Abstract
Mammalian Hedgehog (Hh) signal transduction requires a primary cilium, a microtubule-based organelle, and the Gli–Sufu complexes that mediate Hh signalling, which are enriched at cilia tips. Kif7, a kinesin-4 family protein, is a conserved regulator of the Hh signalling pathway and a human ciliopathy protein. Here we show that Kif7 localizes to the cilium tip, the site of microtubule plus ends, where it limits cilium length and controls cilium structure. Purified recombinant Kif7 binds the plus ends of growing microtubules in vitro, where it reduces the rate of microtubule growth and increases the frequency of microtubule catastrophe. Kif7 is not required for normal intraflagellar transport or for trafficking of Hh pathway proteins into cilia. Instead, a central function of Kif7 in the mammalian Hh pathway is to control cilium architecture and to create a single cilium tip compartment, where Gli–Sufu activity can be correctly regulated.
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Acknowledgements
We thank L. Gunther-Cummins, P. Satir (Albert Einstein College of Medicine, Bronx, NY), K. Uryu (Rockefeller University) for assistance with TEM, and Alan Hall for use of a microscope for TIRF imaging. We thank H. Bazzi, A. Parrish and T. Bestor for helpful comments on the manuscript. We thank B. Tsou and W-J. Wang for technical support and helpful discussion. We thank C.C. Hui (Sick Children’s Hospital) for providing Kif7−/− mice. We thank J. Eggenschwiler (University of Georgia) and A. Salic (Harvard University) for antibodies. We thank N. Lampen for help with scanning electron microscopy and the Memorial Sloan-Kettering Cancer Center core facilities and the Rockefeller University Bio-Imaging Resource Center for help with imaging. We thank R. Hendrickson and H. Erdjument-Bromage for help with mass spectrometry. This work was supported by National Institutes of Health grants NS044385 to K.V. Anderson and GM65933 to T.M. Kapoor and the MSKCC Cancer Center Support Grant P30 CA008748.
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M.H. designed, carried out and analysed experiments and assembled figures. R.S. and M.H. carried out and analysed the microtubule dynamics assays. F.B. carried out IFT assays. T.O. helped with TIRF microscopy. K.F.L. did the TEM. T.M.K. designed, supervised and analysed the microtubule dynamics assays. R.S. and T.M.K. helped write and edit the manuscript. K.V.A. designed, supervised and analysed experiments. M.H. and K.V.A. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Kif7L130P encodes a stable protein that does not localize to cilium tips.
(a) Immunoblot with Kif7 antibody in cell lysates of wild-type, Kif7L130P and Kif7−/− MEFs. (b) Kif7 (green) is absent from Kif7L130P cilia regardless of pathway activation. Acetylated α-tubulin (red) stains cilia and γ-tubulin (blue) stains basal body. Arrows indicate Kif7 at the transition zone. Scale bar, 1 μm. (c) Kif7 localizes to cilium tip in Smo−/− and (d) Gli2−/−Gli3−/− double mutant MEFs. Acetylated α-tubulin (red) stains cilia and γ-tubulin (blue) in (c) and magenta in (d) stains basal body. Scale bar, 5 μm. (e) GM130 (green), a Golgi membrane protein, appears normal in Kif7L130P MEFs. DAPI in blue. Scale bar, 17 μm. (f) In contrast to Kif7 knockdown cells14, normal numbers of centrosomes and cilia are present in wild-type and Kif7L130P MEFs. Acetylated α-tubulin (red) marks axonemal microtubules and γ-tubulin (magenta) marks basal bodies. DAPI is in blue. n = 165 cilia were analysed and were pooled from 4 independent experiments. Error bars represent the s.d., P > 0.0001 by student t-test. Scale bar, 22 μm.
Supplementary Figure 2 Kif7L130P mutant cells have long cilia and abnormal axonemal structure.
(a) The diameter of proximal and distal region was measured from neural tube cilia from e10.5 wild-type and Kif7L130P embryos. The width of proximal cilia was 0.21 ± 0.02 μm in wild-type and 0.20 ± 0.02 μm in Kif7L130P. The width of distal cilia was 0.12 ± 0.02 μm in wild-type and 0.08 ± 0.01 μm in Kif7L130P (n = 50 cilia were measured for each genotype; P < 0.001 by one-way ANOVA analysis). Error bars represent the s.d. (b) Low power s.e.m. of the node of an e8.0 wild-type embryo (black square). (c) Node cilia from e8.0 wild-type and Kif7L130P mutant embryos. Scale bar, 2 μm. (d) TEM images of transverse sections of Kif7L130P neural tubule cilia (4 examples). Arrows indicate singlet microtubules. Scale bars, 50 nm.
