2015-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/655422摘要：細胞的極性化對於細胞的生長、分化、以及單細胞或多細胞生物的型態形成均扮演 重要角色，而有關細胞極性的決定因子可以從細胞内或是細胞外產生。出芽酵母菌 (Saccharomyces cerew+s/ae )是研究細胞極性的一種極佳的模式生物，其具備有三種獨 特的細胞極化之生命週期的表現：包含無性出芽生殖、有性細胞融合生殖、以及侵入性 生長。其中，酵母菌侵入性生長與真菌類的致病能力高度相關，其調控機制與細胞内訊 息傳遞過程已被廣泛地研究。因此，我們實驗室主要長程研究目標乃是探討調控細胞極 性與侵入性生長之分子機制。鳥嘌呤核甘酸結合蛋白的腺苷二磷酸核糖基化因子（ARF)乃是對於細胞囊泡運輸 與細胞骨架重整之主要調控因子。其中，哺乳動物細胞中Arf6參與了多種的細胞功能， 包含細胞貼附、細胞遷移、傷口癒合、細胞邊緣皺褶、以及癌細胞轉移。酵母菌細胞 Arf3p乃是哺乳動物細胞Arf6的同源蛋白質，我們之前的研究結果顯示Arf3p座落於細 胞膜上，並參與了酵母菌出芽生殖過程中的細胞骨架重整以及細胞極性的發育過程。我 們近期的研究成果顯示：在由葡萄糖缺乏所引發之酵母菌侵入性生長的過程中，活化後 的Arf3p可以調控Bud2p的活性。此外，我們也發現當葡萄糖缺乏所引發的酵母菌侵入 性生長的過程中會增加活化態Arf3p，且此過程與其鳥糞嘌呤核苷酸交換因子Yellp無 關。然而，對於酵母菌侵入性生長的過程中Arf3p活化過程及其調控機制仍不清楚。因 此，本研究計晝主要著力於探討酵母菌侵入性生長的過程中調控Arf3p活性的分子機 制，以下是本計晝的研究目標：(一）研究當葡萄糖缺乏時，嶄新鳥糞嘌呤核苷酸交換因子Snflp調控Arf3p活化的分 子機制我們將會研究在葡萄糖缺乏時，Snflp如何活化Arf3p ；以及Snflp轉譯後修飾是否 參與其活化Arf3p的過程中。我們亦會研究在葡萄糖缺乏時，Snflp之相關結合蛋白質 是否會調控Arf3p的活化過程。此外，我們也會去研究哺乳動物細胞同源蛋白質AMPK 是否具有相同功能，可以調控Arf6之活性。(二）探討酵母菌侵入性生長過程中，Ari3p如何調控Flollp之極性化我們將致力於探討在酵母菌侵入性生長過程中，Arf3p調控Flollp於細胞中極性分 布之分子機制。此外，我們將研究酵母菌Arf3p與Flollp之蛋白質交互作用，以及其影 響酵母菌細胞侵入性生長之分子機制。我們也將探討是否Arf3p參與在運輸FL011 mRNA的過程，進而影響侵入性生長。(三）研究細胞内Ari3p之座落位置與其功能之調控機制我們將會研究蛋白質回送分選蛋白質retromer複合體如何調控Arf3p於細胞内之座 落位置；我們亦會研究ARFGAP蛋白質如何調控Arf3p活性以及其細胞内座落位置及 功能；我們也會進一步分析細胞骨架及其結合蛋白質如何調控細胞内Arf3p之座落位置 及其相關功能。本計晝將致力於研究調控細胞極性之相關分子機制，其中包含了調控細胞極性相關 蛋白質的活化過程、以及蛋白質座落位置與其運輸，將會是本計晝的主要研究目標。<br> Abstract: Cell polarization is crucial for cell proliferation, differentiation, and morphogenesis in both unicellular and multicellular organisms. The determinants of polarity can be either internally or externally derived. The yeast Saccharomyces cerevisiae has been used as a model organism for studying the development of polarity, showing three unique life cycle stages of polarized growth: budding, mating, and filamentous growth. The regulation and signaling pathways involved in the invasive growth of yeast have been studied extensively because of their general applicability to fungal pathogenesis. A long-term research goal in my lab is to explore the molecular mechanism in regulating cell polarity and invasive growth.ADP-ribosylation factors (Arfs) are highly conserved small GTPases that are major regulators of vesicular trafficking and cytoskeletal reorganization. We have identified and characterized a yeast plasma membrane-associated Arf3p, and showed that Arf3p is involved in actin organization and polarity development during yeast budding. Yeast Arf3p is the homologue of mammalian Arf6, which is involved in multiple cellular processes, including cell adhesion, migration, wound healing, membrane ruffling, and metastasis. Recently, we reported that a novel spatial activation of Arf3p plays a role in regulating Bud2p activation during glucose depletion-induced invasive growth. We also showed that under glucose depletion-induced invasion conditions in yeast, more Arf3p is activated to the GTP-bound state; however, the activation is independent of a known Arf3p guanine nucleotide-exchange factor (GEF) Yellp. In this proposal, we will investigate the molecular mechanisms underlying regulation of Arf3p signaling in invasive growth and cellular polarity. Specific aims of proposed studies include:1.To study the regulation of Snflp GEF for Arf3p activation in response to glucose depletion. We will examine how Snflp activates Arf3p and identify whether post-translational modification(s) of Snflp is involved in Arf3p activation in response to glucose depletion. We will also examine whether Snflp associated complex participates in regulating Arf3p activity for glucose depletion-induced invasion. Furthermore, we will determine whether mammalian AMPK share similar GEF function toward Arf6 activity.2.To elucidate how Arf3p regulates polarization of Flo11p in invasive growth. We will characterize whether Arf3p regulates polarized localization of Flollp in invasive cells. We will test Arf3p-Flollp interaction in vitro and in v^^vo and examine whether the association of Arf3p and Flollp can determine yeast invasion. We will also examine whether Arf3p controls FLO11 mRNA transport for invasive growth.3.To uncover the machinery for regulating intracellular Arf3p function and localization. We will determine how retromer complex controls Arf3p localization and/or function. We will identify and characterize Arf GAP proteins regulating Arf3p localization and activity. We will also characterize whether actin filament network involved in regulating Arfp3 localization and function.The molecular mechanism governing the individual steps of Arf3p-mediated cellular polarity, which include the activation process, regulation of protein localization and cargos transport, will be the main target of this research腺苷二磷酸核糖基化因子（ARF)鳥嘌呤核甘酸結合蛋白鳥糞嘌呤核甘酸轉換因子 (GEF)蛋白質運送細胞極性侵入性生長ADP-ribosylation factorARFguanine nucleotide-binding proteinGEFprotein transportcell polarityinvasive growth