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  • 學位論文

Atg1與Atg9在細胞自噬上之功能研究

Functional characterization of Atg1 and Atg9 in autophagy

指導教授 : 陳光超

摘要


「細胞自噬」是一種細胞降解物質的途徑,在維持細胞體內平衡和對於如飢餓,缺氧和氧化壓力環境下的細胞反應扮演不可或缺的角色。在這個過程中,細胞質會被一種雙層膜囊泡,稱為細胞自噬體所吞噬,隨後與溶酶體結合,降解所吞噬的大分子,循環再利用。自噬相關基因9(Atg9)是唯一在自噬相關蛋白中發現的膜蛋白,它會在高基式體,晚期內含體,以及細胞自噬體間循環移動,這種循環移動被認為是用來提供膜給正在形成的細胞自噬體。然而,Atg9的生理作用以及調控機制到現在仍然不是很清楚。 在本論文中,我們發現了一種分子馬達-肌凝蛋白-II,能夠調節Atg9的循環移動。我們發現在果蠅中Atg1激酶會經由直接磷酸化一種新的肌球蛋白輕鏈激-Sqa,導致肌凝蛋白-II的活化。在果蠅中,降低Sqa表現量或抑制Myosin-II作用,會抑制細胞自噬體的形成。同樣的,在哺乳類細胞中,我們發現Sqa同源基因-ZIPK和肌凝蛋白-II在自噬作用中也扮演了重要的角色。我們更進一步發現Atg1對肌凝蛋白-II活化的調控,會影響到Atg9的循環移動。因此,這些研究結果提供了證據顯示Atg1會經由活化肌凝蛋白-II控制Atg9的循環移動而調控細胞自噬體的形成,而此訊息調控機轉從果蠅到人類有高度的保留性。總結,我們研究的成果揭露了細胞自噬體如何形成之作用機制。

並列摘要


Autophagy, a highly conserved catabolic process, is essential for cell homeostasis and cellular response to environmental stresses, such as starvation, hypoxia, and oxidative stress. During this process, double-membrane vesicles, autophagosomes, engulf cytoplasmic components and subsequently fuse with lysosomes for degradation and recycling. Autophagy-related gene 9 (Atg9) is the only transmembrane autophagic protein which cycles between the trans-Golgi network, late endosomes and autophagosomes in mammalian cells. Atg9 cycling is proposed to deliver membrane to the forming autophagosomes. However, the precise physiological roles and the regulatory mechanism of Atg9 remain unclear. In this thesis, we identified Atg1-activated myosin-II as a potential motor protein which regulated Atg9 cycling during starvation-induced autophagy. We found that the Ser/Thr kinase Drosophila Atg1, a key initiator of autophagy, promoted the activation of the actin-associated motor protein myosin II through direct phosphorylating of a novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa). Sqa depletion or myosin II inhibition inhibited the formation of autophagosomes in larval fat body cells under starvation conditions. Consistent with the results in Drosophila, we found that ZIPK, the Sqa mammalian homolog, and myosin II played a critical role in the regulation of starvation-induced autophagy in mammalian cells. Furthermore, we found that Atg1–mediated myosin-II activation controlled the cycling of Atg9 when cells were deprived of nutrients. Thus, these findings provided evidence to fill the gaps between Atg1-mediated autophagosome formation and Atg9 cycling. In conclusion, our study expanded our understanding in mechanisms of autophagosome formation.

並列關鍵字

Autophagy Atg1 Atg9 myosin-II cytoskeleton

參考文獻


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