The distribution of subcellular organelles during cell division is essential for the proliferation of eukaryotic cells, yet little is known of its molecular basis or control. Previous studies identified the MDM1 protein (Mdm1p) as a central component mediating mitochondrial distribution during mitosis in yeast. Mdm1p is an intermediate filament-forming protein that defines a cytoplasmic structural system essential for both nuclear and mitochondrial inheritance. The goal of this project is to characterize the function and assembly of the Mdm1p-cytoskeleton. There are four objectives: 1,to analyze dynamics of the Mdm1p- structures within intact cells; 2, to generate and characterize new mutant alleles of mdm1; 3, to characterize the interaction of Mdm1p with mitochondria; and 4, to identify proteins that associate with Mdm1p and play a role in the assembly or function of the Mdm1p- containing structures. A long-range aim is to understand how the function of Mdm1p is coordinated with overall cellular metabolism and the cell division cycle. Study of the Mdm1p-network will involve a combined cellular, biochemical, and genetic approach. Dynamics of the Mdm1p-network will be analyzed in intact cells harboring mutations affecting a variety of other cellular structures or cells treated with various chemical inhibitors. Indirect immunofluorescence microscopic analysis will reveal changes in Mdm1p distribution and allow assessment of assembly and disassembly of the network. New alleles will be created by in vitro mutagenesis of the cloned MDM1 gene, analyzed microscopically to identify those conferring phenotypes different from the original mdm1 allele, and identified by DNA sequence analysis. This component of the study should permit a detailed structure-function analysis of Mdm1p. The interaction of Mdm1p with mitochondria will be studied using binding assays employing purified Mdm1p and isolated mitochondria. Binding studies combined with chemical crosslinking procedures should lead to the identification of mitochondrial proteins that mediate Mdm1p-mitochondrial interactions. Biochemical and genetic approaches will be developed to identify additional cellular components that mediate Mdm1p function and participate in the distribution of mitochondria during mitosis. These studies will reveal new details of the Mdm1p- cytoskeletal system. They should lead to new insights into cellular organization and processes basic to normal cell growth. %%% The appropriate partitioning of cellular components at the time of cell division is critical to the survival of the daughter cells. In prior NSF-supported work, this laboratory identified an essential gene in yeast which plays an important role in partitioning of mitochondria to daughter cells during yeast cell division, and discovered that the gene product is a yeast homologue to animal intermediate filament proteins. This project will extend those studies to the questions of how the yeast intermediate filament-like network is assembled, and how it interacts with mitochondria. The results will provide fundamental and important new knowledge concerning the organization of cytoplasm.
