Abstract
Hormones and neurotransmitters are stored in specialised vesicles and released from excitable cells through exocytosis. During vesicle fusion with the plasma membrane, a transient fusion pore is created that enables transmitter release. The protein dynamin is known to regulate fusion pore expansion (FPE). The mechanism is unknown, but requires its oligomerisation-stimulated GTPase activity. We used a palette of small molecule dynamin modulators to reveal bi-directional regulation of FPE by dynamin and vesicle release in chromaffin cells. The dynamin inhibitors Dynole 34-2 and Dyngo 4a and the dynamin activator Ryngo 1-23 reduced or increased catecholamine released from single vesicles, respectively. Total internal reflection fluorescence (TIRF) microscopy demonstrated that dynamin stimulation with Ryngo 1-23 reduced the number of neuropeptide Y (NPY) kiss-and-run events, but not full fusion events, and slowed full fusion release kinetics. Amperometric stand-alone foot signals, representing transient kiss-and-run events, were less frequent but were of longer duration, similarly to full amperometric spikes and pre-spike foot signals. These effects are not due to alterations in vesicle size. Ryngo 1-23 action was blocked by inhibitors of actin polymerisation or myosin II. Therefore, we demonstrate using a novel pharmacological approach that dynamin not only controls FPE during exocytosis, but is a bi-directional modulator of the fusion pore that increases or decreases the amount released from a vesicle during exocytosis if it is activated or inhibited, respectively. As such, dynamin has the ability to exquisitely fine-tune transmitter release.
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References
Cardenas AM, Marengo FD . Rapid endocytosis and vesicle recycling in neuroendocrine cells. Cell Mol Neurobiol 2010; 30: 1365–1370.
Alabi AA, Tsien RW . Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission. Annu Rev Physiol 2013; 75: 393–422.
Wu LG, Hamid E, Shin W, Chiang HC . Exocytosis and endocytosis: modes, functions, and coupling mechanisms. Annu Rev Physiol 2014; 76: 301–331.
Chanturiya A, Chernomordik LV, Zimmerberg J . Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers. Proc Natl Acad Sci USA 1997; 94: 14423–14428.
Gubar O, Morderer D, Tsyba L, Croise P, Houy S, Ory S et al. Intersectin: the crossroad between vesicle exocytosis and endocytosis. Front Endocrinol 2013; 4: 109.
Xu J, Luo F, Zhang Z, Xue L, Wu XS, Chiang HC et al. SNARE proteins synaptobrevin, SNAP-25, and syntaxin are involved in rapid and slow endocytosis at synapses. Cell Rep 2013; 3: 1414–1421.
Zhang Z, Wang D, Sun T, Xu J, Chiang HC, Shin W et al. The SNARE proteins SNAP25 and synaptobrevin are involved in endocytosis at hippocampal synapses. J Neurosci 2013; 33: 9169–9175.
Yao J, Kwon SE, Gaffaney JD, Dunning FM, Chapman ER . Uncoupling the roles of synaptotagmin I during endo- and exocytosis of synaptic vesicles. Nat Neurosci 2012; 15: 243–249.
Yao LH, Rao Y, Varga K, Wang CY, Xiao P, Lindau M et al. Synaptotagmin 1 is necessary for the Ca2+ dependence of clathrin-mediated endocytosis. J Neurosci 2012; 32: 3778–3785.
Miller SE, Sahlender DA, Graham SC, Honing S, Robinson MS, Peden AA et al. The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM. Cell 2011; 147: 1118–1131.
Sahlender DA, Kozik P, Miller SE, Peden AA, Robinson MS . Uncoupling the functions of CALM in VAMP sorting and clathrin-coated pit formation. PLoS ONE 2013; 8: e64514.
Gonzalez-Jamett AM, Momboisse F, Guerra MJ, Ory S, Baez-Matus X, Barraza N et al. Dynamin-2 regulates fusion pore expansion and quantal release through a mechanism that involves actin dynamics in neuroendocrine chromaffin cells. PLoS ONE 2013; 8: e70638.
Watanabe S, Rost BR, Camacho-Perez M, Davis MW, Sohl-Kielczynski B, Rosenmund C et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 2013; 504: 242–247.
Quan A, Robinson PJ . Repurposing molecular mechanisms of transmitter release: a new job for syndapin at the fusion pore. Focus on "Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells". Am J Physiol Cell Physiol 2014; 306: C792–C793.
Hinshaw JE, Schmid SL . Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding. Nature 1995; 374: 190–192.
Sweitzer SM, Hinshaw JE . Dynamin undergoes a GTP-dependent conformational change causing vesiculation. Cell 1998; 93: 1021–1029.
Collins A, Warrington A, Taylor KA, Svitkina T . Structural organization of the actin cytoskeleton at sites of clathrin-mediated endocytosis. Curr Biol 2011; 21: 1167–1175.
