Regulation of blast cell quiescence by Pten and Tor - 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.
