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The cryo-EM structure of intraflagellar transport trains reveals how dynein is inactivated to ensure unidirectional anterograde movement in cilia

Nature Cell Biology volume 20, pages 1250–1255 (2018)Cite this article

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Abstract

Movement of cargos along microtubules plays key roles in diverse cellular processes, from signalling to mitosis. In cilia, rapid movement of ciliary components along the microtubules to and from the assembly site is essential for the assembly and disassembly of the structure itself1. This bidirectional transport, known as intraflagellar transport (IFT)2, is driven by the anterograde motor kinesin-23 and the retrograde motor dynein-1b (dynein-2 in mammals)4,5. However, to drive retrograde transport, dynein-1b must first be delivered to the ciliary tip by anterograde IFT6. Although, the presence of opposing motors in bidirectional transport processes often leads to periodic stalling and slowing of cargos7, IFT is highly processive1,2,8. Using cryo-electron tomography, we show that a tug-of-war between kinesin-2 and dynein-1b is prevented by loading dynein-1b onto anterograde IFT trains in an autoinhibited form and by positioning it away from the microtubule track to prevent binding. Once at the ciliary tip, dynein-1b must transition into an active form and engage microtubules to power retrograde trains. These findings provide a striking example of how coordinated structural changes mediate the behaviour of complex cellular machinery.

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Fig. 1: Anterograde IFT trains are composed of three different repeating units.
Fig. 2: Train structures of IFT-A and dynein-1b mutants reveal positioning of IFT components.
Fig. 3: Dynein-1b is inhibited by two concurrent mechanisms during anterograde transport.
Fig. 4: Retrograde IFT trains in cryo-electron microscopy.
Fig. 5: At the ciliary tip, dynein-1b appears to be in an open conformation.

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Data availability

The cryo-electron tomography density maps of IFT-A and dynein/IFT-B have been deposited in the EMDataBank (EMDB), accession codes: EMD-4303 and EMD-4304. Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD010699 (Project doi: 10.6019/PXD010699, http://proteomecentral.proteomexchange.org). Additional examples of images are available at figshare (https://figshare.com/s/00171c1da093f5461091, https://figshare.com/s/23fa7e8a449971134fe3) All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank T. Fürstenhaupt for his help in setting up the cryo-TEM facility in Dresden, A. Shevchenko and the mass spectrometry facility, and A. Bogdanova and the protein expression facility of the MPI-CBG for their help with the mutant analyses and plasmid construction. The authors are also grateful to W. Baumeister and his team for sharing the K2align package. Finally, thanks are given to A. Shaposhnykov for the reconstruction of preliminary tomograms, J. Rosenbaum for continuous inspiration, F. Jug and I.K. Patten for fruitful discussions, comments and corrections to the manuscript, and J. Brugués, S. Diez, V. Geyer, A. Hyman, T. Ishikawa, and J. Tabler for comments on the manuscript. This work was supported by the Max Planck Society.

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  1. Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany

    Mareike A. Jordan, Dennis R. Diener, Ludek Stepanek & Gaia Pigino

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Contributions

M.A.J. prepared the samples, acquired data at the microscope, performed image processing, interpreted the data and wrote the manuscript. D.R.D. prepared the samples, performed genetic experiments, acquired data, interpreted data and wrote the manuscript. L.S. prepared samples and acquired fla14 mutant data at the microscope. G.P. designed the experiments, analysed and interpreted the data and wrote the manuscript.

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Correspondence to Gaia Pigino.

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Integrated supplementary information

Supplementary Figure 1 Overall morphology of IFT trains in cilia of Chlamydomonas reinhardtii.

