Evolutionary adaptation and spatial organization of signaling in the Mitotic Exit Network

项目摘要 通过信号级联来感知和处理信息是细胞生命的重要组成部分。几个 在真核生物中，MAP激酶和河马通路等信号级联信号仍然普遍存在。 在不同的生物体中执行不同的功能。尽管人们对这些途径进行了充分的研究，但它们是如何进化到 承担新的职能和适应新的投入仍然知之甚少。有丝分裂退出网络(MEN)，a Ras样GTPase信号级联和河马途径的酵母同源物，提供了一个独特的机会 研究这个问题。在男性中，相同的核心信号组件以不同的方式在 不同的发展轨迹。在酵母有丝分裂期间，通过不对称的细胞分裂发生 这些人被称为萌芽，被固定在纺锤体的极体上(SPB，相当于酵母的中心体)。 并通过其GTP酶TEM1对纺锤体位置做出反应。在减数分裂期间，萌芽被抑制， 因此，不需要感知心轴位置，SPBS不再组织男性信号，而且不清楚 男性激活是否仍然需要TEM1，以及它可能对什么信号做出反应。要了解 男性在不同细胞环境下的适应，这一提议将检验这种适应的假设 部分由MEN激酶CDC15，Tem1的效应蛋白激酶， 有丝分裂和减数分裂之间(目标1)。与男人核心的空间组织的剧烈变化形成对比的是 在有丝分裂和减数分裂之间的成分，男性的效应蛋白，磷酸酶cdc14，保留下来。 在有丝分裂和减数分裂中，核仁在激活之前被隔离。事实上，核仁的这种定位 CDC14从酵母到人类都是保守的。Cdc14在核仁中的隔离作用可以是1)到 确保在激活前严密抑制镉镉14‘S磷酸酶活性或2)将镉镉14定位到 使核仁中的特定底物去磷酸化。为了揭示维持这一点的选择压力 Cdc14的保守核仁定位，这一建议将通过隔离来检验这两个假说。 Cdc14在细胞中的其他位置，并首先在酵母中，然后在哺乳动物细胞中描述其后果。 (目标2)。两个AIMS内的实验将在K99阶段启动，该阶段还包括培训 新的实验系统的候选人，如酵母减数分裂和哺乳动物细胞，以及 发展和实施定量显微镜、邻近标记和光遗传学。此外， 候选人已经组建了一支出色的导师团队，为她提供科学建议，以促进进步 并为她过渡到独立调查员做好准备。总而言之，这项提议将 为了解进化/适应的独立研究生涯奠定坚实的基础 细胞信号的空间组织。

Project Summary Sensing and processing information through signaling cascades is an essential part of cellular life. A few signaling cascades such as the MAP kinase and Hippo pathways are ubiquitous among eukaryotes yet perform different functions across organisms. Although these pathways are well-studied, how they evolve to take on new functions and adapt to new inputs remains poorly understood. The Mitotic Exit Network (MEN), a Ras-like GTPase signaling cascade and yeast homolog of the Hippo pathway, provides a unique opportunity to study this question. In the MEN, the same core signaling components operate in distinct manners under different developmental trajectories. During yeast mitosis which occurs through an asymmetric cell division called budding, the MEN is scaffolded onto the spindle pole bodies (SPB, the yeast equivalent of centrosomes) and responds to spindle position through its GTPase Tem1. During meiosis, where budding is suppressed and thus no need to sense spindle position, MEN signaling is no longer organized at the SPBs, and it is unclear whether Tem1 is still required for MEN activation and what signal it may respond to. To understand the adaptation of the MEN under distinct cellular contexts, this proposal will test the hypothesis that this adaptation is enabled partially by different activation mechanisms of the MEN kinase Cdc15, the effector kinase of Tem1, between mitosis and meiosis (Aim 1). In contrast to the drastic change in spatial organization of the MEN core components between mitosis and meiosis, the effector protein of the MEN, the phosphatase Cdc14, remains sequestered in the nucleolus prior to activation both in mitosis and meiosis. In fact, this nucleolar localization of Cdc14 is conserved from yeast to human. Sequestration of Cdc14 in the nucleolus could function either 1) to ensure tight inhibition of Cdc14’s phosphatase activity prior to activation or 2) to localize Cdc14 to dephosphorylate specific substrates in the nucleolus. To uncover the selection pressure that maintains this conserved nucleolar localization of Cdc14, this proposal will examine these two hypotheses by sequestering Cdc14 elsewhere in the cell and characterize the consequences first in yeast and then in mammalian cells (Aim 2). The experiments within both aims will be initiated during the K99 phase which also includes training of the candidate on new experimental systems such as yeast meiosis and mammalian cells, as well as the development and implementation of quantitative microscopy, proximity labeling and optogenetics. Furthermore, the candidate has assembled an outstanding mentor team to both advise her scientifically to facilitate progress of the project and prepare her for the transition to an independent investigator. Together, this proposal will create a strong foundation for an independent research career in understanding the evolution/adaptation and spatial organization of cellular signaling.