Cell cycle regulation of development

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*** *** *** *** *** *** ***SUMMARY OF THE PROPOSAL******Long-term Objectives:***The complex process of animal development requires precise timing of the cell cycle. My laboratory studies checkpoint mechanisms that regulate cell cycle timing by inhibiting Cdk1. Cdk1 is the major regulator of mitosis and meiosis, two distinct methods that cells use to proliferate at different stages of development. We are using Drosophila (fruit fly) mutants and fluorescent reporters created in my laboratory to analyze the functions of two important Cdk1 inhibitors, called Myt1 and Wee1. Myt1 regulation of Cdk1 is essential for preserving certain important cellular structures during male meiosis, a process where the cell cycle is modified to produce sperm. Wee1 regulation of Cdk1 is essential for slowing down S phase in rapidly developing embryos, a cell cycle stage when the DNA is replicated. Our goal is to better understand these specialized developmental functions of Myt1 and Wee1 by undertaking the following experimental approaches. ***Aims and Short-term Objectives:*** 1 Characterize Myt1 mechanisms that link endomembrane remodeling with centriole engagement: ***Myt1 kinases are membrane-bound Cdk1 inhibitors that evolved in multicellular organisms ~750 million years ago. We are studying male-sterile myt1 mutants to determine their role in meiosis. Drosophila spermatocytes normally arrest for four days in pre-meiotic G2 phase. During this developmental arrest, Myt1 inhibition of Cdk1 prevents cellular defects that can result in aberrant meiotic cell division and sterility. Using new fluorescent reporters and biochemical assays we will characterize Myt functions in vivo, by searching for factors that interact with Myt1 and investigating mechanisms that are important for localization and regulation of Myt1 activity during meiosis. Our results will provide new insights into mechanisms required for sexual reproduction in all multicellular organisms.***2. Characterize Wee1 embryonic checkpoint mechanisms that prevent mitotic catastrophe: ***Wee1 kinases are essential for preventing mitotic catastrophe in both Drosophila and mammalian embryos. Wee1 inhibits Cdk1 by a reversible mechanism called phosphorylation. Cdk1 inhibitory phosphorylation is extremely difficult to detect in early embryos however, suggesting that Wee1 regulates Cdk1 by a novel mechanism. We hypothesize that Wee1 controls Cdk1 activity by de-stabilizing its interactions with essential co-factors called Cyclins. To test this idea we will analyze how mutant and normal forms of Cdk1 interact with Cyclins in both control and wee1 mutant embryos. We will also identify mechanisms that are important for regulating Wee1 localization and activity using genetic screens to identify mutants affecting a transgenic Wee1 reporter. Our results will provide insights into Wee1 mechanisms that control rapid embryonic cell cycles********