Cell cycle regulation of development

* *我的实验室研究通过抑制Cdk1来调节细胞周期时间的检查点机制。cdk1是有丝分裂和减数分裂的主要调节因子，这两种不同的方法是细胞在不同的发育阶段进行增殖的。我们正在使用果蝇（果蝇）突变体和荧光报告在我的实验室创建分析两个重要的Cdk1抑制剂，称为Myt 1和Wee 1的功能。Cdk1的Myt1调节对于在雄性减数分裂期间保留某些重要的细胞结构是必不可少的，减数分裂是细胞周期被修改以产生精子的过程。Wee1对Cdk1的调节对于减缓快速发育胚胎的S期是必不可少的，S期是DNA复制的细胞周期阶段。我们的目标是通过以下实验方法更好地了解Myt 1和Wee 1的这些专门的发育功能。* 目的和短期目标：* 1表征Myt 1机制，该机制将内膜重塑与中心粒接合联系起来： * Myt1激酶是膜结合的Cdk1抑制剂，约7.5亿年前在多细胞生物中进化。我们正在研究雄性不育myt1突变体，以确定它们在减数分裂中的作用。果蝇精母细胞通常在减数分裂前的G2期停滞4天。在这种发育停滞过程中，Cdk1的Myt 1抑制可以防止可能导致异常减数分裂细胞分裂和不育的细胞缺陷。使用新的荧光报告和生化检测，我们将Myt功能在体内，通过寻找与Myt 1相互作用的因素，并调查机制，是重要的本地化和调节的Myt 1活性在减数分裂。我们的研究结果将为所有多细胞生物的有性生殖机制提供新的见解。2.描述防止有丝分裂灾难的Wee1胚胎检查点机制： * Wee1激酶对于防止果蝇和哺乳动物胚胎中的有丝分裂灾难至关重要。Wee1通过称为磷酸化的可逆机制抑制Cdk1。Cdk1抑制磷酸化在早期胚胎中很难检测到，这表明Wee1通过一种新的机制调节Cdk1。我们假设Wee 1通过破坏其与称为细胞周期蛋白的必要辅因子的相互作用的稳定性来控制Cdk 1活性。为了验证这一观点，我们将分析Cdk1的突变体和正常形式如何在对照和wee1突变体胚胎中与细胞周期蛋白相互作用。我们还将确定机制，是重要的调节Wee1的本地化和活动，使用遗传筛选，以确定影响转基因Wee1报告突变体。我们的研究结果将为Wee1控制快速胚胎细胞周期的机制提供见解 **

*** *** *** *** *** *** ***SUMMARY OF THE PROPOSAL******Long-term Objectives:***The complex process of animal development requires precise timing of the cell cycle. My laboratory studies checkpoint mechanisms that regulate cell cycle timing by inhibiting Cdk1. Cdk1 is the major regulator of mitosis and meiosis, two distinct methods that cells use to proliferate at different stages of development. We are using Drosophila (fruit fly) mutants and fluorescent reporters created in my laboratory to analyze the functions of two important Cdk1 inhibitors, called Myt1 and Wee1. Myt1 regulation of Cdk1 is essential for preserving certain important cellular structures during male meiosis, a process where the cell cycle is modified to produce sperm. Wee1 regulation of Cdk1 is essential for slowing down S phase in rapidly developing embryos, a cell cycle stage when the DNA is replicated. Our goal is to better understand these specialized developmental functions of Myt1 and Wee1 by undertaking the following experimental approaches. ***Aims and Short-term Objectives:*** 1 Characterize Myt1 mechanisms that link endomembrane remodeling with centriole engagement: ***Myt1 kinases are membrane-bound Cdk1 inhibitors that evolved in multicellular organisms ~750 million years ago. We are studying male-sterile myt1 mutants to determine their role in meiosis. Drosophila spermatocytes normally arrest for four days in pre-meiotic G2 phase. During this developmental arrest, Myt1 inhibition of Cdk1 prevents cellular defects that can result in aberrant meiotic cell division and sterility. Using new fluorescent reporters and biochemical assays we will characterize Myt functions in vivo, by searching for factors that interact with Myt1 and investigating mechanisms that are important for localization and regulation of Myt1 activity during meiosis. Our results will provide new insights into mechanisms required for sexual reproduction in all multicellular organisms.***2. Characterize Wee1 embryonic checkpoint mechanisms that prevent mitotic catastrophe: ***Wee1 kinases are essential for preventing mitotic catastrophe in both Drosophila and mammalian embryos. Wee1 inhibits Cdk1 by a reversible mechanism called phosphorylation. Cdk1 inhibitory phosphorylation is extremely difficult to detect in early embryos however, suggesting that Wee1 regulates Cdk1 by a novel mechanism. We hypothesize that Wee1 controls Cdk1 activity by de-stabilizing its interactions with essential co-factors called Cyclins. To test this idea we will analyze how mutant and normal forms of Cdk1 interact with Cyclins in both control and wee1 mutant embryos. We will also identify mechanisms that are important for regulating Wee1 localization and activity using genetic screens to identify mutants affecting a transgenic Wee1 reporter. Our results will provide insights into Wee1 mechanisms that control rapid embryonic cell cycles********