Cilia generate three-dimensional waveforms required for cell motility and transport of fluid, mucus, and particles over the cell surface. This movement is driven by multiple dynein motors attached to nine outer doublet microtubules that form the axoneme. The outer and inner arm dyneins are organized into 96-nm repeats tandemly arrayed along the length of the doublets. Motility is regulated in part by projections from the two central pair microtubules that contact radial spokes located near the base of the inner dynein arms in each repeat. Although much is known about the structures and protein complexes within the axoneme, many questions remain about the regulatory mechanisms that allow the cilia to modify their waveforms in response to internal or external stimuli. Here, we used Chlamydomonas mbo (move backwards only) mutants with altered waveforms to identify at least two conserved proteins, MBO2/CCDC146 and FAP58/CCDC147, that form part of a L-shaped structure that varies between doublet microtubules. Comparative proteomics identified additional missing proteins that are altered in other motility mutants, revealing overlapping protein defects. Cryo-electron tomography and epitope tagging revealed that the L-shaped, MBO2/FAP58 structure interconnects inner dynein arms with multiple regulatory complexes, consistent with its function in modifying the ciliary waveform.

To identify proteins specific to the proximal ciliary axoneme, we used iTRAQ to compare short (~2 μm) and full-length (~11 μm) axonemes of Chlamydomonas. Known components of the proximal axoneme such as minor dynein heavy chains and LF5 kinase as well as the ciliary tip proteins FAP256 (CEP104) and EB1 were enriched in short axonemes whereas proteins present along the length of the axoneme were of similar abundance in both samples. The iTRAQ analysis revealed that FAP93, a protein of unknown function, and protein phosphatase 2A (PP2A) are enriched in the short axonemes. Consistently, immunoblots show enrichment of FAP93 and PP2A in short axonemes and immunofluorescence confirms the localization of FAP93 and enrichment of PP2A at the proximal axoneme. Ciliary regeneration reveals that FAP93 assembles continuously but more slowly than other axonemal structures and terminates at 1.03 μm in steady-state axonemes. The length of FAP93 assembly correlates with ciliary length, demonstrating ciliary length-dependent assembly of FAP93. Dikaryon rescue experiments show that FAP93 can assemble independently of IFT transport. In addition, FRAP analysis of GFP-tagged FAP93 demonstrates that FAP93 is stably anchored in the axoneme. FAP93 may function as a scaffold for assembly of other specific proteins at the proximal axoneme.

Ciliary motility requires the spatiotemporal coordination of multiple dynein motors by regulatory complexes located within the 96 nm axoneme repeat. Many organisms can alter ciliary waveforms in response to internal or external stimuli, but little is known about the specific polypeptides and structural organization of complexes that regulate waveforms. In Chlamydomonas, several mutations convert the ciliary waveform from an asymmetric, ciliary-type stroke to a symmetric, flagellar-type stroke. Some of these mutations alter subunits located at the inner junction of the doublet microtubule and others alter interactions between the dynein arms and the radial spokes. These and other axonemal substructures are interconnected by a network of poorly characterized proteins. Here we re-analyze several motility mutants (mbo, fap57, pf12/pacrg) to identify new components in this network. The mbo (move backwards only) mutants are unable to swim forwards with an asymmetric waveform. Proteomics identified more than 19 polypeptides that are missing or reduced in mbo mutants, including one inner dynein arm, IDA b. Several MBO2-associated proteins are also altered in fap57 and pf12/parcg mutants, suggesting overlapping networks. Two subunits are highly conserved, coiled coil proteins found in other species with motile cilia and others contain potential signaling domains. Cryo-electron tomography and epitope tagging revealed that the MBO2 complex is found on specific doublet microtubules and forms a large, L-shaped structure that contacts the base of IDA b that interconnects multiple dynein regulatory complexes and varies in a doublet microtubule specific fashion.

Ciliary motility is critical for the development and health of many organisms. Motility depends on the selective activation and inhibition of the multiple dynein motors along the length and around the circumference of the ciliary axoneme. This chapter focuses on two molecular hubs that are located near the base of the radial spokes (RS) and regulate dynein activity in the axoneme. The first radial spoke (RS1) is associated with inner arm dynein known as I1/f or f dynein. I1/f dynein is attached to the outer doublet microtubule using an intermediate-chain/light-chain (IC/LC) complex at its base and a tether head complex at its motor domains. The I1/f IC/LC complex is also linked to the modifier of inner arm complex. The second radial spoke (RS2) is associated with a calmodulin spoke complex and the nexin–dynein regulatory complex (N-DRC). Nexin was first identified as an interdoublet linker that repeats every 96 nm along the length of the axoneme and was proposed to limit microtubule sliding and thus generate bending during the ciliary beat cycle. The N-DRC is also thought to be a regulator in the signaling pathway between the central apparatus, RS, and dynein arms. Recent high-resolution structural and proteomic studies have provided new insights into the composition, organization, and three-dimensional structure of these regulatory hubs. The implications of these findings for the regulation of ciliary motility are discussed in this chapter.

