Live-cell imaging of endocytosed synaptophysin around individual hippocampal presynaptic active zones

Frontiers in Cellular Neuroscience

Cellular Neuroscience

Junichiro Funahashi¹  Tomoo Hirano¹  Hiromitsu Tanaka² 

[1] Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan;Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan;Department of Developmental Biology, Graduate School of Medicine, Chiba University, Chiba, Japan;

关键词: synaptic vesicle;    endocytosis;    clathrin;    TIRFM;    synaptophysin (SYN);    cytomatrix at the active zone-associated structural protein (CAST);    active zone (AZ);   

DOI  :  10.3389/fncel.2023.1277729

 received in 2023-08-15, accepted in 2023-10-06,  发布年份 2023

来源: Frontiers

【 摘 要 】

In presynaptic terminals 4 types of endocytosis, kiss-and-run, clathrin-mediated, bulk and ultrafast endocytosis have been reported to maintain repetitive exocytosis of neurotransmitter. However, detailed characteristics and relative contribution of each type of endocytosis still need to be determined. Our previous live-cell imaging study demonstrated individual exocytosis events of synaptic vesicle within an active-zone-like membrane (AZLM) formed on glass using synaptophysin tagged with a pH-sensitive fluorescent protein. On the other hand, individual endocytosis events of postsynaptic receptors were recorded with a rapid extracellular pH exchange method. Combining these methods, here we live-cell imaged endocytosed synaptophysin with total internal reflection fluorescence microscopy in rat hippocampal culture preparations. Clathrin-dependent and -independent endocytosis, which was seemingly bulk endocytosis, occurred within several seconds after electrical stimulation at multiple locations around AZLM at room temperature, with the locations varying trial to trial. The contribution of clathrin-independent endocytosis was more prominent when the number of stimulation pulses was large. The skewness of synaptophysin distribution in intracellular vesicles became smaller after addition of a clathrin inhibitor, which suggests that clathrin-dependent endocytosis concentrates synaptophysin. Ultrafast endocytosis was evident immediately after stimulation only at near physiological temperature and was the predominant endocytosis when the number of stimulation pulses was small.

【 授权许可】

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Copyright © 2023 Tanaka, Funahashi and Hirano.