Villanueva J, Torregrosa-Hetland CJ, Garcia-Martinez V, del Mar Frances M, Viniegra S, Gutierrez LM . The F-actin cortex in chromaffin granule dynamics and fusion: a minireview. J Mol Neurosci 2012; 48: 323–327.
Gormal RS, Nguyen TH, Martin S, Papadopulos A, Meunier FA . An acto-myosin II constricting ring initiates the fission of activity-dependent bulk endosomes in neurosecretory cells. J Neurosci 2015; 35: 1380–1389.
Nguyen TH, Maucort G, Sullivan RK, Schenning M, Lavidis NA, McCluskey A et al. Actin- and dynamin-dependent maturation of bulk endocytosis restores neurotransmission following synaptic depletion. PLoS ONE 2012; 7: e36913.
Doreian BW, Fulop TG, Smith CB . Myosin II activation and actin reorganization regulate the mode of quantal exocytosis in mouse adrenal chromaffin cells. J Neurosci 2008; 28: 4470–4478.
Gu C, Yaddanapudi S, Weins A, Osborn T, Reiser J, Pollak M et al. Direct dynamin-actin interactions regulate the actin cytoskeleton. EMBO J 2010; 29: 3593–3606.
Papadopulos A, Gomez GA, Martin S, Jackson J, Gormal RS, Keating DJ et al. Activity-driven relaxation of the cortical actomyosin II network synchronizes Munc18-1-dependent neurosecretory vesicle docking. Nat Commun 2015; 6: 6297.
Graham ME, O'Callaghan DW, McMahon HT, Burgoyne RD . Dynamin-dependent and dynamin-independent processes contribute to the regulation of single vesicle release kinetics and quantal size. Proc Natl Acad Sci USA 2002; 99: 7124–7129.
Holroyd P, Lang T, Wenzel D, De Camilli P, Jahn R . Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells. Proc Natl Acad Sci USA 2002; 99: 16806–16811.
Anantharam A, Bittner MA, Aikman RL, Stuenkel EL, Schmid SL, Axelrod D et al. A new role for the dynamin GTPase in the regulation of fusion pore expansion. Mol Biol Cell 2011; 22: 1907–1918.
Samasilp P, Lopin K, Chan SA, Ramachandran R, Smith C . Syndapin 3 modulates fusion pore expansion in mouse neuroendocrine chromaffin cells. Am J Physiol Cell Physiol 2014; 306: C831–C843.
Song BD, Leonard M, Schmid SL . Dynamin GTPase domain mutants that differentially affect GTP binding, GTP hydrolysis, and clathrin-mediated endocytosis. J Biol Chem 2004; 279: 40431–40436.
Chan SA, Doreian B, Smith C . Dynamin and myosin regulate differential exocytosis from mouse adrenal chromaffin cells. Cell Mol Neurobiol 2010; 30: 1351–1357.
Fulop T, Doreian B, Smith C . Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells. Arch Biochem Biophys 2008; 477: 146–154.
Samasilp P, Chan SA, Smith C . Activity-dependent fusion pore expansion regulated by a calcineurin-dependent dynamin-syndapin pathway in mouse adrenal chromaffin cells. J Neurosci 2012; 32: 10438–10447.
Tsuboi T, McMahon HT, Rutter GA . Mechanisms of dense core vesicle recapture following "kiss and run" ("cavicapture") exocytosis in insulin-secreting cells. J Biol Chem 2004; 279: 47115–47124.
Zhang Z, Hui E, Chapman ER, Jackson MB . Regulation of exocytosis and fusion pores by synaptotagmin-effector interactions. Mol Biol Cell 2010; 21: 2821–2831.
Ngatchou AN, Kisler K, Fang Q, Walter AM, Zhao Y, Bruns D et al. Role of the synaptobrevin C terminus in fusion pore formation. Proc Natl Acad Sci U S A 2010; 107: 18463–18468.
Ferguson SM, Raimondi A, Paradise S, Shen H, Mesaki K, Ferguson A et al. Coordinated actions of actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits. Dev Cell 2009; 17: 811–822.
Park RJ, Shen H, Liu L, Liu X, Ferguson SM, De Camilli P . Dynamin triple knockout cells reveal off target effects of commonly used dynamin inhibitors. J Cell Sci 2013; 126: 5305–5312.
Harper CB, Martin S, Nguyen TH, Daniels SJ, Lavidis NA, Popoff MR et al. Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. J Biol Chem 2011; 286: 35966–35976.
Harper CB, Popoff MR, McCluskey A, Robinson PJ, Meunier FA . Targeting membrane trafficking in infection prophylaxis: dynamin inhibitors. Trends Cell Biol 2013; 23: 90–101.
Hill TA, Gordon CP, McGeachie AB, Venn-Brown B, Odell LR, Chau N et al. Inhibition of dynamin mediated endocytosis by the dynoles—synthesis and functional activity of a family of indoles. J Med Chem 2009; 52: 3762–3773.
McCluskey A, Daniel JA, Hadzic G, Chau N, Clayton EL, Mariana A et al. Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis. Traffic 2013; 14: 1272–1289.
