Coiled-coil-mediated phase separation of Spef1 for non-centrosomal microtubule organization and function

Central-pair microtubules (CP-MTs) are non-centrosomal MTs essential for planar beat pattern of cilia. The CP-MT formation requires the MT-associated protein Spef1, but the underlying molecular mechanism remains unclear. Here, we show that Spef1 undergoes liquid-liquid phase separation (LLPS) to facilitate non-centrosomal MT assembly by enriching tubulins. The LLPS of Spef1 is mediated by its C-terminal coiled-coil (CC) domain. Crystallography reveals that the Spef1-CC domain forms a parallel CC dimer with a unique charge distribution pattern on the surface. The dimerization capacity and charge distribution of Spef1-CC are both critical for controlling in-vitro LLPS. Disruption of the dimerization capacity abolishes ciliary functions of Spef1. In contrast, a charge-changing mutant with attenuated LLPS still supports the CP-MT formation but results in cilia with abnormal beat pattern. Thus, the CC-mediated LLPS of Spef1 provides a mechanistic explanation for its prominent role in controlling non-centrosomal CP-MT organization and function in the axoneme. Significance statement The MT-associated protein Spef1 is a new essential player for the non-centrosomal CP-MT formation in motile cilia and flagella. This study reveals the unexpected LLPS feature of Spef1, leading to forming biomolecular condensates that enrich tubulins to facilitate non-centrosomal MT assembly. Spef1-CC contains a unique charge-distribution pattern, together with its dimerization capacity, contributing to multivalent interactions for initiating LLPS. The LLPS property of Spef1 is important for CP-MT formation and Spef1-mediated ciliary function. The formation of Spef1-LLPS condensates indicates that they work as MT nucleation centers and tubulin sources for the continuous growth of CP-MTs or repairing CP-MTs damaged during ciliary beating, and suggests that LLPS may be a common process for generating and organizing non-centrosomal CP-MTs in the axoneme.