Many axonemal proteins enter cilia and flagella on intraflagellar transport (IFT) trains, which move bidirectionally along the axonemal microtubules. Certain axonemal substructures including the radial spokes and outer dynein arms are preassembled in the cell body and transported as multisubunit complexes into flagella by IFT. Here, we used in vivo imaging to analyze the transport and assembly of DRC2 and DRC4, two core subunits of the nexin-dynein regulatory complex (N-DRC). Tagged DRC2 moved by IFT in mutants lacking DRC4 and vice versa, showing that they do not depend on each other for IFT. Simultaneous imaging of tagged DRC2 and DRC4, expressed from transgenes that rescue a corresponding double mutant, mostly showed transport on separate IFT trains, but occasional cotransports were also observed. The results demonstrate that DRC2 and DRC4 are transported largely independently of each other into flagella. These studies suggest that the N-DRC assembles onto the axoneme by the stepwise addition of subunits.

We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, we used a combination of functional and structural studies, including newly identified Chlamydomonas pacrg mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA b and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional ciliary components that play essential roles in generating coordinated ciliary beating.

The nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the 11 (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a subcomplex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N terminus of DRC4 and C terminus of DRC5. DRC4 is now shown to contribute to the core scaffold of the N-DRC. Our results reveal the overall architecture of N-DRC, with the 3 subunits DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the "functional subunits," namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.

Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat and the highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for preassembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus, IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms (IDAs) to a specific location in the 96 nm repeat. IDA8 encodes flagellar-associated polypeptide (FAP)57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple IDAs and regulatory complexes.

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DRC2 is a subunit of the nexin–dynein regulatory complex linked to primary ciliary dyskinesia. Little is known about the impact of drc2 mutations on axoneme composition and structure. We used proteomic and structural approaches to reveal that DRC2 coassembles with DRC1 to attach the N-DRC to the A-tubule and mediate interactions with other regulatory structures.

Intraflagellar transport (IFT) is essential for the elongation and maintenance of eukaryotic cilia and flagella. Due to the traffic jam of multiple trains at the ciliary tip, how IFT trains are remodeled in these turnaround zones cannot be determined by conventional imaging. Using PhotoGate, we visualized the full range of movement of single IFT trains and motors in Chlamydomonas flagella. Anterograde trains split apart and IFT complexes mix with each other at the tip to assemble retrograde trains. Dynein-1b is carried to the tip by kinesin-II as inactive cargo on anterograde trains. Unlike dynein-1b, kinesin-II detaches from IFT trains at the tip and diffuses in flagella. As the flagellum grows longer, diffusion delays return of kinesin-II to the basal body, depleting kinesin-II available for anterograde transport. Our results suggest that dissociation of kinesin-II from IFT trains serves as a negative feedback mechanism that facilitates flagellar length control in Chlamydomonas.

