Multivalent BBSome-IFT proximities support IFT-A-IFT-B coordination in vivo

Intraflagellar transport (IFT) depends on coordinated movement of IFT-A and IFT-B, yet how these assemblies remain coupled within dynamic ciliary trains is unclear. Here, we combine endogenous fluorescent knock-in tagging, quantitative in vivo FLIM-FRET, structural modeling, and targeted mutagenesis to define IFT organization in C. elegans and mammalian cells. FLIM-FRET mapping identifies proximity networks within IFT-B and places IFT144 near IFT172 and IFT88 in intact cilia. Loss of BBSome function is associated with weaker IFT-A-IFT-B proximity signals and divergent subcomplex transport dynamics, consistent with a contribution of the BBSome to IFT coordination in vivo. Split-GFP complementation places BBSome components near IFT-A and IFT-B, while structural modeling nominates complementary charged surfaces on the IFT46-IFT52 dimer and BBS7/BBS9. Charge-reversal mutations targeting these surfaces impair BBSome recruitment and functional rescue. Together, these findings support a model in which multivalent BBSome-IFT proximities contribute to IFT-A-IFT-B coordination during ciliary transport.