In vivo imaging reveals independent intraflagellar transport of the nexin-dynein regulatory complex subunits DRC2 and DRC4.

Many axonemal proteins enter cilia and flagella on intraflagellar transport (IFT) trains, which move bidirectionally along the axonemal microtubules. Certain axonemal substructures including the radial spokes and outer dynein arms are preassembled in the cell body and transported as multisubunit complexes into flagella by IFT. Here, we used in vivo imaging to analyze the transport and assembly of DRC2 and DRC4, two core subunits of the nexin-dynein regulatory complex (N-DRC). Tagged DRC2 moved by IFT in mutants lacking DRC4 and vice versa, showing that they do not depend on each other for IFT. Simultaneous imaging of tagged DRC2 and DRC4, expressed from transgenes that rescue a corresponding double mutant, mostly showed transport on separate IFT trains, but occasional cotransports were also observed. The results demonstrate that DRC2 and DRC4 are transported largely independently of each other into flagella. These studies suggest that the N-DRC assembles onto the axoneme by the stepwise addition of subunits.

Loss of ARL13 impedes BBSome-dependent cargo export from Chlamydomonas cilia.

The GTPase Arl13b participates in ciliary protein transport, but its contribution to intraflagellar transport (IFT), the main motor-based protein shuttle of cilia, remains largely unknown. Chlamydomonas arl13 mutant cilia were characterized by both abnormal reduction and accumulation of select membrane-associated proteins. With respect to the latter, a similar set of proteins including phospholipase D (PLD) also accumulated in BBSome-deficient cilia. IFT and BBSome traffic were apparently normal in arl13. However, transport of PLD, which in control cells moves by BBSome-dependent IFT, was impaired in arl13, causing PLD to accumulate in cilia. ARL13 only rarely and transiently traveled by IFT, indicating that it is not a co-migrating adapter securing PLD to IFT trains. In conclusion, the loss of Chlamydomonas ARL13 impedes BBSome-dependent protein transport, resulting in overlapping biochemical defects in arl13 and bbs mutant cilia.