Synaptotagmin‐11 inhibits clathrin‐mediated and bulk endocytosis
2015; Springer Nature; Volume: 17; Issue: 1 Linguagem: Inglês
10.15252/embr.201540689
ISSN1469-3178
AutoresChanghe Wang, Yeshi Wang, Meiqin Hu, Zuying Chai, Qihui Wu, Rong Huang, Weiping Han, Claire Xi Zhang, Zhuan Zhou,
Tópico(s)Signaling Pathways in Disease
ResumoArticle20 November 2015free access Source Data Synaptotagmin-11 inhibits clathrin-mediated and bulk endocytosis Changhe Wang Changhe Wang State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China College of Life Sciences, Forestry and Agriculture, Qiqihar University, Qiqihar, China Search for more papers by this author Yeshi Wang Yeshi Wang State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Meiqin Hu Meiqin Hu State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Zuying Chai Zuying Chai State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Qihui Wu Qihui Wu State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Rong Huang Rong Huang State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Weiping Han Weiping Han Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, Agency for Science, Technology, and Research, Singapore City, Singapore Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore Search for more papers by this author Claire Xi Zhang Corresponding Author Claire Xi Zhang State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Center of Parkinson's Disease, Beijing Institute for Brain Disorders, Capital Medical University, Beijing, China Search for more papers by this author Zhuan Zhou Zhuan Zhou State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Changhe Wang Changhe Wang State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China College of Life Sciences, Forestry and Agriculture, Qiqihar University, Qiqihar, China Search for more papers by this author Yeshi Wang Yeshi Wang State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Meiqin Hu Meiqin Hu State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Zuying Chai Zuying Chai State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Qihui Wu Qihui Wu State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Rong Huang Rong Huang State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Weiping Han Weiping Han Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, Agency for Science, Technology, and Research, Singapore City, Singapore Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore Search for more papers by this author Claire Xi Zhang Corresponding Author Claire Xi Zhang State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Center of Parkinson's Disease, Beijing Institute for Brain Disorders, Capital Medical University, Beijing, China Search for more papers by this author Zhuan Zhou Zhuan Zhou State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China Search for more papers by this author Author Information Changhe Wang1,2,‡, Yeshi Wang1,‡, Meiqin Hu1,‡, Zuying Chai1, Qihui Wu1, Rong Huang1, Weiping Han3,4, Claire Xi Zhang 1,5 and Zhuan Zhou1 1State Key Laboratory of Membrane Biology and Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine and PKU-IDG/McGovern Institute for Brain Research and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China 2College of Life Sciences, Forestry and Agriculture, Qiqihar University, Qiqihar, China 3Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, Agency for Science, Technology, and Research, Singapore City, Singapore 4Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore 5Center of Parkinson's Disease, Beijing Institute for Brain Disorders, Capital Medical University, Beijing, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 10 83950074; E-mail: [email protected] EMBO Reports (2016)17:47-63https://doi.org/10.15252/embr.201540689 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Precise and efficient endocytosis is essential for vesicle recycling during a sustained neurotransmission. The regulation of endocytosis has been extensively studied, but inhibitors have rarely been found. Here, we show that synaptotagmin-11 (Syt11), a non-Ca2+-binding Syt implicated in schizophrenia and Parkinson's disease, inhibits clathrin-mediated endocytosis (CME) and bulk endocytosis