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De-Castro, A. R. G.

Publications and source records attributed to De-Castro, A. R. G..

2 recordsLinked to original sources
bioRxiv2023

Hot-wiring dynein-2 uncovers 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 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

cell biology↗
bioRxiv2021

WDR60-mediated dynein-2 loading into cilia powers retrograde IFT and transition zone crossing

The dynein-2 motor complex drives retrograde intraflagellar transport (IFT), playing a pivotal role in the assembly and functions of cilia. However, the mechanisms that regulate dynein-2 motility remain poorly understood. Here, we identify the Caenorhabditis elegans WDR60 homolog (WDR-60) and dissect the roles of this intermediate chain using genome editing and live imaging of endogenous dynein-2/IFT components. We find that loss of WDR-60 impairs dynein-2 recruitment to cilia and its incorporation onto anterograde IFT trains, reducing the availability of the retrograde motor at the ciliary tip. Consistently, we show that less dynein-2 motors power WDR-60-deficient retrograde IFT trains, which move at reduced velocities and fail to exit cilia, accumulating on the distal side of the transition zone. Remarkably, disrupting the transition zones NPHP module almost fully restores ciliary exit of underpowered retrograde trains in wdr-60 mutants. This work establishes WDR-60 as a major contributor to IFT and the NPHP module as a roadblock to dynein-2 passage through the transition zone.

cell biology↗