Regulation of blast cell quiescence by Pten and Tor

项目概要/摘要 静止是干细胞的一个决定性特征，这使得它们能够随着时间的推移而持续存在，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 监管不完善。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物（Pten）和靶基因调节 雷帕霉素（Tor）的合成。优雅In C.在一个特殊的幼虫阶段， 生殖腺中的多能胚细胞受来自环境的信号的引导而保持静止。 在这些性腺胚细胞中，静止期的丧失或维持可以很容易地用荧光标记检测出来。 标记。 我们的实验室最近表明，C。Pten（PTEN-18）的elegans直系同源物调节胚细胞静止 在幼鱼的生殖腺里肿瘤抑制基因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静止 干细胞和癌症干细胞，但我们缺乏对Pten如何做到这一点的理解。平静通常是 很难在体内研究，我们的性腺发生模型提供了一个独特的机会，在活体中研究它， 完整的有机体，拥有无与伦比的遗传分析工具。这项研究的目的是了解如何， 在机制上，Pten使用我们的性腺发生模型促进干细胞静止。 Pten/Pten-18具有多种分子活性：它是一种蛋白磷酸酶，一种脂质磷酸酶，也可 充当“支架”来组装蛋白质复合物。在目标1中，结构/功能研究将用于评估 这些活动中的哪一个是必需的，以调节我们系统中的胚细胞静止。此外，本发明还 为了进一步了解β-18在调节静止中的生物活性， 将表征性腺中的内源性β-18蛋白。目标2将使用组织特异性耗竭 基于初步实验来验证这一假设的实验，即β-18可能促进细胞增殖， 通过对抗生长促进Tor激酶途径的活性来抑制静止。在目标3中， 筛选将用于识别可能在基因通路中与β-18一起起作用的新基因， 安静该提案中的实验将包括活生物体中的荧光成像， 尖端的基因技术这项培训将在哥伦比亚大学进行，为期两周， 多年来，在Iva Greenwald博士的指导下，一位领先的C。优雅的遗传学家， 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.