Regulation of Quiescence in Eukaryotic Cells

真核细胞静止的调节

• 批准号: 8727085
• 负责人: David Gresham
• 金额: $ 29.25万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8727085
• 关键词: Antineoplastic Agents; Biological Assay; Candida albicans; Carbon; Cell Cycle; Cell division; Cell physiology; Cells; Complement; Computing Methodologies; Conflict (Psychology); Coupled; Cryptococcus neoformans; Cyclic AMP-Dependent Protein Kinases; Defect; Development; Diabetes Mellitus; Eukaryota; Eukaryotic Cell; Failure; Fission Yeast; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genetic Programming; Goals; Growth; High-Throughput Nucleotide Sequencing; Human; Image; Infection; Inflammation; Institutes; Label; Laboratories; Libraries; Malignant Neoplasms; Maps; Measures; Messenger RNA; Metabolic; Methods; Microbe; Mitosis; Modeling; New York; Nitrogen; Nutrient; Organism; Pathway interactions; Phosphorus; Physiological; Play; Post-Transcriptional Regulation; Process; Production; Protein Kinase; Proteins; RNA; Recurrence; Regulation; Resistance; Role; Saccharomyces cerevisiae; Saccharomycetales; Signal Pathway; Signal Transduction; Starvation; Testing; Transcript; Translations; Universities; Variant; Yeast Model System; base; cell growth; chemotherapy; clinically relevant; drug standard; experience; gene interaction; genetic analysis; genome-wide; genome-wide analysis; human disease; in vivo; insight; loss of function mutation; mRNA Stability; mRNA Transcript Degradation; mathematical sciences; microbial; mutant; neoplastic cell; novel therapeutics; pathogen; programs; public health relevance; ras Proteins; research study; response; tool; transcriptome sequencing; tumor; tumor microenvironment

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.

描述（由申请人提供）： 受调节的细胞分裂退出和非增殖性静止状态的启动是所有生物体的关键要求。不能维持静止和不适当的增殖细胞生长的重新启动是许多人类癌症的基础。相反，静止肿瘤细胞的亚群可能在耐化疗和肿瘤复发中起关键作用，因为癌症药物通常靶向细胞生长期间的活性过程。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（芽殖酵母）和粟酒裂殖酵母（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进入静止状态以响应营养耗尽，并且能够在长时间的营养饥饿中存活。我们的初步研究表明，启动静止响应定义的营养饥饿积极调节保守的信号通路，包括TORC 1，Ras/蛋白激酶A（PKA）和AMPK途径。在目标1中，我们将定义保守的遗传程序，控制细胞静止，通过量化缺陷的静止可归因于在三个静止诱导条件：碳，氮和磷饥饿的芽殖和裂殖酵母中的每个基因的功能突变的损失。我们将补充这一遗传学方法与野生型和突变细胞的静止的表型标志的研究，以确定在静止突变体缺陷的过程。在目标2中，我们将研究信号通路如何整合环境信息，通过在两个物种的静止条件下使用全基因组遗传相互作用映射确定静止调节通路的靶点和通路之间的相互作用，启动静止程序。这些实验将使我们能够确定保守的功能相互作用，使细胞能够启动静止响应特定的前静止信号，同时接收促生长信号，激活平行的途径。我们假设，协调信号通路的一种手段是通过动态的亚细胞定位的组件，我们将测试这一假设使用突变体中的信号组件被错误定位。在目标3中，我们将使用与RNA-Seq偶联的mRNA的体内代谢标记来定量mRNA合成和降解速率的变化，因为细胞进入静止期。我们将使用这种方法来测试细胞是否会改变特定转录物的稳定性，因为细胞生长放缓，进入静止期。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。通过关注保守的信号通路和调节静止的细胞过程，我们将提高我们对正常和患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将最终实现新的治疗策略，这些策略专门针对各种病理环境中的静止细胞，包括癌症和微生物感染。