1. Scientific merit. Investigations with many organisms and cell lines have identified proteins that affect function and stability of microfilament and microtubule networks or both. Mutations in these proteins can simultaneously weaken the microfilament network and stabilize the microtubule network, indicating a dynamic balance between the two. This balance is regulated by the mitotic cyclins because their increase correlates with fewer microtubules. From this it is postulated that there are dose-dependent negative interactions between microtubules, microfilaments, and the mitotic cyclins. This clearly gives an entry point to understand how the cell-cycle machinery regulates the two major cytoskeletal networks, specifically how are proteins, which affect the stability of the cytoskeleton, activated in specific phases of the cell cycle.
Early embryos are used to study cell cycle and cytoskeleton interactions because the cell cycle only has interphase and mitosis and lacks both gap phases (G1 and G2). The opacity of early Drosophila embryos due to the abundance of yolk has, until recently, made live analysis of the early cycles impossible. The advent of multi-photon microscopy, used in conjunction with green fluorescent protein tagged histone, now allows the precise four-dimensional live analysis of the earliest cycles, their phases and nuclear movement in normal and mutant embryos.
Because development of the early embryo occurs without embryonic transcription, the genome of the mother can be altered and the effect can be assayed in the embryo. Increasing the gene copy number of cyclin B (cycB) from two to six in the mother extends metaphase duration, reduces microtubule configuration and changes many morphological aspects in the embryo without affecting survival. Using this weakened genetic constitution, random mutations in the genome were induced to either reduce "the six cycB phenotype" or worsen it. The screen identified genes that regulate the activity of mitotic cyclins, cytoskeleton dynamics and the onset of anaphase. Cyclin dependent kinase 1 (Cdk1) forms a complex with CycB. To enter mitosis, this complex has to be modified: inhibitory phosphates have to be removed and activating phosphates have to be added. Thus the genetic screen can also find specific kinases and phosphatases that modulate the function of this complex.
2. Broader impacts. This project has promoted the integration of research with undergraduate teaching and learning. The PI regularly teaches formal courses at the University and provides opportunities for students, especially those from underrepresented groups and from other institutions. The "wet" data generated from the project have been incorporated into lectures for undergraduate courses, and participation in the genetic screen gives undergraduates easy access to independent research in genetics and development. Undergraduates, graduate students, technicians and post-docs from this lab have often presented their work at scientific meetings, are first authors on published papers, and have pursued academic careers. This research project has also promoted the infrastructure for research by establishing collaborations with other academic institutions and by using the new technology of two-photon microscopy to make live recordings of Drosophila development. These recordings are a valuable learning tool and are available to the broader public on the Fly Base website. Finally, the project's scientific discoveries will benefit the advances of basic cancer research. The goal of the project is to learn how cell cycle progression and cytoskeleton dynamics are coordinated during the cell cycle so that chromosomes are faithfully transmitted. Loss or gain of chromosomes- aneuploidy, can lead to unregulated cell growth or cell death. In cancer, aneuploid cells can affect the progression of tumor malignancy and resistance to therapy.
