illumination shows that the neuron on the left is brightly fluorescent (infected) while the one on the right is not (uninfected control). (C) Averages of 30 EPSCs evoked by the same stimulation in pairs of neurons, uninfected and infected with scr-mScarlet (top), shRNA KD1-mScarlet (middle), or shRNA KD2-mScarlet (botttom). Both neurons were held at 70 mV then at +40 mV. Right, plots of peak EPSC amplitude at 70 mV for each pair of neurons. In the scr condition, dots are spread around the diagonal, while in the KD1 and KD2 conditions the amplitudes are systematically higher for infected neurons. (D) Same as (C) for neurons co-electroporated with TeNT-LC and GFP. In the neurons expressing TeNT-LC, the amplitude is sytematically smaller than in control neurons.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

Techniques: Transmission Assay, Infection, Control, shRNA, Expressing

Journal: Cell reports

Article Title: The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

doi: 10.1016/j.celrep.2021.109678

Figure Lengend Snippet: Figure 7. Effect of post-synaptic VAMP4 KD and TeNT on LTP (A) Average EPSCs before (black traces) and 20 to 30 min after induction of LTP (color traces) in neurons electroporated with GFP and TeNT-LC (purple) or not (gray). The dotted line shows the peak EPSC before LTP induction. Scale bars, 40 pA and 20 ms. (B) Peak EPSC amplitude normalized to baseline for pairs of neurons transfected with TeNT-LC (purple) or not (gray) (C) Ratio of EPSC amplitude 20 to 30 min after LTP induction to baseline. (D) Same as (A) for neurons transduced with lentivirus expressing scr-mScarlet (blue), shRNA KD1-mScarlet (red), and shRNA KD2-mScarlet (green). Scale bars, 40 pA and 20 ms. (E) Peak EPSC amplitude normalized to baseline for of neurons expressing the corresponding shRNAs. (F) Same as (C) for transduced neurons. ****p < 0.0001. (G) Model of dendritic TfR and AMPAR receptor trafficking.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti VAMP4 Synaptic Systems Cat# 136002, RRID AB_887816 Mouse anti Rab11 BD Biosciences Cat# 610657, RRID AB_397984 Rabbit anti GluA1 Sigma Aldrich Cat# AB1504, RRID AB_2113602 Mouse anti VAMP2 Synaptic Systems Cat# 104211, RRID AB_2619758 Rabbit anti FIP2 Antibodies Online Cat# ABIN6275434, RRID AB_11206004 Chicken anti mScarlet Synaptic Systems Cat# 409006, RRID AB_2725776 Mouse anti actin Sigma Aldrich Cat# A5316, RRID AB_476743 Chemicals, peptides, and recombinant proteins D-APV Abcam Cat# ab120003 Picrotoxin Sigma Cat# P1675 Strychnine hydrochloride Sigma Cat# S8753 Experimental models: Organisms/strains Rat, Sprague Dawley Janvier Labs N/A Oligonucleotides VAMP4 shRNA1 target sequence CTATCTTTATTTAACAACA N/A VAMP4 shRNA2 target sequence GGACCATCTGGACCAAGAT N/A scramble shRNA AATTCTCCGAACGTGTCAC N/A Recombinant DNA SEP-GluA1 Jullié et al., 2014; Rosendale et al., 2017 N/A TfR-SEP Jullié et al., 2014; Rosendale et al., 2017 N/A VAMP4-SEP This paper Addgene 174406 VAMP2-SEP Martineau et al., 2017 N/A TeNT-LC Proux-Gillardeaux et al., 2005 N/A TeNT-LC E234Q Proux-Gillardeaux et al., 2005 N/A VAMP4-HA This paper N/A Homer1c-tdTomato Rosendale et al., 2017 N/A VAMP2-SNAPtag Martineau et al., 2017 N/A VAMP4 KD1 mScarlet This paper Addgene 174407 VAMP4 KD2 mScarlet This paper Addgene 174408 Software and algorithms Metamorph 7.10 https://www.moleculardevices.com/ products/cellular-imaging-systems/ acquisition-and-analysis-software/ metamorph-microscopy N/A MATLAB 2018b https://fr.mathworks.com N/A Custom MATLAB scripts Exo_BD_analysis DOI 10.5281/zenodo.5146169 Igor Pro 6.0 https://www.wavemetrics.com/ N/A imageJ 1.53c http://www.imagej.nih.gov/ij N/A SpineJ 1.0 https://github.com/flevet/SpineJ N/A Cell Reports 36, 109678, September 7, 2021 e1

Techniques: Transfection, Transduction, Expressing, shRNA

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: Distribution and known functions of host SNAREs used in this study.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques:

