ROLE OF CALMODULIN AND SPC110P INTERACTION IN THE PROPER ASSEMBLY OF SPINDLE POLE BODY COMPONENTS

Previously we demonstrated that calmodulin binds to the carboxy termin us of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chr omosome segregation. Immunoelectron microscopy presented here shows th at calmodulin and thus the carboxy terminus of Spc110p localize to the central plaque. We created temperature-sensitive SPC110 mutations by combining PCR mutagenesis with a plasmid shuffle strategy. The tempera ture-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-bindin g site and alone confers a temperature-sensitive phenotype. Calmodulin overproduction suppresses the temperature sensitivity of spc110-220. Furthermore, calmodulin levels at the SPB decrease in the mutant cells at the restrictive temperature. Thus, calmodulin binding to Spc110-22 0p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budde d cells with a G2 content of DNA and suffer considerable lethality. Im munofluorescent staining demonstrates failure of nuclear DNA segregati on and breakage of many spindles. Electron microscopy reveals an aberr ant nuclear structure, the intranuclear microtubule organizer (IMO), t hat differs from an SPB but serves as a center of microtubule organiza tion. The IMO appears during nascent SPB formation and disappears afte r SPB separation. The IMO contains both the 90-kD and the mutant 110-k D SPB components. Our results suggest that disruption of the calmoduli n-Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation.