Molecular mechanism and regulation of microtubule nucleation

Abstract

Composed of αβ-tubulin subunits, microtubules (MTs) form numerous networks that are vital to cellular function. MTs are nucleated in precise locations and times to assemble the cytoskeleton. Yet, the fundamental mechanism for how MTs are nucleated remains to be determined. While γ-tubulin ring complex (γ-TuRC) is known to be the universal MT nucleator, there is evidence that other nucleation factors may exist and remain to be identified. Further, precisely how γ-TuRC and other factors nucleate a MT is poorly understood. Here, using, cell biology, in vitro reconstitution, biochemical and biophysical methods, I identify a novel MT nucleation factor XMAP215 and determine how it nucleates MTs together with γ-TuRC. Finally, I show how MT nucleators are regulated to build specific structures.

While XMAP215 and γ-TuRC possess minimal nucleation activity individually, they synergize for efficient MT nucleation in cytosol and in vitro. XMAP215 directly binds γ-tubulin with its conserved C-terminal domain and recruits αβ-tubulin with its N-terminus. Thus, XMAP215 determines the rate of MT nucleation from γ-TuRC.

By developing a single molecule assay, I directly visualized nucleation of a MT from purified Xenopus laevis γ-TuRC. A high γ-/αβ-tubulin affinity facilitates assembly of a MT from γ-TuRC. Regulation of γ-TuRC’s molecular architecture defines its transition state such that four laterally-associated αβ-tubulin dimers that promote a closed γ-TuRC conformation prior to MT polymerization. Co-nucleation factor XMAP215 strengthens the longitudinal γ-/αβ-tubulin interaction.

To determine how MT nucleation is regulated to assemble cytoskeletal structures, I characterized the nucleation and dynamics of branched MT networks, a process that is critical for cell division. A pathway with rate-limiting, sequential localization of MT nucleation factors precisely regulates the assembly and architecture of branched MT networks.

Collectively, this thesis elucidates the fundamental mechanism of MT nucleation by the principal module composed of γ-TuRC and XMAP215 and how MT nucleation organizes specific cytoskeletal architectures.