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. 1. Fusion and fission are thought to open and close a pore to mediate many vital biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion and fission pores have not been observed and thus proved in live cells. Their regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion and fission pore behaviors in live (neuroendocrine) cells, including pore opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore and fission pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation. 2. Endocytosis generates spherical or ellipsoid-like vesicles from the plasma membrane, which recycles vesicles that fuse with the plasma member during exocytosis in neurons and endocrine secretory cells. Although tension in the plasma membrane is generally considered to be an important factor in regulating endocytosis, whether membrane tension inhibits or facilitates endocytosis remains debated in the endocytosis field, and has been rarely studied for vesicular endocytosis in secretory cells. Here we report that increasing membrane tension by adjusting osmolarity inhibited both the rapid (a few seconds) and slow (tens of seconds) endocytosis in calyx-type nerve terminals containing conventional active zones and in neuroendocrine chromaffin cells. We address the mechanism of this phenomenon by computational modeling of the energy barrier that the system must overcome at the stage of membrane budding by an assembling protein coat. We show that this barrier grows with increasing tension, which may slow down or prevent membrane budding. These results suggest that in live secretory cells, membrane tension exerts inhibitory action on endocytosis.

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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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