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J. Cell Biol.,
Volume 144, Number 5, March 8, 1999 977-987
* Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280; and We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules
(Mts) and spindle dynamics during the mating pathway
in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming
a shmoo projection. Imaging of microtubule dynamics
with green fluorescent protein (GFP) fusions to dynein
or tubulin revealed that the nucleus and spindle pole
body (SPB) became oriented and tethered to the
shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured
at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the
shmoo tip at growth and shortening velocities typical of
free plus end astral Mts (~0.5 µm/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth
and shortening occurred primarily at the shmoo tip, not
the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was
formed between the two shmoo tip bundles, which
slowly shortened (0.23 ± 0.07 µm/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused
SPB ~30 min after SPB fusion. During the first mitosis,
the SPBs separated as the spindle elongated at a constant velocity (0.75 µm/min) into the zygotic bud. There
was no indication of a temporal delay at the 2-µm stage of spindle morphogenesis or a lag in Mt nucleation by
replicated SPBs as occurs in vegetative mitosis implying
a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus
end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid cell fusion in budding yeast may forgo cell cycle checkpoints present in vegetative mitosis.
Department of Cell
Biology, Harvard Medical School, Boston, Massachusetts 02115
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