Denis A. LaRochelle, PhD, Stanford University 1991 - US grants

An integral aspect of all cancers is the requirement for cell division. Cytokinesis is the final step in cell division, where the cell constricts (usually at the equator) to partition the newly duplicated nuclei, along with half of the cytoplasm, into the resulting daughter cells. Many proteins that control cell division have been identified recently, yet those that control cytokinesis remain largely unknown. The long-term goals of the proposed research are to identify the molecules involved in cytokinesis and to understand how these molecules are involved in this process. The strict spatial and temporal requirements of cytokinesis suggests that it must be a very tightly regulated process. Small UTP-binding proteins have been implicated in the regulation of this process yet it is not known how. Recently, the eukaryotic organism Dictyostelium discoideum has emerged as one of the premier model systems for studying cytokinesis. Through a molecular- genetic screen using Dictyostelium we have discovered a novel small GTP- binding protein, named racE, that is absolutely required for cytokinesis. In order to understand how racE is involved in cytokinesis we need to know which domains of this protein are unique to racE and required for it to function in cytokinesis. By generating a number of chimeric proteins combining racE with another closely related small GTP- binding protein, that is not required for cytokinesis, we will identify the domain(s) of racE that are essential to its functioning in cytokinesis. This information will be used to isolate additional proteins that interact specifically with these domains. In this way we can begin to dissect the pathway through which racE is involved in cytokinesis. The genes coding for these racE-interacting proteins will be cloned and sequenced. In addition, their roles in cytokinesis will be investigated through genetic and biochemical means. The identification of the proteins required for cytokinesis, and how they interact with one another, will greatly increase our understanding of how this process is regulated in cells.

Abstract Larochelle 0070241

Modern imaging facilities are essential for biological research in cell and developmental biology. The Department of Biology at Clark University is upgrading their imaging facility with a high quality research-grade compound microscope with phase contrast, differential interference contrast, and epifluorescence optics. In addition, a cooled CCD camera, a computer, and software for capturing, processing, and storing images, will be part of the system. Together these constitute a complete system for the generation and analysis of photomicrographs.

Specific research to be carried out with this system will include the microscopic analysis of cells defective in cell division, the examination of nuclear behavior of certain fungi as a means of gaining insights into fungal evolutionary relationships as well as fungal developmental evolution on a cellular level, the analysis of proteins required in the synthesis and regulation of membrane organelles, and the examination of the role of receptor tyrosine kinase signaling pathways in the growth and development of the Drosophila eye.

Six new faculty members (out of nine total) have joined the Department of Biology within the last seven years. With this turnover also comes an updating and improvement of the current facilities. Although the four co-PIs and their associated lab personnel will be the primary users of the facility, this imaging system will be available for research use by all faculty, graduate students, and advanced undergraduate students in the department. The addition of a research-grade microscopy imaging facility will become a cornerstone for the department. Despite its small size, the Department of Biology has been very successful in involving undergraduate students in research and in preparing them for advanced studies. Since 1989 over 350 students that have received their baccalaureate degrees in the sciences from Clark University have gone on to obtain advance degrees in biology or health-related disciplines. This imaging system will contribute greatly to the ability to train students and will be an integral part of the research programs of the four co-PIs.

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.