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Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia

Abstract

Intracellular transport depends on cooperation between distinct motor proteins. Two anterograde intraflagellar transport (IFT) motors, heterotrimeric kinesin-II and homodimeric OSM-3, cooperate to move cargo along Caenorhabditis elegans cilia. Here, using quantitative fluorescence microscopy, with single-molecule sensitivity, of IFT in living strains containing single-copy transgenes encoding fluorescent IFT proteins, we show that kinesin-II transports IFT trains through the ciliary base and transition zone to a ‘handover zone’ on the proximal axoneme. There, OSM-3 gradually replaces kinesin-II, yielding velocity profiles inconsistent with in vitro motility assays, and then drives transport to the ciliary tip. Dissociated kinesin-II motors undergo rapid turnaround and recycling to the ciliary base, whereas OSM-3 is recycled mainly to the handover zone. This reveals a functional differentiation in which the slower, less processive kinesin-II imports IFT trains into the cilium and OSM-3 drives their long-range transport, thereby optimizing cargo delivery.

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Figure 1: IFT-particle subcomplexes and IFT motors show distinct localization patterns along cilia.
Figure 2: Kinesin-2 motors cooperate to transport IFT trains along cilia.
Figure 3: Single-motor turnarounds result in confinement of kinesin-II close to the base and OSM-3 near the distal segment.
Figure 4: Stochastic simulations of single-motor dynamics reveal the effect of turnaround probability on kinesin-2 distribution.
Figure 5: Mutants with IFT defects reveal kinesin-2 functional specialization.
Figure 6: IFT-particle-subcomplex loading and distribution is altered in the absence of kinesin-II.
Figure 7: ‘Gradual handover’ as a mode of action for same-polarity motor-protein cooperation.

References

  1. Vale, R. D. The molecular motor toolbox for intracellular transport. Cell 112, 467–480 (2003).

    CAS  Article  PubMed  Google Scholar 

  2. Hirokawa, N., Noda, Y., Tanaka, Y. & Niwa, S. Kinesin superfamily motor proteins and intracellular transport. Nat. Rev. Mol. Cell Biol. 10, 682–696 (2009).

    CAS  Article  PubMed  Google Scholar 

  3. Scholey, J. M. Kinesin-2: a family of heterotrimeric and homodimeric motors with diverse intracellular transport functions. Annu. Rev. Cell Dev. Biol. 29, 443–469 (2013).

    CAS  Article  PubMed  Google Scholar 

  4. Encalada, S. E. & Goldstein, L. S. Biophysical challenges to axonal transport: motor-cargo deficiencies and neurodegeneration. Annu. Rev. Biophys. 43, 141–169 (2014).

    CAS  Article  PubMed  Google Scholar 

  5. Hou, Y. Q. & Witman, G. B. Dynein and intraflagellar transport. Exp. Cell Res. 334, 26–34 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Mallik, R., Rai, A. K., Barak, P., Rai, A. & Kunwar, A. Teamwork in microtubule motors. Trends Cell Biol. 23, 575–582 (2013).

    CAS  Article  PubMed  Google Scholar 

  7. Jolly, A. L. & Gelfand, V. I. Bidirectional intracellular transport: utility and mechanism. Biochem. Soc. Trans. 39, 1126–1130 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Derr, N. D. et al. Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold. Science 338, 662–665 (2012).

    CAS  Article  PubMed  Google Scholar 

  9. Snow, J. J. et al. Two anterograde intraflagellar transport motors cooperate to build sensory cilia on C. elegans neurons. Nat. Cell Biol. 6, 1109–1123 (2004).

    CAS  Article  PubMed  Google Scholar 

  10. Pan, X. Y. et al. Mechanism of transport of IFT particles in C. elegans cilia by the concerted action of kinesin-II and OSM-3 motors. J. Cell Biol. 174, 1035–1045 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Rogers, A. R., Driver, J. W., Constantinou, P. E., Jamison, D. K. & Diehl, M. R. Negative interference dominates collective transport of kinesin motors in the absence of load. Phys. Chem. Chem. Phys. 11, 4882–4889 (2009).

