In vitro approaches for the study of microtubule nucleation at the fission yeast spindle pole body

Abstract

Publisher Summary This chapter discusses the in vitro approaches for the study of microtubule nucleation at the fission yeast spindle pole body (SPB). In Saccharomyces cerevisiae , the SPB is a disk-shaped, multilayered structure that is embedded into the nuclear envelope throughout the cell cycle. The isolation procedure requires fractionation and lysis of the nuclei to release the SPB from the nuclear envelope. These isolated preparations have been used for defining the composition and the structure of the SPB. Microtubule-nucleating activity of the budding yeast SPB has been studied, by using the partially purified SPB. This chapter describes a procedure to permeabilize S. pombe cells for studying the mechanisms of spindle elongation and of microtubule nucleation at the SPB. The mitotic SPB, but not the interphase SPB, is capable of nucleating microtubules in vitro . This result indicates that γ-tubulin localized at the SPB is in an inative form during interphase. The interphase SPB can be activated for microtubule nucleation by incubation in mitotic extracts prepared from unfertilized Xenopus eggs. This activation seems to occur through interaction of an activator present in the extracts with γ-tubulin located at the SPB. Finally, the activator has been isolated from the extracts based on the in vitro activity and identified as the ribonucleotide reductase, large subunit R1.