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: Nature Communications

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

doi: 10.1038/s41467-024-55052-w

Figure Lengend Snippet: a Heatmap in which the log 2 Fold Change across time points of members of the kinesin family and motor adaptors is plotted (two-sided t-test, no multiple comparison test was used). b Confocal images of axons of neurons expressing RUSH-LAMP1-V5 and shRNA scramble, or shRNAs against KIF5A-C, KIF1A or ARL8B. c Quantification of the number of LAMP1 compartments in ( b ). n = 26, 21, 18 and 15 neurons, respectively; each from 3 independent experiments ( N = 3; **** p < 0.0001, **** p < 0.0001, *** p = 0.0003) (Scale bar, 5 µm). d – f Confocal image of the soma ( d scale bar, 10 µm) or axon ( f scale bar, 5 µm) of neurons expressing RUSH-LAMP1-V5 and GFP-RAB6A. e Magnified images of the area in ( d ) indicated by orange box (Scale bar, 2 µm). g Quantification of ratios for total axonal RUSH-LAMP1-V5 and RAB6A colocalizing at 1 or 4 h after release. n = 22 and 16 neurons; each from 3 independent experiments ( N = 3). h Confocal images of axons from neurons expressing LAMP1-RFP and GFP-RAB6A. i Quantification of the ratios between total LAMP1 and RAB6A and colocalizing compartments, at the steady state ( n = 13; N = 4 independent experiments) j Representative still images of the axon of a neuron expressing LAMP1-RFP and GFP-RAB6A (Scale bar, 2 µm, repeated with 4 independent experiments). Data are presented as mean values ± SD, plus individual points. Ordinary one-way ANOVA test followed by Tukey’s multiple comparison test in ( c ), and Kruskal-Wallis test followed by Dunn’s multiple comparison test in ( g ). See also Supplementary Fig. . Source data are provided as a Source Data file.

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), rabbit anti-ARL8B (Proteintech Cat# 13049-1-AP, RRID:AB_2059000, 1/500), rabbit anti-KIF5A (Abcam Cat# ab5628, RRID:AB_2132218, 1/100), rabbit anti-KIF5B (Abcam Cat# ab5629, RRID:AB_2132379, 1/100), rabbit anti-KIF5C (Abcam Cat# ab5630, 1/100).

Techniques: Comparison, Expressing, shRNA

Journal: Nature Communications

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

doi: 10.1038/s41467-024-55052-w

Figure Lengend Snippet: a Schematic representation of identified proteins within enriched GO terms. Color coding indicates the time point(s) at which a given protein is enriched (Green 20 min; Red 1 hour; Blue 4 hour). Dashed lines between proteins specify physical interactions analyzed with STRING; grey rectangle depicts proteins involved in both processes. All indicated proteins are statistically significant ( p value < 0.05). Asterisks indicate that the Log fold change enrichment at that time point is smaller than 1. b – e Confocal images of neurons co-labeled for RUSH-LAMP1-V5 and STX6 ( b ), RAB11 ( c ), RAB7 ( d ), and LAMTOR4 ( e ) after 1 h or 4 h of biotin addition. Magnified regions from soma are depicted with orange boxes. Orange arrows indicate the region used to generate intensity profile graphs. Scale bar, 10 µm, and magnified images, 1 µm. Representative images in b – e were repeated in at least 2−3 independent experiments. See also Supplementary Figs. and , and Source Data 1. Source data are provided as a Source Data file.

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), rabbit anti-ARL8B (Proteintech Cat# 13049-1-AP, RRID:AB_2059000, 1/500), rabbit anti-KIF5A (Abcam Cat# ab5628, RRID:AB_2132218, 1/100), rabbit anti-KIF5B (Abcam Cat# ab5629, RRID:AB_2132379, 1/100), rabbit anti-KIF5C (Abcam Cat# ab5630, 1/100).

Techniques: Labeling

Journal: Nature Communications

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

doi: 10.1038/s41467-024-55052-w

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 (two-sided t-test, no multiple comparison test was used). b Live neuron expressing RUSH-LAMP2A-mNG, RUSH-SYT1-Halo and scramble, imaged during 1 h 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 4 h post-release. Kymographs from live cell imaging along the axon every 1 s 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 4 h. n = 15 and 19 neurons; each from 3 independent experiments ( N = 3; **** p < 0.0001, * p = 0.0433, ns p = 0.6092) e Confocal images of neurons expressing RUSH-LAMP1-V5 and EGFP-VAMP4, 1 h 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; each from 4 independent experiments N = 4, **** p < 0.0001) and SYT1 ( n = 27 and 22 cells; each from 3 independent experiments N = 3, **** p < 0.0001) 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 1 h release. Corresponding intensity profile graph on the right. i Neurons expressing RUSH-LAMP2A-mNG and shRNA against VAMP4 or scramble after 1 h release and immunostained for LAMTOR4 with respective intensity profile graphs are shown. j Neurons in ( h ) were labeled for SirLyso and imaged live after 1 h 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 ). Scale bars, 2 µm in ( b ), ( h – j ), 5 µm in ( f ), ( g ), and 10 µm in ( e ). Data are presented as mean values ± SD, plus individual points. Mann-Whitney test was used in ( d ), ( f ), and ( g ). See also Supplementary Figs. and . Representative images in b , e , and i were repeated in at least 3 independent experiments. Source data are provided as a Source Data file.

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), rabbit anti-ARL8B (Proteintech Cat# 13049-1-AP, RRID:AB_2059000, 1/500), rabbit anti-KIF5A (Abcam Cat# ab5628, RRID:AB_2132218, 1/100), rabbit anti-KIF5B (Abcam Cat# ab5629, RRID:AB_2132379, 1/100), rabbit anti-KIF5C (Abcam Cat# ab5630, 1/100).

Techniques: Comparison, Expressing, Live Cell Imaging, shRNA, Control, Labeling, MANN-WHITNEY