The centrosome is responsible for organizing the mitotic spindle in mammalian cells. Malfunctioning spindles can lead to chromosome nondisjunction and genomic instability, a hallmark of cancer cells and the cause of some birth defects. The spindle pole body (SPB) is the functional equivalent of the centrosome in the budding yeast Saccharomyces cerevisiae. The SPB is a trilaminated structure consisting of outer, central and inner plaques. The long term goal of the proposed work is to determine how the central plaque acts to organize and maintain the integrity of the SPB. Genetic and immunochemical analyses have revealed that Spc110p is an essential rod- shaped protein that attaches to the central plaque by its C-terminus and to the inner plaque by its N-terminus. Expression of mutant spc110s and overexpression of wild type SPC110 cause mitotic failure and lethality, indicating that Spc110p plays a critical role in determining the structure and function of the spindle. The experiments described are designed to characterize the functional interactions between Spc110p and components of the central plaque. The primary strategy is to screen for mutations in genes that result in synthetic lethality in combination with mutated spc110. The genes identified will be characterized to determine their role in both SPB assembly and mitotic spindle formation. These genes will be mutated and the phenotypes produced by the mutant alleles assessed. Functional domains will be defined and protein: protein interactions studied in vitro.
