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The molecular structure of mammalian primary cilia revealed by cryo-electron tomography

Abstract

Primary cilia are microtubule-based organelles that are important for signaling and sensing in eukaryotic cells. Unlike the thoroughly studied motile cilia, the three-dimensional architecture and molecular composition of primary cilia are largely unexplored. Yet, studying these aspects is necessary to understand how primary cilia function in health and disease. We developed an enabling method for investigating the structure of primary cilia isolated from MDCK-II cells at molecular resolution by cryo-electron tomography. We show that the textbook ‘9 + 0’ arrangement of microtubule doublets is only present at the primary cilium base. A few microns out, the architecture changes into an unstructured bundle of EB1-decorated microtubules and actin filaments, putting an end to a long debate on the presence or absence of actin filaments in primary cilia. Our work provides a plethora of insights into the molecular structure of primary cilia and offers a methodological framework to study these important organelles.

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Fig. 1: Room temperature ET of MDCK-II primary cilia.
Fig. 2: Cryo-peel off: a method to prepare primary cilia for cryo-ET.
Fig. 3: IFT-B-like polymers are visible in cryo-ET of primary cilia.
Fig. 4: Proteins are present in the microtubule lumen in MDCK-II primary cilia (MIPs).
Fig. 5: Cryo-ET of primary cilia shows decorations of microtubule singlets by EB1.
Fig. 6: Primary cilia contain actin filaments.

Data availability

The cryo-ET density maps have been deposited with the Electron Microscopy Data Bank under accession nos. EMD-10900 (actin filament map) and EMD-10896 (EB1-decorated A-microtubule). Measurements of ciliary length are presented in Supplementary Data 1.

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Acknowledgements

We thank the Electron Microscopy Facility (in particular T. Fürstenhaupt, W. Leng, M. Wilsch-Bräuninger) and the Light Microscope Facility from the Services and Facilities of the Max Planck Institute of Molecular Cell Biology and Genetics (Dresden) for their support. We thank H. Rägel and C. Martin-Lemaitre for their tips on the MDCK-II cell culture, N. Walker for the Imaris tutorial and T.-O. Buchholz for denoising the cryo-ET data. We thank P. Tomancak, F. Jug, D. Diener and J. Brugues for the fruitful discussions and suggestions on the manuscript. We thank O. Gonzales for IT support. We thank the Light Microscopy Core Facility, IMG CAS (Prague) for their support with the confocal imaging. This work was supported by the Max Planck Society and by the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant no. 819826) to G.P. Work in V.V.’s laboratory was supported by the Czech Science Foundation (project no. 20-23165J).

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Contributions

P.K. developed the cryo-peel off method, prepared the samples for FM and EM imaging, acquired and reconstructed room temperature and cryo-tomograms, contributed to the FM data acquisition, analyzed the EM and FM data, prepared the figures, interpreted the results and contributed to writing and revising the manuscript. G.A.V. prepared the samples and contributed to data acquisition of the room temperature tomography, analyzed the cryo-EM data with StA and tomogram segmentation, analyzed the EM and FM data, prepared the figures, interpreted the results and contributed to writing and revising the manuscript. N.T. analyzed the cryo-ET data to average the microtubule singlets, contributed to the preparation of the supplementary figures, contributed to the interpretation of the data and contributed to writing and revising the manuscript. R.M. prepared the samples for FM, contributed to the FM data acquisition and contributed to creating the figures. P.G. and V.V. generated the MDCK-II cells stably expressing mNeonGreen-tagged EB1 and imaged them using confocal microscopy. A.H. contributed to the FM experimental design, provided access to research equipment, contributed to data interpretation and revised the manuscript. G.P. conceived and supervised the project, contributed to the experimental design, data analysis and results interpretation, contributed to writing the manuscript and creating the figures, provided access to crucial research components and provided funding.

Corresponding author

Correspondence to Gaia Pigino.

