Analysis of a spindle pole body mutant reveals a defect in biorientation and illuminates spindle forces.
The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-microm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.
Yoder
Tennessee J
TJ
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
McElwain
Mark A
MA
Francis
Susan E
SE
Bagley
Joy
J
Muller
Eric G D
EG
Pak
Brian
B
O'Toole
Eileen T
ET
Winey
Mark
M
Davis
Trisha N
TN
D000483:Alleles; D015153:Blotting, Western; D002148:Calmodulin-Binding Proteins; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D002875:Chromosomes / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D018386:Kinetochores / Q000378:metabolism; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D008941:Spindle Apparatus; D013696:Temperature; D013997:Time Factors
D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D009687:Nuclear Proteins; C073277:SPC110 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D004247:DNA