Journal: bioRxiv

Article Title: Spatiotemporal proteomics reveals the biosynthetic lysosomal membrane protein interactome in neurons

doi: 10.1101/2024.05.16.594502

Figure Lengend Snippet: a , Heatmap in which the Log 2 Fold Change across time points of axonal synaptic proteins and members of the SNARE complex is plotted. b , Live neuron expressing RUSH-LAMP2A-mNG, RUSH-SYT1-Halo and scramble, imaged during 1h of biotin addition. Still images show part of the Golgi and a budding event. Intensity profile graph in the bottom. c , Neurons expressing RUSH-LAMP2A-mNG and RUSH-SYT1-Halo at 1 and 4h post-release. Kymographs from live cell imaging along the axon every 1s for 180 s are shown. Colocalized anterograde (blue), retrograde (orange) or stationary (grey) trajectories were traced on the right. d , Quantification of the number of trajectories for 1 and 4h. n = 15 and 19 neurons; N=3. e , Confocal images of neurons expressing RUSH-LAMP1-V5 and EGFP-VAMP4, 1h after release. Blue and orange boxes indicate magnified areas shown on the right, with corresponding intensity profile graph. f - g , Confocal images of neurons expressing RUSH-LAMP1-V5 ( f ) or RUSH-Syt1-mNG ( g ) plus shRNA against VAMP4, or scramble. Quantifications of the number of RUSH-LAMP1 (n= 19 and 27 neurons; N=4) and SYT1 (n= 27 and 22 cells; N=3) positive compartments are shown on the right. h , Still images from the soma of neuron expressing RUSH-LAMP2a-mNG, RUSH-SYT1-Halo and shRNA against VAMP4; control scramble in (b). Images show a Golgi budding event after 1h release. Corresponding intensity profile graph on the right. i ) Neurons expressing RUSH-LAMP2A-mNG and shRNA against VAMP4 or scramble. Neurons were fixed after 1h release and stained for LAMTOR4. Magnified images from , and respective intensity profile graphs are shown. j ) Neurons transfected as in (h) were labeled for SirLyso and imaged live after 1h release. Still images from time points indicated in images and respective intensity profile graphs are shown. k ) Temporal intensity profile graph for RUSH-SYT1 and SirLyso from image in (j). See also & . Data are presented as mean values ± SD, plus individual points. ns–not significant; *p<0.05; *** p<0.001; **** p<0.0001. Mann-Whitney test in (d), (f), and (g).

Article Snippet: The following primary antibodies were used in this study: rabbit anti-LAMTOR4 (Cell Signaling, clone D6A4V, Cat# 12284S, RRID: AB_2797870, 1/500), mouse anti-Stx6 (BD Biosciences Cat# 610635, RRID:AB_397965, 1/100), rabbit anti-VAMP4 (Synaptic Systems, Cat# 136002, RRID:AB_887816, 1/100), mouse anti-V5 (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564, 1/1000 for IF and WB), mouse anti-Pan-Neurofascin external (clone A12/18; UC Davis/NIH NeuroMab, Cat# 75-172, RRID: AB_2282826, 0.18 mg/ml), in-house rabbit anti-TRIM46 (1/1000), mouse anti-VTI1B (BD Biosciences Cat# 611404, RRID:AB_398926, 1/250), rabbit anti-GM130 (Abcam Cat# ab52649, RRID:AB_880266, 1/800).

Techniques: Expressing, Live Cell Imaging, shRNA, Staining, Transfection, Labeling, MANN-WHITNEY

Journal: bioRxiv

Article Title: Spatiotemporal proteomics reveals the biosynthetic lysosomal membrane protein interactome in neurons

doi: 10.1101/2024.05.16.594502

Figure Lengend Snippet: a , Confocal images of somas from neurons expressing RUSH-LAMP1-V5 and scramble or VAMP4 shRNA, fixed 1h after release and stained for LAMTOR4 and GM130. c , Still images of the soma of a live neuron expressing RUSH-LAMP2A-mNG and RUSH-SYT1-Halo, and VAMP4 shRNA, 1h after release and labeled with SirLyso. Scale bar, 5 µm in (a) and (b).

