Neurons contact each other mostly by synaptic transmission at synapses. The maintenance of synaptic transmission relies on vesicle endocytosis, which recycles fused vesicles for the second round of exocytosis. My goal is to improve our understanding on the cellular and molecular mechanisms underlying synaptic vesicle endocytosis, which are the building block for the maintenance of synaptic transmission and thus the signaling process of the nervous system. Our progress in the last year is described below. Although the calcium/calmodulin-activated phosphatase calcineurin may dephosphorylate many endocytic proteins, it is not considered a key molecule in mediating the major forms of endocytosis at synapses - slow, clathrin-dependent and the rapid, clathrin-independent endocytosis. Here we studied the role of calcineurin in endocytosis by reducing calcium influx, inhibiting calmodulin with pharmacological blockers and knockdown of calmodulin, and by inhibiting calcineurin with pharmacological blockers and knockout of calcineurin. We measured endocytosis using two advanced, reliable techniques, the capacitance measurement technique and the pHluorin imaging technique at two synapse preparations, the calyx of Held and the cultured hippocampal synapses. We found that inhibition of caclium influx, calmodulin, and calcineurin using the approaches described above significantly inhibited both rapid and slow endocytosis at the large calyx-type synapse in 7n-n10 days old rats and mice, and slow, clathrin-dependent endocytosis at the conventional cultured hippocampal synapse of rats and mice. These recent progress provided the first genetic evidence together with pharmacological evidence suggesting an important role of calmodulin and calcineurin in rapid and slow endocytosis at 7n-n10 days old calyceal synapses and cultured hippocampal synapses (Figs.n1n-n3,n5n-n7). Consistent with results obtained at calyces, where calcium influx triggers endocytosis (Wu et al., 2009;Hosoi et al., 2009), reducing the Ca2+o to 0.1nmM nearly abolished endocytosis at hippocampal synapses (Fig.n4). We therefore concluded that calcium influx during nerve firings activates calmodulin/calcineurin, which initiates and up-regulates slow, clathrin-dependent and rapid, presumably clathrin-independent endocytosis. The calcium/calmodulin/calcineurin signalling pathway as described here may underlie regulation of endocytosis by nerve activity and calcium as reported at many synapses over the last several decades. Our findings raise the question as to how calcium/calmodulin/calcineurin signaling pathway regulates endocytosis. We are currently investigating this urgent question. We expect progress towards addressing this quesiton in the near future.

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Shin, Wonchul; Ge, Lihao; Arpino, Gianvito et al. (2018) Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis. Cell 173:934-945.e12
Wu, Xin-Sheng; Elias, Sharon; Liu, Huisheng et al. (2017) Membrane Tension Inhibits Rapid and Slow Endocytosis in Secretory Cells. Biophys J 113:2406-2414
Wu, Xin-Sheng; Lee, Sung Hoon; Sheng, Jiansong et al. (2016) Actin Is Crucial for All Kinetically Distinguishable Forms of Endocytosis at Synapses. Neuron 92:1020-1035
Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul et al. (2016) Hemi-fused structure mediates and controls fusion and fission in live cells. Nature 534:548-52
Baydyuk, Maryna; Xu, Jianhua; Wu, Ling-Gang (2016) The calyx of Held in the auditory system: Structure, function, and development. Hear Res 338:22-31
Xu, Jianhua; Wu, Xin-Sheng; Sheng, Jiansong et al. (2016) ?-Synuclein Mutation Inhibits Endocytosis at Mammalian Central Nerve Terminals. J Neurosci 36:4408-14
Park, Soonhong; Ahuja, Malini; Kim, Min Seuk et al. (2016) Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV. EMBO Rep 17:266-78
Wen, Peter J; Grenklo, Staffan; Arpino, Gianvito et al. (2016) Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane. Nat Commun 7:12604
Peng, Shiyong; Xu, Jianhua; Pelkey, Kenneth A et al. (2015) Suppression of agrin-22 production and synaptic dysfunction in Cln1 (-/-) mice. Ann Clin Transl Neurol 2:1085-104
Baydyuk, Maryna; Wu, Xin-Sheng; He, Liming et al. (2015) Brain-derived neurotrophic factor inhibits calcium channel activation, exocytosis, and endocytosis at a central nerve terminal. J Neurosci 35:4676-82

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