Cortical Granule Regulation and Function

描述：(申请人提供)皮质颗粒是卵母细胞所特有的 并在受精时分泌其内容物以形成永久的块 多精受精。我们的长期目标是了解 从皮质功能贡献的角度看受精 颗粒。我们选择的卵母细胞是海胆，在那里 大约15,000个皮质颗粒在生物发生过程中是同步的， 移位到表面，对接到质膜，并在 受精。我们将利用海胆的卵母细胞生产数以百万计的 2)手头有编码蛋白质的cdna克隆。 对皮质颗粒的内容物和膜具有特异性；3)能够 分离皮质颗粒和重建功能；4)顺从 卵母细胞体外培养成熟及皮质直观显示 颗粒。在此，我们提出三大目标： 1.测定皮质颗粒蛋白水解酶对 受精。这种酶能裂解鸡蛋的质膜蛋白， 修饰鸡蛋细胞外基质，改变其他皮质颗粒 含量蛋白质。我们将确定蛋白质分解的目标，使我们能够 鉴定对受精很重要的蛋白质并对其进行功能鉴定。 2.皮质颗粒的生物发生和移位。我们将剖析 体内皮质颗粒到达细胞表面的机制 (易位)，使皮质颗粒在底部形成接近完美的单层 质膜，以及皮质颗粒膜蛋白在其中的作用 监管。 3.确定受精时调节胞吐的分子基础。我们 将利用这个卵子中皮质颗粒的预对接状态来识别 调节其胞吐作用的蛋白质。我们将重点研究那些 与SNARE同源物和RAB3相互作用，并解决这两个机制 刺激胞吐作用，以及阻止胞吐作用的分子钳 直到受精。

DESCRIPTION: (provided by applicant) Cortical granules are unique to oocytes and secrete their contents at fertilization to form a permanent block to polyspermy. Our long-term goal is to understand conserved mechanisms of fertilization from a perspective of the functional contribution of cortical granules. Our oocyte of choice for this goal is the sea urchin, where approximately 15,000 cortical granules are synchronous in biogenesis, translocation to the surface, docking to the plasma membrane, and secretion at fertilization. We will make use of the sea urchin oocyte for 1) the millions available from each female; 2) the cDNA clones in hand that encode proteins specific to the contents and membranes of cortical granules; 3) the ability to isolate cortical granules and reconstitute function; and 4) the amenable culture and maturation of oocytes in vitro and direct visualization of cortical granules. Here we propose three major goals: 1. Determine the functional contribution of the cortical granule protease to fertilization. This protease cleaves plasma membrane proteins of the egg, modifies the egg extracellular matrix, and alters other cortical granule content proteins. We will identify the proteolytic targets, enabling us to identify and functionally characterize proteins important for fertilization. 2. Biogenesis and translocation of cortical granules. We will dissect the mechanisms used by cortical granules in vivo to get to the cell surface (translocation), to make a near perfect monolayer of cortical granules at the plasma membrane, and the function of cortical granule membrane proteins in this regulation. 3. Identify the molecular basis for regulated exocytosis at fertilization. We will exploit the pre-docked status of cortical granules in this egg to identify proteins that regulate their exocytosis. We will focus on proteins that interact with SNARE homologues and of rab 3, and address both the mechanism for stimulating exocytosis, as well as the molecular clamp that blocks exocytosis until fertilization.