Ciliary motility is crucial for the development and health of many organisms. Motility depends on the coordinated activity of multiple dynein motors arranged in a precise pattern on the outer doublet microtubules. Although significant progress has been made in elucidating the composition and organization of the dyneins, a comprehensive understanding of dynein regulation is lacking. Here, we focus on two conserved signaling complexes located at the base of the radial spokes. These include the I1/f inner dynein arm associated with radial spoke 1 and the calmodulin- and spoke-associated complex and the nexin-dynein regulatory complex associated with radial spoke 2. Current research is focused on understanding how these two axonemal hubs coordinate and regulate the dynein motors and ciliary motility.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Intraflagellar transport (IFT) is essential for the elongation and maintenance of eukaryotic cilia and flagella. Due to the traffic jam of multiple trains at the ciliary tip, how IFT trains are remodeled in these turnaround zones cannot be determined by conventional imaging. Using PhotoGate, we visualized the full range of movement of single IFT trains and motors in Chlamydomonas flagella. Anterograde trains split apart and IFT complexes mix with each other at the tip to assemble retrograde trains. Dynein-1b is carried to the tip by kinesin-II as inactive cargo on anterograde trains. Unlike dynein-1b, kinesin-II detaches from IFT trains at the tip and diffuses in flagella. As the flagellum grows longer, diffusion delays return of kinesin-II to the basal body, depleting kinesin-II available for anterograde transport. Our results suggest that dissociation of kinesin-II from IFT trains serves as a negative feedback mechanism that facilitates flagellar length control in Chlamydomonas. https://doi.org/10.7554/eLife.28606.001 eLife digest Cilia and flagella are hair-like structures that protrude from nearly every human cell and play a number of roles including transmitting signals and enabling cells to move. These structures lengthen when new material is deposited at their tip by a process called intraflagellar transport (IFT). In this process, protein complexes known as IFT trains carry cargo along tracks that run along the length of each flagellum. Different motor proteins power the IFT trains in different directions: kinesin moves IFT trains from the base to the tip, while dynein moves them back in the opposite direction. When IFT trains arrive at the tip of the flagellum, they release their cargo and undergo a major reorganization process in which the trains switch motors in order to move back to the base. Because the many IFT trains at the tip form a kind of 'traffic jam', standard imaging techniques have been unable to distinguish exactly what happens during this reorganization. A new imaging method called PhotoGate microscopy can track individual molecules inside crowded cells. Chien, Shih et al. have now used this method to visualize the full range of movements made by IFT trains and motors in the flagella of a species of single-celled algae. This revealed that at the tip of the flagellum, IFT trains split apart and mix with each other to assemble into new trains, which move back to the base. In addition, kinesin carries dynein to the tip as inactive cargo, detaches from IFT trains at the tip and diffuses back to the base of the flagellum. This delays kinesin's return and causes it to accumulate in the flagellum, which helps to explain why flagella assemble more slowly as they lengthen: as the flagellum grows longer, less kinesin is available at the base to transport material to the tip. Thus kinesin diffusion helps the algae to regulate the length of their flagella. Further research is now needed to study whether similar mechanisms control the length of flagella in other organisms. Defects in the intraflagellar transport process have been linked to a range of human diseases known as ciliopathies, and so the results presented by Chien, Shih et al. could also help us to uncover the causes and potential treatments for these conditions. https://doi.org/10.7554/eLife.28606.002 Introduction Cilia (or eukaryotic flagella, terms essentially referring to the same organelle) are hair-like organelles that extend from the plasma membrane of nearly all mammalian cells. The core structural component of a cilium is the axoneme, a ring of nine unipolar doublet microtubules surrounding a central pair of singlet microtubules. Cilia play essential roles in cell motility, generate the movement of fluids over multiciliated surfaces, and sense extracellular signals (Satir et al., 2010). To assemble and maintain functional cilia, the IFT machinery (Kozminski et al., 1993) transports axonemal precursors and sensory proteins bidirectionally between the cell body and the ciliary tip. Defects in IFT are linked to a wide range of human diseases including Bardet-Biedl syndrome, retinal degeneration, and polycystic kidney disease (Brown and Witman, 2014). Intraflagellar cargoes interact with multiprotein complexes known as IFT particles that are organized into larger IFT trains as they enter the flagellum (Cole et al., 1998; Lechtreck et al., 2017; Pigino et al., 2009). In most species, the IFT trains are transported anterogradely from the base to the tip of a flagellum by heterotrimeric kinesin-II (Kozminski et al., 1995), but some species also use a second homodimeric kinesin to build more specialized sensory cilia (Snow et al., 2004). Once the trains reach the tip, they are reorganized and transported retrogradely to the ciliary base by dynein-1b (Pazour et al., 1999; Porter et al., 1999; Signor et al., 1999). Along the length of a cilium, the activities of kinesin and