Dynamics And Function Of Cytoplasmic Dynein-2 In Caenorhabditis Elegans Sensory Cilia

The assembly and maintenance of cilia depend on the microtubule motor-driven bidirectional intraflagellar transport(IFT).Although the function of kinesin-2 that drives anterograde IFT has been extensively studied,the composition,dynamics and regulatory mechanisms of cytoplasmic dynein-2 that powers retrograde IFT are unclear.Like cytoplasmic dynein-1,cytoplasmic dynein-2 consists of a pair of ~500 kD motor subunits(or heavy chains)and several accessory subunits,including intermediate chains(ICs),light intermediate chains(LICs)and light chains(LCs).The cytoplasmic dynein-1 accessory subunits may participate in cytoplasmic dynein-2-mediated retrograde IFT.However,because of their essential roles in cell division,their function in ciliogenesis cannot be studied at the post-mitotic stage using conventional germ line knockout.In addition,how cytoplasmic dynein-2 heavy chain moves in vivo,how heavy chain coordinates with accessory subunits during retrograde IFT and how its motility is regulated are not clear.In this study,we established the CRISPR/Cas9-based knock-in and conditional knockout systems to dissect the dynamics and functions of cytoplasmic dynein subunits in C.elegans sensory cilia.Knockout of embryonically essential accessory subunits in the sensory neurons reveals that the intermediate chain DYCI-1,light chain DLC-1 and the dynein regulator LIS-1 are involved in ciliogenesis and the light intermediate chain DLI-1 functions in the dendrites to affect cilium formation indirectly.By labeling the endogenous cytoplasmic dynein-2 heavy chain and multiple IFT proteins with fluorescent tags,we found that the speed of cytoplasmic dynein-2-driven retrograde IFT is relatively slow at the initial and terminating phases,suggesting that the movement of cytoplasmic dynein-2 is tightly regulated.Distinct turnaround behaviors of heavy chain and accessory subunits indicate that cytoplasmic dynein-2 complex is heterogeneous and the incorporation of accessory subunits may be involved in regulating its motility.Consistently,we further showed that the short-rib polydactyly syndrome-related mutant R295 C,which is located at the putative accessory subunit binding region of heavy chain tail domain,affects its motility and frequency.In addition,subcellular localization analysis of truncation mutants revealed that the heavy chain tail domain(rather than the motor domain)and LIC mediate the ciliary entry of cytoplasmic dynein-2.Finally,we identified the C.elegans homolog of the IFT-A component IFT43 by mass spectrometry and found that IFT43 and IFT139 regulate the cytoplasmic dynein-2 motility in a partially redundant fashion.