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. 2020 Jun 17;39(12):e103955.
doi: 10.15252/embj.2019103955. Epub 2020 Apr 27.

Optimal sidestepping of intraflagellar transport kinesins regulates structure and function of sensory cilia

Chao Xie #  1 , Liuju Li #  2 , Ming Li #  1 , Wenxin Shao  1 , Qingyu Zuo  1 , Xiaoshuai Huang  2 , Riwang Chen  2 , Wei Li  3 , Melanie Brunnbauer  4 , Zeynep Ökten  4 , Liangyi Chen  2 , Guangshuo Ou  1
Affiliations

Optimal sidestepping of intraflagellar transport kinesins regulates structure and function of sensory cilia

Chao Xie et al. EMBO J. .

Erratum in

Abstract

Cytoskeletal-based molecular motors produce force perpendicular to their direction of movement. However, it remains unknown whether and why motor proteins generate sidesteps movement along their filamentous tracks in vivo. Using Hessian structured illumination microscopy, we located green fluorescent protein (GFP)-labeled intraflagellar transport (IFT) particles inside sensory cilia of live Caenorhabditis elegans with 3-6-nanometer accuracy and 3.4-ms resolution. We found that IFT particles took sidesteps along axoneme microtubules, demonstrating that IFT motors generate torque in a living animal. Kinesin-II and OSM-3-kinesin collaboratively drive anterograde IFT. We showed that the deletion of kinesin-II, a torque-generating motor protein, reduced sidesteps, whereas the increase of neck flexibility of OSM-3-kinesin upregulated sidesteps. Either increase or decrease of sidesteps of IFT kinesins allowed ciliogenesis to the regular length, but changed IFT speeds, disrupted axonemal ninefold symmetry, and inhibited sensory cilia-dependent animal behaviors. Thus, an optimum level of IFT kinesin sidestepping is associated with the structural and functional fidelity of cilia.

Keywords: Caenorhabditis elegans; cilia; intraflagellar transport; kinesin; torque generation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Visualization of IFT at high spatiotemporal resolutions in GFP knock‐in animals

  1. Schematic depiction of the Caenorhabditis elegans phasmid cilia (left). Two sensory cilia are aligned into the phasmid channel (the dashed box and enlarged in the middle). Each cilium contains a ciliary base, a middle segment (m.s.), and a distal segment (d.s.). Right shows an inverted fluorescence Hessian‐SIM image of GFP‐tagged dynein‐2 heavy chain CHE‐3 in cilia, in which individual puncta were resolved. Scale bar: 1 μm. Also shown in Appendix Fig S4A.

  2. Frame series of an anterograde moving IFT particle from a 7xGFP::CHE‐3 worm. Localization precisions are shown under each frame. Time in millisecond (ms); scale bar, 100 nm. More examples from CHE‐2::7xGFP, DYF‐11::3xGFP, or 7xGFP::CHE‐3; DYF‐11::3xGFP animals are in Appendix Fig S3B and C.

  3. Localization precisions (mean ± SD) of tracks from multi‐GFP‐labeled worms. The number of GFP copies was estimated as the following: The dynein‐2 heavy chain of CHE‐3 forms a dimer, 14 = 2 × 7; IFT particle subunit CHE‐2 is also a dimer, 14 = 2 × 7; IFT particle subunit DYF‐11 can be a tetramer, 12 = 4 × 3; and the genetic cross combined 7xGFP::CHE‐3 with DYF‐11::3XGFP, 26 = 2 × 7 + 4 × 3.

  4. The representative diagram shows the displacement of the cargo within the middle ciliary segment. The y‐axis grid lines are spaced 16 nm. Protein CHE‐3 tagged with 7× GFP was used for tracking experiments. Trace was analyzed using the SIC fitting algorithm (Kalafut & Visscher, 2008).

  5. Histogram of 161 displacements detected in 15 tracks extracted from 12 WT worms. The plus and minus displacements are defined as the upward “jumping” or downward “jumping” in Fig 1D. Protein markers used for tracking experiments are CHE‐3 tagged with 7× GFP, CHE‐2 tagged with 7× GFP, or CHE‐3 tagged with 7× GFP plus DYF‐11 tagged with 3× GFP.

  6. The proportion of the plus displacement, the minus displacement, and dwell (defined as the segments in Fig 1D where there was no displacement detected) during imaging time of 15 tracks extracted from 12 WT animals. Proteins used for tracking experiments are the same as in Fig 1E.