dynein motors are reciprocally coordinated, such that only one type of a motor is active at a time (Shih et al., 2013). As a result, trains move between the tip and base of the cilium without significant pauses or back-and-forth motion (Dentler, 2005; Engel et al., 2009) and switch directions at the turnaround zones (Ishikawa and Marshall, 2011; Laib et al., 2009; Shih et al., 2013). Dynein-1b requires kinesin-II activity to reach the ciliary tip, suggesting that it travels on anterograde trains (Iomini et al., 2001; Pedersen et al., 2006; Rompolas et al., 2007). Retrograde traces of kinesin-II have not been frequently observed in Chlamydomonas, and it remains unclear how kinesin-II returns to the basal body (Engel et al., 2009; Wingfield et al., 2017). Because anterograde and retrograde IFT trains have different sizes and depart at different frequencies (Dentler, 2005; Iomini et al., 2001), IFT trains must be remodeled at the distal tip and the flagellar base. The mechanism underlying the remodeling of IFT complexes at the ciliary tip and base cannot be directly observed by conventional microscopy methods because multiple trains coexist in these turnaround zones (Buisson et al., 2013; Iomini et al., 2001; Wren et al., 2013). In this work, we adapted PhotoGate (Belyy et al., 2017) to control the number of fluorescent IFT trains entering the flagellum of the unicellular algae Chlamydomonas reinhardtii. Using this approach, we monitored the turnaround behavior and remodeling of single IFT trains at the flagellar tip. We also elucidated the mechanisms by which the kinesin and dynein motors are recycled in this process and IFT trains reverse their direction of motion. The dynamics of IFT motor turnover at the tip suggest a new mechanism for how Chlamydomonas controls the length of its flagella. Results IFT trains split apart and mix with each other at the flagellar tip To monitor IFT movement, we tracked the dynamic behavior of IFT27, a core component of the IFT complex B, in a pf18 IFT27-GFP strain (Engel et al., 2009; Qin et al., 2007). This strain has paralyzed flagella (pf) that readily adhere to the glass surface, enabling us to monitor IFT under total internal reflection (TIR) illumination (Engel et al., 2009). Consistent with previous studies (Dentler, 2005; Engel et al., 2009; Iomini et al., 2001; Kozminski et al., 1993; Qin et al., 2007; Shih et al., 2013), IFT trains moved processively along the length of flagella, reversing direction at the flagellar tip and base (Figure 1a, Video 1). Pausing and reversals of anterograde trains before reaching the tip were very rare. The velocity of IFT27-GFP was 2.1 ± 0.4 µm s−1 in the anterograde direction and 3.0 ± 0.7 µm s−1 in the retrograde direction (Figure 1—figure supplement 1, mean ± s.d., N = 80 trains in each direction). Because a large number of GFP-labeled trains accumulated at the tip, the dwell and departure of individual trains at the tip could not be resolved by conventional TIR imaging (Figure 1a). Figure 1 with 3 supplements see all Download asset Open asset IFT trains split apart and mix with each other at the flagellar tip. (a) Kymograph of a surface-immobilized pf18 IFT27-GFP strain shows that IFT trains move bidirectionally along the flagellum, only reversing direction at the tip and the base. Multiple IFT trains accumulate at the flagellar tip. Representative anterograde and retrograde trajectories are shown with yellow and red dashed lines, respectively. (b) Schematic representation of the PhotoGate assay. (1) The distal half of the flagellum is prebleached by moving the powerful gate beam from the flagellar tip to near the base of the flagellum. (2–3) The gate beam is turned off to allow a single anterograde train to enter the flagellum without photobleaching. (4) The beam is then repeatedly turned on for 0.2 s to photobleach the successive trains entering the flagellum and (5) turned off for 0.8 s to image the single fluorescent train within the flagellum. Photobleached trains are not shown. (c–e) Kymographs of one (c), two (d) and three (e) fluorescent anterograde trains entering the flagellum. Anterograde trains pause at the flagellar tip and split into multiple retrograde trains that move back to the base. Arrival of fluorescent anterograde trains and departure of retrograde trains at the tip are shown with red and yellow stars, respectively. Arrows represent repetitive bleaching events near the base of the flagellum. (f) (Left) The number of fluorescent retrograde trains was quantified as a function of one, two or three fluorescent anterograde trains entering the flagellum after photobleaching. (Right) The average number of retrograde trains increased sub-proportionally with the number of fluorescent anterograde trains entering the flagellum. N = 97, 60, 42 train events from top to bottom, in 160 cells, from 13 independent experiments. (g) The number of detectable retrograde trains versus the numbers of incoming anterograde trains in PhotoGate experiments and Monte Carlo simulations (PB: photobleaching). Solid lines represent the fit of data to the power law (y = axn). n is less than one under each condition. Error bars represent s.e.m. (N = 10,000 for simulations). https://doi.org/10.7554/eLife.28606.003 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Tracking of individual IFT trains in Chlamydomonas. IFT movement was visualized by TIR imaging of a surface-adhered pf18 IFT27-GFP cell. Green and blue arrows represent the cell body and flagellar tips, respectively. The size of the window is 15.5 × 27.6 µm. The movie was recorded at 10 frames s−1 and is played in real time. This video corresponds to Figure 1a. https://doi.org/10.7554/eLife.28606.007 To monitor the turnaround behavior of individual IFT trains