Regulation of branching microtubule nucleation

Abstract

The development, maintenance, and propagation of eukaryotic life require the accurate segregation of chromosomes by microtubule-based mitotic and meiotic spindles. To rapidly and accurately form, spindles are nucleated and organized by associated proteins. Branching microtubule nucleation autocatalytically amplifies spindle microtubules from the lattice of pre-existing ones and is essential for generating spindles in higher eukaryotes. Spatiotemporal control of this process is conferred by the RanGTP-importins-α/β pathway and only a single downstream factor, Targeting Protein of xKlp2 (TPX2), is necessary to promote branching microtubule nucleation. However, the molecular mechanism of regulation and activity for TPX2 and other branching factors is unresolved. Recently, cytoskeletal effectors have been postulated to function through liquid-liquid phase separation with filament monomers. Here, I elucidate a molecular mechanism for regulation of branching microtubule nucleation by demonstrating that importins-α/β regulate the phase separation and MT localization of essential branching factors.

TPX2 undergoes liquid-liquid phase separation with the monomers of microtubules, tubulin, on preexisting microtubules. This spatial coordinates branching MT nucleation and enhances TPX2 efficiency. Both TPX2 condensation and function are inhibited by disruption of low affinity interactions via importins-a/β. Additionally, the microtubule localization of the essential branching factor augmin is directly regulated by the RanGTP and importins-α/β. Collectively, these data enhance our biophysical and biochemical understanding of how branching MT nucleation is regulated to achieve accurate spindle assembly and cell division.