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Reconstitution reveals motor activation for intraflagellar transport


The human body represents a notable example of ciliary diversification. Extending from the surface of most cells, cilia accomplish a diverse set of tasks. Predictably, mutations in ciliary genes cause a wide range of human diseases such as male infertility and blindness. In Caenorhabditis elegans sensory cilia, this functional diversity appears to be traceable to the differential regulation of the kinesin-2-powered intraflagellar-transport (IFT) machinery. Here we reconstituted the first, to our knowledge, functional multi-component IFT complex that is deployed in the sensory cilia of C. elegans. Our bottom-up approach revealed the molecular basis of specific motor recruitment to the IFT trains. We identified the key component that incorporates homodimeric kinesin-2 into its physiologically relevant context, which in turn allosterically activates the motor for efficient transport. These results will enable the molecular delineation of IFT regulation, which has eluded understanding since its discovery more than two decades ago.

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Fig. 1: IFT-70(DYF-1) is key to the incorporation of the OSM-3 motor into the QCC.
Fig. 2: IFT-70(DYF-1)-mediated binding of the OSM-3(G444E)–Halo motor to the QCC as measured by MST.
Fig. 3: Colocalization efficiency of the OSM-3(G444E)–Halo motor with its IFT-B complex is solely dependent on the IFT-70(DYF-1) subunit.
Fig. 4: IFT-70(DYF-1)-dependent incorporation into the QCC fully activates OSM-3 in vitro.

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We thank G. Woehlke, E. J. G. Peterman, E. Lorentzen, M. Taschner and F. Müller-Planitz for discussions throughout this work, T.-H. Ho for technical assistance and A. Oberhofer and F. Müller-Planitz for critically reading the manuscript. This work was supported by European Research Council Grant 335623 (to Z.Ö.). We apologize to our colleagues whose work could not be cited owing to space limitations.

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Nature thanks R. Vale and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Authors and Affiliations



M.A.A.M. and Z.Ö. designed the experiments. M.A.A.M. performed the experiments and analysed the data. W.L.S. wrote all the customized MATLAB routines. Z.Ö. and M.A.A.M. wrote the manuscript.

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Correspondence to Zeynep Ökten.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Overview of recombinant constructs and their proposed assembly in vitro.

Schematic of the presumptive IFT-B core complex from C. elegans (corresponding nomenclature of the subunits in C. elegans is shown in brackets). Subunits known to autonomously form sub-complexes in C. reinhardtii are colour-coded. The OSM-3 motor is shown in green. Subunits of the IFT-B core complex that are proposed to interfere with OSM-3 function in vivo are highlighted with black circles.

Extended Data Fig. 2 Interaction of OSM-3(G444E)–Halo with QCC and TCC.

a, Calculated molar mass of QCC subunits and the OSM-3(G444E)–Halo motor along with their expected sum (left). The SDS–PAGE analyses of the elution peaks in Fig. 1 (middle and right) show that OSM-3(G444E)–Halo co-elutes with the complex in the presence of the IFT-70(DYF-1) subunit (middle) but does not co-elute with the complex in the absence (right) of the IFT-70(DYF-1) subunit. b, Overlay of the elution profiles of the TCC with and without the OSM-3(G444E)–Halo motor and of the OSM-3(G444E)–Halo motor alone (left). Note that the left shoulder of the TCC + OSM-3(G444E)–Halo complex overlaps with the elution profile of the OSM-3(G444E)–Halo motor, and the right shoulder with the TCC. Consistently, the molar masses determined for the TCC + OSM-3(G444E)–Halo under peak 1 correspond to the OSM-3(G444E)–Halo motor and peak 2 to the TCC, respectively (middle versus right). c, Left, the calculated molar mass of the TCC subunits and the OSM-3(G444E)–Halo along with their expected sum. Middle and right, the SDS–PAGE analyses of the elution peaks shown in b. Data are representative of three independent experiments. The identities of all subunits were confirmed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. Asterisks in a and c indicate the location of Hsp70 protein.

Extended Data Fig. 3 Overview of the IFT-B subunits functionalized with C-terminal tags and their photobleaching properties.

The subunits were functionalized either with a GFP or SNAP tag for fluorescence labelling. All subunits displayed mostly single-step photobleaching consistent with non-aggregated, single subunits after functionalization. N, the number of events obtained from three different slides in three independent experiments.

Extended Data Fig. 4 Pairwise colocalization efficiency of IFT-B subunits.