    CAS  Article  PubMed  Google Scholar 

  12. Bieling, P., Kronja, I. & Surrey, T. Microtubule motility on reconstituted meiotic chromatin. Curr. Biol. 20, 763–769 (2010).

    CAS  Article  PubMed  Google Scholar 

  13. Furuta, K. et al. Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors. Proc. Natl Acad. Sci. USA 110, 501–506 (2013).

    CAS  Article  PubMed  Google Scholar 

  14. Norris, S. R. et al. A method for multiprotein assembly in cells reveals independent action of kinesins in complex. J. Cell Biol. 207, 393–406 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Ishikawa, H. & Marshall, W. F. Ciliogenesis: building the cell’s antenna. Nat. Rev. Mol. Cell Biol. 12, 222–234 (2011).

    CAS  Article  PubMed  Google Scholar 

  16. Pedersen, L. B. & Rosenbaum, J. L. Intraflagellar transport (Ift): role in ciliary assembly, resorption and signalling. Curr. Top. Dev. Biol. 85, 23–61 (2008).

    CAS  Article  PubMed  Google Scholar 

  17. Pigino, G. et al. Electron-tomographic analysis of intraflagellar transport particle trains in situ. J. Cell Biol. 187, 135–148 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Cole, D. G. et al. Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J. Cell Biol. 141, 993–1008 (1998).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Nachury, M. V., Seeley, E. S. & Jin, H. Trafficking to the ciliary membrane: how to get across the periciliary diffusion barrier? Annu. Rev. Cell Dev. Biol. 26, 59–87 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Reiter, J. F., Blacque, O. E. & Leroux, M. R. The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO Rep. 13, 608–618 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Bhogaraju, S. et al. Molecular basis of tubulin transport within the cilium by IFT74 and IFT81. Science 341, 1009–1012 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Craft, J. M., Harris, J. A., Hyman, S., Kner, P. & Lechtreck, K. F. Tubulin transport by IFT is upregulated during ciliary growth by a cilium-autonomous mechanism. J. Cell Biol. 208, 223–237 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Hao, L. M. et al. Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments. Nat. Cell Biol. 13, 790–453 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Marshall, W. F. & Rosenbaum, J. L. Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control. J. Cell Biol. 155, 405–414 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Cole, D. G. et al. Novel heterotrimeric kinesin-related protein purified from sea-urchin eggs. Nature 366, 268–270 (1993).

    CAS  Article  PubMed  Google Scholar 

  26. Walther, Z., Vashishtha, M. & Hall, J. L. The Chlamydomonas Fla10 gene encodes a novel kinesin-homologous protein. J. Cell Biol. 126, 175–188 (1994).

    CAS  Article  PubMed  Google Scholar 

  27. Kozminski, K. G., Beech, P. L. & Rosenbaum, J. L. The Chlamydomonas kinesin-like protein Fla10 is involved in motility associated with the flagellar membrane. J. Cell Biol. 131, 1517–1527 (1995).

    CAS  Article  PubMed  Google Scholar 

  28. Pazour, G. J., Dickert, B. L. & Witman, G. B. The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly. J. Cell Biol. 144, 473–481 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Porter, M. E., Bower, R., Knott, J. A., Byrd, P. & Dentler, W. Cytoplasmic dynein heavy chain 1b is required for flagellar assembly in Chlamydomonas. Mol. Biol. Cell 10, 693–712 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Signor, D. et al. Role of a class DHC1b dynein in retrograde transport of IFT motors and IFT raft particles along cilia, but not dendrites, in chemosensory neurons of living Caenorhabditis elegans. J. Cell Biol. 147, 519–530 (1999).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Ou, G. S., Blacque, O. E., Snow, J. J., Leroux, M. R. & Scholey, J. M. Functional coordination of intraflagellar transport motors. Nature 436, 583–587 (2005).

    CAS  Article  PubMed  Google Scholar 

  32. Perkins, L. A., Hedgecock, E. M., Thomson, J. N. & Culotti, J. G. Mutant sensory cilia in the nematode Caenorhabditis-elegans. Dev. Biol. 117, 456–487 (1986).

