Optimal sidestepping of intraflagellar transport kinesins regulates structure and function of sensory cilia
2020; Springer Nature; Volume: 39; Issue: 12 Linguagem: Inglês
10.15252/embj.2019103955
ISSN1460-2075
AutoresChao Xie, Liuju Li, Ming Li, Wenxin Shao, Qingyu Zuo, Xiaoshuai Huang, Riwang Chen, Wei Li, Melanie Brunnbauer, Zeynep Ökten, Liangyi Chen, Guangshuo Ou,
Tópico(s)Microtubule and mitosis dynamics
ResumoArticle27 April 2020free access Optimal sidestepping of intraflagellar transport kinesins regulates structure and function of sensory cilia Chao Xie Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China Search for more papers by this author Liuju Li State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China Search for more papers by this author Ming Li Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China Search for more papers by this author Wenxin Shao Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China Search for more papers by this author Qingyu Zuo Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China Search for more papers by this author Xiaoshuai Huang State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China Search for more papers by this author Riwang Chen State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China Search for more papers by this author Wei Li School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Melanie Brunnbauer Physik Department E22, Technische Universitat Munchen, Garching, Germany Search for more papers by this author Zeynep Ökten orcid.org/0000-0002-3848-3488 Physik Department E22, Technische Universitat Munchen, Garching, Germany Search for more papers by this author Liangyi Chen Corresponding Author [email protected] orcid.org/0000-0003-1270-7321 State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China Search for more papers by this author Guangshuo Ou Corresponding Author [email protected] orcid.org/0000-0003-1512-7824 Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China Search for more papers by this author Chao Xie Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China Search for more papers by this author Liuju Li State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China Search for more papers by this author Ming Li Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China Search for more papers by this author Wenxin Shao Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China Search for more papers by this author Qingyu Zuo Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China Search for more papers by this author Xiaoshuai Huang State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China Search for more papers by this author Riwang Chen State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China Search for more papers by this author Wei Li School of Medicine, Tsinghua University, Beijing, China Search for more papers by this author Melanie Brunnbauer Physik Department E22, Technische Universitat Munchen, Garching, Germany Search for more papers by this author Zeynep Ökten orcid.org/0000-0002-3848-3488 Physik Department E22, Technische Universitat Munchen, Garching, Germany Search for more papers by this author Liangyi Chen Corresponding Author [email protected] orcid.org/0000-0003-1270-7321 State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China Search for more papers by this author Guangshuo Ou Corresponding Author [email protected] orcid.org/0000-0003-1512-7824 Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China Search for more papers by this author Author Information Chao Xie1,‡, Liuju Li2,‡, Ming Li1,‡, Wenxin Shao1, Qingyu Zuo1, Xiaoshuai Huang2, Riwang Chen2, Wei Li3, Melanie Brunnbauer4, Zeynep Ökten4, Liangyi Chen *,2 and Guangshuo Ou *,1 1Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, School of Life Sciences and MOE Key Laboratory for Protein Science, Tsinghua University, Beijing, China 2State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing, China 3School of Medicine, Tsinghua University, Beijing, China 4Physik Department E22, Technische Universitat Munchen, Garching, Germany ‡These authors contributed equally to this work as first authors *Corresponding author. Tel: +86-10-62764959; E-mail: [email protected] *Corresponding author. Tel: +86-10-62794766; E-mail: [email protected] EMBO J (2020)39:e103955https://doi.org/10.15252/embj.2019103955 Correction(s) for this article Optimal sidestepping of intraflagellar transport kinesins regulates structure and function of sensory cilia15 September 2020 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis Kinesin sidestepping