Regulation of blast cell quiescence by Pten and Tor

项目摘要/摘要 静止状态是干细胞的一个决定性特征，这使它们能够在一段时间内持续存在而不会丢失 发展潜力。干细胞和其他多能细胞在休眠静止期之间交替 以及细胞分裂和分化的活跃期。然而，我们对这些转变的理解是 监管是不完整的。该项目的长期目标是研究如何在 静止和分化受磷酸酶和张力蛋白同源基因(Pten)和靶基因的调控 雷帕霉素(Tor)，使用线虫线虫。在线虫中，在一个特殊的幼虫阶段 Dauer说，性腺中的多能母细胞受到来自环境的信号的指示，保持静止。 这些性腺母细胞的静止状态的丧失或维持可以很容易地用荧光检测出来。 记号笔。 我们的实验室最近发现线虫Pten的同源基因(DAF-18)调节原始细胞的静止 在达尔幼虫的性腺中。肿瘤抑制基因Pten是哺乳动物发育所必需的基因， 而Pten的缺失会导致许多不同类型的癌症。PTEN还调节成年哺乳动物的安静 干细胞和癌症干细胞，但我们对Pten是如何做到这一点缺乏了解。宁静通常是 在活体内研究非常困难，我们的性腺发生模型为在生活中研究它提供了一个独特的机会， 完整的有机体，拥有无与伦比的遗传分析工具。这项研究的目标是了解， 从机制上讲，使用我们的性腺发生模型，Pten促进干细胞静止。 PTEN/DAF-18具有多种分子活性：它是一种蛋白质磷酸酶，一种脂肪磷酸酶，还可以 充当组装蛋白质复合体的‘脚手架’。在目标1中，将使用结构/功能研究来评估 在我们的系统中，DAF-18‘S对母细胞静止的调节需要这些活动中的哪一个。另外， 为了进一步了解DAF-18‘S调节安静的生物活性，我们对其亚细胞定位进行了研究。 性腺中的内源性DAF-18蛋白将被表征。目标2将使用组织特异性耗竭 在初步实验的基础上，验证这一假设的实验，即DAF-18可能促进细胞 通过对抗促进生长的Tor激酶途径的活性而静止。在Aim 3中，强大的基因 筛选将被用来识别可能与DAF-18一起调节遗传途径的新基因 宁静。这项计划中的实验将在活体中加入荧光成像，并 尖端基因技术。该培训将在哥伦比亚大学进行，为期两年 多年来，在伊娃·格林沃尔德博士的指导下，伊娃·格林沃尔德博士是一位著名的线虫遗传学家，在 培养成功的研究人员。

Project Summary/Abstract Quiescence is a defining feature of stem cells, which allows them to persist over time without losing developmental potential. Stem cells and other multipotent cells alternate between dormant quiescent periods and active periods of cell division and differentiation. However, our understanding of how these transitions are regulated is incomplete. The long-term objective of this project is to investigate how transitions between quiescence and differentiation are regulated by the genes Phosphatase and tensin homolog (Pten) and Target of Rapamycin (Tor), using the nematode worm C. elegans. In C. elegans, during a special larval stage called dauer, multipotent blast cells in the gonad are directed by signals from the environment to remain quiescent. The loss or maintenance of quiescence in these gonad blasts can easily be detected using fluorescent markers. Our laboratory recently showed that the C. elegans ortholog of Pten (DAF-18) regulates blast cell quiescence in the gonad of dauer larvae. The tumor suppressor Pten is an essential gene for mammalian development, and loss of Pten can cause many different types of cancer. Pten also regulates quiescence in adult mammalian stem cells and cancer stem cells, but we lack an understanding of how Pten does this. Quiescence is usually very difficult to study in vivo, and our gonadogenesis model provides a unique opportunity to study it in a living, intact organism, with unparalleled tools for genetic analysis. The goal of this research is to understand how, mechanistically, Pten promotes stem cell quiescence, using our gonadogenesis model. Pten/DAF-18 has multiple molecular activities: it is a protein phosphatase, a lipid phosphatase, and can also act as a ‘scaffold’ to assemble protein complexes. In Aim 1, structure/function studies will be used to assess which of these activities is required for DAF-18’s regulation of blast cell quiescence in our system. Additionally, to further understand DAF-18’s biological activity in regulating quiescence, the subcellular localization of endogenous DAF-18 protein in the gonad will be characterized. Aim 2 will use tissue-specific depletion experiments to test this hypothesis, based on preliminary experiments, that DAF-18 may promote cellular quiescence by opposing the activity of the growth-promoting Tor kinase pathway. In Aim 3, powerful genetic screens will be used to identify new genes that may act in a genetic pathway with DAF-18 to regulate quiescence. The experiments in this proposal will incorporate fluorescent imaging in living organisms and cutting-edge genetic techniques. This training is to be conducted at Columbia University over the course of two years, under the guidance of Dr. Iva Greenwald, a leading C. elegans geneticist with an excellent record of training successful researchers.