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Extended data

Extended Data Fig. 1 Assessment of structural measurements performed on averaged data of microtubule singlets from MDCKII primary cilia.

a, Fourier Shell Correlation (FSC) curve of the average electron density map from MDCKII microtubule singlets depicting the resolution associated with typical criteria (FSC = 0.5 and 0.143). b, Power spectrum of the singlet microtubule average showing tubulin monomer and dimer repeats.

Extended Data Fig. 2 Immunofluorescence microscopy of EB1 in primary cilia.

A1-3 Immunofluorescence staining showing EB1 (green) and acetylated tubulin (magenta) along the axoneme of peeled-off cilia. B1-3 and C1-3 EB1 (green) is also present along cell-attached cilia of wildtype cells and of cells stably expressing mNeonGreen-tagged EB1 (mNG-EB1). In B, cilia were stained with an antibody against EB1 (green) while in C, EB1 was imaged directly through the fluorescence signal of the mNG tag (mNG-EB1, green). Magenta, acetylated tubulin; Green, EB1; (-), cilium base.

Extended Data Fig. 3 Vesicles were found in the vicinity of ciliary membranes and ciliary filaments.

(a-g), Longitudinal cryo-tomographic slices through peeled-off MDCKII primary cilia showing the presence of vesicles (V) in the proximity of the cilium, often tethered to the ciliary membrane by thin connections (C). (b-g). Half of the identified vesicles were found along regions of the cilium that contained filaments (l). Magenta arrowheads indicate examples of filaments located in the vicinity of vesicles (a,d-j). (h-j), Vesicles were found associated with the ciliary membrane, and in the proximity of filaments, also in tomograms from fixed and plastic embedded cell-attached cilia. k, Number of cryo-tomograms containing membrane and filament associated vesicles. l, Quantification of vesicles with diverse interactions with ciliary membrane and filaments in cryo-tomograms. C, vesiclemembrane connections; MT, microtubule; M, membrane, V, vesicle.

Supplementary information

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Supplementary Tables 1–3.

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Supplementary Video 1

Proximodistal tomographic sections through resin-embedded MDCK-II primary cilium from the ciliary base towards the ciliary shaft depicting the early migration of a doublet towards the center of the axoneme.

Supplementary Video 2

Proximodistal sections through a resin-embedded MDCK-II primary cilium depicting the rotation of the inner junction of a microtubule doublet around the ciliary central axis, measured across a portion of the proximal end of the doublet region.

Supplementary Video 3

Proximodistal cryo-tomographic slices through a MDCK-II primary cilium depicting the location of each microtubule singlet seam (colored dots).

Supplementary Video 4

Proximodistal cryo-tomographic slices through a MDCK-II primary cilium showing the termination of a microtubule singlet and the presence of two IFT-B polymers.

Supplementary Video 5

Longitudinal cryo-tomographic slices through a MDCK-II primary cilium depicting the presence of two IFT-B polymers.

Supplementary Video 6

Longitudinal slices through a cryo-CARE-denoised tomogram depicting the presence of EB1 singlet decoration and F-actin within the primary cilium of MDCK-II cells.

Supplementary Video 7

Confocal microscopy of MDCK-II cells stably expressing mNeonGreen-tagged EB1. EB1 signal is visible in the cilium and in the cytoplasm (EB1 comets). In the cilium, the mNG-EB1 signal is stronger at the base and progressively decreases towards the tip, probably because of the reduced number of microtubules towards the ciliary tip.

Supplementary Video 8

Fitting of a deposited structure of F-actin (EMD-6448) (gray surface) in the subtomogram-averaged model of F-actin from the primary cilium (magenta mesh).

Supplementary Data 1

Measurements of ciliary length under different experimental conditions: cilia attached to cells imaged by confocal immunofluorescence microscopy, peeled-off cilia on glass slides imaged by immunofluorescence microscopy and peeled-off cilia imaged by cryo-EM.

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Kiesel, P., Alvarez Viar, G., Tsoy, N. et al. The molecular structure of mammalian primary cilia revealed by cryo-electron tomography. Nat Struct Mol Biol 27, 1115–1124 (2020). https://doi.org/10.1038/s41594-020-0507-4

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