Direct observation of branching MT nucleation in living animal cells.

Abstract

Centrosome-mediated microtubule (MT) nucleation has been well-characterized; however, numerous non-centrosomal MT nucleation mechanisms exist. The branching MT nucleation pathway envisages that the {gamma}-tubulin ring complex ({gamma}-TuRC) is recruited to MTs by the augmin complex to initiate nucleation of new MTs. While the pathway is well-conserved at a molecular and functional level, branching MT nucleation by core constituents has never been directly observed in animal cells. Here, multi-color TIRF microscopy was applied to visualize and quantitatively define the entire process of branching MT nucleation in dividing Drosophila cells. The stereotypical branching nucleation event entailed augmin first binding to a \"mother\" MT, recruitment of {gamma}-TuRC after 16s, followed by nucleation 15s later of a \"daughter\" MT at a 36{degrees} branch angle. Daughters typically remained attached throughout their ~40s lifetime unless the mother depolymerized past the branch point. Assembly of branched MT arrays, which did not require D-TPX2 (Drosophila TPX2) or evident regulation by a RanGTP gradient, enhanced localized RhoA activation during cytokinesis.