Animal development is a fascinating biological process that requires precisely choreographed cell divisions and cell movements followed by terminal cell differentiation during embryogenesis to form adults with properly specified tissues and organs. Conserved regulatory mechanisms called cell cycle checkpoints are used to spatially and temporally coordinate these distinct aspects of development. My laboratory studies checkpoints that inhibit the activity of Cdk1, a cyclin-dependent kinase that regulates mitosis in all eukaryotic cells, transiently delaying mitosis until cells are ‘ready’ to divide much like a ‘brake’ is used to regulate the speed of a vehicle entering a tight curve. These checkpoints function during S and G2 phases by up-regulating Wee1 and Myt1 kinases (to inhibit Cdk1 by phosphorylation) and down-regulating Cdc25 phosphatases (which remove Cdk1 inhibitory phosphorylation). Although well characterized in yeast and cultured cells, cell cycle regulation is very dynamic during animal development and remains poorly understood. We address this fundamental problem using mutants and transgenic strains to study the essential functions of three major cell cycle regulators (Wee1, Myt1 and Cdk1) during Drosophila development.
1. How does Wee1 regulate the rapid embryonic cleavage cycles to prevent mitotic catastrophe? Maternal Wee1 kinases are essential for checkpoints that prevent mitotic catastrophe during rapid nuclear cleavage divisions of early embryogenesis in Drosophila. Curiously, Cdk1 inhibitory phosphorylation is extremely difficult to detect at this stage, suggesting that Wee1 is regulating Cdk1 by a novel mechanism. To test potential mechanisms for controlling Cdk1 activity in embryonic cell cycles we have developed tools for expressing mutant and transgenic forms of Cdk1 in wild type and wee1 mutants and using targeted mutagenesis to identify functional domains important for Wee1 activity and interactions with Cdk1. The specialized mechanisms that control the cell cycle in early Drosophila embryos may also be relevant to human biology, since Wee1 also prevents mitotic catastrophe during early vertebrate development and defects in Wee1 regulation have been implicated in human cancer and neurodegenerative disease.
2. How do organelle checkpoints coordinate G2 phase arrest with M phase during meiosis and terminal cell fate differentiation? Myt1 kinases are animal-specific Cdk1 inhibitors that localize to the endoplasmic reticulum and Golgi apparatus. Myt1 is essential for a prolonged G2 phase pre-meiotic arrest in many different organisms and for regulating the dynamic behavior of the ER and Golgi during entry and exit from mitosis. Neither of these functions is well understood molecularly, however. Drosophila Myt1 is essential for male meiosis, making this an excellent model system for studying these questions. We have identified distinct organelle checkpoints regulated by Myt1 that are crucial for during a prolonged pre-meiotic arrest and are now developing genetic and biochemical approaches to fully characterize these functions in vivo. Myt1 is also required for ensuring that cells undergo terminal differentiation correctly at certain stages of development. We are collaborating with the Gho lab in Paris, France to model this aspect of Myt1 function, using live imaging and reporter assays to precisely characterize its role during sensory organ development.
3. What is the role of Cdk1 inhibitory phosphorylation during normal aging? Human Wee1 has been implicated in cancer and neurodegeneration, however little is known about the mechanisms involved. We became interested in the role played by cell cycle regulation in adult flies after noticing progressively worse locomotor activity deficits in aging wee1 mutants compared with controls. Most adult Drosophila tissues are post-mitotic, so conventional pre-mitotic checkpoints seem unnecessary, however stem cell regulation or non-canonical checkpoint mechanisms might be required. To test these ideas we are using mutants, transgenes and an automated activity monitor system to analyze how loss of Wee1 and Myt1 affects specific adult tissues as they age. Insights gained from these experiments may help us to understand how defects in these conserved regulators can contribute to human disease.
Partially inhibitable (C) versus non-inhibitable (D) transgenic Cdk1 have very different phenotypes when expressed during Drosophila wing and eye development. From: Ayeni et al., 2014.
Localization of EGFP-Myt1 (green), a germline-specific organelle called the fusome (red) and DNA (blue) in early Drosophila spermatocytes. Varadarajan, et al., (PhD thesis, 2015).