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  • 學位論文

Synaptotagmin III在PC12中 藉由鈣離子結合至C2AB區塊調控胞吐作用的動態變化

Synaptotagmin III modulates the kinetics of regulated exocytosis by Ca2+ binding to the C2AB domains in PC12 cells

指導教授 : 王致恬
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摘要


Synaptotagmin (Syt) 是一個至少擁有17種成員的蛋白家族,他們被視為一種感應鈣離子的蛋白。在神經細胞中,Syt可以藉由他們的C2A與C2B和鈣離子結合,進而影響神經傳導物質的釋放。先前已經有研究指出,Syt I能夠藉由與鈣離子結合來調控由鈣離子調控胞吐作用(Ca2+-dependent exocytosis)的動態變化。在本論文中我們將探討Syt III在鈣離子調控胞吐作用的動態變化中所扮演的角色。因為我們先前發現,Syt III蛋白在囓齒動物視網膜發育過程中的關鍵時期(P4-P6)的視網膜節細胞與視神經中大量表現,而此時期視網膜會經由自發性的放電反應來調控視野分離,由此推測Syt III在發育過程中可能會藉由調控胞吐作用來影響神經傳導素的釋放並影響發育中雙眼動物的視野分離;然而,目前Syt III如何調控作用的詳細機制仍然尚未明瞭。本論文中,我們將Syt III第386和520個胺基酸利用點突變的方式以降低Syt III其C2A與C2B和鈣離子結合的能力,為了進一步的了解對胞吐作用產生的影響,我們利用單一囊泡安培測定法(Amperometry)直接偵測在神經內分泌細胞(neuroendocrine cell)中神經傳導素的釋放,並且結合了免疫螢光染色及免疫沉澱法來探討Syt III在緻密核心囊泡胞吐作用中的分子機制。 我們的研究結果顯示,Syt III蛋白的主要表現於細胞膜上而非緻密核心囊泡,並且Syt III有增加總胞吐作用的趨勢,而在刺激發生後的中、後期紀錄中,我們發現胞吐作用的釋放頻率明顯高於控制組及突變組(Syt III-C2AB*),並且,Syt III可增加full fusion在刺激發生的中、後期紀錄中發生的頻率,而在突變的組別則有著和Syt III相反的結果。除此之外,Syt III明顯減少融合孔(fusion pore)的開啟時間,而突變組則會增加融合孔(fusion pore)的開啟時間。在我們提出的胞吐作用動態的模型中,我們發現Syt III會同時增加速率常速kc與kd,表示Syt III會藉由促進緻密核心囊泡離開fusion pore state (O)往dilation state (D)及close state (C)來降低融合孔(fusion pore)的開啟時間,而Syt III-C2AB*則藉由抑制緻密核心囊泡離開fusion pore state (O)往dilation state (D)及close state (C)來增加融合孔(fusion pore)的開啟時間。利用免疫沉澱的方式,我們發現Syt III 在沒有與鈣離子結合的情況下能夠與SN25交互作用,但在有鈣離子的情況下,Syt III與鈣離子結合後會加強和SN25的交互作用。 綜合上述,我們可以得知Syt III能夠藉由其C2A與C2B和鈣離子結合來影響胞吐作用的動態變化,除此之外,Syt III可能會藉由和鈣離子結合來促進緻密核心囊泡往細胞膜的方向移動,藉此來增加刺激發生後的中、後期胞吐作用的發生,並且我們推測Syt III 會藉由與鈣離子結合來增加與SN25的交互作用去減少融合孔(fusion pore)的開啟時間。因此,Syt III可能作為一個細胞膜上的感應蛋白,來調控密核心囊泡胞吐作用的動態變化。

並列摘要


Synaptotagmin (Syt) protein family consists of at least seventeen isoforms. Most of them have been reported as vesicular Ca2+ sensors by Ca2+ binding to their C2AB domains. For example, Syt I plays a role in regulating the kinetics of vesicular exocytosis. However, the functions of other isoforms remain to be identified. Previously, we found that Syt III is upregulated in retinal output neurons (retinal ganglion cells, RGCs) and optic nerves during a critical period (postnatal day P6 in rats) of visual circuit development. However, how Syt III functions in regulated exocytosis remains unclear. Here, we investigated if Syt III involves in Ca2+-regulated exocytosis by Ca2+ binding to its C2AB domains. We used PC12 cells as the model to examine Syt III’s function in regulated exocytosis, by combining molecular perturbation, immunofluorescence staining, and amperometry (a single-vesicle technique to measure real-time exocytosis). By mutating aspartate to asparagine at the site 386 and 520 of Syt III, we blocked the Ca2+ binding ability in the C2AB domains (D386, 520N, designated Syt III-C2AB*). After overexpressing control vector, wild-type Syt III, or Syt III-C2AB* in PC12 cells, we analyzed single exocytotic events by amperometry. We found that Syt III was poorly colocalized with dense-core vesicles (DCVs) but localized to plasma membrane. Moreover, overexpressing Syt III increased secretion rate after long-time stimulus compared to control or Syt III-C2AB*, suggesting that Syt III may involve in vesicle recruitment by Ca2+ binding to its C2AB domains. From co-immunoprecipitation, we found that binding to Ca2+ can enhance the interaction between Syt III and SN25, accounting for a decrease in the prespike foot (PSF) duration. These results suggested that in contrast to the conventional role of Syt family, Syt III functions as a “plasma membrane” Ca2+ sensor in modulating the kinetics of regulated exocytosis, via Ca2+ binding to its C2AB domains.

參考文獻


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