Collaborative role of two distinct cilium-specific cytoskeletal systems in driving Hedgehog-responsive transcription...

In vertebrate Hedgehog (Hh) signaling, the precise output of the final effectors, GLI (glioma-associated oncogene) transcription factors, depends on the primary cilium. Upon pathway initiation, generating the precise levels of the activator form of GLI depends on its concentration at the cilium tip. The mechanisms underlying this critical step in Hh signaling are unclear. We developed an assay to visualize GLI2, the primary GLI activator isoform, at single-particle resolution within the cilium. We found that GLI2 is a cargo of intraflagellar transport (IFT) machinery. Anterograde-biased IFT loading of GLI2 in a restricted time window following pathway activation results in the tip accumulation of GLI2. Unexpectedly, we found that the conserved Hh regulator KIF7, a nonmotile kinesin, is important for the temporal control of IFT-mediated GLI2 transport and retention of GLI2 at the cilium tip. Our findings underscore a design principle where a cilia-specific cytoskeletal transport system and an Hh pathway-specific cytoskeletal protein collaboratively regulate GLI2 trafficking for Hh signaling.