Targeted dynein inhibition generates flagellar beating

SUMMARY Motile cilia and flagella are highly conserved organelles that are essential for the normal development and health of many eukaryotes including humans. To reveal the molecular mechanism of motility, we used cryo-electron tomography of active sea urchin sperm flagella to directly visualize the macromolecular complexes and their structural changes during flagellar beating. We resolved distinct conformations of dynein motors and regulators, and showed that many of them are distributed in bend-direction-dependent fashion in active flagella. Our results provide direct evidence for the conformational switching predicted by the “switch-point-hypothesis”. However, they also reveal a fundamentally different mechanism of generating motility by inhibiting dyneins, rather than activating them, causing an asymmetric distribution of force and thus bending. Our high-resolution structural and biochemical analyses provide a new understanding of the distinct roles played by various dyneins and regulators in ciliary motility and suggest a molecular mechanism for robust beating in an all-or-none manner. HIGHLIGHTS Direct visualization of the switching mechanism for flagellar motility. Flagellar beating is generated by oscillating the side of dynein inhibition. I1 dynein, I1-tether and N-DRC are key regulators that are involved in switching. Structural insights into distinct roles of dynein isoforms in flagellar motility.