Three-dimensional electron microscopy analysis of ndc10-1 mutant reveals an aberrant organization of the mitotic spindle and spindle pole body defects in Saccharomyces cerevisiae.
Kinetochore components play a major role in regulating the transmission of genetic information during cell division. Ndc10p, a kinetochore component of the essential CBF3 complex in budding yeast is required for chromosome attachment to the mitotic spindle. ndc10-1 mutant was shown to display chromosome mis-segregation as well as an aberrant mitotic spindle (Goh and Kilmartin, 1993). In addition, Ndc10p localizes along the spindle microtubules (Muller-Reichert et al., 2003). To further understand the role of Ndc10p in the mitotic apparatus, we performed a three-dimensional electron microscopy (EM) reconstruction of mitotic spindles from serial sections of cryo-immobilized ndc10-1 mutant cells. This analysis reveals a dramatic reduction in the number of microtubules present in the half-spindle, which is connected to the newly formed spindle pole body (SPB) in ndc10-1 cells. Moreover, in contrast to wild-type (WT) cells, ndc10-1 cells showed a significantly lower signal intensity of the SPB components Spc42p and Spc110p fused with GFP, in mother cell bodies compared with buds. A subsequent EM analysis also showed clear defects in the newly formed SPB, which remains in the mother cell during anaphase. These results suggest that Ndc10p is required for maturation of the newly formed SPB. Intriguingly, mutations in other kinetochore components, ndc80-1 and spc24-1, showed kinetochore detachment from the spindle, similar to ndc10-1, but did not display defects in SPBs. This suggests that unattached kinetochores are not sufficient to cause SPB defects in ndc10-1 cells. We propose that Ndc10p, alongside its role in kinetochore-microtubule interaction, is also essential for SPB maturation and mitotic spindle integrity.
Romao
Maryse
M
Institut Curie UMR144 CNRS, 26 rue d'Ulm, 75248 Paris cedex 05, France.
Tanaka
Kozo
K
Sibarita
Jean-Baptiste
JB
Ly-Hartig
Nga Thi Bach
NT
Tanaka
Tomoyuki U
TU
Antony
Claude
C
D020090:Chromosome Segregation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D018386:Kinetochores / Q000737:chemistry / Q000473:pathology; D008854:Microscopy, Electron; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*; D008941:Spindle Apparatus / Q000473:pathology
C081071:CBF2 protein, S cerevisiae; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins