Role and Mechanisms of Microtubule Nucleation in Spindle Assembly

点击翻译按钮获取中文摘要

Project Summary Cell division is orchestrated by the mitotic spindle, composed of hundreds of thousands of microtubules (MT). Since the discovery of the MT building block tubulin 50 years ago, scientists have puzzled over how the mitotic spindle assembles via MTs and executes chromosome segregation despite a MT turnover of seconds. Now we know that spindle assembly relies largely on MT nucleation. Yet, when, where, and how MTs are nucleated, and how they are subsequently incorporated into the bipolar spindle, remains unclear. Based on my discovery of branching MT nucleation, my laboratory contributed to understanding how several essential factors, namely the protein complex augmin, the phase-separating protein TPX2, and the nucleator g- TuRC, conduct this reaction. Meanwhile it has been shown that this mechanism creates a majority of MTs in a spindle. As a result of this work, we are in an ideal position to investigate how branching MT nucleation is incorporated into spindle assembly to produce a continuous MT framework supporting chromosome segregation. We will pursue three aims: Aim 1: Determine where, when, and how MTs form in the vicinity of chromosomes. We will observe exactly where and when MTs form at purified chromosomes in Xenopus egg extract. We developed a novel assay to visualize MT nucleation from chromosomes, a direct visualization that is difficult to do in living cells. Further, we will define the contribution toward MT generation of the RanGTP pathway, branching MT nucleation, the chromosomal passenger complex, and the kinetochore. We hypothesize that branching MT nucleation is the main source of MTs from chromosomes. Aim 2: Elucidate how importins regulate onset of branching microtubule nucleation. RanGTP releases the spindle assembly factor TPX2 from importins, which then stimulates branching MT nucleation. Previous studies assumed that TPX2 exists as a monomer. In contrast, we recently showed that the active form of TPX2 undergoes a liquid liquid phase separation (LLPS), and importins inhibit this TPX2 condensate. How importins achieve inhibition of TPX2’s LLPS is not only important for MT assembly but also widely relevant in cell biology, as few studies have described how inhibition of LLPS can regulate cellular function. We will further assess whether the second essential branching factor, augmin, is also regulated by RanGTP, a pathway that would provide additional control for cell-cycle regulation. Aim 3: Provide mechanistic insight into the core branching factor augmin. To determine the structural basis of augmin-medidated branching MT nucleation, we will solve the single particle cryo-EM structure of augmin. This work will reveal the location and fold of augmin’s eight subunits. Using structure-function analysis, we will investigate the functional interfaces through which augmin binds to MTs and g-TuRC, besides interrogating how augmin and TPX2 interact. Achieving these aims will help answer open and pressing questions in cell biology about how MT nucleation occurs in the correct location and at the correct time to assemble the mitotic spindle.