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Vertebrate Smoothened functions at the primary cilium

Abstract

The unanticipated involvement of several intraflagellar transport proteins in the mammalian Hedgehog (Hh) pathway has hinted at a functional connection between cilia and Hh signal transduction1,2. Here we show that mammalian Smoothened (Smo), a seven-transmembrane protein essential for Hh signalling3, is expressed on the primary cilium. This ciliary expression is regulated by Hh pathway activity; Sonic hedgehog or activating mutations in Smo promote ciliary localization, whereas the Smo antagonist cyclopamine inhibits ciliary localization. The translocation of Smo to primary cilia depends upon a conserved hydrophobic and basic residue sequence homologous to a domain previously shown to be required for the ciliary localization of seven-transmembrane proteins in Caenorhabditis elegans4. Mutation of this domain not only prevents ciliary localization but also eliminates Smo activity both in cultured cells and in zebrafish embryos. Thus, Hh-dependent translocation to cilia is essential for Smo activity, suggesting that Smo acts at the primary cilium.

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Figure 1: Smo is expressed on nodal primary cilia.
Figure 2: Hh pathway activity regulates the ciliary localization of Smo.
Figure 3: Identification of a conserved Smo ciliary localization motif.
Figure 4: CLDSmo is inactive.

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Acknowledgements

We are grateful to F. Schaufele for microscopy assistance, and K. Liu for advice about MDCK cell culture. This work was assisted by valuable conversations with P.-T. Chuang, W. Marshall and A. Peterson. Support for microscopy was provided by the NIH. Other support was provided by the NIH (D.Y.R.S.), the Sandler Foundation and a March of Dimes Basil O'Connor Award (J.F.R.).

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Correspondence to Jeremy F. Reiter.

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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

α-Smo antibodies are specific. (JPG 1234 kb)

Supplementary Figure 2

Ciliary localization of Smo occurs in Shh-expressing nodal cells. (JPG 207 kb)

Supplementary Figure 3

Smo localization to cilia in populations of MDCK cells. (JPG 1575 kb)

Supplementary Figure 4

The presence of cilia correlates with Hedgehog responsiveness, and Shh induces the movement of Smo to the primary cilia of different cell types. (PDF 948 kb)

Supplementary Notes

This file contains Supplementary Figure Legends, Supplementary Table S1 and an additional reference. (DOC 28 kb)

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Corbit, K., Aanstad, P., Singla, V. et al. Vertebrate Smoothened functions at the primary cilium. Nature 437, 1018–1021 (2005). https://doi.org/10.1038/nature04117

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