CAMSAP2 condensates drive a γ-TuRC-independent pathway for non-centrosomal microtubule nucleation

Microtubule nucleation is commonly viewed as a γ-tubulin ring complex (γ-TuRC)-templated process ^1–3 , yet many differentiated cells build extensive non-centrosomal microtubule arrays of unclear origin ^4–6 . Although spontaneous tubulin nucleation has long been observed in vitro as a γ-TuRC-independent nucleation process ^7–10 , its mechanism and cellular relevance have remained unclear. Here we show that CAMSAP2, a microtubule minus-end-binding protein, links spontaneous nucleation to non-centrosomal microtubule organization. Cryo-electron microscopy (cryo-EM), high-speed atomic force microscopy (hsAFM), and molecular dynamics simulations reveal that CAMSAP2 lowers the nucleation barrier by straightening tubulin oligomers and promoting lateral protofilament interactions that drive sheet formation and closure into microtubules. hsAFM captures stepwise nucleation and early growth, revealing that tubulin rings can serve as productive intermediates rather than dead-end depolymerization products. CAMSAP2 further self-organizes through liquid–liquid phase separation (LLPS) to concentrate tubulin and assembly intermediates, thereby promoting efficient microtubule formation at non-centrosomal microtubule-organizing centers. In HeLa cells, dispersed CAMSAP2 condensates act as γ-TuRC-independent microtubule-organizing centers alongside centrosomal asters, supporting a model in which spontaneous nucleation contributes to non-centrosomal microtubule biogenesis.