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

γ-tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-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 γ-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 γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 inhibited CAMSAP-driven microtubule detachment by suppressing CAMSAP binding to γ-TuRC-anchored minus ends. CDK5RAP2 also improved γ-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 γ-TuRC and the nucleated microtubule to promote minus-end elongation and release, whereas CDK5RAP2 improves the fit between γ-TuRC and 13-protofilament microtubules and enhances nucleation.