BIOLOGY OF CORTICAL GRANULES

皮质颗粒是卵母细胞所特有的，在受精过程中发挥作用 通过分泌它们的内含物来形成一个永久性的多精受精障碍。我们 将研究皮质颗粒的生物学，有两个主要目标：l） 了解卵母细胞调节细胞器的机制 生物发生和蛋白质储存; 2）了解它们的调节 受精时的分泌物。为这些目标选择的卵母细胞来自 海胆大约有15,000个皮质颗粒 在细胞表面的细胞外泌，以响应精子的结合。它 是唯一的卵母细胞，1）cDNA克隆已被分离， 编码皮质颗粒特异性的内容物和膜蛋白; 2)皮质颗粒可以功能性形式分离，和3） 皮质颗粒的体外培养、成熟和直接可视化 都是可能的皮质颗粒不同于大多数其他 分泌囊泡，因为它们保持停靠在细胞表面， 几个星期，他们是非回收，他们包含了十几个不同的 在囊泡内亚区室化的蛋白质。三 提出了具体目标： 1.我们将确定皮质内容蛋白质的决定因素， 这些颗粒是靶向进入皮质颗粒所需的。我们将 利用皮质颗粒特异性蛋白SFE 9的cDNA， 蛋白质报告系统中的ovperoxidase和hyalin，以确定 最小序列对于适当的细胞器靶向是必需的。 因为囊泡在形态上和生化上 不同的区域，我们还将研究蛋白质所必需的信号 利用蛋白质特异性地在囊泡内进行亚区室化 在每个区域划分。 2.我们将探讨皮层颗粒的机制和调控 移位至卵母细胞表面。我们将根据 这一过程的选择性，即对皮质颗粒的选择性， 它们的移位时间，以及细胞器中使用的途径 运动此外，我们还将研究用于创建 卵母细胞表面有完整的单层皮质颗粒。 3.我们将确定调节皮质激素分泌的机制， 受精时的颗粒我们将检验皮质的假设 颗粒含有独特的膜蛋白群体， 通过使用单克隆抗体对这种囊泡的专门生物学 对皮质颗粒膜和测试同源体细胞 参与调节分泌功能的细胞蛋白。

Cortical granules are unique to oocytes and function during fertilization by secreting their contents to form a permanent block to polyspermy. We will study the biology of cortical granules with two major goals: l) to understand the mechanisms used by oocytes to regulate organelle biogenesis and protein storage; and 2) to understand their regulated secretion at fertilization. The oocyte of choice for these goals is from the sea urchin, where approximately 15,000 cortical granules are poised at the cell surface to exocytose in response to either sperm binding. It is the only oocyte for which l) cDNA clones have been isolated that encode content and membrane proteins specific to the cortical granules; 2) the cortical granules can be isolated in a functional form, and 3) in vitro culture, maturation and direct visualization of cortical granules are each possible. Cortical granules are different from most other secretory vesicles in that they remain docked at the cell surface for weeks, they are non recycling, and they contain over a dozen different proteins which are subcompartmentalized within the vesicle. Three specific aims are proposed: 1. We will identify determinants on the content proteins of cortical granule that are required for targeting into cortical granules. We will utilize cDNAs to the cortical granule specific proteins SFE 9, ovoperoxidase and hyalin in a protein reporter system to determine what minimal sequence is necessary for appropriate organelle targeting. Because the vesicle contains two morphologically and biochemically distinct regions, we will also examine the signals necessary for protein subcompartmentalization within the vesicle using proteins specifically compartmentalized in each region. 2. We will examine the mechanism and regulation of cortical granule translocation to the oocyte cell surface. We will determine the basis for selectivity of this process, i.e. selectivity for cortical granules, for their timing of translocation, and for the pathways used in organelle movement. In addition, we will examine the mechanism used to create a perfect monolayer of cortical granules at the oocyte cell surface. 3. We will identify the mechanism(s) of regulated secretion of cortical granules at fertilization. We will test the hypothesis that cortical granules contain a unique population of membrane proteins to direct the specialized biology of this vesicle by use of monoclonal antibodies against cortical granule membranes and by testing homologues of somatic cell proteins that are involved in regulated secretory function.