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

描述(由申请人提供)：在酵母极性和细胞器特性的产生中的膜自组织真核细胞的一个核心特征是细胞内的区隔和组织：它允许酶和底物浓度的局部增加，促进关键的生化反应，同时将细胞的其他部分与潜在的有害过程隔离。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的、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，如特定的磷脂酰肌醇(PI)或GTP酶种类。这些结构是如何产生的，它们的分子身份是如何稳定建立的？积极的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)利用酵母中没有的一种新的磷脂酰肌醇(PI)在酿酒酵母中创建合成的膜细胞器或极化结构。酵母缺乏在高等真核生物中发现的PI(3，4，5)P3。我们将重点针对产生或降解该物种的脂蛋白激酶和磷酸酶，以确定哪些途径是建立极化的PI(3，4，5)P3膜域或PI(3，4，5)P3标记的细胞器所必需的。通过有控制地引入一个新的PI物种，我们将把PI在细胞器鉴定中的作用与其他细胞成分分开，并阐明细胞器生物发生过程中的最低要求和分泌途径的进化/多样化。2)利用光控制的CDC42(一种小的GTP酶)和它的全长GTP酶Cdc24的募集，定量分析了酵母自然极化的产生。内源酵母的极化结构以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却知之甚少。使用一个新开发的光激活蛋白质招募系统(来自LIM和Voigt实验室)，我们将精确地调节CDC42和CDC24的时空招募。这种方法将使我们能够控制和观察极化的动力学，并剖析有助于在稳定的膜组织状态之间进行阶梯式切换的调节反馈途径。 与公众健康相关：膜的自组织在维持正常的细胞过程和隔离生化反应方面发挥着关键作用。稳定膜结构域的破坏，如上皮细胞的极性缺陷，可导致多囊肾病等病理改变。我们的目标是确定和表征膜自组织的指导原则，包括空间和时间招募和正反馈所起的相对作用。

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.