Quan A, McGeachie AB, Keating DJ, van Dam EM, Rusak J, Chau N et al. Myristyl trimethyl ammonium bromide and octadecyl trimethyl ammonium bromide are surface-active small molecule dynamin inhibitors that block endocytosis mediated by dynamin I or dynamin II. Mol Pharmacol 2007; 72: 1425–1439.
Robertson MJ, Deane FM, Robinson PJ, McCluskey A . Synthesis of Dynole 34-2, Dynole 2-24 and Dyngo 4a for investigating dynamin GTPase. Nat Protoc 2014; 9: 851–870.
Gu C, Chang J, Shchedrina VA, Pham VA, Hartwig JH, Suphamungmee W et al. Regulation of dynamin oligomerization in cells: the role of dynamin-actin interactions and its GTPase activity. Traffic 2014; 15: 819–838.
Trouillon R, Ewing AG . Amperometric measurements at cells support a role for dynamin in the dilation of the fusion pore during exocytosis. ChemPhysChem 2013; 14: 2295–2301.
Keating DJ, Dubach D, Zanin MP, Yu Y, Martin K, Zhao YF et al. DSCR1/RCAN1 regulates vesicle exocytosis and fusion pore kinetics: implications for Down syndrome and Alzheimer's disease. Hum Mol Genet 2008; 17: 1020–1030.
Zanin MP, Phillips L, Mackenzie KD, Keating DJ . Aging differentially affects multiple aspects of vesicle fusion kinetics. PLoS ONE 2011; 6: e27820.
Meunier FA, Osborne SL, Hammond GR, Cooke FT, Parker PJ, Domin J et al. Phosphatidylinositol 3-kinase C2alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis. Mol Biol Cell 2005; 16: 4841–4851.
Meunier FA, Feng ZP, Molgo J, Zamponi GW, Schiavo G . Glycerotoxin from Glycera convoluta stimulates neurosecretion by up-regulating N-type Ca2+ channel activity. EMBO J 2002; 21: 6733–6743.
Cardinale J, Paul G, Sbalzarini IF . Discrete region competition for unknown numbers of connected regions. IEEE Trans Image Process 2012; 21: 3531–3545.
Perrais D, Kleppe IC, Taraska JW, Almers W . Recapture after exocytosis causes differential retention of protein in granules of bovine chromaffin cells. J Physiol 2004; 560: 413–428.
Galas MC, Chasserot-Golaz S, Dirrig-Grosch S, Bader MF . Presence of dynamin—syntaxin complexes associated with secretory granules in adrenal chromaffin cells. J Neurochem 2000; 75: 1511–1519.
Albillos A, Dernick G, Horstmann H, Almers W, Alvarez de Toledo G, Lindau M . The exocytotic event in chromaffin cells revealed by patch amperometry. Nature 1997; 389: 509–512.
Ales E, Tabares L, Poyato JM, Valero V, Lindau M . Alvarez de Toledo G. High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism.[see comment]. Nat Cell Biol 1999; 1: 40–44.
Villanueva J, Torres V, Torregrosa-Hetland CJ, Garcia-Martinez V, Lopez-Font I, Viniegra S et al. F-actin-myosin II inhibitors affect chromaffin granule plasma membrane distance and fusion kinetics by retraction of the cytoskeletal cortex. J Mol Neurosci 2012; 48: 328–338.
Chow RH, von Ruden L, Neher E . Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells. Nature 1992; 356: 60–63.
Gong LW, Hafez I, Alvarez de Toledo G, Lindau M . Secretory vesicles membrane area is regulated in tandem with quantal size in chromaffin cells. J Neurosci 2003; 23: 7917–7921.
Marks B, Stowell MH, Vallis Y, Mills IG, Gibson A, Hopkins CR et al. GTPase activity of dynamin and resulting conformation change are essential for endocytosis. Nature 2001; 410: 231–235.
Acknowledgements
This work was supported by grants and a Senior Research Fellowship (569596 to FAM) from the National Health and Medical Research Council Australia (PJR, FAM, and AM), Australian Research Council Discovery Project (DJK and FAM) a LIEF grant to FAM (LE0882864), and grants from the Children’s Medical Research Institute, Newcastle Innovation, the Ramaciotti Foundation, the Australian Cancer Research Foundation and the Ian Potter Foundation.
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Some of the authors’ institutions, Newcastle Innovation Ltd and Children’s Medical Research Institute, hold trademarks for the Dynole, Dyngo and Ryngo compounds and make the compounds commercially available via Abcam (Cambridge, UK).
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Jackson, J., Papadopulos, A., Meunier, F. et al. Small molecules demonstrate the role of dynamin as a bi-directional regulator of the exocytosis fusion pore and vesicle release. Mol Psychiatry 20, 810–819 (2015). https://doi.org/10.1038/mp.2015.56
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DOI: https://doi.org/10.1038/mp.2015.56
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