Article Snippet: The following primary antibodies were used in this study: rabbit anti-LAMTOR4 (Cell Signaling, clone D6A4V, Cat# 12284S, RRID: AB_2797870, 1/500), mouse anti-Stx6 (BD Biosciences Cat# 610635, RRID:AB_397965, 1/100), rabbit anti-VAMP4 (Synaptic Systems, Cat# 136002, RRID:AB_887816, 1/100), mouse anti-V5 (Thermo Fisher Scientific Cat# R960-25, RRID:AB_2556564, 1/1000 for IF and WB), mouse anti-Pan-Neurofascin external (clone A12/18; UC Davis/NIH NeuroMab, Cat# 75-172, RRID: AB_2282826, 0.18 mg/ml), in-house rabbit anti-TRIM46 (1/1000), mouse anti-VTI1B (BD Biosciences Cat# 611404, RRID:AB_398926, 1/250), rabbit anti-GM130 (Abcam Cat# ab52649, RRID:AB_880266, 1/800).

Techniques: Expressing, shRNA, Staining, Labeling

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: PeerJ

Article Title: Phosphorylation of the N-terminus of Syntaxin-16 controls interaction with mVps45 and GLUT4 trafficking in adipocytes

doi: 10.7717/peerj.15630

Figure Lengend Snippet: HeLa cells expressing HA-GLUT4-GFP were transfected with plasmids encoding the N-terminal 33 amino acids of Sx16 fused to protein A (WT) or the same peptide with an F10A mutation, or empty plasmid (control). 48 h after transfection, cells were stimulated with or without 1 µM insulin for 60 min and fixed. Cell surface GLUT4 was immuno-stained using anti-HA (pseudo-coloured blue) in non-permeabilised cells. After washing, cells were permeabilised and cells expressing Protein-A fusions identified using a distinct secondary antibody (pseudo-coloured red). Signal from GFP is pseudo-coloured green. (A) Data from a typical experiment; protein-A positive cells are indicated with a white asterisk in some panels so that transfected cells can be readily identified. (B) >50 cells in each condition from four separate biological replicates were quantified to allow calculation of the HA/GFP ratio; values are expressed relative to Basal control (non-transfected) cells. An asterisk (*) indicates a significant reduction in insulin-stimulated surface/total ratio compared to control and F10A insulin-stimulated cells ( p = 0.02 ANOVA).

Article Snippet: Antibodies used: anti-GLUT4 (Thermo Fisher Scientific Cat# PA1-1065, RRID:AB_2191454), anti-Sx16 (Synaptic Systems Cat# 110 162, RRID:AB_887799), anti-mVps45 (Novus Cat# NB100-2431, RRID:AB_2272935), anti-VAMP4 (Synaptic Systems Cat# 136 002, RRID:AB_887816), anti-SNAP23 (Synaptic Systems Cat# 111 202, RRID:AB_887788) and anti-Syntaxin 4 (Synaptic Systems Cat# 110 042, RRID:AB_887853).

Techniques: Expressing, Transfection, Mutagenesis, Plasmid Preparation, Staining

Journal: PeerJ

Article Title: Phosphorylation of the N-terminus of Syntaxin-16 controls interaction with mVps45 and GLUT4 trafficking in adipocytes