at the flagellar tip, we developed the one-dimensional PhotoGate assay (Belyy et al., 2017) to track single fluorescent complexes at the flagellar tip. In this assay, fluorescent trains located at distal parts of a flagellum were initially photobleached by moving a focused laser beam from the tip of the flagellum to near its base. We next opened the 'gate' by turning off the focused beam until a single fluorescent train entered the flagellum. The gate beam was repeatedly turned on for 0.2 s at 1 Hz to photobleach any additional anterograde trains entering the flagellum (Figure 1b, Video 2). Under these conditions, less than 1% of anterograde IFT trains were able to pass the gate unbleached. This approach revealed the full range of movement of single fluorescent IFT trains within the flagellum. IFT movement can be divided into three stages: anterograde movement toward the tip, pausing at the tip, and returning to the base by retrograde transport. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Observing the dynamics of single IFT trains at the flagellar tip using the PhotoGate. The distal half of the flagellum on the right of a surface-adhered pf18 IFT27-GFP cell is photobleached by an intense laser beam. The gate beam is turned off to allow a single anterograde train to enter the bleached region and turned on repeatedly at the base of the flagellum to bleach the subsequent anterograde trains. Two retrograde trains emanate from a single fluorescent anterograde train at the tip. Frames with the gate beam on are removed for illustration purposes. The movie was recorded at 10 frames s−1 and is played in real time. Cyan and magenta arrows represent anterograde and retrograde particles, respectively. Pre-bleaching frames are marked by red borders. The position of the PhotoGate is marked by a red line. This video corresponds to Figure 1c. https://doi.org/10.7554/eLife.28606.008 We directly observed that a single anterograde train splits into multiple retrograde trains at the tip (Figure 1c–e, Figure 1—figure supplement 2a). On average, 2.4 retrograde trains were detected departing from the tip after the arrival of a single fluorescent anterograde train (Figure 1f, N = 97), consistent with higher frequencies of retrograde IFT trains than anterograde IFT trains (Dentler, 2005; Iomini et al., 2001; Qin et al., 2007). However, the number of retrograde trains per fluorescent anterograde train in PhotoGate assays (2.4, Figure 1c) was significantly higher (Welch's t-test, p=10−20) than the ratio of retrograde to anterograde train frequencies (1.15, Figure 1a) (Dentler, 2005; Iomini et al., 2001; Reck et al., 2016). These observations suggested that IFT complexes from different anterograde trains recombine with each other to form retrograde trains at the tip. To test this possibility, we closed the gate after two or three fluorescent anterograde trains entered the flagellum (Figure 1d,e, Videos 3 and 4) and measured the number and return frequency of retrograde trains departing from the tip. If individual trains split and return without mixing with each other, the number and frequency of fluorescent retrograde trains would be proportional to the number of fluorescent anterograde trains. In contrast, we observed 2.4, 3.6, and 4.2 returning trains on average for one, two, and three incoming trains, respectively (N = 97, 60, 42, Figure 1f). The return frequencies for one, two, and three incoming fluorescent trains were 0.57, 0.71, and 0.76 s−1, respectively. Because the increase was sub-proportional with the number of anterograde trains, we concluded that the fluorescent complexes in the anterograde trains disassemble and mix with a pool of 'dark' complexes from the other photobleached trains at the tip before they reorganize into retrograde trains (Figure 1f). Monte Carlo simulations revealed that our conclusions are not markedly affected by the limited number of IFT27-GFPs per anterograde train (~6) or GFP photobleaching under TIR illumination (0.07 s−1, Figure 1g, Figure 1—figure supplement 3, see Materials and methods). Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Tip return dynamics of two fluorescent anterograde trains using the PhotoGate. A flagellum of a surface-adhered pf18 IFT27-GFP cell is photobleached. Two fluorescent anterograde IFT trains are allowed pass through the gate without photobleaching and subsequent anterograde trains were photobleached by the gate beam. After the anterograde trains reach the tip, three retrograde trains return with fluorescent signal from these trains. The movie was recorded at 10 frames s−1 and is played in real time. Cyan and magenta arrows represent anterograde and retrograde particles, respectively. The position of the PhotoGate is marked by a red line. This video corresponds to Figure 1d. https://doi.org/10.7554/eLife.28606.009 Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Tip return dynamics of three fluorescent anterograde trains using the PhotoGate. A flagellum of a surface-adhered pf18 IFT27-GFP cell is photobleached. Three fluorescent anterograde IFT trains are allowed pass through the gate without photobleaching and subsequent anterograde trains were photobleached by the gate beam. The frames in which the gate beam was on were deleted for illustration purposes. After the anterograde trains reach the tip, four retrograde trains return with fluorescent signal from these trains. The movie was recorded at 10 frames s−1 and is played in real time. Cyan and magenta arrows represent anterograde and retrograde particles, respectively. Pre-bleaching frames are marked by red borders. The position of the PhotoGate is marked by a red line. This video corresponds to Figure 1e. https://doi.org/10.7554/eLife.28606.010 IFT tip turnaround is regulated