Hedgehog-induced ciliary trafficking of kinesin-4 motor KIF7 requires intraflagellar transport but not KIF7’s microtubule binding

The kinesin-4 motor KIF7 is a conserved regulator of the Hedgehog signaling pathway. In vertebrates, Hedgehog signaling requires the primary cilium, and KIF7 and Gli transcription factors accumulate at the cilium tip in response to Hedgehog activation. Unlike conventional kinesins, KIF7 is an immotile kinesin and its mechanism of ciliary accumulation is unknown. We generated KIF7 variants with altered microtubule binding or motility. We demonstrate that microtubule binding of KIF7 is not required for the increase in KIF7 or Gli localization at the cilium tip in response to Hedgehog signaling. In addition, we show that the immotile behavior of KIF7 is required to prevent ciliary localization of Gli transcription factors in the absence of Hedgehog signaling. Using an engineered kinesin-2 motor that enables acute inhibition of intraflagellar transport, we demonstrate that kinesin-2 KIF3A/KIF3B/KAP mediates the translocation of KIF7 to the cilium tip in response to Hedgehog pathway activation. Together, these results suggest that KIF7’s role at the tip of the cilium is unrelated to its ability to bind to microtubules.