Regulation of blast cell quiescence by Pten and Tor

项目总结/文摘

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.