by dynein activity and extracellular Ca2+ To understand how IFT trains are processed at the tip, we analyzed the time between the arrival of an anterograde train and the departure of fluorescent retrograde trains (referred to as tip resting time) at the tip (Figure 2a). When only a single fluorescent anterograde IFT27-GFP train was left unbleached near the base of the flagellum, the tip resting time of the first retrograde train was 3.1 ± 0.3 s (mean ± s.e.m., N = 97, Figure 2b), comparable to that of IFT cargos (Craft et al., 2015; Reck et al., 2016; Wren et al., 2013). Tip resting time was independent of flagellar length (Figure 2—figure supplement 1). If IFT tip turnaround was rate-limited by a single process, we would expect a single exponential distribution of tip resting times. However, the tip resting time histogram of first retrograde trains fit well to a Gamma distribution with a shape parameter of 3 and a rate constant of ~1 s−1, indicating that tip turnaround of IFT trains occurs rapidly through a multistep process (Figure 2b). We also observed that the tip resting time of first, second, third, and fourth fluorescent retrograde trains increases linearly (Figure 2c). Because the average time between successive retrograde trains (Δt = 1.7 s) is the same, we concluded that the tip departure is a purely stochastic process. The linear fit to the tip resting times has a y-intercept of 1.3 s (Figure 2c), revealing that the departure of the first train takes longer than Δt. Therefore, the complexes dwell at the tip through another rate limiting process before they can depart from the tip. Figure 2 with 1 supplement see all Download asset Open asset Tip turnaround of IFT trains is a multistep process regulated by dynein activity and extracellular Ca2+. (a) The schematic describes the definition of tip resting time, remodeling time, and departure time measured from the kymographs. Arrival of the first fluorescent anterograde train and the departure of retrograde trains are shown with red and yellow stars, respectively. Tip resting time and departure time are only shown for the first retrograde train. Tip remodeling time is assumed to be the same for each train. Δt represents the time between the departure of successive retrograde trains. (b) The tip resting time histogram of the first retrograde IFT27-GFP train (dark grey) and all of the trains (light grey) emanating from a single anterograde train. The histogram of the first retrograde trains was fitted to a Gamma function (red curve). α and λ are shape and rate parameters, respectively. (c) The linear fit to the average tip resting time reveals Δt between successive trains. The y-intercept (black dashed line) represents the tip remodeling time. Errors represent standard error of the linear fit. (d) Average tip resting times of the first, second, third, and fourth retrograde IFT27-GFP trains coming out of an anterograde train for cells in TAP media (red, N = 97) and calcium-depleted media (blue, N = 44). Errors represent standard error of the linear fit. (e) IFT27-GFP tip resting times in 0.5 mM EGTA and 0.1 mM ciliobrevin D treated cells. The line within the boxplot represents the mean. The outer edges of the box represent standard deviation. N = 97, 241, 44, 109, 34, and 48 retrograde trains from left to right, in a total of 20 independent experiments (Welch's t-test, *p<0.05, ***p<0.001, as compared to no treatment for first retrograde trains). https://doi.org/10.7554/eLife.28606.011 When two or three fluorescent anterograde trains were allowed to pass through the gate, Δt became shorter (1.4 and 1.3 s, Welch's t-test, p=0.03 and 0.02 for two and three trains, respectively; Figure 2c). This is because providing a higher number of fluorescent GFPs available at the tip increases the likelihood of retrograde trains to have at least one fluorescent GFP. Remarkably, the y-intercept remained constant when we allowed one to three more fluorescent anterograde trains to enter the flagellum (Figure 2c), suggesting that this duration corresponds to processing and breakdown of anterograde trains at the tip (referred to as tip remodeling time). The time between tip remodeling and departure of IFT trains is defined as the tip departure time (Figure 2a). We also tested whether extracellular calcium and the dynein inhibitor ciliobrevin D (Firestone et al., 2012) affect the duration of IFT tip turnaround. Calcium regulates pausing of IFT trains along the flagellum (Collingridge et al., 2013; Shih et al., 2013), and disrupting calcium-dependent kinesin-II phosphorylation causes abnormal accumulations of IFT proteins at the ciliary tip (Liang et al., 2014). When extracellular calcium in media (0.34 mM) was chelated using 0.5 mM EGTA, the tip departure time increased to 2.8 s (Welch's t-test, p=0.01), whereas tip remodeling time (1.4 s) remained unaltered (Figure 2d,e). Therefore, calcium has minimal effect on the breakdown of anterograde trains, but may have a regulatory role in the assembly or departure of retrograde trains. Addition of 0.1 mM ciliobrevin D to media results in 50% reduction in the frequency of retrograde and anterograde trains, and 50% and 28% reduction in retrograde and anterograde train velocities, respectively (Shih et al., 2013). In this case, the tip resting time of IFT27 increased over two-fold (Figure 2e, p<10−4), suggesting that rapid turnover of IFT trains depends on dynein activity. Kinesin-II dissociates from IFT trains at the tip We next turned our attention to the movement of the IFT motors and their exchange at the flagellar tip. Dynein-1b was tagged with GFP at its light intermediate chain (D1bLIC), which assembles into the dynein-1b complex and rescues d1blic mutant phenotypes (Reck et al., 2016). In the d1blic::D1bLIC-GFP