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

描述（由申请人提供）：酵母极性和细胞器特性产生中的膜自组织细胞内区室化和组织是真核细胞的核心特征：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，可识别的膜亚结构。大多数不同的细胞器或膜结构域含有独特的身份标志物，如特定的磷酸肌醇（PI）或GTP酶种类。这些结构是如何产生的，它们的分子身份又是如何稳定建立的？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）在S.使用通常在酵母中不存在的新的磷酸肌醇（PI）种类，在酿酒酵母中进行。酵母缺乏在高等真核生物中发现的PI种类PI（3，4，5）P3。我们将专注于空间靶向的脂质激酶和磷酸酶，产生或降解这种物种，以确定哪些途径是必要的，要么建立一个极化PI（3，4，5）P3膜结构域或PI（3，4，5）P3标记的细胞器。通过引入一个新的PI物种在一个受控的方式，我们将分离的PI在细胞器的身份从其他细胞成分的作用，并阐明在细胞器的生物合成和分泌途径的进化/多样化的过程中的最低要求。2)定量分析使用Cdc 42（一种小的GTdR）及其GEF Cdc 24的光控募集产生的天然酵母极化。内源性酵母极化结构的标志是活性形式的GTdR Cdc 42，但精确的反馈架构，导致稳定的极性建立知之甚少。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Cdc 42和Cdc 24的时空募集。这种方法将使我们能够控制和观察极化的动力学，并剖析调节反馈途径，促进稳定的膜组织状态之间的步进式切换。 公共卫生相关性：膜自组织在维持正常细胞过程和隔离生化反应中起着至关重要的作用。稳定膜结构域的破坏，如上皮细胞中的极性缺陷，导致病理学，如多囊肾病。我们的目标是确定和表征膜自组织的指导原则，包括空间和时间的招聘和正反馈所发挥的相对作用。

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.