doi: 10.7717/peerj.15630

Figure Lengend Snippet: HeLa cells expressing HA-GLUT4-GFP were transfected with plasmids encoding either HA-mVps45 (wild-type) or HA-mVps45-V107R (a mutant which prevents the interaction of the Sx16 N-terminus with mVps45 –see text). 48 h after transfection, cells were incubated in serum-free media for 2 h, fixed and cell surface GLUT4 immuno-stained using anti-HA (pseudo-coloured blue) prior to permeabilization. Subsequently, cells were permeabilised and stained using anti-mVps45 which detects both endogenous and over-expressed mVps45 (pseudo-coloured red; note that the use of HA-tagged mVps45 constructs and HA-tagged GLUT4 precluded this as a means to distinguish cells over-expressing mVps45 species). Signal from GFP is pseudo-coloured green. (A) Data from a typical experiment; white asterisk represent cells expressing higher than average anti-mVps45 immunoreactivity and which are therefore assumed to be over-expressing the indicated species. Data from a typical experiment is shown. (B) Levels of HA-GLUT4-GFP were not significantly altered upon over-expression of either wild-type or mVps45-V107R, which were expressed at similar levels (both are recognised by anti-HA); the approximate positions of molecular weight markers are shown (in kDa). (C) Quantification of the HA/GFP ratio from fields of cells such as those shown in (A). Fields of cells transfected with mVps45-V107R exhibited increased HA/GFP ratios compared to cells transfected with wild-type mVps45 ( ∗ p < 0.001 ANOVA). Wild-type transfected cells were indistinguishable from non-transfected controls (ns; p = 0.33 ANOVA). Each point on the graph is from a single field of cells; data from three independent biological experiments is presented.

Article Snippet: Antibodies used: anti-GLUT4 (Thermo Fisher Scientific Cat# PA1-1065, RRID:AB_2191454), anti-Sx16 (Synaptic Systems Cat# 110 162, RRID:AB_887799), anti-mVps45 (Novus Cat# NB100-2431, RRID:AB_2272935), anti-VAMP4 (Synaptic Systems Cat# 136 002, RRID:AB_887816), anti-SNAP23 (Synaptic Systems Cat# 111 202, RRID:AB_887788) and anti-Syntaxin 4 (Synaptic Systems Cat# 110 042, RRID:AB_887853).

Techniques: Expressing, Transfection, Mutagenesis, Incubation, Staining, Construct, Over Expression, Molecular Weight

Journal: PeerJ

Article Title: Phosphorylation of the N-terminus of Syntaxin-16 controls interaction with mVps45 and GLUT4 trafficking in adipocytes

doi: 10.7717/peerj.15630

Figure Lengend Snippet: 3T3-L1 adipocytes were infected with lentivirus delivering either wild-type Sx16, or Sx16-T7A or Sx16-T7D as outlined in Methods . (A) DeGlc uptake was assayed after incubation with or without 1 µM insulin for 30 min. Shown are the means of three independent experiments in which basal and insulin-stimulated deGlc uptake rates were measured in quadruplicate at each condition and are presented as a % of the insulin-stimulated rate in control (non-infected) cells. Over-expression of Sx16-T7D consistently impaired insulin-stimulated deGlc uptake; p = 0.02 compared to control cells (ANOVA). No other differences were observed between groups. (B) Shown are lysates from a typical dataset immunoblotted with anti-Sx16, anti-GLUT4, anti-mVps45 and anti-GAPDH. Note that over-expressed Sx16 and mutants thereof consistently migrate faster than the endogenous protein (indicated by * on the figure), the approximate positions of molecular weight markers are indicated (in kDa). We saw no significant differences in levels of expression of the different Sx16 mutants across all experiments of this type. (C) 3T3-L1 adipocytes expressing either Sx16-WT, T7A or T7D (as indicated) were treated with or without 1 µM insulin for 30 min and lysates separated on SDS-PAGE and immunoblotted using antibodies that recognise phosphorylated Akt or total Akt, as shown. Data from a typical experiment is shown.

Article Snippet: Antibodies used: anti-GLUT4 (Thermo Fisher Scientific Cat# PA1-1065, RRID:AB_2191454), anti-Sx16 (Synaptic Systems Cat# 110 162, RRID:AB_887799), anti-mVps45 (Novus Cat# NB100-2431, RRID:AB_2272935), anti-VAMP4 (Synaptic Systems Cat# 136 002, RRID:AB_887816), anti-SNAP23 (Synaptic Systems Cat# 111 202, RRID:AB_887788) and anti-Syntaxin 4 (Synaptic Systems Cat# 110 042, RRID:AB_887853).

Techniques: Infection, Incubation, Over Expression, Molecular Weight, Expressing, SDS Page