CAMSAP-driven microtubule release from γ-TuRC and its regulation by nucleation-promoting factors

Abstract

{gamma}-tubulin ring complex ({gamma}-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from {gamma}-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here, we reconstituted this process in vitro using purified components. We found that all CAMSAP proteins could bind to the minus-ends of {gamma}-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends, but not the minus-end tracking protein CAMSAP1 induced microtubule release from {gamma}-TuRC. CDK5RAP2, a {gamma}-TuRC-interactor, and CLASP2, a regulator of microtubule growth, stimulated {gamma}-TuRC-dependent microtubule nucleation, but only CDK5RAP2 inhibited CAMSAP-driven microtubule detachment by suppressing CAMSAP binding to {gamma}-TuRC-anchored minus ends. CDK5RAP2 also improved {gamma}-TuRC selectivity for 13-rather than 14-protofilament microtubules in microtubule-capping assays. Our results support a model whereby CAMSAPs exploit an imperfect attachment between {gamma}-TuRC and the nucleated microtubule to promote minus-end elongation and release, whereas CDK5RAP2 improves the fit between {gamma}-TuRC and 13-protofilament microtubules and enhances nucleation.