    CAS  Article  PubMed  Google Scholar 

  33. Starich, T. A. et al. Mutations affecting the chemosensory neurons of Caenorhabditis elegans. Genetics 139, 171–188 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Evans, J. E. et al. Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans. J. Cell Biol. 172, 663–669 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Brunnbauer, M. et al. Regulation of a heterodimeric kinesin-2 through an unprocessive motor domain that is turned processive by its partner. Proc. Natl Acad. Sci. USA 107, 10460–10465 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Imanishi, M., Endres, N. F., Gennerich, A. & Vale, R. D. Autoinhibition regulates the motility of the C. elegans intraflagellar transport motor OSM-3. J. Cell Biol. 174, 931–937 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Verhey, K. J., Dishinger, J. & Kee, H. L. Kinesin motors and primary cilia. Biochem. Soc. Trans. 39, 1120–1125 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. Malicki, J. & Besharse, J. C. Kinesin-2 family motors in the unusual photoreceptor cilium. Vision Res. 75, 33–36 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Ludington, W. B., Wemmer, K. A., Lechtreck, K. F., Witman, G. B. & Marshall, W. F. Avalanche-like behavior in ciliary import. Proc. Natl Acad. Sci. USA 110, 3925–3930 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Milic, B., Andreasson, J. O. L., Hancock, W. O. & Block, S. M. Kinesin processivity is gated by phosphate release. Proc. Natl Acad. Sci. USA 111, 14136–14140 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Engel, B. D. et al. The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function. J. Cell Biol. 199, 151–167 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Pedersen, L. B., Gelmer, S. & Rosenbaum, J. L. Dissecting the molecular mechanisms of intraflagellar transport in Chlamydomonas. Curr. Biol. 16, 450–459 (2006).

    CAS  Article  PubMed  Google Scholar 

  43. Mueller, J., Perrone, C. A., Bower, R., Cole, D. G. & Porter, M. E. The FLA3 KAP subunit is required for localization of kinesin-2 to the site of flagellar assembly and processive anterograde intraflagellar transport. Mol. Biol. Cell 16, 1341–1354 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Williams, C. L. et al. MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis. J. Cell Biol. 192, 1023–1041 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. Orozco, J. T. et al. Movement of motor and cargo along cilia. Nature 398, 674 (1999).

    CAS  Article  PubMed  Google Scholar 

  46. Kozminski, K. G., Johnson, K. A., Forscher, P. & Rosenbaum, J. L. A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc. Natl Acad. Sci. USA 90, 5519–5523 (1993).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. Schroeder, H. W. et al. Force-dependent detachment of kinesin-2 biases track switching at cytoskeletal filament intersections. Biophys. J. 103, 48–58 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. Hoeprich, G. J., Thompson, A. R., McVicker, D. P., Hancock, W. O. & Berger, C. L. Kinesin’s neck-linker determines its ability to navigate obstacles on the microtubule surface. Biophys. J. 106, 1691–1700 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. Trivedi, D., Colin, E., Louie, C. M. & Williams, D. S. Live-cell imaging evidence for the ciliary transport of rod photoreceptor opsin by heterotrimeric kinesin-2. J. Neurosci. 32, 10587–10593 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. Jiang, L. et al. Heterotrimeric kinesin-2 (KIF3) mediates transition zone and axoneme formation of mouse photoreceptors. J. Biol. Chem. 290, 12765–12778 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. O’Hagan, R. et al. The tubulin deglutamylase CCPP-1 regulates the function and stability of sensory cilia in C. elegans. Curr. Biol. 21, 1685–1694 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Buisson, J. et al. Intraflagellar transport proteins cycle between the flagellum and its base. J. Cell Sci. 126, 327–338 (2013).

    CAS  Article  PubMed  Google Scholar 

  53. Williams, C. L. et al. Direct evidence for BBSome-associated intraflagellar transport reveals distinct properties of native mammalian cilia. Nat. Commun. 5, 5813 (2014).

