The temporal and spatial distribution of microtubules in eukaryotic cells is controlled by discrete organelles, known as microtubule-organizing centers (MTOCs). MTOCs exhibit remarkable structural variation among different organisms but have similar functions of organizing interphase microtubule arrays and determining the bipolarity of the mitotic spindle; as such, they are crucial to the fidelity of cellular reproduction and cytoplasmic organization.
The specific aims of this proposal are to study the structure and function of caltractin, a calcium-binding protein that is a conserved component of MTOCs in divergent species. Caltractin, also known as """"""""centrin,"""""""" has been cloned at the DNA level from a number of different organisms including yeast, algae, frog, mouse and human. Based on amino acid sequence analysis these proteins have been found to represent a new subfamily within the superfamily of EF-hand calcium- binding proteins that includes calmodulin and troponin C. Genetic analyses indicate that caltractin in the alga Chlamydomonas reinhardtii and its yeast homolog in Saccharomyces cerevisiae, Cdc31p are required for the normal duplication and segregation of the basal body complex and spindle pole body, the major MTOC in the respective cell types. In this proposal, the localization and expression of human caltractin in HeLa cells, and the consequences of disrupting caltractin function in these cells will be studied to determine if, as in Chlamydomonas and yeast cells, the protein plays a role in the duplication and segregation of the centrosome, the major MTOC in animal cells. Protein structure/function relationships will be examined by studying the consequences of expressing site-specific mutations in caltractin in stable transformants of Chlamydomonas. Genetic and biochemical approaches will be used to identify other genes and gene products that affect the basal body complex in Chlamydomonas and proteins that may interact with caltractin. And finally, the in vitro functional and structural features of wild-type and mutant forms of algal and human caltractin expressed and purified from bacteria will be characterized.
The aim of these in vitro studies is to identify features that distinguish caltractin from other closely related calcium-binding proteins, and to define at high resolution by NMR spectroscopy and potentially, X- ray crystallography, the consequences of calcium-binding on protein conformation. The long term objectives of this project are to define the mechanisms at the molecular level that regulate the assembly and function of MTOCs. Protein components of MTOCs that are characterized to be of fundamental importance in their physiology and/or replication constitute potential novel targets for developing new therapeutic approaches to influence microtubule function.