Errors in chromosome segregation contribute to the development of cancer by causing aneuploidy and loss of heterozygosity. Such errors also cause several human disabilities and half of all spontaneous human abortions. Proper segregation of chromosomes requires correct formation, function and regulation of the mitotic spindle. The centrosome is a key structure responsible for organizing the mitotic spindle in animal cells. The functional equivalent of the centrosome in the yeast Saccharomyces cerevisiae is the spindle pole body (SPB). The SPB is a cylindrical structure embedded in the nuclear envelope. The outer layer or plaque organizes the microtubules that extend into the cytoplasm; the inner plaque organizes nuclear microtubules, which form the mitotic spindle. Spc110p/Nuf1p is an essential component of the SPB and attaches the inner plaque to the central core of the SPB. The N- terminus of Spc110p is embedded in the inner plaque. Binding of calmodulin to the C-terminus is required for proper assembly of spindle pole components. The central region of Spc110p is a long coiled-coil predicated to oligomerize. Oligomerization and insertion of Spc110p into the SPB is a late step in assembly of a new SPB and a prerequisite to spindle assembly. This proposal focuses on the regulation, oligomerization, and assembly of Spc110p. We will describe the changes in the structure of Spc110p induced by calmodulin binding and study the role of calmodulin in oligomerization and assembly. Spc110p is phosphorylated as the bipolar spindle forms and dephosphorylated as cells enter anaphase. This phosphorylation requires the kinase Mps1p already shown to be required for spindle pole body duplication. Mps1p phosphorylates Spc110p in vitro on three peptides in the N-terminus. The sites of phosphorylation will be identified and mutagenized to determine the function of phosphorylation by Mps1p. The role of the N-terminus of Spc110p in assembling the proteins involved in nucleating spindle microtubules will be explored. Components that interact with the C-terminus of Spc110p will be identified by two genetic screens. The function of these components at the SPB will be determined by a mutational analysis. We will work towards developing assays for spindle pole body assembly by studying the incorporation of Spc110p and its interacting partners into the spindle pole body.
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