The function of the nervous system relies on synaptic transmission. Synaptic transmission is mediated by calcium-triggered vesicle fusion, followed by vesicle endocytosis that recycles vesicles. Although significant progress has been made in understanding these processes, much remains unknown. My goal is to advance our understanding of these synaptic signaling processes. The progress of the last year is described below. 1. Vesicle fusion with the plasma membrane generates an Ω-shaped membrane profile. Its pore is thought to dilate until flattening (full-collapse), followed by classical endocytosis to retrieve vesicles. Alternatively, the pore may close (kiss-and-run), but the triggering mechanisms and its endocytic roles remain poorly understood. Here, using confocal and STED imaging of dense-core vesicles, we find that fusion-generated Ω-profiles may enlarge or shrink while maintaining vesicular membrane proteins, which is entirely unexpected from the classical models. Closure of fusion-generated Ω-profiles, which produces various sizes of vesicles, is the dominant mechanism mediating rapid and slow endocytosis within 1-30 s. Strong calcium influx triggers dynamin-mediated closure. Weak calcium influx does not promote closure, but facilitates the merging of Ω-profiles with the plasma membrane via shrinking rather than full-collapse. These results establish an exo-endocytosis model, termed Ω-exo-endocytosis, in which the fusion-generated Ω-profile may shrink to merge with the plasma membrane, change in size, or change in size then close in response to calcium, which is the main mechanism to retrieve dense-core vesicles. This new model challenges the current exo-endocytosis model. 2. We provided a comprehensive review for Annual Review of Physiology on the progress over the last four decades regarding exocytosis modes, and the coupling between exocytosis and endocytosis. 3. The role of presynaptic glycine receptors (GlyRs) is largely unclear. In collaboration with dr. Li Zhang, we demonstrate that presynaptic GlyRs are an emerging therapeutic target for dominant familial startle disease and other diseases with GlyR deficiency. The presynaptic GlyR deficiency can be rescued by DH-CBD, a nonpsychoactive cannabinoid. 4. How the mixed lineage kinase domain-like protein, MLKL, induces necroptosis is unclear. In collaboration with Dr. Zheng-Gang Liu, we found that MLKL forms a homotrimer through its N-terminal coiled-coil domain and locates to the cell plasma membrane to activate its downstream target, the transient receptor potential melastatin related 7 (TRPM7), which mediates calcium influx to generate necroptosis. Hence, our study reveals a crucial mechanism of MLKL-mediated TNF-induced necroptosis. 5. Fibroblasts can be converted to neurons by forced expression of transcription factors. However, the mechanisms underlying this conversion remain unclear. In collaboration with Dr. Qin Yang, we show that the efficiency of neuronal conversion of embryonic human fibroblasts aged in culture is lower than that in cells in early culture stages. Moreover, depletion of p16(Ink4a) and p19(Arf) involved in the activation of cellular senescence is sufficient to convert human fibroblast and epithelial cells into neurons. The induced neurons express neuron-specific proteins, generate action potentials and neurotransmitter receptor-mediated currents. Genome-wide transcriptional analysis shows that the induced neurons have a profile different from fibroblasts and similar to that of control neurons induced by established methods. We further noted that expression of p53 blocks the neuronal conversion, whereas expression of human telomerase reverse transcriptase (hTERT) induces it. Our results indicate that overcoming senescence is a crucial step for neuronal conversion of somatic cells.

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Yuan, Jie; Zhang, Fan; Hallahan, Dennis et al. (2018) Reprogramming glioblastoma multiforme cells into neurons by protein kinase inhibitors. J Exp Clin Cancer Res 37:181
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
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
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
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
Wu, Ling-Gang; Hamid, Edaeni; Shin, Wonchul et al. (2014) Exocytosis and endocytosis: modes, functions, and coupling mechanisms. Annu Rev Physiol 76:301-31
Chiang, Hsueh-Cheng; Shin, Wonchul; Zhao, Wei-Dong et al. (2014) Post-fusion structural changes and their roles in exocytosis and endocytosis of dense-core vesicles. Nat Commun 5:3356

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