9403689 Wolniak The goal of this project is to investigate the involvement of three protein kinases in a signaling cascade that triggers sister chromatid separation at the onset of anaphase during mitosis. The proposed experiments will be performed on stamen hair cells of the spider plant ,Tradescantia virginiana because they exhibit a rate of progression from nuclear envelope breakdown to anaphase onset that is remarkably predictable and useful as a temporal assay for mitotic progression. The proposed experiments are based on a body of published work from the PI's laboratory that shows the regulatory cascade to consist of two or three successive waves of protein kinase activity, punctuated by an interval in mid-metaphase when protein phosphatase activity is elevated. Protein phosphatase activation is a necessary requirement for synchronous entry into anaphase. In this proposal a series of microinjections in living cells with a series of peptides that serve as activators, inhibitors and substrates of specific protein kinases will provide an assessment of the timing of involvement of p34cdc2 protein kinase, calcium calmodulin dependent protein kinase II, and calcium dependent but calmodulin-independent protein kinase (cdpk) in the regulation of progression through metaphase. All three of these protein kinases are present in plant cells, and the experiments include the utilization of the best available oligopeptide probes for the detection, inhibition or activation. The microinjection protocol developed by the PI permits the introduction of a known quantity of charged or uncharged molecule (MW < 6,000, though probably much larger) into the cytosolic compartment of a living stamen hair cell at a known time point during prometaphase or metaphase. The injection protocol, by itself, has no significant event on the metaphase transit time of these cells. These experiments will contribute to our understanding of the regulatory mechanisms that mediate progr ession through metaphase and trigger entry into anaphase. %%% Mitosis is the process that facilitates the equal partitioning of the replicated chromosomes to the incipient daughter cells that will separate from each other as one cell becomes two. In this proposal experiments are presented to test for the involvement of a set of protein phosphorylating enzymes in the regulation of progression through a specific cell division stage that is called mitosis, with a particular focus on sister chromatid separation, the morphological marker for entry into the anaphase stage of mitosis. The model system utilized in these experiments is the stamen hair cell from the spiderworth plant, Tradescantia virginiana. These plant cells progress through mitosis at a highly predictable rate, and they have been proven to be uniquely suited for studies of the timing of regulatory events involved in mitotic progression. In the current experiments living cells will be injected with known quantities of synthesized peptides that will affect the activities of specific protein kinases in known ways. Some of these peptides are kinase activators, while others are inhibitors or substrates for these enzymes. Because the cell can be injected at known time points during metaphase, their effect on the protein kinases at different times can provide clues about the environment of these enzymes in the regulatory cascade that controls the progression through metaphase and entry into anaphase. Results from this project will contribute to our overall understanding of the regulatory mechanisms thatcontrol mitotic progression in intact cells. ***
