Determining Temporal Localization of Branching Factors for Spindle Assembly

Abstract

Branching microtubule nucleation is a pathway responsible for spindle assembly and chromosome segregation during mitosis. Two proteins required for branching are Augmin and γ-tubulin. While the functions of these proteins in the context of branching are well understood, their spatial distribution in interphase and their colocalization with each other has not been thoroughly characterized. In this thesis, an optimized and replicable protocol for the synchronization and fixation of mitotic cells, and for fluorescent labeling of relevant proteins, MTs, and DNA is reported. After imaging these cells, the distribution of Augmin and γ-tubulin in interphase, their colocalization with each other and with spindle MTs, and their enrichment to specific subcellular regions was investigated. Among other findings, γ-tubulin was observed to be highly mobile in early mitosis, as it is substantially recruited to the growing spindle in prophase. Augmin’s association with spindle MTs and with γ-tubulin, meanwhile, remains generally consistent throughout mitosis. These results corroborate previously established processes underlying spindle assembly, such as RanGTP regulation. They also demonstrate evidence for poorly understood aspects of branching, such as the binding sequence of Augmin and γ-tubulin at the branch site and provide future avenues for future investigations into branching MT nucleation.