SPACA9 Acts as a Molecular Staple Modulating Microtubule Dynamic Instability

Motile cilia rely on highly stable axonemal microtubules reinforced by microtubule inner proteins (MIPs) that form a network within their lumen, yet the functions of individual MIPs remain poorly understood. Here, we characterize the conserved MIP sperm acrosome-associated protein 9 (SPACA9), which localizes to respiratory cilia and sperm flagella. Using in vitro reconstitution assays, we show that human SPACA9 (hSPACA9) acts as a molecular staple: it stabilizes protofilaments at growing microtubule ends, and inhibits dynamic instability. Surprisingly, these effects do not confer resistance to motor-induced lattice damage, indicating that regulation of microtubule dynamics can be uncoupled from mechanical resilience. Mechanistically, we identify an unstructured C-terminal region that is sufficient for microtubule binding and recapitulates the effects on dynamics. Together, our findings reveal functional specialization among MIPs and provide a mechanistic framework for how lumenal proteins tune distinct properties of axonemal microtubules.