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 CAMSAPs 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, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

γ-微管蛋白环复合物（γ-TuRC）是主要的微管成核因子。成核后，微管可以从 γ-TuRC 中释放并被其他蛋白质（例如 CAMSAP）稳定，但负端调节途径之间的生化串扰尚不清楚。在这里，我们使用纯化的成分在体外重建了这个过程。我们发现所有 CAMSAP 都可以与 γ-TuRC 附着的微管的负端结合。CAMSAP2 和 CAMSAP3 装饰和稳定生长的负端，但不修饰和稳定负端跟踪蛋白 CAMSAP1，诱导 γ-TuRC 释放微管。CDK5RAP2（一种 γ-TuRC 相互作用物）和 CLASP2（一种微管生长调节剂）强烈刺激 γ-TuRC 依赖性微管成核，但只有 CDK5RAP2 抑制 CAMSAP 与 γ-TuRC 锚定负端的结合及其释放。在微管加帽测定中，CDK5RAP2 还提高了含 γ-微管蛋白复合物对 13-而不是 14-原丝微管的选择性。细胞中的敲除和过表达实验表明，CDK5RAP2 抑制与微管组织中心分离的 CAMSAP2 结合微管的形成。我们得出的结论是，CAMSAP 可以从 γ-TuRC 中释放新成核的微管，而成核促进因子可以差异性地调节这一过程。