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: A, Localization of endogenous SNAREs in HeLa cells infected for 24 h with C. trachomatis serovar D was assessed using specific antibodies. Vamp3 and Vamp7 encircle the inclusion (arrows), while Sec22 does not (arrowheads). SNAREs are shown on the left, host and bacterial DNA in the second column, and merged images in the third column. Distribution of the SNAREs in uninfected cells is shown on the right. B, Cells were transfected with different GFP-SNARE constructs 8 h before infection. The inclusion was labelled with anti-IncA antibodies followed with TRITC-coupled secondary antibodies. GFP-Vamp8 or GFP-Vamp7 encircle the inclusion membrane (arrows), while GFP, GFP-Vamp4 or GFP-Vamp7 deleted from its SNARE domain (GFP-Longin) do not (arrowheads). GFP-tagged SNAREs are shown on the left, IncA in the second column, and merged images in the third column. Distribution of the SNAREs in uninfected cells is shown on the right. C, Quantification of SNARE recruitment to the inclusion. Cells were scored as positive when the entire circumference of the inclusion was surrounded by the SNARE protein. Means and standard deviations of at least two independent experiments are shown.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques: Infection, Transfection, Construct, Membrane

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: A, Distribution of GFP was observed in fields with an inclusion, in cells transfected with GFP-Vamp4 (top) or GFP-Vamp8 (bottom), and infected. Two representative panels are shown for each construct. Arrowheads point to gold particles less than 50 nm from the inclusion membrane. I, inclusion, B, bacteria. B, Equivalent numbers of gold particles were counted in both sets of images and their localization relative to the inclusion membrane was assessed. Levels of significance are indicated by p-values comparing the distribution of GFP-Vamp4 and GFP-Vamp8 for each category.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques: Transfection, Infection, Construct, Membrane, Bacteria

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: A, GFP-SNARE proteins (V3 = Vamp3, V4 = Vamp4, V7 = Vamp7, V8 = Vamp8, Longin = Vamp7 without its SNARE motif) were immunoprecipitated from HeLa cells co-expressing IncA or empty vector (pQE). The top left panel (anti-His blot) shows the level of expression of IncA in cell lysates, and the bottom right panel (anti-GFP blot) shows immunoprecipitation of GFP-tagged proteins. IncA co-immunoprecipitated with Vamp3, Vamp7 and Vamp8, and very little with Vamp4 (top right panel, anti-IncA blot). B, Purified IncA-His and purified GST-Vamp3, GST-Vamp4 or GST-Vamp8 were inserted in two populations of liposomes, mixed, and GST-SNAREs were pulled-down using glutathione agarose beads. Pull-down complexes were resolved on SDS-PAGE gels and analyzed after staining with Coomassie.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques: Immunoprecipitation, Expressing, Plasmid Preparation, Purification, Liposomes, SDS Page, Staining

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: GFP-SNARE proteins (V3 = Vamp3, V4 = Vamp4, V7 = Vamp7, V8 = Vamp8, Longin = Vamp7 without its SNARE motif) were immunoprecipitated from HeLa cells co-expressing CT813-His* or empty vector (pQE). The top left panel (anti-His blot) shows the level of expression of CT813-His* in cell lysates, and the bottom right panel (anti-GFP blot) shows immunoprecipitation of GFP-tagged proteins. CT813-His* co-immunoprecipitated with Vamp7 and Vamp8 (top right panel, anti-His blot), and not with Vamp3, Vamp4, GFP or GFP-Longin. Absence of immunoprecipitation with Vamp3 might be due to insufficient expression level of both CT813-His* and GFP-Vamp3, which were consistently low in these experiments.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques: Immunoprecipitation, Expressing, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: SNARE Protein Mimicry by an Intracellular Bacterium

doi: 10.1371/journal.ppat.1000022

Figure Lengend Snippet: A, GFP-SNARE proteins were immunoprecipitated from HeLa cells co-expressing IncA wild-type, mutated in motif Nter (T126R) or in both SNARE-like motifs (TRQR), or empty vector (pQE) as control. The top left panel (anti-IncA blot) shows the level of expression of IncA in cell lysates, and the bottom right panel (anti-GFP blot) shows immunoprecipitation of GFP-tagged proteins. IncA wild-type and T126R immunopecipitated with the SNAREs, and the double mutant did not (anti-IncA blot,top right panel). B, Cells transfected for 18 hrs with the indicated His-tagged IncA constructs were infected for 20 h with C. trachomatis serovar D before fixation. Coverslips were stained with anti-His antibodies and anti-EfTu antibodies to label the bacteria. Histograms show the percentage of cells (n>100) with a normal inclusion for each population of transfected cells, in one experiment representative of three (means and standard deviations of two independent counts are shown). Only inclusions that looked intact were scored; in cells transfected with IncA wild-type and IncAT126R, and, to a lesser extent, IncAQ244R and IncAT126RQ244R (TRQR), many disrupted inclusions were also observed.

Article Snippet: Antibodies against Vamp4 were kindly provided by Dr. Andrew A. Peden (Cambridge Institute for Medical Research, Cambridge), rabbit anti-Vamp3, rabbit anti-Vamp8 and mouse anti-Vamp7 and vectors coding for GFP-Vamp3, GFP-Vamp7, GFP-Longin and GFP-Vamp8 were kindly provided by Dr Thierry Galli (Institut Jacques Monod, Paris), rabbit anti-Sec22 was described previously , plasmid coding for GFP-Vamp4 was a gift of Dr Ludger Johannes (Institut Curie, Paris).

Techniques: Immunoprecipitation, Expressing, Plasmid Preparation, Control, Mutagenesis, Transfection, Construct, Infection, Staining, Bacteria