第三型突觸連結蛋白調控胞外體運輸所扮演的角色

The role of Synaptotagmin III in regulating trafficking of extracellular vesicles

余妮晏(Ni-Yen Yu)

指導教授 ： 王致恬

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902087

摘要

胞外體 (Extracellular vesicles, EVs) 為具有脂質雙層膜構造且可被細胞釋放的顆粒，並且根據其生成與釋放方式分成不同亞型。胞外體內部已知包含許多核酸、脂質與蛋白質，可以反映出細胞的生理狀態，因此胞外體又被稱為細胞間的溝通者。先前研究指出，胞外體的釋放是藉由鈣離子的調控，此種模式和鈣離子主導的囊泡胞吐作用相似。然而，目前並不清楚調控鈣離子主導的胞外體釋放的分子機制。鈣離子突觸結合蛋白 (Synaptotagmins, Syts) 目前已有17種不同亞型在哺乳動物中被發現，並且已知Syts在鈣離子主導的胞吐作用中藉由5個鈣離子與其結合位C2AB結合可扮演感測器的角色。其中第三型鈣離子突觸結合蛋白(Syt III)為已知特別具有6個鈣離子結合位的亞型，藉由鈣離子結合位可調控胞吐作用。並且，在本實驗室先前的研究中，我們過量表現Syt III在PC12細胞中，比較野生型 (wild-type Syt III) 與降低鈣離子結合能力的突變組 (Syt III C2AB*)，發現野生型會促進胞外體標記分子CD63的釋放。因此，我們假設Syt III會藉由鈣離子結合位調控細胞中胞外體的運輸。在本研究中，我們用西方墨點法、免疫螢光染色與電子顯微鏡來證明此假設。首先，我們過量表現野生型與突變組在PC12細胞內並給予普通溶液或高鉀溶液 (KCl) 刺激，將收集到的溶液進行超高速離心後回溶進行電子顯微鏡觀察或西方墨點法分析。我們發現經過超高速離心後收集到的外泌體大小約為20~120 奈米，且胞外體上有表現胞外體標記分子CD63或CD9。接著，我們分析了給予KCl去極化刺激或給予普通溶液的細胞，發現野生型細胞中CD9的蛋白量相比於突變組在KCl刺激後明顯下降，顯示鈣離子結合到Syt III的C2AB結合位調控了有表現CD9的胞外體的運輸。我們也發現給予野生型細胞高鉀溶液並經過離心進行西方墨點分析，CD63的蛋白量在野生組相較突變組多，顯示Syt III可能會調控胞外體的運輸。其二，我們也利用免疫螢光染色分析細胞中CD9的螢光亮度，發現在給予KCl刺激後，野生型細胞內CD9螢光亮度較給予普通溶液增加，但是此現象在突變組並沒有出現，顯示鈣離子結合到Syt III結合位C2AB調控了KCl去極化所引發的胞外體生成。最後，我們利用免疫墨點 (dot blotting) 分析Syt III在胞外體膜構造上的拓撲 (topology)，發現在胞外體上的Syt III為C端朝向細胞外，N端朝向胞外體內部。從上述結果，我們推論KCl去極化刺激下，Syt III是藉由鈣離子結合到結合位C2AB調控胞外體的生成與釋放。

關鍵字

胞外體 ； 鈣離子突觸結合蛋白 ； 胞外體標記分子 ； 超高速離心 ； 電子顯微鏡 ； 拓撲

並列摘要

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.

並列關鍵字

Extracellular vesicles ； Synaptotagmin ； The markers of extracellular vesicles ； Super-speed gradient centrifugation ； Electron microscopy ； Topology

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