(a,b) Comparison of IFT trains from room temperature (RT) electron tomography (ET) of resin-embedded samples and cryo-ET of plunge frozen samples enables differentiation between anterograde and retrograde IFT trains in cryo-ET. (a), Anterograde IFT trains have a tight repetitive structure and they appear electron dense in both RT14 and cryo-ET. (b), Retrograde IFT trains are less dense and appear as elongated zig-zag like structures with a broader repeat in both RT14 and cryo-ET. They appear much shorter than standing trains14, although they share a similar repeat. The retrograde train in the first cryo-ET panel is also shown in Fig. 4a. The orientation of the tip is indicated by a plus. (c-e), Repeating distances of IFT particles are visualized in power spectra. Averages (top), show the 6-nm (c), 18-nm (d) and 11-nm (e) repeats of IFT-B, dynein and IFT-A, respectively. The corresponding power spectra (bottom) show layer lines at the respective frequencies and their multiples. In (e), a 6-nm satellite layer line, which identifies the IFT-B repeat, is visible near the second order line of IFT-A at 5.5 nm (arrowhead). This shows that IFT-A has a repeating distance that is slightly smaller than two repeats of IFT-B. (f,g), Two separate averages from IFT particles were generated with picking distances of 18 and 11 nm. While the 6-nm repeat of IFT-B was fully resolved in the 18-nm repeat average (grey), only parts of it were visible in the 11-nm average of IFT-A (yellow). To join both averages and show the complete train structure, the densities that appeared in both averages were used for the registration of the two models (overlapping of grey and yellow densities, black arrowheads in g). (f), Side view corresponding to Fig. 1j. The connection between IFT-A and -B (arrowhead) only emerges in the 18-nm average but is not visible in the 11-nm average. This indicates a flexible linkage between IFT-A and B, which could explain the non-integral ratio of their 6- and 11-nm repeats. (g), Orientation corresponding to Fig. 1l. Scale bars, 100 nm (a,b) 150 nm (c-e).

Supplementary Figure 2 Characterization of the mutants ift139-2 and dhc1b-3.

(a), Mass spectrometry of ‘membrane and matrix’ fractions shows a reduction of all IFT-A proteins in the ift139-2 mutant and of the dynein heavy chain DHC1b in the dhc1b-3 mutant. The values of each sample were normalized to the total amount of IFT-B proteins to facilitate a direct comparison of IFT-A and IFT-B proteins. Values were divided by protein molecular weight. In the ift139-2 mutant, the amount of IFT139 was reduced to 5% of wild-type. Other IFT-A proteins were reduced to 10-30%. These proteins could form the short irregular stumps that sporadically associated with anterograde IFT trains in tomograms (Supplementary Fig. 3c, yellow arrowheads). IFT-dynein DHC1b was reduced to 36%, explaining the presence of some flexible IFT trains consisting of only IFT-B. In the dhc1b-3 mutant, DHC1b was reduced to 4% at the restrictive temperature. In accordance with the proteomic analysis, we never observed trains with the 18-nm repeat structure of dynein in cryo-tomograms. IFT-A proteins were slightly enriched by ~30%, possibly due to inhibited retrograde transport and accumulation of IFT material in cilia. This experiment was performed twice with similar results. (b), Immunoblotting of ‘membrane and matrix’ fractions showed that IFT139 was depleted in ift139-2 cilia, whereas the IFT-B proteins IFT81 and IFT57 remained abundant. The ciliary membrane protein FMG1-B was used as a loading control. (c-f), Transformation of the mutant ift139-2 with the wild-type IFT139 gene rescued the presence of IFT-A in anterograde IFT trains. (c), Immunoblotting showed that IFT139 was restored in cilia of the rescued mutant ift139-2::IFT139. The axonemal protein RSP3 was used as a loading control. (d), Tomographic slices of an anterograde IFT train in the rescued mutant ift139-2::IFT139. Dynein-1b repeats are marked with blue arrowheads; the row of IFT-B particles is indicated by green arrowheads; the row of restored IFT-A particles is indicated by yellow arrowheads. The orientation of the tip is labeled by a plus. (e), Two different Z sections (top and bottom rows) of subtomogram averages of IFT-A from ift139-2, the rescued mutant ift139-2::IFT139, and the wild-type CC-125. Averages included 231 particles (ift139-2), 232 particles (ift139-2::IFT139) and 1450 particles (wt). Particles for the average from ift139-2::IFT139 derive from tomograms of three individual cells; all 9 trains analyzed showed IFT-A particles. While IFT-A is missing in the average of ift139-2, its density is restored in ift139-2::IFT139. (f), Phase contrast microscopy of Chlamydomonas cells shows characteristic blebs (arrows) at the distal parts of cilia from the ift139-2 mutant that have been reported previously for temperature sensitive IFT139 mutants19,41]. Transformation with the wild-type IFT139 gene rescued this phenotype. Blebs on the flagella of the ift139-2 were consistently seen in numerous experiments, and were counted and directly compared with wild type and ift139-2::IFT139 in one experiment that yielded: number of blebs/number of flagella: wild-type cc4533 = 0/180; ift139-2 = 146/200; ift139-2::IFT139 = 0/200. Scale bars, 50 nm (d), 10 nm (e) and 4 µm (f).

Supplementary Figure 3 IFT trains of IFT-A and dynein-1b mutants reveal positioning of IFT components.