    CAS  Article  PubMed  Google Scholar 

  54. Burghoorn, J. et al. Mutation of the MAP kinase DYF-5 affects docking and undocking of kinesin-2 motors and reduces their speed in the cilia of Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 104, 7157–7162 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. Liang, Y. W. et al. FLA8/KIF3B phosphorylation regulates kinesin-II interaction with IFT-B to control IFT entry and turnaround. Dev. Cell 30, 585–597 (2014).

    CAS  Article  PubMed  Google Scholar 

  56. Verhey, K. J. & Hammond, J. W. Traffic control: regulation of kinesin motors. Nat. Rev. Mol. Cell Biol. 10, 765–777 (2009).

    CAS  Article  PubMed  Google Scholar 

  57. Sirajuddin, M., Rice, L. M. & Vale, R. D. Regulation of microtubule motors by tubulin isotypes and post-translational modifications. Nat. Cell Biol. 16, 335–344 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. Wedaman, K. P., Meyer, D. W., Rashid, D. J., Cole, D. G. & Scholey, J. M. Sequence and submolecular localization of the 115-kD accessory subunit of the heterotrimeric kinesin-II (KRP(85/95)) complex. J. Cell Biol. 132, 371–380 (1996).

    CAS  Article  PubMed  Google Scholar 

  59. Frokjaer-Jensen, C. et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 40, 1375–1383 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  60. Brenner, S. Genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Brust-Mascher, I., Ou, G. S. & Scholey, J. M. Measuring rates of intraflagellar transport along Caenorhabditis elegans sensory cilia using fluorescence microscopy. Method Enzymol. 524, 285–304 (2013).

    CAS  Article  Google Scholar 

  62. Jaqaman, K. et al. Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5, 695–702 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

During initial stages of the project, B.P. was a visiting student in the Scholey Laboratory, Department of MCB at UC Davis. We thank S. Açar, L. Hao (UC Davis), D. Cheerambathur and A. Desai (UC San Diego) for discussion; S. Mitani (NPB, Japan) for the tm3433 deletion mutant; E. Kroezinga for biochemical support; P. Noordeloos for technical support; J. Girard and J. Mijalkovic for critical reading of the manuscript (VU University Amsterdam). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We acknowledge financial support from the Netherlands Organisation for Scientific Research (NWO) via a Vici, an NWO-Groot and an ALW Open Program grant, via the STW research programme ‘Nanoscopy’, the FOM programme ‘Barriers in the Brain’, a grant from NanoNextNL of the Government of the Netherlands and 130 partners (E.J.G.P.), and from an NIH grant no. GM50718 (J.M.S.).

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Authors

Contributions

B.P., J.M.S. and E.J.G.P. initiated research. B.P. created reagents. B.P. and E.J.G.P. designed experiments. B.P., P.M. and F.O. performed experiments and analysed data. All authors contributed to data interpretation, with P.M. particularly contributing to the simulations and kymograph analysis and F.O. to the single-molecule analysis. B.P., J.M.S. and E.J.G.P. wrote the manuscript. All authors read the manuscript.

Corresponding author

Correspondence to Erwin J. G. Peterman.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 2 IFT-particle subcomplexes and IFT-motors show distinct localization patterns along cilia (related to Fig. 1).

(a) Representative two-color time-averaged fluorescence image of kinesin-II (green) and transition zone marker MKS-6 (magenta) (top), and corresponding fluorescence-intensity profile (bottom) along the lower cilium (same data as inset Fig. 1d) from 20 worms. Scale bar, 1 μm. (b,c) Average fluorescence-intensity profiles obtained from time-averaged fluorescence images (22.5 s each) of multiple different phasmid cilia. Line thickness represents 95% confidence interval for the mean (CIM). (b) Kinesin-II pooled from 15 worms, 30 phasmid cilia. (c) OSM-3 pooled from 19 worms, 32 phasmid cilia. (ac) B—Base, TZ—Transition Zone, PS—Proximal Segment and DS—Distal Segment.

