Biogenesis and Function of Cortical Granule Contents

皮质颗粒是卵和卵母细胞特有的分泌囊泡。在受精时，它们分泌其内容物以形成对多精受精的永久阻断并为早期胚胎发育提供保护。在这个应用程序中，我们将解决三个问题：什么是在皮质颗粒？，它能做什么？它是怎么进去的我们将使用海胆卵和卵母细胞来回答这些问题，因为在这种动物中，我们可以获得大约106个卵和1个卵母细胞/雌性，并且因为每个卵母细胞中的大约15，000个皮质颗粒在生物发生、易位到表面、对接到质膜以及响应精子或孤雌激活的分泌中是同步的。该系统为研究皮层颗粒生物发生和功能的分子机制提供了一个独特的机会。海胆卵母细胞也是唯一的卵母细胞，其中1）cDNA克隆已被分离，编码内容物和膜蛋白特异性的皮质颗粒; 2）皮质颗粒可以分离的功能形式;和3）在体外培养和成熟的卵母细胞和直接可视化的皮质颗粒是可能的。除了囊泡生物发生步骤的同步性之外，皮质颗粒与大多数其他分泌囊泡的不同之处在于它们是不可回收的，并且含有十几种不同的蛋白质，这些蛋白质是皮质颗粒特有的，并且在囊泡内亚区室化。提出了三个具体目标：1。识别皮质颗粒的内容物。我们将集中在三个受精被膜蛋白：proteohaisin，p90和p63的cDNA克隆。这些是主要的包膜蛋白，必须迅速相互作用，并与卵黄层形成一个不可穿透的层在受精后几秒钟内。描述皮质颗粒蛋白的功能和调节。我们将确定受精包膜结构的机制，一个专门的细胞外基质，通过鉴定皮质颗粒蛋白相互作用的领域，负责包膜的建设。然后，我们将使用这些信息来检查层次结构，并与卵黄层的相互作用的身份，以了解快速浓缩的受精卵的分子机制。确定内容物是如何选择性地包装成皮质颗粒的。我们将利用新鉴定的cDNA克隆来确定皮质颗粒生物发生的机制。选择性蛋白质靶向皮质颗粒将使用重组，标记皮质颗粒蛋白质进行研究。标签将包括myc表位和绿色荧光蛋白，我们将遵循野生型和修改皮质颗粒蛋白序列在皮质颗粒生物发生的命运。我们还将使用这些标记的蛋白质，结合生物素化的氨基酸标记，以测定皮质颗粒生物发生。由于我们最近在海胆卵母细胞体外成熟方面取得的成功，我们也将能够在体内研究皮质颗粒蛋白的功能。

Cortical granules are secretory vesicles unique to eggs and oocytes. At fertilization they secrete their contents to form both a permanent block to polyspermy and to provide protection for early embryonic development. In this application we will address three questions: What is in the cortical granule?, What does it do?, and How does it get in there? We will use sea urchin eggs and oocytes to answer these questions because in this animal we can obtain approximately 106 eggs and 1 ~ oocytes per female, and because the approximately 15,000 cortical granules in each oocyte are synchronous in biogenesis, in translocation to the surface, in docking to the plasma membrane, and in secretion in response to sperm or parthenogenic activation. This system offers a unique opportunity to examine the molecular mechanism of cortical granule biogenesis and flinction. The sea urchin oocyte is also the only oocyte in which 1) 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 fimctional form; and 3) in vitro culture and maturation of oocytes and direct visualization of cortical granules is possible. In addition to the synchrony of vesicle biogenic steps, cortical granules are different from most other secretory vesicles in that they are nonrecycling, and contain over a dozen different proteins that are specific to cortical granules and are subcompartmentalized within the vesicle. Three specific aims are proposed:1. Identify the contents of the cortical granules. We will focus on the cDNA cloning of the three fertilization envelope proteins: proteohaisin, p90 and p63. These are major envelope proteins that must quickly interact with each other and with the vitelline layer to form an impenetrable layer within seconds of fertilization.2. Characterize the function and regulation of the cortical granule proteins. We will determine the mechanism of fertilization envelope construction, a specialized extracellular matrix, by identiiying domains of cortical granule protein interactions that are responsible for envelope construction. We will then use this information to examine the hierarchy, and identity of interactions with the vitelline layer to understand the molecular mechanism for the rapid condensation of the fertilization envelope.3. Determine how the contents are packaged selectively into cortical granules. We will make use of the newly identified cDNA clones to determine the mechanism of cortical granule biogenesis. Selective protein targeting into cortical granules will be studied using recombinant, tagged cortical granule proteins. The tags will include the myc epitope and the green fluorescent protein, and we will follow the fates of wild-type and modified cortical granule protein sequences during cortical granule biogenesis. We will also use these tagged proteins, in conjunction with biotinylated amino acid markers, to assay cortical granule biogenesis. Because of our recent success with in vitro maturation of sea urchin oocytes, we will also be able to study cortical granule protein function in vivo.