PKC--CYTOPLASMIC REGULATOR OF EARLY DEVELOPMENT

了解生殖生物学需要了解 发生在关键的发育转变过程，如减数分裂， 恢复受精和原肠胚形成 一个最 这些过程的广泛保守的方面是动态重塑， 细胞结构 事实上，细胞结构的巨大变化 本质上所有的发展过程，无论是正常的（例如， 受精、器官发生和细胞分化）和病理学 (e.g.瘤形成、转移和致畸）。 这种方法， 细胞结构的变化是精心策划的，然而， 理解，尽管这种理解会产生好处， 出生缺陷和癌症的治疗。 的长期目标 拟议的研究是描述生物化学机制， 蛋白激酶C（PKC）调节细胞结构的变化， 早期两栖动物的发展 PKC是一种细胞内信号酶， 与人类和其他动物的肿瘤发生密切相关。 生物，因此，阐明其在两栖动物发展中的作用， 可能具有广泛的相关性。 两栖动物系统也有重要的 优于其他系统。 首先，广泛的文献资料 结构变化和细胞内信号传导的时间 事件伴随着早期两栖动物的发展，因此，潜在的 两者之间的相关性可以容易地辨别。 第二，大 两栖类卵母细胞的大小和从单个卵母细胞中获得的大量卵母细胞， 动物使该系统非常实用的生化和 超微结构研究 拟议的研究将利用这些 优势，以确定涉及的生化和分子机制， 非洲爪蟾早期发育过程中PKC介导的事件。 的 本提案中描述的实验旨在回答 以下问题：1）PKC是否介导细胞质的关键变化 在减数分裂成熟和卵的恢复过程中的结构 激活？2)PKC的内源性底物是什么？ 它们被磷酸化了吗？3)有哪些 PKC底物的细胞内定位？4)当在不受干扰的情况下， 发展，是否发生潜在的PKC激活事件？5)有哪些 内源性PKC激活剂的时空分布， 抑制剂？6)肌球蛋白或肌球蛋白轻链的磷酸化 (MLC)通过PKC介导肌球蛋白向皮质网络的募集， 引起皮层收缩？7)肌球蛋白或MLC的磷酸化是否 PKC调节收缩环的形成？这些问题 包括细胞的生物化学和机械细胞学方面 结构，因此应该提供相当深入的了解 一种特定的细胞内信号分子，PKC， 介导细胞结构的变化。

Understanding the biology of reproduction requires understanding of processes which occur at key developmental transitions such as meiotic resumption, fertilization, and gastrulation. One of the most widely-conserved aspects of these processes are dynamic remodelings of cellular architecture. Indeed, dramatic changes in cell architecture are intrinsic to virtually all developmental processes, both normal (eg., fertilization, organogenesis, and cell differentiation) and pathological (e.g. neoplasia, metastasis, and teratogenesis). The means by which such changes in cellular structure are orchestrated, however, are poorly understood, in spite of the benefits such understanding would yield to treatment of birth defects and cancer. The long term goal of the proposed research is to characterize the biochemical mechanisms by which protein kinase C (PKC) regulates changes of cell structure that accompany early amphibian development. PKC, an intracellular signalling enzyme, has been firmly implicated in tumorigenesis in humans and other organisms, thus, elucidation of its role in amphibian development is likely to have broad relevance. The amphibian system also has important advantages over other systems. First, an extensive literature documents the timing of the architectural changes and the intracellular signalling events which accompany early amphibian development, therefore, potential correlations between the two can be readily discerned. Second, the large size of amphibian oocytes and the large quantity obtainable from a single animal makes the system very practical for biochemical and ultrastructural studies. The proposed studies will exploit these advantages to define biochemical and molecular mechanisms involved in PKC-mediated events during the early development of Xenopus laevis. The experiments described in this proposal are designed to answer the following questions: 1) Does PKC mediate crucial changes in cytoplasmic architecture during the resumption of meiotic maturation and egg activation? 2) What are the endogenous substrates of PKC and when during unperturbed development are they phosphorylated? 3) What are the intracellular locations of PKC substrates? 4) When, during unperturbed development, do potential PKC activating events occur? 5) What are the spatial and temporal distributions of endogenous PKC activators and inhibitors? 6) Does phosphorylation of myosin or myosin light chains (MLC) by PKC mediate recruitment of myosin into the cortical network and induce cortical contraction? 7) Does phosphorylation of myosin or MLC by PKC regulate the formulation of the contractile ring? These questions encompass both biochemical and mechanistic cytological aspects of cell structure and should therefore provide considerable insight into the mechanisms by which a specific intracellular signalling molecule, PKC, mediates changes in cell structure.