Supplementary Figure 3 Kinesin-2 motors cooperate to transport IFT-trains along cilia (related to Fig. 2).

(a,b) Representative anterograde (a) and retrograde (b) Fourier-filtered kymographs of IFT-A particle subcomplexes and corresponding train velocity (black) and train intensity (orange). (c) Representative retrograde Fourier-filtered kymograph of IFT-B particle subcomplexes and corresponding train velocity. (d) Average IFT-B intensity for (left) anterograde and (right) retrograde trains. (e) Velocities of retrograde transport of kinesin-II (green) and OSM-3 (magenta). (ae) Minus (−) and plus (+) denote MT-polarity, horizontal scale bars, 1 μm; vertical scale bars, 2 s. Dashed lines represent mean ± SD, line thickness represents 95% CIM. For number of kymograph trajectories included and number of worms imaged to obtain representative images see Supplementary Fig. 6a–d.

Supplementary Figure 4 Single-motor turnarounds result in confinement of kinesin-II close to the base and OSM-3 near the distal segment (related to Fig. 3).

(a) All localizations of the kinesin-II anterograde-to-retrograde (A-to-R) trajectory of Fig. 3b in main text; a single kinesin-II motor initially moves in the anterograde direction (green), suddenly switches direction, to move in retrograde direction (grey), being transported by IFT-dynein. (b) All localizations of the kinesin-II retrograde-to-anterograde (R-to-A) trajectory of Fig. 3d in main text; a single kinesin-II motor first moves in the retrograde direction (grey), transported by IFT-dynein, waits at the base, and next moves in the opposite, anterograde direction (green). (c) All localizations of the OSM-3 A-to-R trajectory of Fig. 3f in main text; a single OSM-3 motor initially moves in the anterograde direction (magenta), suddenly switches direction into the retrograde direction (grey), transported by IFT-dynein. (d) All localizations of the OSM-3 R-to-A trajectory of Fig. 3h in main text; a single OSM-3 motor initially moves in the retrograde direction (grey), carried by IFT-dynein, and suddenly switches direction just before the base, moving in the anterograde direction (magenta). (e) One representative single-particle trajectory (left) and corresponding images (right, indicated by open symbols in trajectories) of an IFT-B particle subcomplex A-to-R turnaround from 10 worms, 20 phasmid cilia. (f) All localizations of the IFT-B A-to-R trajectory of Supplementary Fig. 3e; Scale bars, 0.5 μm.

Supplementary Figure 5 Mutants with IFT-motor defects and transition-zone defects reveal that kinesin-II navigates IFT-trains into the proximal segment, where OSM-3 takes over and drives fast, long-distance transport (related to Fig. 5).

(a) Fluorescence-intensity profiles obtained from time-averaged fluorescence images (22.5 s each) of multiple different phasmid cilia. Comparison of OSM-3 in kinesin-II mutant background (kap-1; grey, pooled 25 worms, 46 phasmid cilia) and wild-type OSM-3 (magenta, pooled from 19 worms, 32 phasmid cilia, from Supplementary Fig. 1c) reveals a three-fold difference in OSM-3 intensity at the base. (b) Representative super-resolution (SR) images obtained by accumulating (Σ) localizations (blue dots) of multiple single-molecule trajectories (grey lines) for OSM-3 (top right; from 20 worms, 31 phasmid cilia) and OSM-3 in a kinesin-II mutant background (kap-1; bottom right; from 11 worms, 18 phasmid cilia), see also Supplementary Fig. 6i. (c) Representative anterograde Fourier-filtered kymograph of OSM-3 in a kinesin-II mutant background (kap-1). (d) Average OSM-3 intensity for anterograde trains in C. elegans strains lacking (kap-1; grey) and containing (magenta) functional kinesin-II. (e) Representative super-resolution images for kinesin-II (top right; from 12 worms, 15 phasmid cilia) and kinesin-II in an OSM-3 mutant background (osm-3; bottom right; from 20 worms, 34 phasmid cilia, see also Supplementary Fig. 6j. (f) Representative (from 20 worms) two-color time-averaged fluorescence image of kinesin-II (green) and transition zone marker MKS-6 (magenta) (top), and corresponding fluorescence-intensity profile (bottom) along the lower cilium. Scale bar, 1 μm. The fluorescence-intensity profile of kinesin-II is normalized to its maximum and that of MKS-6 is scaled relative to MKS-6 in wild type (Supplementary Fig. 1a). (g) Average fluorescence-intensity profiles obtained from time-averaged fluorescence images (22.5 s each) of multiple phasmid cilia. Comparison of kinesin-II in MKSR-1 mutant background (mksr-1; grey, pooled from 22 worms, 42 phasmid cilia) and wild-type kinesin-II (green, pooled from 15 worms, 30 phasmid cilia, from Supplementary Fig. 1b) reveals a decrease of kinesin-II in the TZ but an increase in the PS. (h) Representative anterograde Fourier-filtered kymograph of OSM-3 in a kinesin-II and MKSR-1 double-mutant background (kap-1;mksr-1). (b,e) Scale bars, 0.25 μm. (c,h) Minus (−) and plus (+) denote MT-polarity, horizontal scale bar, 1 μm; vertical scale bar, 2 s. (a,d,g) Dashed lines are means ± SD, line thickness represents 95% CIM. For number of kymograph trajectories included and number of worms imaged to obtain representative kymographs see Supplementary Fig. 6d-g, i, j.

