RT Journal Article SR Electronic T1 Collaborative role of two distinct cilium-specific cytoskeletal systems in driving Hedgehog-responsive transcription factor trafficking JF bioRxiv FD Cold Spring Harbor Laboratory SP 2024.09.26.615198 DO 10.1101/2024.09.26.615198 A1 Ku, Pei-I A1 Sreeja, Jamuna S A1 Chadha, Abhishek A1 Williams, David S A1 Engelke, Martin F A1 Subramanian, Radhika YR 2024 UL http://biorxiv.org/content/early/2024/10/01/2024.09.26.615198.abstract AB Calibrated transcriptional outputs in cellular signaling require fine regulation of transcription factor activity. In vertebrate Hedgehog (Hh) signaling, the precise output of the final effectors, the GLI (Glioma-associated-oncogene) transcription factors, depends on the primary cilium. In particular, the formation of the activator form of GLI upon pathway initiation requires its concentration at the distal cilium tip. However, the mechanisms underlying this critical step in Hh signaling are unclear. We developed a real-time imaging assay to visualize GLI2, the primary GLI activator isoform, at single particle resolution within the cilium. We observed that GLI2 is a cargo of Intraflagellar Transport (IFT) machinery and is transported with anterograde bias during a restricted time window following pathway activation. Our findings position IFT as a crucial mediator of transcription factor transport within the cilium for vertebrate Hh signaling, in addition to IFT’s well-established role in ciliogenesis. Surprisingly, a conserved Hh pathway regulator, the atypical non-motile kinesin KIF7, is critical for the temporal control of GLI2 transport by IFT and the spatial control of GLI2 localization at the cilium tip. This discovery underscores the collaborative role of a motile and a non-motile cilium-specific cytoskeletal system in determining the transcriptional output during Hh signaling.Competing Interest StatementThe authors have declared no competing interest.