Hot-wiring dynein-2 establishes roles for IFT-A in retrograde train assembly and motility

Intraflagellar transport (IFT) trains, built around IFT-A and IFT-B complexes, are carried by opposing motors to import and export ciliary cargo. While transported by kinesin-2 on anterograde IFT trains, the dynein-2 motor adopts an autoinhibitory conformation until it needs to be activated at the ciliary tip to power retrograde IFT. Growing evidence has linked the IFT-A complex to retrograde IFT; however, its roles in this process remain unknown. Here, we use CRISPR-Cas9-mediated genome editing to disable the dynein-2 autoinhibition mechanism in Caenorhabditis elegans and assess its impact on IFT with high-resolution live imaging and photobleaching analyses. Remarkably, this dynein-2 “hot-wiring” approach reignites retrograde motility inside IFT-A-deficient cilia without triggering tug-of-war events. In addition to providing functional evidence that multiple mechanisms maintain dynein-2 inhibited during anterograde IFT, our data establish key roles for IFT-A in mediating motor-train coupling during IFT turnaround, promoting retrograde IFT initiation, and modulating dynein-2 retrograde motility. [Display omitted] •Hot-wiring mutations enable dynein-2 to undergo retrograde movement in IFT-A-deficient cilia•Disabling dynein-2 autoinhibition does not lead to tug-of-war during anterograde IFT•IFT-A promotes retrograde IFT initiation and efficient dynein-2 motility•IFT-A mediates dynein-2 coupling to retrograde IFT trains Gonçalves-Santos et al. show that hot-wiring C. elegans dynein-2, by disabling its autoinhibition mechanism, bypasses the need for IFT-A in the initiation of dynein-2 retrograde motility without causing tug-of-war during anterograde IFT. Notably, in the absence of IFT-A, hot-wired dynein-2 uncouples from retrograde trains and moves much slower.