PKC--CYTOPLASMIC REGULATOR OF EARLY DEVELOPMENT

理解生殖的生物学需要理解 发生在关键发育转变阶段的过程，如减数分裂 恢复、受精和原肠形成。其中最 这些过程中广泛保守的方面是动态重构 蜂窝架构。事实上，细胞架构的戏剧性变化 几乎所有发育过程都是固有的，既正常(例如， 受精、器官发生和细胞分化)和病理学 (例如，肿瘤形成、转移和畸胎)。通过这种方式 然而，细胞结构的变化是精心策划的，效果很差。 理解，尽管这样的理解将产生以下好处 治疗出生缺陷和癌症。中国的长期目标是 建议的研究是描述生物化学机制，通过它 蛋白激酶C(PKC)调节伴随的细胞结构变化 早期的两栖动物发育。PKC，一种细胞内的信号酶， 已经确定与人类和其他人的肿瘤发生有关 因此，对其在两栖动物发育中的作用的阐明是 可能具有广泛的关联性。两栖系统也有重要的 相对于其他系统的优势。第一，广泛的文献资料 结构变化的时间和细胞内的信号 因此，伴随着早期两栖动物发育的事件，可能 两者之间的关联很容易辨别出来。第二，大的 两栖类卵母细胞的大小和从单个卵母细胞获得的大量 动物实验使该系统在生物化学和生物化学方面非常实用。 超微结构研究。拟议的研究将利用这些 确定涉及的生化和分子机制的优势 非洲爪哇早期发育过程中PKC介导的事件。这个 本提案中描述的实验旨在回答 以下问题：1)PKC是否介导细胞质的重要变化 减数分裂成熟和卵子恢复过程中的结构 激活？2)PKC的内源性底物是什么？在 不受干扰的发展它们被磷酸化了吗？3)什么是 PKC底物的细胞内位置？4)在不受干扰的情况下 发展，是否会发生潜在的PKC激活事件？5)什么是 内源性PKC激活物的时空分布和 抑制剂？6)肌球蛋白或肌球蛋白轻链的磷酸化 PKC(MLC)介导肌球蛋白向皮质网络的募集 诱导皮质收缩？7)肌球蛋白或MLC的磷酸化 PKC调控收缩环的形成？这些问题 包括细胞的生物化学和机械细胞学方面 结构，因此应该能提供对 一种特定的细胞内信号分子PKC， 调节细胞结构的变化。

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.