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Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae
P Maddox1, E Chin, A Mallavarapu
1Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA. pmaddox@email.unc.edu
Abstract:
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 (approximately 0.5 micrometer/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 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer 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.
Insights
Budding yeast mating uses microtubule dynamics for nuclear tethering and fusion. The first post-fusion mitosis may bypass cell cycle checkpoints, similar to higher eukaryotes.
Area of Science:
- Cell Biology
- Microscopy
- Yeast Genetics
Background:
- Budding yeast Saccharomyces cerevisiae undergoes a mating pathway involving cell cycle arrest and projection formation (shmoo).
- Cytoplasmic microtubules (Mts) play crucial roles in nuclear positioning and cell division.
Purpose of the Study:
- To investigate cytoplasmic astral microtubule and spindle dynamics during the yeast mating pathway.
- To understand the mechanisms of nuclear tethering, fusion, and early zygotic mitosis.
Main Methods:
- Time-lapse digital imaging microscopy was employed.
- Green fluorescent protein (GFP) fusions to dynein and tubulin were used to visualize microtubule dynamics.
- Fluorescent fiduciary marks were utilized to track microtubule growth and shortening.
Main Results:
- Microtubule-dependent search and capture oriented and tethered the nucleus to the shmoo tip.
- Microtubule plus end assembly/disassembly at the shmoo tip drove nuclear pushing and pulling.
- Post-fusion, microtubules facilitated nuclear fusion (karyogamy) and zygotic bud formation.
- The first zygotic mitosis showed rapid spindle elongation without typical cell cycle checkpoint delays.
Conclusions:
- The shmoo tip serves as a model for studying microtubule plus end dynamic attachments.
- Early zygotic mitosis in budding yeast may lack the cell cycle checkpoints observed in vegetative growth.