in dorsal root ganglion neurons. The frequency of both types of endocytic event increases in Syt11 knockdown neurons, while the sizes of endocytosed vesicles and the kinetics of individual bulk endocytotic events remain unaffected. Specifically, clathrin-coated pits and bulk endocytosis-like structures increase on the plasma membrane in Syt11-knockdown neurons. Structural–functional analysis reveals distinct domain requirements for Syt11 function in CME and bulk endocytosis. Importantly, Syt11 also inhibits endocytosis in hippocampal neurons, implying a general role of Syt11 in neurons. Taken together, we propose that Syt11 functions to ensure precision in vesicle retrieval, mainly by limiting the sites of membrane invagination at the early stage of endocytosis. Synopsis This study identifies Syt11 as an inhibitory regulator for neuronal endocytosis. Syt11 inhibits both clathrin-mediated and bulk endocytosis probably at the stage of membrane invagination. Syt11 inhibits clathrin-mediated endocytosis (CME) and bulk endocytosis in mammalian neurons. Syt11 limits the formation of clathrin-coated pits and and bulk endocytosis-like structures on the plasma membrane. Membrane localization and AP-2-binding ability of Syt11 are critical for CME. The Syt11 C2A domain is not necessary for CME while both C2 domains are required for bulk endocytosis. Introduction During neurotransmission, precise and efficient exocytosis-coupled endocytosis is critical for neurons to recapture and reuse vesicle components and to keep the area of plasma membrane constant, especially during a sustained neuronal activity 123. Several modes of endocytosis operate to recycle vesicle proteins and replenish vesicle pools 12345. Clathrin-mediated endocytosis (CME), the best-characterized pathway, is the predominant route of vesicle retrieval after exocytosis. Elevated neuronal activity also elicits bulk endo-cytosis when large areas of plasma membrane are internalized 124. To understand the high efficiency of exo-endocytotic coupling in neurons, the regulation of exocytotic and endocytotic pathways has been extensively studied for the past four decades. Ca2+ has been shown to trigger endocytosis via voltage-dependent Ca2+ channels and the activation of calmodulin and calcineurin 678910. Endocytic machinery and major vesicle proteins, such as synaptotagmin (Syt) 1 111213, synaptophysin 14, and the three SNARE proteins such as synaptobrevin 2, SNAP25, and syntaxin 1015161718 have all been shown to promote exo-endocytosis. However, the mechanisms underlying the precision and fidelity of vesicle retrieval remain elusive. Recent studies have identified Syt11 as a candidate gene for susceptibility to schizophrenia and a risk locus for Parkinson's disease 192021. However, its function in neurons remains unknown. Syt11 belongs to a family of type I membrane proteins with evolutionarily conserved cytoplasmic tandem C2 domains, C2A and C2B 222324. Members of the Syt family are well-characterized Ca2+ sensors for SNARE-dependent vesicle fusion during neurotransmitter release and hormone secretion 23242526. Interestingly, Syt1 and Syt4 have also been shown to function in exocytosis-coupled endocytosis 11121327. Among the 17 mammalian Syt isoforms, Syt4 and Syt11 are classified as anomalies because they harbor an aspartate-to-serine substitution in a Ca2+ coordination site of the C2A domain and do not bind Ca2+ biochemically 222428. In rat brain, Syt11 is an abundant isoform at the mRNA level 29, implying an important function in the nervous system. Using membrane capacitance (Cm) recording, confocal imaging, and electron microscopy, we demonstrate here that Syt11 specifically inhibits clathrin-mediated and bulk endocytosis. We propose that Syt11 serves as a clamp for endocytosis to ensure precision in vesicle retrieval. Results Syt11 inhibits endocytosis To investigate the function of Syt11 in neurons, especially in exocytosis and endocytosis, we used an shRNA-based knockdown (KD) approach. Syt11 was efficiently and specifically silenced by three shRNAs (Fig 1A, shSyt11-1, 73 ± 14% reduction; shSyt11-2, 85 ± 11%; and shSyt11-3, 45 ± 14%; n = 4, P < 0.01), while its closest homolog Syt4, the exocytotic