Figure 2
Figure 2. Sidesteps of anterograde IFT particles

  1. Frame series shows the off‐axis movement of an IFT particle (#1). A neighboring particle (#2) serves as a fiduciary marker. Scale bar: 200 nm.

  2. Representative two‐dimensional IFT tracks from WT, kinesin‐II (klp‐11), and osm‐3 null animals. Red and violet circles indicate the start and end of the tracks, respectively. Proteins used for tracking experiments are CHE‐3 tagged with 7× GFP or DYF‐11 tagged with 3× GFP.

  3. Time‐averaged mean‐square displacement (MSD) versus lag time (t lag) of 10–15 tracks from WT and IFT kinesin mutant animals. Proteins used for tracking experiments are CHE‐3 tagged with 7× GFP, CHE‐3 tagged with 7× GFP plus DYF‐11 tagged with 3× GFP, CHE‐2 tagged with 7× GFP, or DYF‐11 tagged with 3× GFP. Error bars indicate standard errors.

  4. Angle histogram (rose plot) of the deviation angles of displacement in 10–15 tracks from WT and mutant animals. The deviation angle is defined as the angle between the displacement vector and the overall direction of motion. Deviation to the right or left of the overall direction is between 0° and 180° or between 0° and −180°, respectively. Deviations between 90°–180° and −90° to −180° are backstepping (orange). Proteins used for tracking experiments are the same as in Fig 1E.

  5. Average deviation angles in 10–15 tracks from WT and mutant animals. Comparisons were performed with the WT. Proteins used for tracking experiments are the same as in Fig 1E. *P < 0.05, **P < 0.01, ****P < 0.0001 by Student's t‐tests. Error bars indicate standard deviations.

  6. Representative tracks of the recombinant Caenorhabditis elegans kinesin‐II and OSM‐3‐kinesin moving along suspended MTs in the optical trap assay. Appendix Fig S3E depicts the experiment scheme.

  7. Representative TEM images of the middle ciliary segments from WT and IFT kinesin mutant animals. Red arrowheads indicate ectopic singlet microtubules. Blue arrowheads indicate ectopic doublet microtubules. The schematics below depict the phenotype of each image. Scale bars: 100 nm.

Figure 3
Figure 3. GS insertion of OSM‐3 increased sidestepping

  1. Design of OSM‐3 constructs with flexible necks. Top, poly‐GS repeats (orange) were knocked into the endogenous osm‐3 locus. Bottom, schematic of osm‐3 gene model, and sgRNA sequence.

  2. Representative two‐dimensional IFT tracks from WT and OSM‐3(1 or 3xGS) animals. Red and violet circles indicate the start and end of the tracks, respectively. Proteins used for tracking experiments are the same as in Fig 1E.

  3. Time‐averaged mean‐square displacement (MSD) versus lag time (t lag) of 10–15 tracks from WT and mutant worms. Proteins used for tracking experiments are the same as in Fig 1E. Error bars indicate standard errors.

Figure 4
Figure 4. GS insertion of OSM‐3 reduced IFT speeds and perturbed ciliary ultrastructure

  1. Histogram of anterograde IFT speeds of 3xGFP::CHE‐3 at the middle (top) and distal (bottom) ciliary segments (top). The plots were fit by a Gaussian distribution. Comparisons were performed between the WT and osm‐3 mutants. ***P < 0.001 by Student's t‐tests. Error bars indicate standard errors.

  2. Schematic depiction of the Caenorhabditis elegans amphid and phasmid cilia. Each cilium contains a ciliary base, a middle segment (m.s.), and a distal segment (d.s.).

  3. Amphid (top) and phasmid (bottom) cilia in the WT and osm‐3 GS knock‐in animals. Cilia are marked with 7xGFP::CHE‐3. Arrowheads indicate the ciliary base. Arrows indicate the junctions between the middle and distal segments. Scale bars: 5 μm.

  4. Cilium length (mean ± SD) in WT and osm‐3 mutant animals. Comparisons were performed between the WT and osm‐3 mutants. N = 24–32. n.s., not significant by Student's t‐tests. Error bars indicate standard errors.

  5. Quantifications of dye‐filling defects from WT and osm‐3 mutant animals. N ≥ 100.

  6. Representative TEM images of the middle ciliary segments from osm‐3(1xGS) and osm‐3(3xGS) worms. Red arrowheads indicate ectopic singlet microtubules. Blue arrowheads indicate ectopic doublet microtubules. The schematics below depict the phenotype of each image. Scale bars: 100 nm.

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