spc110–226 forms normal spindles
early, but spindle integrity is compromised later. A population of
synchronous spc110–226 cells was obtained from strain
HSY83 by centrifugal elutriation and shifted to 37°C. At regular
intervals samples were removed and processed for either electron
microscopy or immunofluorescence. t = 0 min is the
midpoint of S phase. (A) Electron micrographs depicting short complete
spindles at t = 12 min. Bar, 0.2 μm. (B) Spc98p
staining and Spc110–226p staining of spc110–226 mutant
cells reveal two dots of colocalized staining representing the poles of
a short complete spindle 43 min after the midpoint of S phase. The
cells were stained with affinity-purified anti-Spc110p antibodies
(1:500) (Friedman et al., 1996) and mouse monoclonal
anti-98 kDa antibodies (1:10; gift of J. Kilmartin). The secondary antibodies were
a mixture of rhodamine-conjugated affinity-purified sheep IgG
anti-mouse Ig (20 μg/ml) and fluorescein-conjugated goat anti-rabbit
antibody (1:1000; Boehringer Mannheim Corp.). Bar, 2 μm. (C) Spc98p
staining and Spc110–226p staining of spc110–226 mutant
cells reveal three dots of colocalized staining and thus the presence
of an aberrant structure 103 min after the midpoint of S phase in the
synchronous shift experiment. Samples were processed for
immunofluorescence as described in Figure 6B. Bar, 2 μm. (D) Timing
of the appearance of three dots of Spc98p staining in the
spc110–226 synchronous shift experiment. ▪, fraction
of the cell population that has replicated at least one-half of its
genome; •, fraction of spc110–226 cells with two dots
of anti-98 kDa staining; ▴, fraction of spc110–226
cells with three or more dots of anti-98 kDa staining.