strain, D1bLIC moved continuously in the anterograde and retrograde directions at velocities similar to that of the IFT trains (Reck et al., 2016) (Figure 3a, Figure 1—figure supplement 1). Kinesin-II was tagged with GFP at its non-motor subunit KAP that localizes kinesin-II to the flagellar base (Mueller et al., 2005). In the fla3::KAP-GFP strain, KAP moved primarily in the anterograde direction to the flagellar tip at a similar speed to anterograde IFT27 (Figure 3b, Figure 1—figure supplement 1). Unlike D1bLIC, retrograde traces of KAP were not frequently observed (Engel et al., 2009; Wingfield et al., 2017), suggesting that kinesin-II dissociates from IFT trains at the tip (Engel et al., 2009; Engel et al., 2012). Figure 3 with 2 supplements see all Download asset Open asset PhotoGate reveals the tip turnaround behavior of IFT motors. (a) In a conventional TIR assay, anterograde and retrograde D1bLIC-GFP traces were clearly visible, but the tip behavior of individual trains could not be discerned. (b) In a conventional TIR assay, KAP-GFP was observed to move anterogradely, but the retrograde transport of KAP was rarely observed. (c) PhotoGate imaging of D1bLIC-GFP shows that D1bLIC trains move to the tip anterogradely, split into multiple trains, and return to the base retrogradely. Red and yellow stars indicate arrival to and departure of D1bLIC-GFP from the tip, respectively. (d) The tip resting time histogram of D1bLIC-GFP. Tip resting time of the first retrograde trains are fit to a Gamma distribution (red curve; 95% c.i. for α is 1.30–2.52 and for λ is 0.69–1.47 s−1). N = 60 anterograde trains in 60 cells over nine independent experiments. (e) Averaged tip resting time of the first, second, third, and fourth retrograde D1bLIC-GFP particles returning from the tip (mean ± s.e.m.). (f) Kymograph analysis of a KAP-GFP cell imaged by PhotoGate. KAP undergoes active transport in the anterograde direction, pauses at the flagellar tip, and diffuses back to the flagellar base. (g) The tip resting time histogram of KAP-GFP. The red curve represents a fit of first train resting times to a Gamma distribution. N = 95 anterograde trains in 47 cells over four independent experiments (95% c.i. for α is 1.87–3.19 and for λ is 0.88–1.60 s−1). (h) MSD analysis of KAP-GFP diffusion after it leaves the flagellar tip reveals the average diffusion constant (N = 27, mean ± s.e.m.). (i) High-resolution tracking of a KAP-GFP particle reveals the two-dimensional trajectory during anterograde transport and diffusion. The red curve is the polynomial fit to the trace. (j) The residual plot to the trace in (i) reveals lateral fluctuations (σ) during anterograde transport (black) and diffusion (blue). https://doi.org/10.7554/eLife.28606.013 PhotoGate assays revealed that D1bLIC-GFP has similar tip turnaround dynamics to IFT27-GFP (Figure 3c, Figure 1—figure supplement 2b, Video 5). After arrival of a single anterograde D1bLIC-GFP train at the tip, we detected on average 2.5 retrograde D1bLIC trains. The average tip resting time until the departure of the first retrograde train was 1.8 ± 0.2 s (mean ± s.e.m., N = 60, Figure 3d), with additional ~1.1 ± 0.1 s between subsequent departure events (Figure 3e). PhotoGate imaging of KAP-GFP cells showed that single KAP-GFP trains moved anterogradely to the tip and rested at the tip for 2.2 ± 0.2 s (N = 95). Unlike D1bLIC, individual KAP-GFP particles moved away from the tip by rapid saltatory motion (Figure 3f,g, Figure 1—figure supplement 2c, Video 6). Mean square displacement (MSD) analysis showed that KAP undergoes one-dimensional diffusion at 1.68 ± 0.04 µm2 s−1 (mean ± s.e.m., N = 27 traces) within the flagellum after it departs from the tip (Figure 3h), consistent with the values measured for tubulin and EB1 that undergo diffusion within the ciliary space (Craft et al., 2015; Harris et al., 2016). The tip resting time of KAP remained nearly constant at different steady-state flagellar lengths (Figure 2—figure supplement 1) and was shorter than that of IFT27, indicating that departure of kinesin from the tip is independent of flagellar length and departure of retrograde trains (Figure 2—figure supplement 1). Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Tip return dynamics of D1bLIC-GFP. In a surface-adhered d1blic::D1bLIC-GFP cell, one D1bLIC-GFP particle is allowed to pass through the gate without photobleaching and two fluorescent retrograde particles return to the base. The size of the window is 30.2 × 31.1 µm. The movie was recorded at 10 frames s−1 and is played in real time. Cyan and magenta arrows represent anterograde and retrograde particles, respectively. Pre-bleaching frames are marked by red borders. The position of the PhotoGate is marked by a red line. This video corresponds to Figure 3c. https://doi.org/10.7554/eLife.28606.016 Video 6 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg KAP-GFP dissociates from IFT trains at the tip. In the fla3::KAP-GFP strain, one fluorescent KAP-GFP particle is allowed to pass through the gate without photobleaching. The KAP-GFP particle undergoes diffusive motion after arriving at the flagellar tip. The size of the window is 26.6 × 16.6 µm. The movie was recorded at 10 frames s−1 and is played in real time. Cyan and magenta arrows represent anterograde and diffusing particles, respectively. The position of the PhotoGate is marked by a red line. This video corresponds to Figure 3f. https://doi.org/10.7554/eLife.28606.017 Unlike IFT trains, the majority (89%, N = 95) of KAP-GFPs simultaneously departed from the tip in a single step (Figure 3—figure supplement 1). Because each anterograde train contains multiple (6) KAP-GFPs on average (Engel et al., 2009), this observation indicates that kinesin-II