has been observed in vitro, but its physiological relevance has not been investigated in vivo. In this study, optimal sidestepping of intraflagellar transport (IFT) kinesins in Caenorhabditis elegans sensory neurons is shown to regulate proper axoneme symmetry and animal chemosensation. IFT particles take sidesteps along axoneme microtubules, demonstrating torque generation of IFT motors in a living animal. Coordinated movement of kinesin-II and OSM-3 kinesin generates an intermediate level of sidestepping. Aberrant sidestepping allows ciliogenesis, but disrupts axonemal nine-fold symmetry Aberrant sidestepping inhibits sensory cilia-dependent chemosensation. Introduction An efficient intracellular transport system depends on the unidirectional translocation of cargo molecules by members of the myosin, kinesin, and dynein family motor proteins (Hartman et al, 2011; Scholey, 2013; Reck-Peterson et al, 2018; Sweeney & Holzbaur, 2018). Intriguingly, these linear motor proteins do not always follow a strictly linear path along actin filaments or microtubules (MTs). They also produce force perpendicular to their direction of movement, generating sidesteps or off-axis movement along their filamentous tracks (Ricca & Rock, 2010; Brunnbauer et al, 2012; Ferro et al, 2019). Torque generation of motor proteins was first demonstrated by the purified Tetrahymena axonemal dynein, which rotates the microtubules around their axes while translocating them (Vale & Toyoshima, 1988). Subsequently, in vitro studies revealed that such torsional and axial force generation appears to be a prevalent feature for all three families of molecular motors (Nishizaka et al, 1993; Yajima & Cross, 2005; Beausang et al, 2008; Yajima et al, 2008; Ricca & Rock, 2010; Brunnbauer et al, 2012; Can et al, 2014; Ferro et al, 2019). However, it remains unclear whether motor proteins take sidesteps and generate torque in a living cell. Detection of sidesteps of motor proteins in a living organism requires observing the motor or cargo molecules with an unprecedented combination of temporal and spatial resolution. In particular, the spatial resolution needs to be higher than the diameter of the cytoskeletal tracks, and the temporal resolution should be sufficient to follow the stepping behaviors of motor proteins in the cellular environment of physiological adenosine 5′-triphosphate concentration and temperature. Here, we developed an ultrafast live super-resolution imaging system to track multi-GFP-labeled intraflagellar transport (IFT) particles being carried by MT motors along the sensory cilia of Caenorhabditis elegans with 3–6-nanometer accuracy and 3.4-ms resolution. IFT is a microtubule-based transport system that drives the assembly and maintenance of cilia, which are essential for cell motility and signal perception in virtually all eukaryotic cells (Reiter & Leroux, 2017; Khan & Scholey, 2018; Breslow & Holland, 2019). During IFT, anterograde kinesin-2 and retrograde IFT dynein motors bidirectionally transport IFT particles that deliver functional cargo molecules, including axoneme precursors, to the site of assembly within cilia (Scholey, 2013; Taschner & Lorentzen, 2016). Sidestepping behavior is proposed to be advantageous for motor proteins to evade macromolecular obstacles such as cytoskeleton-associated proteins and stationary organelles in the tracks. The model predicts that the reduction of the sidestepping of motor proteins will cause traffic jams on the cytoskeleton and slow down intracellular transport. Still, the underlying functional significance of torque generation has not been directly examined in vivo. In our study, we deleted the torque-generating kinesin-II and reduced its sidestepping behavior; however, we did not detect traffic jams within cilia and found an acceleration of IFT in the formation of the full-length cilia. Instead of the obstacle avoidance model, we propose that an optimum level of the sidestepping of IFT kinesins is associated with proper axoneme symmetry and animal chemosensation. Results Development of an ultrafast super-resolution imaging system for IFT in Caenorhabditis elegans We first sought to label IFT protein machinery with the highest possible fluorescence signal and the lowest background in C. elegans. Using the CRISPR-Cas9-based genome editing method (Appendix Fig S1A), we inserted fluorescence tags into the genomic loci of the subunits of IFT particles (CHE-2 or DYF-11) or IFT dynein heavy chain (CHE-3; the anterograde cargo for IFT kinesin). The endogenous expression level minimizes non-specific background