Supplementary Figure 6 IFT-particle subcomplex loading and distribution is altered in the absence of kinesin-II (related to Fig. 6).

(a) Average fluorescence-intensity profiles obtained from time-averaged fluorescence images (22.5 s each) of multiple different phasmid cilia. IFT-A in kinesin-II mutant background (kap-1; grey, pooled from 22 worms, 44 phasmid cilia), and wild-type IFT-A (orange, pooled from 25 worms, 50 phasmid cilia). (b) Coefficient of variation (standard deviation divided by the mean) of kymographs obtained from image stacks of IFT-B in C. elegans strains lacking (kap-1; grey, n = 40 kymographs from 17 worms, 40 phasmid cilia) and containing (blue, n = 52 kymographs from 19 worms, 52 phasmid cilia) functional kinesin-II show that kymographs of C. elegans strains lacking kinesin-II are less regular. (c) Coefficient of variation (standard deviation divided by the mean) of kymographs obtained from image stacks of IFT-A in C. elegans strains lacking (kap-1; grey, n = 36 kymographs from 22 worms, 36 phasmid cilia) and containing (orange, n = 50 kymographs from 25 worms, 50 phasmid cilia) functional kinesin-II show that kymographs of C. elegans strains lacking kinesin-II are less regular. (d) Cumulative probability distribution of the durations of single particle subcomplexes escaping from the transition zone. Distributions are obtained by defining a region of interest and measuring the time it takes for a particle subcomplex to cross the boundary in anterograde direction (inset). IFT-B particle subcomplexes escaped the transition zone in 0.6 ± 0.1 s (mean ± s.e.m., n = 45 IFT-B particle subcomplexes from 10 worms, 19 phasmid cilia) in the presence of kinesin-II and in 0.6 ± 0.1 s (mean ± s.e.m., n = 45 IFT-B particle subcomplexes from 25 worms, 38 phasmid cilia) lacking functional kinesin-II (kap-1). Dashed lines are mean ± SD, line thickness represents 95% CIM.

Supplementary Figure 7 Histograms of the amount of kymograph trajectories used to analyze train dynamics and intensities, the distribution and number of localizations used to build the super-resolution (SR) images, and in vivo comparison of eGFP and mCherry intensity (related to multiple figures and experimental procedures (see legend)).