proteins Syt1, synaptobrevin 2, SNAP25, and complexins 1 and 2, as well as the endocytic proteins clathrin heavy chain and adaptor protein 2, remained unaffected (Fig 1A, n = 4 for each protein, P > 0.05). Real-time Cm recording was used to monitor exocytosis and endocytosis in the somata of dorsal root ganglion (DRG) neurons (Appendix Fig S1A and B), which release ATP and neuropeptides via clear and dense-core vesicles 303132. In response to a 200-ms depolarizing pulse from −70 to 0 mV, Syt11 KD (using shSyt11-2) caused a reduction in the post-stimulation Cm jump, while the subsequent Cm decay was greatly accelerated, indicating fast exo-endocytosis (Fig 1B–G). The Ca2+ currents (Fig 1H) were not affected by Syt11 KD. Furthermore, the basal Cm, which reflects the size of the neuronal soma, was similar in KD and control neurons (control: 36.55 ± 3.30 pF, n = 21; KD: 39.29 ± 2.1 pF, n = 58; P = 0.3907). Therefore, the reduced Cm jump may reflect impaired exocytosis or faster endocytosis during stimulation. Cm overshoot, representing excessive membrane retrieval, was recorded in 45% of KD neurons, but absent from control neurons (Fig 1D and E; Appendix Fig S1C–E), indicating unbalanced endocytosis following exocytosis in the absence of Syt11. Both scrambled shRNA (Sc; Fig 1B and E–H) and untransfected neurons (Ctrl, Fig 1E–H) served as controls in the Syt11 KD experiments. These phenomena also occurred in Syt11 KD neurons with the other two shRNAs (Fig 1E–H) and were completely reversed by expressing an RNAi (shSyt11-2)-resistant form of Syt11 (Rescue, Fig 1E–H), indicating that the findings in Syt11 KD neurons were a direct result of Syt11 deletion. Since shSyt11-2 showed the highest knockdown efficiency among the three shRNAs, we used it in the subsequent experiments. The kinetic analysis of endocytic Cm decay revealed two exponential components (time constant (τ), τfast = 1.86 ± 0.26 s, τslow = 6.38 ± 0.57 s, n = 52) in most KD neurons; both components were faster than in control neurons and were reversed by Syt11 rescue (Appendix Fig S2A–D), indicating that at least two modes of endocytosis were accelerated in Syt11 KD neurons. Although Syt4 has been reported to regulate both exocytosis and exo-endocytosis 273334, it failed to rescue the endocytic effects of Syt11 KD, while further reducing the Cm jump (Appendix Fig S2E and F). Figure 1. Syt11 knockdown accelerates exo-endocytosis and the subsequent vesicle replenishment A. KD efficiency of three different Syt11 shRNAs in DRG neurons. Cultured DRG neurons were infected with control or shRNA-expressing lentiviruses at DIV 1, and Western blotting for Syt11, Syt1, Syt4, synaptobrevin 2 (Syb2), SNAP25, complexins 1, 2 (Cpx1, Cpx2), clathrin, adaptor protein 2 (AP-2), and β-actin was performed at DIV 6–7. n = 4 independent experiments. B, C. Representative Cm traces induced by 200-ms pulse depolarization (arrows) in DRG neurons. DRG neurons were transfected with plasmids expressing shSyt11-2 (Syt11 KD, KD) or scrambled shRNA (Sc), and Cm recording was performed 5 days after transfection. Endo-5s represents the Cm decay 5 s after stimulation. Insets show Ca2+ currents recorded in the same neurons. D. Representative Cm overshoot recorded from a Syt11 KD neuron. About 45% of the KD neurons showed an excessive membrane retrieval after a 200-ms depolarizing pulse. E. Averaged Cm traces recorded from Control (Ctrl), Sc, KD (from all three shRNAs, n = 39 for shSyt11-2, n = 7 for shSyt11-1, and n = 14 for shSyt11-3), and rescued (with the shSyt11-2-resistant form of Syt11) DRG neurons. F–H. Statistics of Cm jumps, Endo-5s, and Ca2+ current recorded from DRG neurons as in (B–E). Data were collected from 4 (Sc, Rescue), 8 (Ctrl), and 16 (KD) independent experiments. I. Normalized Cm changes induced by two pulses of 200-ms depolarization (arrows) at a 1-s interval. Data were normalized to the capacitance jump induced by the first depolarization. J. Paired-pulse ratios of ΔCm with different interstimulus intervals as in (I). DRG neurons were stimulated with two 200-ms pulses at different intervals as indicated, and the paired-pulse ratio was calculated by normalizing the ΔCm in response to the second stimulation to that induced by the first one. Data information: All data are presented as mean ± s.e.m. One-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint To determine whether the rapid endocytosis in Syt11 KD neurons could lead to faster vesicle recycling, we performed paired-pulse stimulation. The paired-pulse ratios were larger in the KD neurons, especially with short pulse intervals (Fig 1I and J), supporting a faster vesicle replenishment process in the absence of Syt11. We next made Cm recordings in DRG neurons with a train of 10 pulses at 1 Hz to investigate whether Syt11 KD also facilitates the sustained exocytosis during tonic transmission. Strikingly, we found that control neurons failed to release after three successive 200-ms pulses, while the KD neurons showed a stable Cm jump with each pulse and maintained the release probability even at the 10th pulse (Appendix Fig S2G). Consistently, when the Cm jumps in response to individual pulses were normalized to that induced by the first pulse, they showed increased values in KD neurons (Appendix Fig S2H). Thus, Syt11 KD accelerates exo-endocytosis and vesicle pool replenishment, which then facilitates the recovery of exocytosis during the sustained neurotransmission. Next, we performed FM1-43 uptake assays to confirm the endocytic phenotype of Syt11 KD neurons, which took up twice as much FM1-43 dye as control neurons when stimulated with 100 mM K+ for 30 s (Fig 2A and B). Furthermore, the fluorescence increase was greater at shorter stimulation times as the dye uptake plateaued within 1 min in Syt11 KD neurons (Fig 2C), consistent with the faster endocytosis recorded by Cm. Importantly, the accelerated FM1-43 uptake due to Syt11 KD was not limited to DRG neurons, but was also found in hippocampal neurons (Fig 2G). To investigate whether Syt11 also inhibits endocytosis during synaptic transmission, FM4-64 was loaded into hippocampal neurons and then unloaded with a train of 800 action potentials at 40 Hz. We found that Syt11 KD increased the dischargeable FM dye in the presynaptic boutons of hippocampal neurons (Fig 2H and I; Appendix Fig S3A), indicating an inhibitory effect of Syt11 on presynaptic endocytosis as well. When the dischargeable FM dye was normalized to the amount taken up, the discharge kinetics was similar in KD and control neurons (Appendix Fig S3B), indicating no effect on exocytosis in KD terminals. These results indicated a general role of Syt11 in neuronal endocytosis. Interestingly, FM1-43 uptake was also enhanced under resting conditions in Syt11 KD DRG neurons (Fig 2D and E), although the basal intracellular Ca2+ concentration ([Ca2+]i) was not altered (Fig 2F). These findings suggested that Syt11 is involved in both stimulus-coupled and constitutive endocytosis. Figure 2. Both stimulus-coupled endocytosis and constitutive endocytosis are accelerated by Syt11 knockdown A, B. FM1-43 uptake into DRG neurons stimulated by 100 mM K+ for 30 s. The right-shifted cumulative frequency in (B) indicates the increased uptake level in KD neurons. Scale bars, 5 μm. C. Quantification of KCl-evoked FM1-43 uptake with different stimulation times. D. Representative micrographs showing the increased constitutive FM 1-43 uptake (for 5 min) in KD neurons. Scale bars, 5 μm. E. Quantification of constitutive FM1-43 uptake with different loading times. F. Basal [Ca2+]i in DRG neurons measured with Fura-2. G. FM1-43 uptake by Syt11 KD (RFP-positive) and control (RFP-negative) hippocampal neurons stimulated by 100 mM K+ for 2 min. Quantitative data are shown in the lower-right panel. Scale bars, 20 μm. H. Representative images showing the preloaded FM4-64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz. Scale bars, 5 μm. I. Time course of FM4-64 unloading from control, Syt11 KD, and rescued nerve terminals within the same field of view (six coverslips from at least three biological repeats each). Data information: All data are presented as mean ± s.e.m. of 3–4 independent experiments. Kolmogorov–Smirnov test for (B, G), one-way ANOVA for (I), Student's t-test for (C, E, F), *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 2 [embr201540689-sup-0002-SDataFig2.zip] Download figure Download PowerPoint