Microtubule and spindle pole body regulation in the budding yeast mitotic spindle

Abstract

The microtubule organizing center (MTOC) is a specialized structure with a main function in microtubule (MT) nucleation and organization. In higher eukaryotes, the main MTOC is known as the centrosome, and its functional equivalent in yeast is the spindle pole body (SPB). During mitosis, the cells build the mitotic spindle, an MT-based structure that mediates accurate chromosome segregation, which is essential to prevent aneuploidy and cancer. In yeast, the assembly and function of the mitotic spindle depend on the SPB, microtubule-associated proteins (MAPs), and motor proteins. In this thesis, we aim to advance our understanding of the general principles that regulate the SPBs, and spindle MTs in budding yeast.

In Paper I, we developed a method to separate the old (from the previous cell cycle) and newly synthesized SPB component Spc110 and identified age-specific phosphorylation residues in Spc110. We combined Recombination Induced Tag Exchanged – a genetic method to label old and new proteins differentially- with affinity purification. Using mass spectrometry analyses, we identified two phosphosites, S11 and S36, in old Spc110. We explore the function of these two phosphosites in non-phosphorylatable Spc110 mutants. Cells expressing Spc110S11A showed a distinct spindle phenotype, where tubulin intensity is higher and distributed asymmetrically. Furthermore, the double mutant Spc110S11AS36A was slightly delayed in cell cycle progression and re-entry in G1. Thus, we propose Spc110 phosphorylation at S11 regulates MT dynamics, whereas together with S36 regulates timely cell cycle progression in budding yeast.

In Paper II, we explored the role of the MT plus-end tracking protein (+TIP) Bik1 in the nucleus of budding yeast. Bik1 has a nuclear and a cytoplasmic pool, and we found that nuclear Bik1 localizes to the kinetochores in a cell cycle-dependent manner, peaking at metaphase. To explore the role of Bik1 at kinetochores, we added a Nuclear Export Signal (NES) to generate a Bik1-NES mutant that excludes Bik1 from the nucleus without disrupting its cytoplasmic pool. The Bik1-NES mutant had a slower cell-cycle progression, characterized by prolonged metaphase. Furthermore, we demonstrated that the kinetochores in Bik1-NES cells are frequently unclustered and mispositioned towards the spindle midzone in metaphase. By applying proximity-dependent methods, we identified kinesins Cin8 and Kip1 as Bik1 interactors. Bik1 and Cin8 cooperate to regulate kinetochore-MT dynamics for chromosome congression. Hence, the study uncovers a novel role of kinetochore Bik1 in cell cycle progression and chromosome congression.

In Paper III, we examined the turnover of Spc110 using cycloheximide (CHX) chase experiments. The results indicated that Spc110 has a very short half-life. Performing CHXchase experiments with the autophagy-defective mutant atg1D, we showed that autophagy is not essential for Spc110 degradation. On the other hand, Spc110 contains a potential Esp1 recognition site (the yeast homolog of Separase). Mutation of this Esp1 recognition site on Spc110 results in partial Spc110 stabilization. Hence, we speculate that Esp1 might be involved in Spc110 degradation.