Phosphorylation-dependent regional motility of the ciliary kinesin OSM-3

Kinesin motor proteins, vital for intracellular microtubule-based transport, display region-specific motility within cells, a phenomenon that remains molecularly enigmatic. This study focuses on the localized activation of OSM-3, an intraflagellar transport kinesin crucial for the assembly of ciliary distal segments in Caenorhabditis elegans sensory neurons. Fluorescence lifetime imaging microscopy unveiled an extended, active conformation of OSM-3 in the ciliary base and middle segments, where OSM-3 is conveyed as cargo by kinesin-II. We demonstrate that NEKL-3, a never in mitosis kinase-like protein, directly phosphorylates the motor domain of OSM-3, inhibiting its in vitro activity. NEKL-3 and NEKL-4, localized at the ciliary base, function redundantly to restrict OSM-3 activation. Elevated levels of protein phosphatase 2A at the ciliary transition zone or middle segments triggered premature OSM-3 motility, while its deficiency resulted in reduced OSM-3 activity and shorter cilia. These findings elucidate a phosphorylation-mediated mechanism governing the regional motility of kinesins.