(a) IFT-B, (left) anterograde trains (corresponding to Fig. 1c; Table 1; Fig. 2b, d, e; Supplementary Fig. 2d; Fig. 6b, d) and (right) retrograde trains (corresponding to Supplementary Fig. 2c, d). (b) IFT-A, (left) anterograde trains (corresponding to Fig. 1b; Table 1; Supplementary Fig. 2a) and (right) retrograde trains (corresponding to Supplementary Fig. 2b). (c) Kinesin-II, (left) anterograde trains (corresponding to Fig. 1d; Table 1; Fig. 2c, d, e; Fig. 5h (top), i) and (right) retrograde trains (corresponding to Fig. 2f; Supplementary Fig. 2e; Fig. 5j). (d) OSM-3, (left) anterograde trains (corresponding to Fig. 1e; Table 1; Fig. 2c, d, e; Fig. 5c; Supplementary Fig. 4d) and (right) retrograde trains (corresponding to Fig. 2f; Supplementary Fig. 2e). (e) Anterograde OSM-3 in a kinesin-II mutant background (kap-1; corresponding to Table 1; Fig. 5a–c, k; Supplementary Fig. 4c, d). (f) Anterograde (left) (corresponding to Table 1; Fig. 5h (bottom), i) and retrograde (right) (corresponding to Fig. 5j) kinesin-II in an MKSR-1 mutant background (mksr-1). (g) Anterograde OSM-3 in a kinesin-II and MKSR-1 double-mutant background (kap-1;mksr-1; corresponding to Fig. 5k; Supplementary Fig. 4h). (h) Anterograde IFT-B in a kinesin-II mutant background (kap-1; corresponding to Table 1; Fig. 6c, d). (ik) SR-images (top and bottom) and projection of localizations along long image axis (middle, area for each trace normalized), Related to Fig. 6 and Supplementary Fig. 4. (i) OSM-3, from Supplementary Fig. 4b; (j) Kinesin-II, from Supplementary Fig. 4e; (k) IFT-B, from Fig. 6e. Scale bar: 0.25 μm. (l) Fluorescence intensities of eGFP-tagged (EJP13) and mCherry-tagged (EJP85) KAP-1 as a function of excitation intensity. Intensities were obtained by integrating a cilium region of interest (1.7 μm2) over the first ten frames of an image series and averaging over 12 to 20 phasmid cilia (6 to 14 worms for each data point) for each imaging condition. Error bars represents s.e.m. Linear fits are used to calculate a mCherry to eGFP fluorescence intensity correction factor of 2.2, Related to ‘Quantification of fluorescence intensities’ in Methods.

Supplementary Table 1 C. elegans strains used in this study.

Supplementary information

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Dynamics of IFT-B in C. elegans strain EJP76.

Video plays at twice real time (time indicated). Scale bar, 2 μm. Related to Fig. 1c and Fig. 2b and Supplementary Fig. 2c. (MOV 43 kb)

Dynamics of kinesin-II (green) and OSM-3 (magenta) in C. elegans strain EJP42.

Video plays at twice real time (time indicated). Scale bar, 2 μm. Related to Fig. 2c, f. (MOV 36 kb)

Single-molecule dynamics of kinesin-II in C. elegans strain EJP13.

Video plays at twice real time (time indicated). Scale bar, 1 μm. Related to Fig. 3b, d. (MOV 293 kb)

All single-molecule localizations of Supplementary Video 3 of kinesin-II in C. elegans strain EJP13.

Video plays at twice real time (time indicated). Scale bar, 1 μm. Related to Fig. 3b, d. (MOV 313 kb)

Dynamics of OSM-3 in the absence of functional kinesin-II (kap-1) in C. elegans strain EJP22.

Video plays at twice real time (time indicated). Scale bar, 2 μm. Related to Fig. 5a–c. (MOV 49 kb)

Dynamics of kinesin-II in the absence of OSM-3 (osm-3) in C. elegans strain EJP41.

Video plays at twice real time (time indicated). Scale bar, 2 μm. Related to Fig. 5d, e. (MOV 32 kb)

Dynamics of kinesin-II in transition-zone mutant C. elegans strain EJP64.

Video plays at twice real time (time indicated). Scale bar, 2 μm. Related to Fig. 5g–j. (MOV 43 kb)

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Prevo, B., Mangeol, P., Oswald, F. et al. Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia. Nat Cell Biol 17, 1536–1545 (2015). https://doi.org/10.1038/ncb3263

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