fluorescence. To increase the signal, we knocked seven copies of the GFP11 tag into the loci and used a ciliated neuron-specific promoter Pdyf-1 to express GFP1-10 in 7XGFP11 knock-in animals. GFP1-10 and GFP11 self-complemented in the ciliated neurons and illuminated IFT proteins with proportionally enhanced green fluorescence (Kamiyama et al, 2016; Jia et al, 2019). We also constructed knock-in animals in which three copies of GFP were inserted into the sites of the C. elegans genome (Appendix Fig S1A). Next, we combined fluorescence markers by the genetic cross and achieved fluorescence labeling of IFT particles with up to 42 copies of GFP molecules (Appendix Fig S1A and B). We determined whether fluorescence labeling affected ciliary structure or functionality by measuring cilium length, IFT speeds, and the capacity of the animal to uptake fluorescence dye through sensory cilia (Dyf). Dye-filling defects of C. elegans indicate the abnormality of sensory neurons to contact with the environment and suggest defective ciliary structure and function (Perkins et al, 1986). Expression of GFP with copy numbers more than 26 shortened cilium length, disrupted IFT, and completely abolished Dyf. In contrast, the ciliary structure and function in the animals with 14 GFP copies or less were indistinguishable to those of non-labeling animals (Appendix Figs S1 and S2). Thus, we used the knock-in animals expressing 12 or 14 copies of GFP or animals carrying 26 copies of GFP within the normal cilia in our imaging experiments. We chose to study IFT in the middle ciliary segments of the phasmid PHA/B neurons because this region allows the visualization of an individual cilium in the animal (Fig 1A, Movie EV1; Inglis et al, 2007). The C. elegans typically grow at 15–25°C, and we performed our measurements at 20°C. Fluorescence images of IFT particles labeled with 12–26 GFP molecules were visualized with structured illumination microscopy based on Hessian matrixes (Hessian-SIM), employing the second-order partial derivative matrixes to minimize artifacts of SIM, and operating at a rolling reconstruction of a 3.4-ms rate per frame (Huang et al, 2018). Super-resolved fluorescence puncta could be fitted with a Gaussian function with an overall 3–6-nm precision (Fig 1B and C, Appendix Fig S3A–C). By projecting the time-lapse fluorescence images of IFT particles, we found four peaks of fluorescence intensity along the width of the middle ciliary segments in C. elegans (Appendix Fig S4A–C). To understand the number of fluorescence tracks, we modeled the laser illumination of an axoneme that contains nine doublet MTs. From most imaging angles, we detected four doublets on the focal plane (Appendix Fig S4D–G, Movie EV2). The spatial resolution of Hessian-SIM is 88 nm (Huang et al, 2018), allowing us to distinguish MT doublet tracks. Considering that the diameter of the cilium is 300–400 nm, we suggest that IFT particles could be tracked on individual axonemal doublet MTs. Figure 1. Visualization of IFT at high spatiotemporal resolutions in GFP knock-in animals 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. 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. 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. 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). 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. 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. Download figure Download PowerPoint Sidestepping behavior of IFT in cilia Two-dimensional Gaussian-fit localization showed that IFT particles underwent displacements in the anterograde direction. Because GFPs are labeled on the cargo molecules, not on the motors themselves, the "jumping" in Fig 1D reflects the displacement of the cargo, and we are not able to determine the step size of the motorheads. In agreement with in vitro kinesin assays (Yildiz et al, 2004), we detected some 8-nm sizes within 161 displacements of the cargo; however, the majority of sizes were more substantial than 8 nm (Fig 1D and E). During 88% of imaging time, IFT particles did not move (Fig 1F); the large portion of dwelling time indicates that 3.4-ms per frame imaging rate is not fast enough to follow each stepping behavior of IFT kinesins in vivo. The displacement of IFT particles must consist of multiple steps of IFT kinesins. The transport paths revealed the sidestepping behavior of IFT particles along axoneme MTs in live animals. Figure 2A and B and Movie EV3 showed that the IFT particle deviated ~ 40 nm toward the right and then turned back to the left while moving from the ciliary base to the tip. The ~ 40 nm deviation is larger than the pixel size of 32.5 nm in our imaging step. As expected, we could directly detect the sidestepping from the original images (Fig 2A, yellow line, 10.2 ms), confirming the trajectories generated by the Gaussian fit (Fig 2B). This IFT particle may take sidesteps between two most lateral protofilaments of the doublet MT, whose diameter is about 45 nm. Importantly, a neighboring IFT particle (Fig 2A, green line) did not undergo an apparent off-axis movement in the course of our imaging, making it a fiduciary marker to exclude motion artifacts from the failure of animal anesthetization or the microscopic stage drift. Figure 2. Sidesteps of anterograde IFT particles 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. 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. Time-averaged mean-square displacement (MSD) versus lag time (tlag) 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. 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. 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. 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. 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. Download figure Download PowerPoint The anterograde IFT along the middle ciliary segment in C. elegans is driven by the coordinated action of a heterotrimeric kinesin-II and a homodimeric OSM-3-kinesin in the kinesin-2 family (Ou et al, 2005). To gain insights into how different motor proteins cooperate to generate sidesteps in vivo, we assayed IFT in the C. elegans mutant animals carrying putative null alleles of kinesin-II (klp-11[tm324]) or osm-3(p802). In the absence of kinesin-II, OSM-3 alone moves the cargo and builds the full length of sensory cilia (Ou et al, 2005). Interestingly, sidesteps of IFT particles were significantly reduced compared with those in WT cilia (Fig 2B, middle, Movie EV4). In the absence of OSM-3, kinesin-II alone forms the middle ciliary segment (Ou et al, 2005), and in stark contrast to WT or kinesin-II mutant cilia, sidesteps of IFT particles were much more pronounced (Fig 2B, right, Movie EV5). We performed the mean-square displacement analysis of multiple trajectories. OSM-3-kinesin alone in kinesin-II mutant cilia moved most directionally, whereas kinesin-II alone in osm-3 mutants moved at the least directional fashion. The combination of OSM-3 and kinesin-II in WT animals generated an intermediate directionality (Fig 2C). To better characterize the sidestepping behavior of IFT kinesins, we quantified the deviation of each trajectory from the overall direction of motion (Fig 2D). The average deviation angle of IFT particles in klp-11 mutants is smaller than that of WT (Fig 2E), which indicates that IFT particles took less sidestepping in the absence of kinesin-II. In contrast, IFT particles in osm-3 mutant cilia underwent more sidestepping than WT, which supports the notion that kinesin-II generates more sidestepping than OSM-3-kinesin. The deviation angles that are larger than 90° indicate backstepping, and IFT particles took more backstepping in osm-3 mutant cilia but less backstepping in klp-11 mutants than that in WT (Fig 2D and Appendix Fig S3D). These data are in agreement with IFT speeds that were previously determined using spinning disk confocal microscopy and kymography. In essence, kinesin-II alone moves IFT cargo at 0.5 μm/s, OSM-3 alone moves at 1.3 μm/s, and the cooperation of kinesin-II and OSM-3 generates an intermediate 0.7 μm/s speed in WT cilia (Ou et al, 2005). The ability of motor proteins to take sidesteps itself would, in principle, give rise to torque because such movement can generate off-axis force. As such, sidesteps can be used as a readout for torque generation even though torque is difficult to be directly measured in vivo. Notably, sidestepping will generate torque only if they occur in the same helical direction. If the motor is equally likely to step right or left, this will not lead to a net torque generation at the macroscopic scale. The different levels of sidesteps in WT and IFT kinesin mutant cilia (Fig 2A–E) suggest that kinesin-II displays considerable torque, while OSM-3-kinesin moves torque less. In support of the in vivo observation (Fig 2A–E), recombinantly expressed kinesin-II motor from C. elegans spiraled around a freely suspended MT with a characteristic pitch (Brunnbauer et al, 2012), which indicates that this motor exerts off-axis force and thus would be able to generate torque during cargo transport. While the optical trapping experiments are useful to determine the helical directionality of the motors, the