The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms.

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.

We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed "reactivated cell models." ATP-induced reactivation of wild-type cells resulted in the forward swimming of ∼90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function. © 2016 Wiley Periodicals, Inc.

Cryo-electron tomography (cryo-ET) has reached nanoscale resolution for in situ three-dimensional imaging of macromolecular complexes and organelles. Yet its current resolution is not sufficient to precisely localize or identify most proteins in situ; for example, the location and arrangement of components of the nexin-dynein regulatory complex (N-DRC), a key regulator of ciliary/flagellar motility that is conserved from algae to humans, have remained elusive despite many cryo-ET studies of cilia and flagella. Here, we developed an in situ localization method that combines cryo-ET/subtomogram averaging with the clonable SNAP tag, a widely used cell biological probe to visualize fusion proteins by fluorescence microscopy. Using this hybrid approach, we precisely determined the locations of the N and C termini of DRC3 and the C terminus of DRC4 within the three-dimensional structure of the N-DRC in Chlamydomonas flagella. Our data demonstrate that fusion of SNAP with target proteins allowed for protein localization with high efficiency and fidelity using SNAP-linked gold nanoparticles, without disrupting the native assembly, structure, or function of the flagella. After cryo-ET and subtomogram averaging, we localized DRC3 to the L1 projection of the nexin linker, which interacts directly with a dynein motor, whereas DRC4 was observed to stretch along the N-DRC base plate to the nexin linker. Application of the technique developed here to the N-DRC revealed new insights into the organization and regulatory mechanism of this complex, and provides a valuable tool for the structural dissection of macromolecular complexes in situ.

The green alga Chlamydomonas reinhardtii is a popular unicellular organism for studying photosynthesis, cilia biogenesis, and micronutrient homeostasis. Ten years since its genome project was initiated an iterative process of improvements to the genome and gene predictions has propelled this organism to the forefront of the omics era. Housed at Phytozome, the plant genomics portal of the Joint Genome Institute (JGI), the most up-to-date genomic data include a genome arranged on chromosomes and high-quality gene models with alternative splice forms supported by an abundance of whole transcriptome sequencing (RNA-Seq) data. We present here the past, present, and future of Chlamydomonas genomics. Specifically, we detail progress on genome assembly and gene model refinement, discuss resources for gene annotations, functional predictions, and locus ID mapping between versions and, importantly, outline a standardized framework for naming genes.