Supplementary Figure 3 Defects of tubulin modification in Kif7−/− mutant MEFs.
Tubulin acetylation (red) and glutamylation (green) are reduced at the distal segment of cilia in Kif7−/− MEFs compared to wild type. Scale bar, 1 μm.
Supplementary Figure 4 Kif7 associates with microtubule in vivo.
(a) Protein domain analysis of mouse Kif7 and KIF4. Motor domain is in green, neck in pink and coiled-coil in purple. Note the long linker between the neck and the first coiled-coil of Kif7, which may inhibit its ability to act as a processive motor. Arrows and numbers indicate the positions of respective domains in amino acid. (b) Co-immunoprecipitation of Flag- and GFP-tagged full-length Kif7 in 293T cells. (c) Expression of C-terminal GFP-tagged Kif7-full length and Kif7560 constructs in HEK293T cells visualized by confocal fluorescent microscope. DAPI in blue. Scale bar, 8 μm. (d) Deletion analysis of Kif7 for in vivo microtubule bundling activity and dimerization.
Supplementary Figure 5 Purified Kif7560-GFP directly binds to microtubules.
(a) Purified Kif7560-GFP for in vitro analysis. Asterisk indicates bacterial protein Arna (Bifunctional polymyxin resistance protein Arna). (b) 70 nM Kif7560-GFP binds to X-rhodamine-labelled microtubules in the presence of 1 mM MgATP or 2 mM AMP-PNP. Scale bar, 5 μm.
Supplementary Figure 6 Induced microtubule destabilization by purified Kif7560-GFP.
Examples of fragmented microtubules observed when X-rhodamine-labeled microtubules were incubated with 500 nM of Kif7560-GFP in the presence of 1 mM MgATP.
Supplementary Figure 7 Kif7L130P mutant cilia have defects in IFT dynamics.
(a–b) Representative kymographs generated from time lapse imaging of wild-type and (C–D) Kif7L130P primary cilium (Supplementary Videos 1 and 2). (a) and (c) are taken from Fig. 3. The base and tip of the cilium are indicated with arrowheads. Asterisks indicate stationary IFT88 puncta associated with the initiation of retrograde trafficking. Horizontal scale bar (distance), 1 μm. Vertical scale bar (time), 7.5 s. Enlarged portion of kymographs are highlighted (yellow rectangles). Asterisks show the position of stationary IFT88 puncta. Our kymograph analysis shows IFT88–GFP (4/200 retrograde trains) switches tracks in the wild-type cilia very rarely, whereas significantly more trains (79/214 retrograde trains) switch tracks in Kif7 mutants. (e) Numbers of stationary IFT88–GFP puncta per cilium in wild-type and Kif7L130P (n = 50 cilia were imaged for each condition and were pooled from 5 independent experiments; P < 0.0001 by student t-test). The error bars represent s.d.
Supplementary Figure 8 Uncropped western blots referring to Supplementary Figs 1a and 4b.
Supplementary Fig. 1a Kif7 protein level in wild-type, Kif7L130P and Kif7−/− MEFs.γ-tubulin (asterisk) was used as loading control. Supplementary Fig. 4b co-immunoprecipitation of Flag- and GFP-tagged full-length Kif7 in 293T cells. Membranes were blotted with anti-Flag and anti-GFP antibodies, respectively.
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Live imaging of IFT88–GFP in wild-type cilia.
IFT visualized by time-lapse imaging of a wild-type primary cilium expressing IFT88–GFP. Images are taken every 250 ms for 30 s. Movie shows 10 frames s−1. (MOV 1813 kb)
Live imaging of IFT88–GFP in Kif7L130P mutant cilia.
IFT visualized by time-lapse imaging of a Kif7L130P primary cilium expressing IFT88–GFP. Images are taken every 250 ms for 30 s. Movie shows 10 frames s−1. (MOV 1607 kb)
Effect of Kif7560-GFP on dynamic microtubules.
Kif7560-GFP (70 nM; 1 mM MgATP) and dynamic X-rhodamine-microtubule visualized by time-lapse TIRF microscopy. Images are taken every 5 s for 15 min. Movie shows 15 frames s−1. (MOV 8959 kb)
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He, M., Subramanian, R., Bangs, F. et al. The kinesin-4 protein Kif7 regulates mammalian Hedgehog signalling by organizing the cilium tip compartment. Nat Cell Biol 16, 663–672 (2014). https://doi.org/10.1038/ncb2988
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DOI: https://doi.org/10.1038/ncb2988
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