請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76532
標題: | 探討突觸囊泡專屬蛋白Synapsin Ia如何調控緻密核心囊泡的釋放 Exploring how the synaptic vesicle-specific protein Synapsin Ia regulates the release of dense-core vesicles |
作者: | Hui-Ju Yang 楊蕙如 |
指導教授: | 王致恬(Chih-Tien Wang) |
關鍵字: | Synapsin Ia,胞吐作用,緻密核心囊泡,融合孔動態,Synaptophysin, Synapsin Ia,Exocytosis,Dense-core vesicles,Fusion pore kinetics,Synaptophysin, |
出版年 : | 2019 |
學位: | 博士 |
摘要: | 神經傳導物質的釋放與接受驅動神經系統中的化學性突觸傳導。神經傳導物質主要被包覆在兩種截然不同的囊泡中-緻密核心囊泡(dense-core vesicles)和突觸囊泡(synaptic vesiclse),雖然此兩種囊泡的釋放都是藉由相同的機制-鈣離子調控胞吐作用;然而,Synapsin (Syn) Ia卻為唯一一種只特定存在於突觸囊泡的專屬蛋白,其藉由自身的磷酸化對調控突觸囊泡的補給至為重要。在適當的刺激下,Syn Ia會被磷酸化,進而將突觸囊泡帶至釋放位置,因此,Syn Ia的磷酸化會促進突觸囊泡的釋放。儘管已在含有緻密核心囊泡的突觸末端中發現有大量的Syn Ia,但目前仍完全未知的是Syn Ia是否也會(或如何)去影響緻密核心囊泡的胞吐作用。因此,為了在單一囊泡的層級釐清Syn Ia如何調控緻密核心囊泡的釋放,我們利用單一囊泡安培測定法(single-vesicle amperometry)直接偵測在transfected PC12細胞中,正腎上腺素從緻密核心囊泡的釋放。研究結果顯示,Syn Ia 可以藉由其磷酸化調控緻密核心囊泡胞吐作用的動態變化;除此之外,Syn Ia還可以調節融合孔(緻密核心囊泡融合時的中間體)的動態,藉此影響緻密核心囊泡融合孔開啟狀態時的穩定性。由於已知Syn I不存在於緻密核心囊泡,推測Syn Ia應該是藉由和某特定蛋白結合,進而調控緻密核心囊泡的胞吐作用動態。為了進一步找出這個特別的蛋白,我們結合生物資訊、免疫共沉澱以及蛋白質交互作用檢測技術,證明Syn I能夠和Synaptophysin (Syp)在細胞內進行交互作用;除此之外,在同一個細胞中,Syn I能夠和Syp直接性且原位的交互作用。最後,我們發現Syn I和Syp的交互作用不是因為蛋白質表現量的改變而影響,而是倚賴Syn Ia的磷酸化。因此,這些結果除了揭開了突觸囊泡專屬蛋白Syn Ia如何去調控緻密核心囊泡胞吐作用的動態,還提供了關於Syn Ia在緻密核心囊泡和突觸囊泡共釋放機制上的角色;這些結果引導出一個新觀念的突破,並賦予神經傳導物質釋放的多樣性。 Neurotransmitters release and reception mediate chemical synaptic transmission. Neurotransmitters are packaged into two distinct classes of vesicles, “dense-core vesicles” (DCVs) and “synaptic vesicles” (SVs). The secretion from both DCVs and SVs share the common exocytotic machinery, i.e., Ca2+ regulated exocytosis. Instead, Synapsin (Syn) Ia is a SV-specific SV protein, essential for recruiting SVs by phosphorylation. Upon the appropriate stimulus, Syn Ia undergoes phosphorylation, thus recruiting SVs to release sites. Therefore, the phosphorylation of Syn Ia up-regulates the release of SVs. Although Syn Ia is also abundant in the axon terminals containing DCVs, whether (or how) Syn Ia regulates the DCV release remains completely unknown. To determine the Syn Ia’s regulation of DCV exocytosis at the single-vesicle level, we directly measured the NE release from DCVs by performing single-vesicle amperometry in transfected PC12 cells following molecular perturbation. We showed that Syn Ia can regulate the dynamics of DCV exocytosis in a phosphorylation-dependent manner. In addition, Syn Ia can modulate the kinetics of fusion pores, the intermediates during DCV fusion, suggesting that Syn Ia regulates the stabilization of opening DCV fusion pores. Since Syn I rarely localized to DCVs, Syn Ia may interact certain protein to regulate DCV exocytosis. To further predict and identify the candidate Syn Ia-interacting proteins, we combined bioinformatics, co-immunoprecipitation, and proximity ligation assay. As the results, we identified that the in vivo interaction between Syn Ia and Synaptophysin (Syp). In addition, Syn Ia may directly interact with Syp in situ in the same cell. Finally, we showed that the in vivo interaction between Syn Ia and Syp was not attributed to the change in expression levels, but dependent on the phosphorylation of Syn Ia. In conclusion, our results not only unveil how the SV-specific protein Syn Ia regulates the dynamics of DCV exocytosis, but also provide a new conceptual advance regarding the co-release mechanism of DCVs and SVs, conferring the versatility of neurotransmitter release. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76532 |
DOI: | 10.6342/NTU201901181 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2024-07-05 |
顯示於系所單位: | 基因體與系統生物學學位學程 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-108-D03b48004-1.pdf | 9.24 MB | Adobe PDF | 檢視/開啟 |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。