Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

微管双联体 (MTD) 包含附着在完整圆柱形微管侧面的不完整微管（B 管）。这些复合微管在整个生命树的纤毛中得到保守。然而，MTD在体内形成和维持的机制仍然知之甚少。在这里，我们将微管相关蛋白 9 (MAP9) 鉴定为 MTD 相关蛋白。我们证明，线虫MAPH-9（一种MAP9同源物）存在于MTD组装过程中，并且仅定位于MTD，这种偏好部分是由微管蛋白多谷氨酰化介导的。我们发现 MAPH-9 的缺失会导致超微结构 MTD 缺陷，包括 B 管缩短和/或压扁，原丝数量减少、轴丝运动速度失调和纤毛功能紊乱。因为我们发现哺乳动物直系同源物 MAP9 定位于培养的哺乳动物细胞和小鼠组织中的轴丝，所以我们提出 MAP9/MAPH-9 在调节纤毛运动和支持轴丝 MTD 结构中发挥保守作用。