Fig 2: AP1-mediated sorting of VAMP4 to endolysosomes regulates its abundance in the SV pool.(A, C, and E) Representative images display hippocampal neurons transfected with VAMP4-EGFP and mCherry, a-synuclein A53T, or red fluorescent protein (RFP)–bassoon (Bsn) (A), VAMP4-EGFP–transfected neurons with and without 30 µM SMIFH2 (C) and VAMP4-EGFP cotransfected with either AP1 shRNA (AP1 KD) or a scrambled (Scr) control (E). Scale bars, 5 µm. (B, D, and F) Synaptic VAMP4 expression under all conditions described in (A), (C) and (E). (B) n = 40 (mCherry), n = 51 (a-synuclein A53T), and n = 53 (bassoon) cells from three independent preparations, ****P < 0.001 and **P = 0.0038, Kruskal-Wallis test with Dunn’s multiple comparisons test (P values adjusted). (D) n = 47 (control) and n = 61 (SMIFH) cells from three independent preparations (F), n = 18 coverslips (scrambled and AP1 KD) from four independent preparations, ***P = 0.0009 and **P = 0.0085, Mann-Whitney test. (G and H) Hippocampal neurons were transfected with either wild-type or VAMP4 L25A–FT. (G) Representative images display fluorescence in the blue (new protein), red (old protein), or merged channel. Scale bar, 5 µm. (H) Blue/red ratio [n = 30 (wild-type) and n = 39 (L25A) cells from four independent preparations, ****P < 0.001, Mann-Whitney test]. (I to K) Hippocampal neurons cotransfected with VAMP4-pHluorin and or shRNAs against either AP1 or CHC (or scrambled). Representative images (I) (scale bar, 5 µm) and quantification of VAMP4-pHluorin expression (J) in either scrambled or CHC KD synapses [n = 12 (scrambled), n = 14 (CHC KD) coverslips from three independent preparations, *P = 0.011, Mann-Whitney test]. (K) VAMP4-pHluorin fusion events [n = 12 (scrambled), n = 10 (AP1 KD), and n = 14 (CHC KD) coverslips from three independent preparations, ****P < 0.0001 and **P < 0.01, Kruskal-Wallis test with Dunn’s multiple comparisons test].

Fig 3: The effect of knockdown of VAMP isoforms on HA–GLUT4–GFP translocation.HeLa cells expressing HA–GLUT4–GFP were grown and transfected with 200 nM scrambled control sequence (SCR), VAMP2, 4 or 8 SMARTpool siRNA as described. Cells were serum-starved (basal) before being treated with or without 1 µM insulin (insulin) for 20 min. Cells were fixed and surface HA was stained as described. (A) Immunofluorescence images of a typical field for each condition are shown. (B) The fold-change in the HA/GFP ratio with insulin-stimulation. Values were compared using Student’s t-test (*p < 0.05 and **p > 0.05) and are means ± SD of 16 random fields of view, taken from four independent experiments. In this analysis, the HA/GFP ratio in the absence of insulin is set equal to 1 for each siRNA. VAMP2 knockdown significantly inhibited insulin-simulated HA–GLUT4–GFP translocation, *p = 0.01. The apparent reduction in translocation upon VAMP4 knockdown did not reach statistical significance. (C) Quantifies the basal HA/GFP ratio for each VAMP knockdown compared to that observed in SCR siRNA treated cells; values represent the means ± SD of 16 fields of view, taken from four independent experiments **p < 0.05.

Fig 4: SNAP25 co‐localizes better with 4‐day‐old antibody‐labeled Synaptotagmin 1 molecules A, BTwo‐color STED analysis of changes in synaptic vesicle protein levels during the transition from the releasable state to the inactive state. Living neurons were incubated with Atto647N‐conjugated Synaptotagmin 1 lumenal domain antibodies for 1 h at 37°C, to label the actively recycling vesicles, as in Fig 1A, and were then fixed and co‐immunostained for different proteins of interest directly, or were placed in a cell culture incubator for 3–4 days, to enable the antibody‐labeled molecules to enter the inactive pool, before fixation and co‐immunostaining. The samples were embedded in melamine and cut in ultrathin (50 nm) sections, before two‐color STED imaging, as in Fig 2B. Exemplary images are shown in (A) for SNAP25 and in (B) for Syntaxin 1, with the protein of interest signal next to the signal from the live tagging of Synaptotagmin 1, and a merged image of both, for day 0 (releasable vesicles) and day 4 (inactive vesicles). Scale bar: 1 μm.CWe analyzed the amount of fluorescence corresponding to the protein of interest that overlapped with the Synaptotagmin 1 signal (i.e., the two signals were presented within the same voxels, which are substantially below the synaptic vesicle volume in this experiment). The only protein whose levels changed significantly is SNAP25 (SNAP25, n (day 0) = 4, n (day 4) = 3, *P = 0.0124, t(5) = 3.8200; Syntaxin 1, n (day 0) = 3, n (day 4) = 3, P = 0.8850, t(4) = 0.1541; VGlut 1/2, n (day 0) = 2, n (day 4) = 3, P = 0.1986, t(3) = 1.6447; vATPase, n (day 0) = 3, n (day 4) = 4, P = 0.7340, t(5) = 0.3594; VAMP2, n (day 0) = 4, n (day 4) = 4, P = 0.8837, t(6) = 0.1527; Synaptotagmin 1, n (day 0) = 3, n (day 4) = 3, P = 0.1604, t(4) = 1.7208; Syntaxin 16, n (day 0) = 4, n (day 4) = 4, P = 0.7406, t(6) = 0.3468; VAMP4, n (day 0) = 3, n (day 4) = 3, P = 0.9863, t(4) = 0.0183; Synapsin I/II, n (day 0) = 3, n (day 4) = 3, P = 0.6638, t(4) = 0.4685; at least 10 neurons sampled per experiment). Statistical significance was evaluated using unpaired t‐tests. All data represent the mean ± SEM.Data information: Imaging was performed with a Leica SP5 STED microscope.Source data are available online for this figure.

Fig 5: Endolysosomal sorting of VAMP4 regulates its abundance in the SV pool.(A and B) Hippocampal neurons cotransfected with VAMP4-pHluorin and either wild-type (WT), constitutively active (Q67L), or dominant-negative (T22N) mCherry-rab7. Scale bar, 5 µm. Representative images (A) and bar graph (B) display synaptic VAMP4-pHluorin expression. n = 23 (mCherry), n = 12 (mCherry-rab7), n = 13 (Q67L), and n = 18 (T22N) coverslips from four independent preparations, ****P < 0.001, Kruskal-Wallis test with Dunn’s multiple comparisons test. (C) Effect of T22N mCherry-rab7 overexpression on sypHy synaptic expression. n = 21 (mCherry) or n = 16 (T22N mCherry-rab7) coverslips from four independent preparations, ***P < 0.0002, Mann-Whitney test. (D) Fusion events reported by VAMP4-pHluorin upon stimulation with 400 APs at 40 Hz under the conditions shown in (A) and (B), n = 23 (mCherry), n = 12 (mCherry-rab7), n = 13 (Q67L), and n = 18 (T22N) coverslips from four independent preparations, *P = 0.0129, Kruskal-Wallis test with Dunn’s multiple comparisons test.