smooth helical movement of the bead is observed when many motor proteins carry the bead. Using the same optical tweezer setup (Appendix Fig S3E), we showed that kinesin-II displayed a pronounced pitch (Fig 2F, upper panel, Movie EV6), which is consistent with our early results (Brunnbauer et al, 2012). However, OSM-3-kinesin appeared to take much less sidesteps and thus moved in the straighter fashion than kinesin-II (Fig 2F, lower panel, Movie EV7). Therefore, these in vitro data correlate with our in vivo findings. Perturbation of sidestepping affects axonemal ultrastructure and animal behavior Why do kinesin-II and OSM-3 cooperate to produce an intermediate level of sidesteps in WT cilia? In the kinesin-II mutants, OSM-3-kinesin builds up the full length of cilia, but sidesteps of IFT particles are significantly reduced (Fig 2B–E). We wondered whether the decrease of torque generation by kinesin-II deletion might perturb the ciliary ultrastructure. To test this idea, we performed transmission electron microscopy (TEM) of high-pressure frozen, freeze-substituted WT and kinesin-II mutant animals (Fig 2G). Our observation from serial sections confirmed that the kinesin-II animals developed the distal and middle ciliary segments, as revealed by fluorescence microscopy (Appendix Fig S5A). Importantly, the organization of axonemal doublet microtubules was defective in klp-11(tm324) mutant cilia. In the WT middle ciliary segments, nine doublets symmetrically distribute and surround a variable number of singlet microtubules (Fig 2G). However, four out of 19 examined cilia from three klp-11(tm324) mutant animals only contained eight doublets on the periphery in the middle segments, and the last doublets spiraled into the axonemal lumen, revealing an impaired ninefold symmetry of axonemal MTs. Evans et al (2006) performed TEM analysis of kinesin-II mutant cilia, but the perturbation of the axoneme organization was unknown. Inspired by our current observations, we revisited the TEM images in the early publication (Evans et al, 2006). We uncovered the same spiraling of a doublet MT into axoneme lumen in the middle ciliary segment of kap-1 mutant, which deletes another subunit of heterotrimeric kinesin-II (Appendix Fig S5B). Our TEM further confirmed the same doublet spiraling defect in kap-1 mutant cilia (Appendix Fig S5C, 16 out of 43 cilia from four animals). These results show that the decrease of sidesteps of IFT particles by removal of a torque-generating kinesin-II disrupted ninefold symmetry of axoneme. Intriguingly, loss of kinesin-II function in sea urchin embryos caused a similar inward collapse of outer doublet MTs (Appendix Fig S5D; Morris & Scholey, 1997), suggesting that inhibition of kinesin-II led to a relatively widespread defect across species. We also found that the singlet MT abnormally appears between the doublet MTs in the kinesin-II null in 2/19 micrographs. The frequency is less than the ectopic singlet insertion between doublets in osm-3 null cilia (Fig 2G; 21 out of 27 cilia from three animals), which indicates that the doublet MT spiraling phenotype is caused by deleting kinesin-II, whereas the ectopic singlet insertion is the primary defect by inhibiting OSM-3-kinesin. To assess the more direct regulation of axoneme symmetry by torque generation of IFT kinesins, we sought to engineer an OSM-3-kinesin that takes increased sidesteps and, in turn, generates more torque. The early structural and optical trap studies show that the stability of kinesin's neck domain governs the torque-generating properties, and the neck region but not the neck linker length determines the sidestepping behavior (Yildiz et al, 2008; Brunnbauer et al, 2012). Kinesin-1 typically moves torque-free along one protofilament of the MT. Insertion of flexible glycine–serine (GS) extensions between the neck linker and neck allows the motorheads to switch the protofilament and spiral around the MT (Brunnbauer et al, 2012). Using the same strategy, we inserted the flexible GS motif into the OSM-3 neck domain. We first determined the adequate number of GS insertion by transgenesis in C. elegans. We transformed the plasmids expressing OSM-3 that contain 1–5 copies of GS motifs into osm-3(p802) null allele and assayed for the ciliary rescue effects (Appendix Figs S5E and F, and S6). The expression of WT osm-3 gene rescued all the defects on the ciliary length, fluorescence dye uptake, and IFT speeds; however, OSM-3 with five copies of GS repeats failed to recover, and three copies of GS inserts parti
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