The Mn-motif protein MAP6d1 assembles ciliary doublet microtubules

Most eukaryotic cells have cilia that serve vital functions in sensing, signaling, motility. The core architecture of cilia is an array of microtubule doublets, which consist of a complete A-tubule and an incomplete B-tubule. The mechanisms governing the assembly of this complex structure remain poorly understood. Here, using total internal reflection fluorescence microscopy and cryo-electron tomography, we investigate the role of MAP6d1, a brain-specific protein containing microtubule lumen-targeting Mn-motifs. We show that MAP6d1 assembles stable microtubule doublets by recruiting tubulin dimers onto the lattice of the A-tubule to initiate the nucleation of the B-tubule. MAP6d1 also promotes the formation of luminal protofilaments in singlet and doublet microtubules, a previously undescribed phenomenon that likely enhances microtubule stability. In neurons, MAP6d1 localises to the proximal part of primary cilia via its Mn-motif, with its loss resulting in shortened cilia, a characteristic of ciliopathies. MAP6d1 is thus the first microtubule-associated protein found to assemble microtubule doublets, uncovering new functions for Mn-motif proteins in neurons.