Regulation of microtubule nucleation and attachment to spindle pole bodies

Abstract

The precise regulation and coordination of the mitotic spindle is vital for accurate chromosomal segregation within a dividing cell. The centrosome is the microtubule organizing center of the cell, responsible for nucleating and organizing the microtubules of the mitotic spindle. In yeast, the centrosome functional equivalent is called the spindle pole body. The studies presented here aimed to understand how microtubules are nucleated at the spindle pole body and also studied the regulation and mechanical strength of the yeast spindle pole body in vitro. In yeast, the γ-tubulin small complex, a highly conserved heterotetramer essential for microtubule nucleation, is composed of two copies of Tub4, and one copy each of Spc97 and Spc98. In this study, a comprehensive mutagenesis technique coupled with high-throughput sequencing was used to identify regions of Spc97 and Spc98 that were essential for the formation of the γ-tubulin small complex and mutations that influenced the organization of the mitotic spindle. Regions essential for structure and function of the complex were mapped onto the protein sequence for both Spc97 and Spc98 and temperature sensitive mutants identified by the mutagenic screen were isolated and their unique nucleation phenotypes characterized. Improvements were made to historical spindle pole body purification methods to facilitate a variety of in vitro biochemical and biophysical assays that had previously been hindered by technological constraints. Higher spindle pole body yields, purity, and reproducibility enabled a re-evaluation of the yeast spindle pole body cell cycle phosphoproteome. High confidence phosphorylation assignments were collected and compared to previously published data sets to reduce the ambiguity in the phosphoproteome data set while also expanding the data set to analyze the novel phosphorylation profile of the spindle pole body in G1/S. In vivo characterization of the high confidence phosphorylation sites revealed a combination of phosphorylation events in Spc97 that were essential for cell viability. The adaptability of the spindle pole body purification methods also allowed for the purification of genetic mutants of the spindle pole body, which were studied by biophysical assays. The strength of the microtubule attachment to the spindle pole body was probed by a laser trapping technique to determine how mutations in spindle pole body component Spc110 affected the physical integrity of the spindle pole body. It was shown that mutations in Spc110 decreased the force at which the microtubule was pulled from the spindle pole body, providing mechanical evidence for in vivo phenotypes of mitotic spindle failure. Together, these experiments interrogated several aspects of the yeast spindle pole body in vivo and in vitro to better understand the requirements for and regulation of microtubule nucleation.