Cryo-ET and MD simulations reveal that dynein-2 is tuned for binding to the A-tubule of the ciliary doublet.

Eukaryotic cilia and flagella are thin structures present on the surface of cells, playing vital roles in signaling and cellular motion. Cilia assembly depends on intraflagellar transport (IFT) along doublet microtubules (doublets). Unlike dynein-1, which works on cytoplasmic singlet microtubules, dynein-2 works on the doublets inside cilia. Previous studies have shown that retrograde IFT, driven by dynein-2, occurs on the A-tubule of the doublet, suggesting an elusive mechanism by which dynein-2 recruits retrograde IFT to the A-tubule. Here, we investigated the molecular basis of this mechanism using cryo-electron tomography (cryo-ET), molecular dynamics (MD) simulations, and biochemical analysis. Our biochemical assays revealed that the microtubule-binding domain of dynein-2 exhibits a higher affinity for the ciliary doublets than dynein-1. Cryo-ET further visualized the preferential binding of dynein-2 to the A-tubule of the doublet. MD simulations suggest that dynein-2 prefers the tyrosinated tubulin lattice as is present in the A-tubule. These findings reveal a recruitment mechanism of retrograde IFT by dynein-2, providing new insights into the spatial and functional specialization of ciliary doublets.