(a-c), Slices through a tomogram of an anterograde train of the ift139-2 mutant show the 18-nm (a) and 6-nm (b) repeats, whereas the 11-nm repeat (c) was missing, indicating that IFT-A proteins are located within the 11-nm repeat structure. Sporadic stumps are indicated (yellow arrowheads) that could arise from remaining IFT-A proteins or cargos. (d), Views of the isosurface rendering of the average obtained from ift139-2 mutant trains. Averages were generated from 132 (dynein/IFT-B) and 231 (IFT-A) particles from 13 tomograms of individual cells. (e-g), At the restrictive temperature of 34°C, trains from the dhc1b-3 mutant lacked the 18-nm repeat (e), whereas the 6-nm (f) and 11-nm (g) repeats were present, identifying the 18-nm repeat as dynein-1b. (h), Views of the isosurface rendering of the average obtained from dhc1b-3 mutant trains. Averages were generated from 206 (dynein/IFT-B) and 300 (IFT-A) particles from 4 tomograms of individual cells. (i-k), Cross-sectional views of IFT particles averaged with an 18-nm picking distance. Z-projections of 100 tomographic slices (140 nm) show the signal from structures that do not align according to the 18-nm repeat. Structures are labelled in the lower panels. The 6-nm repeat structure (green) is well resolved in all averages. A blurred density of the 11-nm repeat (yellow) is visible in the wild-type (i) and in the dhc1b-3 mutant at restrictive temperature (k) but not in the ift139-2 mutant (j). This shows that the 11-nm repeat structure contains IFT-A proteins. Wild-type and ift139-2 mutant models show the 18-nm repeat structure (blue and grey in i and j), which was missing in the dhc1b-3 mutant, showing that this repeat contains dynein-1b. (l), IFT trains from ift139-2 show an irregular arrangement. Upper panel: The row of IFT-B particles (green arrowheads) shows a curve that is unusual for wild-type trains. Centre panel: In this train, dyneins (blue arrowheads) do not decorate the whole length of IFT-B particles (green arrowheads). Where the row of dynein ends, IFT-B particles deviate from their straight path (white arrowhead). This indicates that both dynein and IFT-A provide stability to the train morphology. Lower panel: IFT train consisting of only IFT-B particles (green arrowheads). The membrane adapts to the smaller size of the train and appears to be nearer to the microtubule doublet. (m-p), Cilia of fla14-2 were short and had to be isolated to be imaged in EM. Anterograde trains were rarely observed in these cilia, where IFT particles tend to accumulate in a seemingly disorganized manner. The train shown here was not associated with a microtubule doublet but remained tightly attached to the membrane. (m-o), Tomographic slices of the train (left) and of the corresponding averages (right) show the absence of the 18-nm dynein repeat (m), whereas the 6-nm IFT-B (n) and 11-nm IFT-A (o) repeats are present. (p), Isosurface of the train structure shows the absence of dynein. Orientations correspond to Fig. 1j, l, m. Averages were generated from 15 (dynein/IFT-B) and 23 (IFT-A) particles from one tomogram. (The orientation of the ciliary tip is indicated by a plus. G, glycocalyx; M, membrane; MTd, microtubule doublet with A and B tubule). Scale bars, 50 nm (a-c, e-g, l, m-o left), 20 nm (i-k) and 10 nm (m-o, right).

Supplementary Figure 4 Close-up view of dynein-1b, the putative position of kinesin-2 and potential cargo binding sites in an anterograde IFT train.