Syt11-knockdown-induced fast endocytosis is dynamin dependent Dynamin plays global and essential roles in endocytosis in mammalian cells 353637. We then determined whether the fast endocytosis in Syt11 KD neurons is dynamin dependent. Dynasore, a potent inhibitor of dynamin 383940, was included in the intracellular solution and dialyzed into patched DRG neurons. Cm traces were recorded 1–3 min and 5–10 min after whole-cell dialysis. The fast Cm decay in KD neurons was completely blocked after 5-min treatment with 100 μM dynasore (Fig 3A and B). In addition, 10 μM dynole-34-2™, another specific inhibitor of dynamin 4142, similarly inhibited the fast endocytosis (Fig 3D and E). As a control, 0.1% DMSO had no effect on the Cm decay (Fig 3G and H). These results suggested that the fast endocytosis in Syt11 KD neurons is dynamin dependent. We found that after the fast endocytosis was blocked by dynasore or dynole, the Cm jumps in Syt11 KD neurons were similar to those in controls (Fig 3C and F). These data indicated that the reduction in the Cm jumps in Syt11 KD neurons (Fig 1E and F) is due to faster endocytosis during stimulation, but not a defect in exocytosis. Figure 3. Syt11-knockdown-induced fast endocytosis is dynamin dependent A. Averaged ∆Cm traces induced by 200-ms depolarization in the presence and absence of dynasore. Endocytic inhibition by dynasore (Dyna) was estimated by comparison of Cm traces recorded during 1–3 min (KD) and 5–10 min (KD + Dyna and Ctrl + Dyna) after whole-cell dialysis. B, C. Endo-5s and Cm jumps recorded as in (A). D. Averaged ∆Cm traces induced by 200-ms depolarization in the presence and absence of dynole-34-2™ (Dynole). Cm traces and endocytic rate were measured as in (A) except that 10 μM dynole-34-2™ with 0.1% DMSO was dialyzed into patched cells. E, F. Endo-5s and Cm jumps as in (D). G, H. Averaged ∆Cm traces and Endo-5s recorded from KD neurons under control conditions (0.1% DMSO). I. KCl-evoked FM1-43 uptake in DRG neurons after 10-min pre-incubation with 0.1% DMSO (control) or with 100 μM dynasore. Data information: All data are presented as mean ± s.e.m. of three independent experiments. One-way ANOVA for (B, E), Student's t-test for (C, F, H, I), **P < 0.01, ***P < 0.001; NS, not significant. Download figure Download PowerPoint The dynamin dependence of fast endocytosis was also confirmed by FM1-43 uptake assays. Ten-minute pre-incubation with 100 μM dynasore in the extracellular solution, but not with 0.1% DMSO, strongly inhibited the accelerated FM1-43 uptake in KD cells (Fig 3I). Taken together, the results showed that Syt11 inhibits a dynamin-dependent endocytosis. Since Syt11 functions in the endocytic pathway, we then investigated its subcellular localization in DRG neurons. Available antibodies were tested with various protocols, but no specific immunocytochemical signals were detectable. We hence expressed Myc-tagged Syt11 and found punctate staining in both the somata and axons of DRG neurons (Fig 4A, top left panel). Myc-Syt11 partly co-localized with dynamin, the endocytic coat protein clathrin and its adaptor AP-2, the trans-Golgi network marker TGN46, the vesicle marker synaptobrevin 2 (VAMP2), and the recycling endosome marker transferrin receptor, but rarely with the lysosome marker LAMP1 (Fig 4). These results support the notion that Syt11 functions in vesicle recycling pathways. Figure 4. Syt11 localizes to vesicle recycling pathways DRG neurons expressing Myc-Syt11 were immunostained for Myc-Syt11, dynamin (Dyn), AP-2, clathrin (CL), TGN46, synaptobrevin 2 (Syb2), and LAMP1. For dynamin, the box along the plasma membrane was straightened, enlarged, and is shown in the upper right panels with arrows indicating co-localized puncta. An enlarged inset of the inside of the cell (box) is shown in the lower panel. For the localization of the transferrin receptor, EGFP-transferrin receptor (TfR) was expressed in DRG neurons. Scale bars, 10 μm. Analysis of co-localization of Myc-Syt11 with dynamin, AP-2, clathrin, TGN46, synaptobrevin 2, transferrin receptor, and LAMP1. n = 3 independent experiments. Data information: All data are presented as mean ± s.e.m. of three independent experiments. Source data are available