The gamma-Tubulin Ring Complex promotes mitotic spindle integrity and acts as a microtubule minus-end cap during mitosis

Abstract

{gamma}-TuRC is the primary microtubule (MT) nucleator in eukaryotic cells. Vertebrate {gamma}-TuRC is composed of {gamma}-tubulin and the {gamma}-Tubulin Complex Proteins (GCPs): GCP2, GCP3, GCP4, GCP5, and GCP6. {gamma}-TuRC localizes to MT-Organizing Centers (MTOCs) and promotes MT nucleation by providing a structural template that mirrors the 13-protofilament symmetry of the MT lattice. To understand the contribution of individual {gamma}-TuRC subunits in mitotic spindle dynamics, we endogenously tagged GCP2, GCP4 or GCP6 with an Auxin-Inducible Degron (AID) tag, enabling precise and rapid depletion of each protein. When depletion occurred before mitotic entry, we observed that cells arrested in prometaphase and that loss of any single subunit resulted in displacement of all {gamma}-TuRC components from spindle poles. In cells with preformed spindles, depletion triggered rapid spindle collapse, demonstrating that {gamma}-TuRC remains essential for the maintenance of spindle integrity. Remarkably, depletion of KIF2A, a MT-depolymerizing kinesin, rescued spindle collapse after {gamma}-TuRC loss, suggesting that KIF2A activity contributes to the spindle instability observed in the absence of {gamma}-TuRC. These findings indicate a dual role of {gamma}-TuRC in mitosis: acting not only as a critical MT nucleation factor for initiation of spindle assembly but also as a stabilizing and capping structure at the minus ends of spindle MTs to preserve spindle integrity.