Characterization of a Novel Regulatory Protein Required for Cytokinesis

Clark University, Worcester MA

Investigators

Abstract

This project is concerned with the mechanism whereby a cell divides into two. In general, cell division in animal cells ("cytokinesis") results in the production of two daughter cells that are nearly identical to each other. The process of cytokinesis ensures that the newly duplicated nuclei, along with half of the cytoplasm, are equally partitioned into the resulting daughter cells. This process occurs at a very specific time in the cell cycle, and at a very specific location, usually the equator. However, how the timing and location of cytokinesis are regulated at the molecular level has remained largely unknown. Dr. Larochelle has generated mutant Dictyostelium discoideum vegetative cells and screened these cells for defects in cytokinesis. This approach had been used successfully in the past to identify racE and lvsA, two unique genes that are required for normal cytokinesis. Dr. Larochelle has now isolated a new cell line that is defective in cytokinesis due to a disruption in a novel gene, which is the focus of this research project. The specific aims of this research are to characterize the specific cytokinesis defect that results from the disruption of this novel gene, determine the location and mechanism of how the encoded protein functions in cytokinesis by conducting a careful analysis of the various domains that compose this protein, identify and characterize proteins that interact with this novel protein, and provide training for graduate and undergraduate students, as well as high school students and teachers, in the areas of cell biology and molecular biology. By making use of the ease with which one can manipulate the Dictyostelium genome, coupled with the Dictyostelium genome sequencing project, the experiments will combine molecular genetics with cell biology to dissect the exact nature through which this newly identified gene regulates cytokinesis. The potential to identify additional proteins that participate in the control if this important cellular event is suggested through the presence of multiple signaling domains (a phosphatase domain, a rho-related domain, two kinase domains, and eight WD-40 repeats) in the newly identified protein. These domains will be examined in isolation and coupled with neighboring domains, as well as through deletion analysis, to dissect their role in cytokinesis regulation. Preliminary results, in which one of the kinase domains was over-expressed in wild-type cells, resulting in a cytokinesis defect, is a promising indicator that this approach will be successful. Because Dictyostelium is easily cultured and is highly amenable to molecular genetic manipulation, while at the same time exhibiting the same cellular behaviors observed in cells from higher eukaryotic organisms, it is ideally suited for the training of budding scientists in the areas of cell and molecular biology. The development of a strong research program, in an atmosphere where high school, undergraduate, and graduate students interact with each other and faculty on a daily basis, will provide unique training opportunities for all participants.