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.

Department of Biology Stanford University Stanford CA 94305 USA

Department of Biology Stanford University Stanford CA 94305 USA; Department of Genetics Stanford University School of Medicine Stanford CA 94305 USA

Howard Hughes Medical Institute Department of Biology Stanford University Stanford CA 94305 USA

Institut Curie Université PSL CNRS UMR3348 Orsay France; Université Paris Saclay CNRS UMR3348 Orsay France

Institute of Biotechnology Czech Academy of Sciences BIOCEV 25250 Vestec Prague West Czech Republic

The College of New Jersey Ewing NJ 08628 USA

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bioRxiv. 2023 Feb 23:2023.02.23.529616. doi: 10.1101/2023.02.23.529616. PubMed

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