Microtubule and chromosome dynamics during mitosis in budding yeast

Abstract: As a cell divides, DNA must be replicated and faithfully segregated between the mother and daughter cells. This segregation is facilitated by the mitotic spindle, assembled to pull sister chromatids apart as the cell divides. In budding yeast, spindle pole bodies nucleate microtubules that make up the mitotic spindle, position it at the site of division, and physically link chromosomes to opposing poles via the kinetochores. The chromosomes are held together by cohesin, which is also involved in the architecture of chromatin. In this thesis, I have explored mechanisms controlling microtubule dynamics, kinetochore positioning and chromosome dynamics during mitotic cell division in budding yeast. Bik1 is a microtubule-associated protein shown to play a role in the cytosol to position the spindle before anaphase. In paper I, we have characterized the nuclear function of Bik1 and identified a novel role in clustering kinetochores prior to spindle elongation. Cells lacking nuclear Bik1 have a delayed cell cycle progression, with prolonged metaphase, and fail to cluster kinetochores. We also connect this function to the nuclear kinesin Cin8, which has previously been described to regulate kinetochore microtubule dynamics in metaphase. The spindle pole body anchors microtubule nucleating γ-tubulin complexes using two different receptors, Spc72 in the cytosol and Spc110 in the nucleus. In paper II, we have isolated ‘old’ Spc110, originating from the previous cell cycle, and mapped its phosphorylation sites. These analyses revealed that old Spc110 is phosphorylated at serine 36 and at a novel site, serine 11. Non-phosphorylatable mutant strains revealed that these sites influence microtubule dynamics and cell cycle progression. The Spc110S11A mutant strain frequently had brighter spindle microtubules with asymmetric distribution of α-tubulin. Furthermore, Spc110S11A S36A cells had slightly delayed cell cycle progression and spindle disassembly. The cohesin complex has been shown to shape the chromosomes into loops in budding yeast through a mechanism known as loop extrusion. This phenomenon has primarily been studied using genome-wide sequencing techniques, which report detailed population averages of contact frequencies throughout the genome. How chromosomes of individual cells are affected, and whether this looping affects physical compaction remains poorly understood. In paper III we have generated a microscopy-based system to study chromosome dynamics in single yeast cells by fluorescently tagging specific chromosomal loci. We then used this system to investigate how physical distances between the fluorescently marked loci change after inhibiting loop extruding cohesin. This study revealed that loop extrusion does not significantly affect physical distances but may limit the dynamic movement of chromosomes. In conclusion, these studies reveal novel mechanisms controlling spindle and chromosome dynamics during mitotic cell division: 1) We have uncovered a new role of Bik1 at the spindle. 2) We have mapped phosphorylation sites in old Spc110 and characterized a novel site. 3) We have created a system to study chromosome dynamics in single cells and found that loop extrusion does not significantly compact mitotic yeast chromosomes.