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

Nature Cell Biology volume 17, pages 1536–1545 (2015)Cite this article

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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.

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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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  1. Pierre Mangeol

    Present address: Present address: Biology Institute of Marseille, Aix-Marseille University, 13288 Marseille Cedex 9, France.,

  2. Pierre Mangeol and Felix Oswald: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physics and Astronomy and LaserLaB Amsterdam, VU University Amsterdam, De Boelelaan 1081 1081 HV Amsterdam, The Netherlands,

    Bram Prevo, Pierre Mangeol, Felix Oswald & Erwin J. G. Peterman

  2. Department of Molecular and Cellular Biology, University of California at Davis, Davis, California 95616, USA

    Jonathan M. Scholey

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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.

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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. (a–c) 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). (a–e) 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). (i–k) 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.
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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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