The role of Synaptotagmin III in regulating trafficking of extracellular vesicles

• Main Authors: Ni-Yen Yu, 余妮晏
• Other Authors: 王致恬
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/uy9qu6

碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 107 === Extracellular vesicles (EVs) are membrane-derived lipid bilayer structures with several subtypes, which are distinguished according to their sizes and cargos, such as different lipids, nucleic acids, or proteins. EVs can be released by cells, reflecting the physiological state of originating cells or serving as communication signals between cells. Several studies have shown that EV release can be regulated by calcium, similar to the triggering signal for calcium-dependent exocytosis of intracellular vesicles. However, the candidate protein coupling calcium signal to the EV release remains largely unknown. The Syt protein superfamily has been found to serve as the calcium sensor during calcium-dependent vesicular exocytosis, by calcium binding to two C2 domains (C2A and C2B). We have previously found that upon KCl depolarization, overexpressing Syt III or Syt III C2AB* (a mutant harboring the weakened calcium-binding ability in the C2AB domains) differentially changes the subcellular localization of CD63 (the EV marker) in PC12 cells. Therefore, we speculated that Syt III may regulate EV trafficking through calcium binding to its C2AB domains. However, there is no direct evidence for this up to date. In this study, we aim to determine the role of Syt III-C2AB in regulating EV trafficking from PC12 cells. First, after transfecting cells with Syt III or Syt III C2AB*, we treated cells with normal buffer or the KCl buffer for depolarizing cells. Different EV subtypes were harvested by gradient centrifugation to yield microvesicles (MVs) or exosomes. Subsequent electron microscopy and western blot analysis confirmed the EVs collected by centrifugation. EVs collected by this protocol ranged in size between 20 to 120 nm in diameter. Further, we found that after KCl depolarization, the CD9 signals in cell lysates were decreased in Syt III-overexpressing cells compared to the cells without KCl treatment. Western blot analysis also showed an increase in the CD63 signals in cells overexpressing Syt III after KCl depolarization, suggesting the potential role of Syt III in regulating EV trafficking. Moreover, confocal imaging analysis revealed that in the same batch of cells overexpressing Syt III, KCl depolarization increased the intracellular CD9 fluorescent signals compared with normal buffer, suggesting that Syt III may promote the formation of CD9-containing organelles upon KCl depolarization. In addition, by analyzing the topology of released EVs, we found that Syt III localized to the membrane of the EVs, with its C-terminal end towards the extracellular space. Therefore, these data suggested that Syt III plays a crucial role in regulating both biogenesis and trafficking of EVs in PC12 cells.