Size control is a fundamental property of cells that involves coordinated regulation of growth and division, processes that are frequently dysfunctional in human diseases such as cancer. The long-term goal of this research is to establish the mechanistic principles that govern size control. We have exploited a uniquely informative model for cell-size regulation, the unicellular alga Chlamydomonas reinhardtii. Using Chlamydomonas we have identified key components of a size checkpoint pathway whose behavior suggests a mechanism for coupling cell size to cell cycle activation. The genetically identified components of this checkpoint each have human counterparts and include proteins of the retinoblastoma (RB) tumor suppressor pathway, a cyclin dependent kinase, CDKG1, that phosphorylates RB, and an RNA binding protein, TNY1. We hypothesize that the activity of the kinase, CDKG1, is held in check by TNY1 as a means of coupling cell size to cell cycle entry, and that the balance of these two activities controls the cell cycle by switching on and off the RB pathway. The findings from this Proposal will provide fundamental knowledge regarding mechanisms of cell growth and division control that are relevant for understanding similar processes in humans.
The Specific Aims of this Proposal are as follows:
Aim 1. Investigate the role of CDKG1 as sizer protein: We will i) determine how CDKG1 abundance and activity are controlled during the cell cycle;ii) identify and characterize CDKG1 phosphorylation sites on its substrate, the RB related protein MAT3;iii) determine the temporal requirement for CDKG1 in the cell cycle using conditional alleles.
Aim 2. Determine the mechanism by which TNY1 represses the cell cycle: TNY1 is produced once per cell cycle and then becomes diluted as cells grow during G1. We will test the role of TNY1 as a threshold regulator of cell size that opposes the activity of CDKG1. We will i) establish whether TNY1 regulates CDKG1 through binding its mRNA;ii) test dosage effects of TNY1;iii) identify endogenous TNY1 RNA targets;and iv) establish whether targeting TNY1 to a mRNA brings expression of the encoded protein under control of TNY1 and/or the cell cycle.
Aim 3. Identify new size pathway genes through suppressor screens: tny1 null mutants and CDKG1 over- expressing (CDKG1-OE) lines both have small-cell phenotypes that provide a sensitized genetic background in which to identify interacting components of size control. i) We will use these lines in forward screens to find suppressors of their small-cell phenotypes. These suppressors are predicted to encode either targets or interacting proteins. ii) We will use secondary screens to order the mutants in the size pathway and to identify candidates for further investigation and cloning.
Many human diseases such as cancer are linked to deregulated growth and cell cycle control and involve changes in cell size homeostasis. This proposal aims to establish the mechanisms by which cell size is linked to cell cycle progression in the model unicellular eukaryote, Chlamydomonas reinhardtii. The pathways that control this process are conserved in humans and have direct relevance for understanding human cell biology and disease.
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