PT - JOURNAL ARTICLE AU - Gonçalves-Santos, Francisco AU - De-Castro, Ana R. G. AU - Rodrigues, Diogo R. M. AU - De-Castro, Maria J. G. AU - Gassmann, Reto AU - Abreu, Carla M. C. AU - Dantas, Tiago J. TI - Hot-wiring dynein-2 uncovers roles for IFT-A in retrograde train assembly and motility AID - 10.1101/2023.05.10.539247 DP - 2023 Jan 01 TA - bioRxiv PG - 2023.05.10.539247 4099 - http://biorxiv.org/content/early/2023/05/10/2023.05.10.539247.short 4100 - http://biorxiv.org/content/early/2023/05/10/2023.05.10.539247.full AB - 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 used CRISPR/Cas9-mediated editing to disable the dynein-2 autoinhibition mechanism in Caenorhabditis elegans, and assessed 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 uncover key roles for IFT-A in: mediating motor-train coupling during IFT turnaround; promoting retrograde IFT initiation; and modulating dynein-2 retrograde motility.Highlights- Hot-wiring mutations enable dynein-2 to undergo retrograde movement in IFT-A-deficient cilia- Disabling dynein-2 autoinhibition reveals that multiple mechanisms restrain dynein-2 activity during anterograde IFT- IFT-A promotes retrograde IFT initiation and efficient dynein-2 motility- IFT-A mediates dynein-2 coupling to retrograde IFT trainsCompeting Interest StatementThe authors have declared no competing interest.