Cortical Granule Regulation and Function

描述：（由申请人提供）皮质颗粒是卵母细胞特有的 并在受精时分泌它们的内容物，形成一个永久的屏障， 多精受精我们的长期目标是了解 从皮层功能贡献的角度来看受精 颗粒。我们为此目的选择的卵母细胞是海胆， 大约15，000个皮质颗粒在生物发生中是同步的， 转运到表面，对接到质膜，并分泌到 受精我们将利用海胆卵母细胞， 2）现有的编码蛋白质的cDNA克隆 特异于皮质颗粒的内容物和膜; 3） 分离皮质颗粒并重建功能;以及4） 卵母细胞的体外培养和成熟以及皮质的直接可视化 颗粒。我们在此提出三大目标： 1.确定皮质颗粒蛋白酶的功能贡献， 受精这种蛋白酶切割卵的质膜蛋白， 修饰卵细胞外基质，并改变其他皮质颗粒 蛋白质含量我们将确定蛋白水解的目标，使我们能够 鉴定和功能表征对受精重要蛋白质。 2.皮质颗粒的生物发生和移位。我们将解剖 皮质颗粒在体内到达细胞表面的机制 （易位），在皮质颗粒的表面形成近乎完美的单层。 质膜，以及皮层颗粒膜蛋白在这一过程中的作用。 调控 3.确定受精时调节胞吐作用的分子基础。我们 将利用这个卵子中皮质颗粒的预对接状态来识别 调节胞吐作用的蛋白质。我们将重点关注那些 与SNARE同源物和rab 3相互作用，并解决 刺激胞吐作用，以及阻断胞吐作用的分子钳 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.