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 in two aspects. 1. Calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells. The calcium channel identity is unclear. While the plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, which triggers endocytosis and thus couples endocytosis to exocytosis. This hypothetic mechanism is considered to conserve from sea urchin eggs to neurons. Here we found that vesicular membrane, when inserted to the plasma membrane upon exocytosis, did not generate detectable calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs, including the P/Q-type, at the plasma membrane provided the calcium influx to trigger rapid and slow endocytosis, and thus couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells where VDCCs control exocytosis and exocytosis is coupled to endocytosis. 2. Endocytosis overshoot, which retrieves more membrane than vesicles just being exocytosed, occurs at nerve terminals and non-neuronal secretory cells. The mechanism that retrieves the overshoot membrane pool and the role of this pool remain largely unknown. We addressed this issue at the rat calyx of Held nerve terminal with capacitance measurements. We found that every calyx contained an overshoot pool 1.8 times the readily releasable pool. Retrieval of this pool required large calcium influx, and was inhibited by blockers of calcium/calmodulin-activated calcineurin and dynamin, suggesting the involvement of calcineurin and dynamin in endocytosis overshoot. Depletion of the overshoot pool slowed down compensatory endocytosis, whereas recovery of the overshoot pool via exocytosis that deposited stranded vesicles to the plasma membrane led to recovery of compensatory endocytosis, suggesting that the overshoot pool enhances endocytosis efficiency. These results suggest that the overshoot pool exists at every nerve terminal, is of limited size arising from vesicles stranded at the plasma membrane, is retrieved via calcium/calmodulin/calcineurin and dynamin signalling pathway, and can enhance endocytosis efficiency. Potential mechanisms for how the endocytosis overshoot pool enhances endocytosis efficiency are discussed.

Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2012
Total Cost
$1,610,334
Indirect Cost
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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
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
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
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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