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

Goncalves-Santos, F.; De-Castro, A. R. G.; Rodrigues, D. R. M.; De-Castro, M. J. G.; Gassmann, R.; Abreu, C. M. C.; Dantas, T. J.

doi:10.1101/2023.05.10.539247

bioRxiv · 10.1101/2023.05.10.539247

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

Goncalves-Santos, F.·De-Castro, A. R. G.·Rodrigues, D. R. M.·De-Castro, M. J. G.·Gassmann, R.·Abreu, C. M. C.·Dantas, T. J.

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Abstract

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 trains

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Goncalves-Santos, F., De-Castro, A. R. G., Rodrigues, D. R. M., De-Castro, M. J. G., Gassmann, R., Abreu, C. M. C., Dantas, T. J.. 2023-05-10. Hot-wiring dynein-2 uncovers roles for IFT-A in retrograde train assembly and motility. https://doi.org/10.1101/2023.05.10.539247

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