Membrane self-organization in generation of yeast polarity and organelle identity

描述（由申请人提供）：酵母极性和细胞器身份生成中的膜自组织细胞内区隔化和组织是真核细胞的核心特征：它允许局部增加酶和底物浓度，促进关键的生化反应，同时将细胞的其他部分从潜在的有害过程中分离出来。我们感兴趣的是确定细胞如何在不断变化的细胞环境中保持稳定，可识别的膜亚结构。大多数不同的细胞器或膜结构域包含独特的身份标记，如特定的磷酸肌苷（pi）或GTPase种类。这些结构是如何产生的，它们的分子特性又是如何稳定建立的？积极反馈如何有助于建立这种独特的身份？我们的具体目标是：1)利用酵母中不常见的一种新型磷酸肌苷（PI）在酿酒酵母中合成膜细胞器或极化结构。酵母缺乏高等真核生物中存在的PI(3,4,5)P3。我们将重点关注产生或降解该物种的脂质激酶和磷酸酶的空间靶向，以确定哪些途径是建立极化PI(3,4,5)P3膜结构域或PI(3,4,5)P3标记细胞器所必需的。通过以受控方式引入新的PI物种，我们将分离PI在细胞器身份中的作用与其他细胞成分，并阐明细胞器生物发生过程中的最低要求以及分泌途径的进化/多样化。2)通过光控募集Cdc42（一种小GTPase）及其GEF Cdc24，定量分析天然酵母极化的产生。内源性酵母极化结构以GTPase Cdc42的活性形式为标志，但导致稳定极性建立的精确反馈结构尚不清楚。使用新开发的光激活蛋白募集系统（来自Lim和Voigt实验室），我们将精确调节Cdc42和Cdc24的时空募集。这种方法将使我们能够控制和观察极化动力学，并剖析促进稳定膜组织状态之间步进式切换的调节反馈途径。

DESCRIPTION (provided by applicant): Membrane self-organization in generation of yeast polarity and organelle identity Intracellular compartmentalization and organization is a central feature of eukaryotic cells: it permits local increases in enzyme and substrate concentrations, facilitating critical biochemical reactions while isolating other parts of the cell from potentially harmful processes. We are interested in determining how cells maintain stable, recognizable membrane substructures within the constantly shifting cellular environment. Most distinct organelles or membrane domains contain unique markers of identity such as specific phosphoinositides (PIs) or GTPase species. How do these structures arise and how are their molecular identities stably established? How does positive feedback contribute to establishment of this distinct identity? Our specific aims are to: 1) create synthetic membrane organelles or polarized structures in S. cerevisiae using a novel phosphoinositide (PI) species not normally found in yeast. Yeast lack the PI species PI(3,4,5)P3, found in higher eukaryotes. We will focus on spatially targeting the lipid kinases and phosphatases that produce or degrade this species in order to determine which pathways are necessary to either establish a polarized PI(3,4,5)P3 membrane domain or a PI(3,4,5)P3 tagged organelle. By introducing a new PI species in a controlled manner, we will separate the role of PIs in organelle identity from that of other cellular components and illuminate the minimal requirements in the process of organelle biogenesis and the evolution/diversification of the secretory pathway. 2) Quantitatively analyze the generation of natural yeast polarization using light-controlled recruitment of Cdc42 (a small GTPase) and its GEF, Cdc24. Endogenous yeast polarized structures are marked by the active form of the GTPase Cdc42, but the precise feedback architectures that lead to stable polarity establishment are poorly understood. Using a newly developed light-activated protein recruitment system (from the Lim and Voigt labs) we will precisely modulate the spatio-temporal recruitment of Cdc42 and Cdc24. This approach will allow us to control and observe the kinetics of polarization and to dissect the regulatory feedback pathways facilitating step-like switching between stable membrane organization states. PUBLIC HEALTH RELEVANCE: Membrane self-organization plays a crucial role in the maintenance of normal cellular processes and sequestration of biochemical reactions. Disruption of stable membrane domains, such as a polarity defect in epithelial cells, leads to pathology such as polycystic kidney disease. Our goal is determination and characterization of the guiding principles of membrane self-organization, including the relative roles played by spatial and temporal recruitment and positive feedback.