Role of calmodulin and Spc110p interaction in the proper assembly of spindle pole body components

• Published in: The Journal of cell biology 1996-04, Vol.133 (1), p.111-124
• Main Authors: Sundberg, H.A. (University of Washington, Seattle, WA.), Goetsch, L, Byers, B, Davis, T.N
• Format: Article
• Language: English
• Subjects: ADN; Antibodies; CALMODULINA; CALMODULINE; Cell cycle; Cells; Cellular biology; Diploidy; Electron microscopy; FENOTIPOS; Genes; Genetic mutation; IMMUNOFLUORESCENCE; IMMUNOLOGIE; INMUNOFLUORESCENCIA; INMUNOLOGIA; Microtubules; MITOSE; MITOSIS; MUTACION INDUCIDA; MUTANT; MUTANTES; MUTATION PROVOQUEE; ORGANITE CELLULAIRE; ORGANULOS CITOPLASMICOS; PHENOTYPE; Plasmids; PROTEINAS AGLUTINANTES; PROTEINE DE LIAISON; SACCHAROMYCES CEREVISIAE; SEGREGACION; SEGREGATION; Spindle pole body; ULTRAESTRUCTURA; ULTRASTRUCTURE; Yeast; Yeasts
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.133.1.111

Previously we demonstrated that calmodulin binds to the carboxy terminus of Spc110p, an essential component of the Saccharomyces cerevisiae spindle pole body (SPB), and that this interaction is required for chromosome segregation. Immunoelectron microscopy presented here shows that 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 temperature-sensitive allele spc110-220 differs from wild type at two sites. The cysteine 911 to arginine mutation resides in the calmodulin-binding 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 Spc110-220p is defective at the nonpermissive temperature. Synchronized mutant cells incubated at the nonpermissive temperature arrest as large budded cells with a G2 content of DNA and suffer considerable lethality. Immunofluorescent staining demonstrates failure of nuclear DNA segregation and breakage of many spindles. Electron microscopy reveals an aberrant nuclear structure, the intranuclear microtubule organizer (IMO), that differs from an SPB but serves as a center of microtubule organization. The IMO appears during nascent SPB formation and disappears after SPB separation. The IMO contains both the 90-kD and the mutant 110-kD SPB components. Our results suggest that disruption of the calmodulin-Spc110p interaction leads to the aberrant assembly of SPB components into the IMO, which in turn perturbs spindle formation