Calmodulin localizes to the spindle pole body of Schizosaccharomyces pombe and performs an essential function in chromosome segregation.
The essential calmodulin genes in both Saccharomyces cerevisiae and Schizosaccharomyces pombe were precisely replaced with genes encoding fusions between calmodulin and the green fluorescent protein (GFP). In living budding yeast the GFP-calmodulin fusion protein (GFP-Cmd1p) localized simultaneously to sites of cell growth and to the spindle pole body (SPB), the yeast analog of the centrosome. Having demonstrated proper localization of GFP-calmodulin in budding yeast, we examined the localization of a fusion between GFP and calmodulin (GFP-Camlp) in fission yeast, where calmodulin had not been localized by any method. We find GFP-Camlp also localizes both to sites of polarized cell growth and to the fission yeast SPB. The localization of calmodulin to the SPB by GFP fusion was confirmed by indirect immunofluorescence. Antiserum to S. pombe calmodulin labeled the ends of the mitotic spindle stained with anti-tubulin antiserum. This pattern was identical to that seen using antiserum to Sad1p, a known SPB component. We then characterized the defects in a temperature-sensitive S. pombe calmodulin mutant. Mutant cam1-E14 cells synchronized in S phase completed DNA synthesis, but lost viability during transit of mitosis. Severe defects in chromosome segregation, including hypercondensation, fragmentation, and unequal allocation of chromosomal material were observed. Immunofluorescence analysis of tubulin revealed a population of cells containing either broken or mislocalized mitotic spindles, which were never observed in wild-type cells. Taken together with the subcellular localization of calmodulin, the observed spindle and chromosome segregation defects suggest that calmodulin performs an essential role during mitosis at the fission yeast SPB.
Moser
M J
MJ
Department of Biochemistry, University of Washington, Seattle 98195, USA.
Flory
M R
MR
Davis
T N
TN
D002147:Calmodulin / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D015825:Chromosomes, Fungal / Q000378:metabolism*; D004271:DNA, Fungal / Q000032:analysis; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008870:Microtubules / Q000737:chemistry / Q000648:ultrastructure; D008938:Mitosis; D009154:Mutation; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D012568:Schizosaccharomyces / Q000737:chemistry / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000737:chemistry* / Q000648:ultrastructure; D013696:Temperature; D014404:Tubulin / Q000032:analysis
D002147:Calmodulin; D004271:DNA, Fungal; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins