Cell cycle regulation of development

长期目标： 动物发育的复杂过程需要精确的细胞周期时间。我的实验室研究通过抑制CDK1来调节细胞周期计时的检查点机制。CDK1是有丝分裂和减数分裂的主要调节者，这是细胞在不同发育阶段用于增殖的两种不同方式。我们正在使用果蝇(果蝇)突变体和我实验室创造的荧光记者来分析两种重要的CDK1抑制剂Myt1和Wee1的功能。Myt1对CDK1的调节对于在雄性减数分裂过程中保存某些重要的细胞结构是必不可少的，这是一个细胞周期被修改以产生精子的过程。Wee1对CDK1的调控对于减缓快速发育的胚胎的S期至关重要，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Myt1和Wee1的这些专门的发育功能。 目标和短期目标： 1描述将内膜重塑与中心粒结合联系起来的Myt1机制： Myt1激酶是一种膜结合的CDK1抑制剂，大约7.5亿年前在多细胞生物体中进化而来。我们正在研究雄性不育的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G2期停滞4天。在这种发育停滞过程中，Myt1对CDK1的抑制可以防止细胞缺陷，这种缺陷可能导致异常的减数分裂和不育。通过寻找与Myt1相互作用的因子，并研究在减数分裂过程中Myt1活性定位和调节的重要机制，我们将使用新的荧光报告程序和生化分析来表征MYT在体内的功能。我们的结果将为所有多细胞生物体有性繁殖所需的机制提供新的见解。 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是必不可少的。Wee1通过一种称为磷酸化的可逆机制抑制CDK1。然而，在早期胚胎中很难检测到CDK1抑制磷酸化，这表明Wee1通过一种新的机制调节CDK1。我们假设Wee1通过破坏其与被称为细胞周期蛋白的基本辅助因子的相互作用来控制CDK1的活性。为了测试这一想法，我们将分析在对照和Wee1突变胚胎中，突变和正常形式的CDK1是如何与细胞周期蛋白相互作用的。我们还将通过基因筛选来识别影响转基因Wee1报告基因的突变体，从而确定对调节Wee1定位和活性至关重要的机制。我们的结果将为控制快速胚胎细胞周期的Wee1机制提供见解

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