Pairs of differentially labelled subunits of the IFT-B sub-complexes were incubated and analysed for their colocalization efficiency. The columns (bottom) represent the percentage of colocalized spots in the corresponding colocalized images (top). All assayed combinations of the labelled subunits displayed significant colocalization efficiencies demonstrating that C-terminal functionalization of the subunits does not interfere with their complex formation capabilities. Data are mean ± s.d. from three independent experiments. Scale bars, 3 μm. C. elegans nomenclature is used in this figure owing to space limitations.

Source Data

Extended Data Fig. 5 Colocalization of the heterotrimeric KLP11–KLP20–KAP motor with the IFT-B complex.

Neither QCC (top) nor TCC (bottom) of the IFT-B complex displayed efficient colocalization with the KLP11–KLP20–KAP motor. The IFT-81 subunit of TCC was fluorescently labelled with a SNAP tag and the IFT-52(OSM-6) subunit of QCC was GFP tagged. Data are mean ± s.d. from three independent experiments. Scale bars, 3 μm.

Source Data

Extended Data Fig. 6 The IFT-70(DYF-1)-dependent activation does not alter the processivity of the OSM-3 motor.

ac, OSM-3(G444E)–Halo (a), and the IFT-70(DYF-1)-activated OSM-3–Flag (b), and OSM-3–SNAP (c) motors display similar processivity that is independent of the presence of the IFT-70(DYF-1) subunit and the QCC. N, the number of events obtained from three different flow chambers in three independent experiments. Run length was fit to a single exponential ± confidence interval.

Extended Data Fig. 7 OSM-3–SNAP motor containing the wild type stalk colocalized with QCC in an IFT-70(DYF-1)-dependent manner.

a, Neither TCC nor QCC lacking DYF-1 efficiently colocalize with the OSM-3–SNAP motor. However in the presence of the IFT-70(DYF-1) subunit, the QCC efficiently colocalizes with OSM-3–SNAP (81 ± 8%). b, Consistently, OSM-3–SNAP showed robust colocalization (82 ± 6%) with the IFT-70(DYF-1) subunit but not with IFT-52(OSM-6), IFT-88(OSM-5) or IFT-46(DYF-6) subunits. IFT-81 from TCC was fluorescently labelled with a SNAP tag and IFT-52(OSM-6) from QCC and QCC lacking DYF-1 were GFP-tagged. Data are mean ± s.d. of three independent experiments. Scale bars, 3 μm. C. elegans nomenclature is used in the figure owing to space limitations.

Source Data

Supplementary information

Supplementary Information

The respective protein sequences of the OSM-3 motor constructs used in this study are listed in constructs

Reporting Summary

Supplementary Figures

This file contains Supplementary Figure 1 which shows the uncropped SDS-PAGE analyses of the respective protein purifications shown in Extended Data Fig. 2

Video 1: IFT-70(DYF-1)-dependent activation of OSM-3(G444E)-Halo.

Movement of OSM-3(G444E)-Halo motor alone (top, left) and its co-movement with the IFT-70(DYF-1) subunit (top, right) and with QCC (bottom, right). Removal of the IFT-70(DYF-1) subunit from QCC, dissociates the motor from its complex and the motor moves alone (bottom, left). IFT-70(DYF-1) from QCC and IFT-52(OSM-6) from QCC w/o DYF-1 were GFP-tagged in all videos (1-3). The video is sped up 4X. n=3 independent experiments. Scale bar: 10 µm

Video 2: IFT-70(DYF-1) activates the autoinhibited motor.

OSM-3-SNAP containing the wild type stalk is incapable of directional movement alone, and displays diffusion (top, left). Presence of IFT-70(DYF-1) and QCC advances the motor to a unidirectional and processive state (top and bottom right). The processivity as well as colocalisation is lost by the removal of the IFT-70(DYF-1) subunit from the QCC (bottom, left). Frequency of OSM-3 movement was increased ~an order of magnitude by IFT-70(DYF-1) or the QCC (OSM-3-SNAP: 1.4*10−2 min−1µm−1; OSM-3-SNAP + IFT-70(DYF-1): 9.4*10−2 min−1µm−1; OSM-3-SNAP + QCC: 8.7*10−2 min−1µm−1). The video is sped up 4X. n=3 independent experiments. Scale bar: 10 µm

Video 3: IFT-70(DYF-1) activates the autoinhibited OSM-3-Flag.

In the presence of the unlabelled OSM-3-Flag motor, both IFT-70(DYF-1) and QCC display directional movement (top, left vs. right) but not in the absence of the IFT-70(DYF-1) subunit (bottom, left). The video is sped up 4X. n=3 independent experiments. Scale bar: 10 µm

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Mohamed, M.A.A., Stepp, W.L. & Ökten, Z. Reconstitution reveals motor activation for intraflagellar transport. Nature 557, 387–391 (2018).

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