online for this figure. Source Data for Figure 4 [embr201540689-sup-0003-SDataFig4.zip] Download figure Download PowerPoint Syt11 inhibits clathrin-mediated endocytosis Clathrin-mediated endocytosis and bulk endocytosis are both dynamin-dependent mechanisms of vesicle retrieval during neuronal activity 1234. To test whether Syt11 functions in CME, Alexa Fluor-conjugated transferrin uptake was measured in DRG neurons with Syt11 KD or overexpression. We found that transferrin uptake was enhanced in Syt11 KD neurons, but suppressed in neurons overexpressing Myc-Syt11 or Syt11 (Fig 5A and B). These results indicated that Syt11 inhibits constitutive CME. Figure 5. Syt11 inhibits clathrin-mediated endocytosis Transferrin (Tf, red) uptake in Syt11 KD (GFP-positive) and control neurons. Quantitative data are on the right. Scale bars, 20 μm. Tf uptake in Syt11-overexpressing and control neurons. Quantitative data are on the right. Scale bars, 20 μm. Normalized ∆Cm induced by 200-ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double-exponential decay function (solid black and red, fitted curves). Fast and slow time constants of endocytosis in Syt11 KD neurons recorded as in (C). KCl-evoked FM1-43 uptake in DRG neurons after 1-h pre-incubation with 0.1% DMSO or 100 μM MDC in the bath solution. Data information: All data are presented as mean ± s.e.m. of 4–5 independent experiments. One-way ANOVA for (A, B), Student's t-test for (D, E, G), **P < 0.01, ***P < 0.001; NS, not significant. Source data are available online for this figure. Source Data for Figure 5 [embr201540689-sup-0004-SDataFig5.zip] Download figure Download PowerPoint We next determined whether stimulus-coupled CME is also altered in Syt11 KD neurons. Monodansylcadaverine (MDC), an inhibitor of CME 4344, was included in the intracellular solution and dialyzed into patched cells. Cm traces were obtained 1–3 min and 5–10 min after whole-cell dialysis. MDC partly inhibited the accelerated Cm decay in Syt11 KD neurons (Fig 5C). When the Cm traces were fit to a double-exponential decay function, two phases of endocytosis were revealed (Fig 5C). The initial fast phase had a time constant of 1.44 ± 0.48 s and was unaffected by MDC (Fig 5D). In contrast, the subsequent slow phase in Syt11 KD cells was significantly inhibited by MDC (Fig 5D, KD, τ = 5.78 ± 1.23 s; KD + MDC, τ = 12.32 ± 1.69 s), suggesting that CME is involved in the slow phase of vesicle retrieval under 200-ms depolarization. Consistently, FM1-43 uptake assays also showed a partial block of the accelerated endocytosis in Syt11 KD neurons after pre-incubation with 100 μM MDC (Fig 5E). To further confirm the function of Syt11 in CME, Pitstop 1, a specific clathrin inhibitor 45, was dialyzed into patched cells during Cm recordings. As expected, the Syt11 KD-induced fast endocytosis was also attenuated by the application of Pitstop 1 (Appendix Fig S4). Although the dynamin and clathrin inhibitors may have off-target effects 4647, our results point to the involvement of Syt11 in stimulus-coupled CME. Syt11 inhibits bulk endocytosis Since the endocytic Cm decay revealed that two different kinetic forms of dynamin-dependent endocytosis were accelerated in Syt11 KD neurons, while the slow mode of endocytosis was reversed by blocking CME (Figs 1, 3 and 5; Appendix Fig S2), we speculated that an additional mode of the exocytosis-coupled endocytic pathway, which is dependent on dynamin and faster than CME, is modulated by Syt11 expression. Bulk endocytosis provides a high-capacity pathway for vesicle retrieval during elevated neuronal activity and peaks immediately after stimulation 1346. To investigate a possible contribution of bulk endocytosis to the accelerated endocytic activity, we analyzed the Cm traces recorded in Syt11 KD and control neurons in detail. Bulk endocytosis was reflected as a brief downward capacitance shift of ~20–500 fF with a decay rate > 50 fF/100 ms, according to the standard protocols 648 (Fig 6A and B). We found that the frequency of bulk endocytotic events was ~5-fold higher in Syt11 KD neurons with a peak immediately after a 200-ms stimulus pulse (Fig 6C and D). The
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