(a-d), IFT-B shows connections to the microtubule doublet that could be the anterograde motor kinesin-2. (a), View of IFT-B from the microtubule side (looking toward the membrane of the cilium) onto one 18-nm repeat that includes three repeats of IFT-B (green). An arrow indicates a connection between IFT-B and the dynein tail (grey). Globular domains of IFT-B (yellow stars) are visible in close proximity to the microtubule (c). (b), View from the membrane onto IFT-B and dynein, corresponding to the view in Fig. 1k. IFT-A is not shown here, thereby exposing the structure of IFT-B. To refine the structure of connections to the microtubule, smaller masks were built and applied to the reference for alignment (outlined in grey). (c), Close-up view of the globular domains that show a connecting density (magenta) to the microtubule doublet (MTd), which is probably the anterograde motor kinesin-2. For orientation of the lower panel, compare with Fig. 1j, where the position of kinesin-2 is marked by a pink dotted line. (d), The opposite end of IFT-B contains a pointed domain near the microtubule doublet. Earlier studies of plastic-embedded cilia, which showed two connections of IFT trains to the B-microtubule, suggested a second motor at this position12,14. However, the train structure is more flexible in this region and subtomogram averaging could not reveal an obvious connection to the microtubule (dotted line and question mark). The position of IFT-A is marked with yellow dashes. (e-h), Structure of the auto-inhibited dynein-1b in anterograde IFT trains. (e), Slice of the subtomogram average showing densities of both dynein stalks (marked with arrowheads). (f), The atomic model of human cytoplasmic dynein-2 (monomeric crystal structure PDB 4RH721, in dimeric conformation as visualized in ref22) is superimposed onto the average. The orientation of the ciliary base is indicated by a minus. (g,h), Comparison of the 3D structure of a single IFT dynein-1b dimer (g) with the human cytoplasmic dynein-1c23 (single particle density EMD-3705, Gaussian filtered, with the atomic model of the motor domains from PDB 5NVU) (h) revealed a kink between the tail and the motor domains in the IFT dynein-1b structure (dashed lines). This kink appears to be stabilized by a contact point between the tail and one of the AAA+ rings (star) that could correspond to the density of the regulatory light chains LC8 and Tctex, which are thought to associate with the dynein neck domain in humans23. MTBD: microtubule binding domain, NDD: N-terminal dimerization domain. (i-k), Potential cargo binding sites can be proposed on anterograde IFT trains. Extra densities that bind to the train antipodal to the dynein are visible in some anterograde trains. (i), Comparison between a tomographic slice through an anterograde train as seen from the membrane side (right) and the train 3D model with the same orientation (left). A row of extra densities with variable size and shape (white arrowheads) is visible below the IFT-B repeat (green). (j), Three selected orthogonal slices through the same train show variously shaped densities connected to IFT-B (white arrowheads). These densities could represent IFT cargos. The red cross in (i) indicates the slicing plane corresponding to the first two orthogonal slices in (j). The red crosses in both (i) and (j) also indicate the end of IFT-B. (k), Extra densities (cargos?) appear poorly reinforced after subtomogram averaging (white arrowheads and dashed lines). Scale bars, 10 nm (e,f,i-k).

Supplementary Figure 5 Model of inhibition and activation of IFT dynein in the cilium.

When transported as a cargo on anterograde IFT trains (1), the motor domains of dynein (blue) point toward the tip and face towards the membrane of the cilium, thereby keeping the microtubule binding domains away from the microtubule tracks (only one dynein tail domain is shown in the magnified view, grey). Due to this positioning, a tug-of-war with the anterograde kinesin (magenta) is avoided. Furthermore, the dynein AAA+ rings are stacked and the stalks are crossed. This conformation is known to have reduced motor activity22, therefore contributing to the inhibition of the motor during anterograde transport. Both mechanisms combined guarantee unperturbed processive anterograde transport in the cilium. We also propose that this inhibited conformation ensures stable loading and unloading of dyneins and prevents premature retrograde motility when anterograde trains disassemble at the tip. At the tip (2), dyneins are released in an open conformation that might be associated with reorganizing IFT trains. This conformation is thought to be a transitional state between the inhibited and the active forms and has impaired retrograde motility23. To power retrograde IFT (3), dyneins assume the active conformation with parallel motor domains.

Supplementary Figure 6 Unprocessed scans of immunoblots.

Unprocessed scans of all shown immunoblots. Presented areas are indicated with boxes and their corresponding antibodies.

Supplementary information

Supplementary Information

Supplementary Figures 1–6, and Supplementary Table and Supplementary Video legends.

Reporting Summary

Supplementary Table 1

List of antibodies used in this study, with used dilutions.

Supplementary Table 2

Cryo-EM data collection, refinement and validation statistics.

Supplementary Video 1

Raw tomogram of a wild-type IFT train.

Supplementary Video 2

Guided tour through the electron density of IFT, obtained by sub-tomogram averaging.

Supplementary Video 3

View of the 3D model of an anterograde IFT train.

Supplementary Video 4

Raw tomogram of an anterograde IFT train from the ift139-2 mutant.

Supplementary Video 5

Raw tomogram of an anterograde IFT train from the dhc1b-3 mutant at restrictive temperature.

Supplementary Video 6

Fitting of the atomic model of dynein-1b in its auto-inhibited form.

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Jordan, M.A., Diener, D.R., Stepanek, L. et al. The cryo-EM structure of intraflagellar transport trains reveals how dynein is inactivated to ensure unidirectional anterograde movement in cilia. Nat Cell Biol 20, 1250–1255 (2018). https://doi.org/10.1038/s41556-018-0213-1

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