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標題: | 第三型突觸連結蛋白在PC12細胞與發育視網膜神經元中
調控胞吐作用的機制 Cellular and Molecular Mechanisms of Synaptotagmin III-Regulated Exocytosis in PC12 Cells and Developing Retinal Neurons |
作者: | Yu-Hung Tsai 蔡毓紘 |
指導教授: | 王致恬(Chih-Tien Wang) |
關鍵字: | 第三型突觸連結蛋白,緻密核心囊泡,SNAP-25,溶?體,外泌體, Synaptotagmin III,dense-core vesicle,SNAP-25,lysosome,exosome, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | Sytnaptotagmins (Syts) 的功能是扮演一個鈣離子感受器,進而去調控神經及神經內分泌細胞中胞吐作用的動態變化。Syts 至少有17種亞型已在哺乳動物中被發現,並且不同亞型的C2A及C2B擁有不同跟鈣離子結合的親和力。我們先前已發現Syt III (一種擁有6個鈣離子結合位的亞型)會在大鼠出生後4-6天(視覺網絡發育的關鍵時期),突然在視網膜神經節細胞大量表現,由此可知Syt III在發育中的神經系統中可能扮演一個調控神經突觸活性的角色。此外,我們先前過量表現Syt III在PC12細胞中,結果緻密核心囊泡釋放的頻率和控制組與突變組(Syt III-C2AB*, 一種降低跟鈣離子結合的能力的突變)相比明顯地增加。然而,Syt III如何調控囊泡釋放的詳細分子機制目前還不清楚。在本論文中,我們過量表現Syt III或Syt III-C2AB*在PC12細胞中,於過量表現後第三天,利用免疫染色及內源性免疫沉澱來進行研究。結果發現Syt III僅和緻密核心囊泡部分重疊,但在被KCl刺激去極化後,Syt III卻與細胞膜高度重疊。另外,Syt III會和SNAP-25或Syntaxin (t-SNAREs,位在細胞膜上的蛋白質)交互作用,此結果與免疫染色一致,並且,阻斷跟鈣離子結合能力(Syt III-C2AB*)會降低Syt III跟t-SNAREs交互作用的量。我們還發現了被KCl刺激後,過量表現Syt III會降低跟溶酶體重疊;相反地,和外泌體重疊的比例卻增加。並且,在KCl刺激下,外泌體的釋放受到鈣離子跟C2AB結合來調控。然而,以上的結果並未在發育中大鼠視網膜被初步發現。這些結果顯示了在PC12細胞中,Syt III會透過跟SNAP-25或Syntaxin交互作用,進而調控囊泡融合孔的動態變化。除此之外,Syt III還有可能透過鈣離子跟C2AB結合而調控溶酶體及外泌體的釋放。 Synaptotagmins (Syts) function as Ca2+ sensors that regulate the kinetics of exocytosis in neurons and neuroendocrine cells. At least 17 Syt isoforms have been found in mammals and possess variable calcium-binding affinities in C2 domains (C2A and C2B). Our previous studies found that Syt III (a particular isoform with six calcium-binding sites) is significantly up-regulated in P4-P6 (a critical period of visual circuit development) rat retinal ganglion cells, suggesting that Syt III may play a unique role in regulating synaptic activity in the developing nervous system. In addition, overexpressing Syt III in PC12 cells increases the frequency of DCV release after KCl depolarization compared to control or Syt III-C2AB* (a mutant harboring the abolished calcium-binding sites). However, the molecular mechanism underlying Syt III’s regulation of vesicle release remains elusive. In this study, we overexpressed Syt III or Syt III-C2AB* in PC12 cells. At 72 hr post transfection, we performed immunoflurorescence staining and endogenous co-immunoprecipitation. We found that Syt III was poorly colocalized with dense-core vesicles (DCVs) but highly colocalized to plasma membrane after KCl depolarization. Moreover, abolishing the calcium-binding sites in Syt III decreased its interaction with SNAP-25 or Syntaxin (two t-SNAREs), consistent with the localization of Syt III to plasma membrane. We also found that Syt III down-regulated the localization to lysosome upon KCl depolarization. By contrast, overexpressing Syt III enhanced the colocalization ratios of Syt III overlapping with exosomes. Furthermore, exosome export was regulated by Ca2+-binding to the C2AB domains of Syt III upon KCl depolarization. Nevertheless, these effects were not shown in developing rat retinas. These results suggested that Syt III regulates the kinetics of fusion pores by interacting with SNAP-25 or Syntaxin, and this interaction acts through calcium binding to its C2AB domains. In addition, Syt III may also modulate the release of lysosomes or exosomes by Ca2+-binding to the C2AB domains. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7749 |
DOI: | 10.6342/NTU201702315 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 分子與細胞生物學研究所 |
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