Identification and Mechanism of a Novel Microtubule Nucleation Factor

Abstract

Microtubule (MT) cytoskeleton forms diverse structures such as the mitotic spindle and cellular appendages. These defined MT architectures are created by spatiotemporally regulating MT dynamics, transport and most importantly, nucleation. Since the identification of γ-tubulin (γ-TB) in 1989, it was thought to be the sole MT nucleating protein in the cell. However, purified γ-TB complexes display very low nucleation capacity and upon knockdown of γ-TB, the cell can still form MTs. Therefore, it has long been speculated that nucleation factors other than γ-TB may exist. However, what protein factors are critical for nucleating MTs and how this process occurs in the cell has remained a mystery.We discovered a novel protein that nucleates MTs. By using an isolated mitotic spindle assembly pathway, branching MT nucleation, we resolved individual MTs and directly observed every MT nucleation event. In the absence of this protein, no MTs emerge at all, thus resembling the phenotype of γ-TB depletion. The recombinant protein rescues the phenotype in a concentration-dependent manner, indicating that the protein has bonafide nucleation activity. Next, we identified the domains required for the nucleation activity of this protein by generating truncation constructs. In addition, we defined its role in relation to γ-TB and other proteins involved in MT nucleation. In summary, we identified a novel and critical MT nucleation factor and elucidated its mechanism, which provides insight into how MT nucleation occurs to assemble MT structures in the cell.