Regulation of Quiescence in Eukaryotic Cells

描述(由申请人提供)： 有规律地退出细胞分裂和启动非增殖性静止状态是所有生物体的关键要求。未能保持增殖细胞生长的静止和不适当的重新启动是许多人类癌症的基础。相反，静止的肿瘤细胞亚群可能在化疗抵抗和肿瘤复发中发挥关键作用，因为抗癌药物通常针对细胞生长过程中活跃的过程。同样，静止的病原微生物通常对标准药物治疗不敏感。我们将使用单细胞真核微生物酿酒酵母(芽生酵母)和裂殖酵母(裂殖酵母)来鉴定调节细胞静止的保守网络。微生物和一些肿瘤细胞在营养耗尽后进入静止状态，能够在长时间的营养饥饿中存活下来。我们的初步研究表明，在特定的营养饥饿反应中，沉默的启动受到保守的信号通路的积极调控，包括TORC1、RAS/蛋白激酶A(PKA)和AMPK通路。在目标1中，我们将定义控制细胞静止的保守遗传程序，通过量化在三种静止诱导条件下芽酵母和分裂酵母中每个基因的功能突变导致的静止缺陷：碳、氮和磷饥饿。我们将通过研究野生型和突变型细胞中静止的表型特征来补充这种遗传方法，以识别静止突变体中有缺陷的过程。在目标2中，我们将研究信号通路如何整合环境信息来启动静止程序，方法是利用两个物种在静止条件下的全基因组遗传作图来识别静止调节通路的靶标和途径之间的相互作用。这些实验将使我们能够识别保守的功能相互作用，使细胞能够对特定的静止期信号做出反应，同时接收激活平行通路的促生长信号。我们假设，协调信号通路的一种方法是通过其组件的动态亚细胞定位，我们将使用信号组件被错误定位的突变体来验证这一假设。在目标3中，我们将使用与RNA-Seq结合的mRNAs的体内代谢标记来量化细胞进入静止状态时mRNA合成和降解率的变化。我们将使用这种方法来测试当细胞生长放缓并进入静止状态时，细胞是否会改变特定转录本的稳定性。然后，我们将使用计算方法确定mRNA降解变异的保守决定因素。通过重点研究调节静止的保守信号通路和细胞过程，我们将加强我们对正常和疾病人类细胞以及微生物病原体中静止的理解。对细胞静止的详细了解最终将使新的治疗策略能够特别针对包括癌症和微生物感染在内的各种病理环境中的静止细胞。

DESCRIPTION (provided by applicant): Regulated exit from cell division and initiation of a non-proliferative quiescent state is a criticl requirement in all organisms. Failure to maintain quiescence and inappropriate reinitiation of proliferative cell growth underlies many human cancers. Conversely, subpopulations of quiescent tumor cells may play critical roles in resistance to chemotherapy and tumor recurrence as cancer drugs typically target processes active during cell growth. Similarly, quiescent pathogenic microbes are frequently insensitive to standard drug treatments. We will use the single-celled eukaryotic microbes, Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) to identify the conserved networks that regulate cell quiescence. Microbes and some tumor cells enter quiescent states in response to nutrient depletion and are able to survive for prolonged periods of nutrient starvation. Our preliminary studies demonstrate that initiation of quiescence in response to defined nutrient starvation is actively regulated by conserved signaling pathways including the TORC1, Ras/Protein kinase A (PKA) and AMPK pathways. In Aim 1 we will define the conserved genetic program that controls cell quiescence by quantifying the defect in quiescence attributable to loss of function mutations in each gene in both budding and fission yeast in three quiescence-inducing conditions: carbon, nitrogen and phosphorous starvation. We will complement this genetic approach with studies of the phenotypic hallmarks of quiescence in wildtype and mutant cells to identify processes defective in quiescent mutants. In Aim 2 we will study how signaling pathways integrate environmental information to initiate the quiescence program by identifying targets of quiescence-regulating pathways and interactions between pathways using genome-wide genetic interaction mapping in quiescent conditions in both species. These experiments will allow us to identify conserved functional interactions that enable the cell to initiate quiescence n response to specific pro-quiescence signals while simultaneously receiving pro-growth signals that activate parallel pathways. We hypothesize that one means of coordinating signaling pathways is by dynamic subcellular localization of their components and we will test this hypothesis using mutants in which signaling components are mislocalized. In Aim 3 we will quantify variation in mRNA synthesis and degradation rates as cells enter quiescence using in vivo metabolic labeling of mRNAs coupled with RNA-Seq. We will use this method to test whether cells alter the stability of specific transcripts as cell growth slows and they enter quiescence. We will then identify conserved determinants of mRNA degradation variation using computational methods. By focusing on conserved signaling pathways and cellular processes that regulate quiescence we will enhance our understanding of quiescence in both normal and diseased human cells as well as microbial pathogens. A detailed understanding of cell quiescence will ultimately enable new therapeutic strategies that specifically target quiescent cells in a variety of pathological settings including cancer and microbial infections.