Delivery of intraflagellar transport proteins to the ciliary base and assembly into trains.

Anterograde intraflagellar transport (IFT) trains, composed of IFT-B, IFT-A, and BBSome subcomplexes, are responsible for transporting ciliary proteins into the cilium. How IFT subcomplexes reach the ciliary base and assemble into IFT trains is poorly understood. Here, we perform quantitative single-molecule imaging in chemosensory cilia to uncover how IFT subcomplexes arrive at the base, organize in IFT trains, and enter the cilium. We find that BBSomes reach the base via diffusion where they either associate with assembling IFT trains or with the membrane surrounding the base. In contrast, IFT-B and IFT-A reach the base via directed transport most likely on vesicles that stop at distinct locations near the base. Individual subcomplexes detach from the vesicles into a diffusive pool and associate to assembling trains. Our results show that IFT-B is first incorporated into IFT trains, followed by IFT-A, and finally BBSomes, indicating that the assembly of IFT trains is a highly regulated, step-wise process.