BIOLOGY OF CORTICAL GRANULES

皮质颗粒是卵母细胞独有的，在受精过程中发挥作用。 通过分泌它们的内容形成永久的块来阻止多精受精。我们 将研究皮质颗粒生物学有两大目标：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)了解它们的调控 受精时的分泌物。这些目标所选择的卵母细胞来自 海胆，在那里大约有15,000个皮质颗粒 在细胞表面排出胞液以响应两种精子的结合。它 是唯一一个分离到L)cdna克隆的卵母细胞 编码皮质颗粒特有的内容物和膜蛋白； 2)皮质颗粒可以以功能形式分离，3)在 皮质颗粒的体外培养、成熟及直观观察 每一个都是可能的。皮质颗粒与大多数其他颗粒不同 分泌小泡在细胞表面保持停靠状态 几个星期，它们是非回收的，它们包含十几种不同的 囊泡内细分的蛋白质。三 提出了具体目标： 1.确定大脑皮层蛋白质含量的决定因素 定位为皮质颗粒所需的颗粒。我们会 利用cDNA对皮质颗粒特异蛋白SFE 9进行编码， 卵细胞过氧化物酶和透明质酸在蛋白质报告系统中确定 最小的序列是适当的细胞器靶向所必需的。 因为小泡在形态上和生化上包含两个 不同的区域，我们还将研究蛋白质所需的信号 利用特定蛋白质在囊泡内进行亚室隔化 在每个地区被隔开。 2.我们将研究皮质颗粒的机制和调节 移位到卵母细胞表面。我们将确定以下基础： 这一过程的选择性，即对皮质颗粒的选择性 它们的转位时间，以及细胞器中使用的途径 有动静。此外，我们还将研究用于创建 卵母细胞表面有一层完整的皮质颗粒。 3.明确皮质醇调节分泌的机制(S) 受精时的颗粒。我们将检验这一假设，即大脑皮质 颗粒含有一组独特的膜蛋白，可以引导 利用单抗对该小泡的特殊生物学研究 抗皮质颗粒膜和通过检测体细胞的同系物 参与调节分泌功能的细胞蛋白质。

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.