Chlamydomonas ARMC2/PF27 is an obligate cargo adapter for IFT of radial spokes

Intraflagellar transport (IFT) carries proteins into flagella but how IFT trains interact with the large number of diverse proteins required to assemble flagella remains largely unknown. Here, we show that IFT of radial spokes in Chlamydomonas requires ARMC2/PF27, a conserved armadillo repeat protein associated with male infertility and reduced lung function. Chlamydomonas ARMC2 was highly enriched in growing flagella and tagged ARMC2 and the spoke protein RSP3 comigrated on anterograde trains. In contrast, a cargo and an adapter of inner and outer dynein arms moved independently of ARMC2, indicating that unrelated cargoes distribute stochastically onto the IFT trains. After concomitant unloading at the flagellar tip, RSP3 attached to the axoneme whereas ARMC2 diffused back to the cell body. In armc2/pf27 mutants, IFT of radial spokes was abolished and the presence of radial spokes was limited to the proximal region of flagella. We conclude that ARMC2 is a cargo adapter required for IFT of radial spokes to ensure their assembly along flagella. ARMC2 belongs to a growing class of cargo-specific adapters that enable flagellar transport of preassembled axonemal substructures by IFT.

Oscillatory movement of a dynein-microtubule complex crosslinked with DNA origami

During repetitive bending of cilia and flagella, axonemal dynein molecules move in an oscillatory manner along a microtubule (MT), but how the minus-end-directed motor dynein can oscillate back and forth is unknown. There are various factors that may regulate the dynein activities, e.g., the nexin-dynein regulatory complex, radial spokes, and central apparatus. In order to understand the basic mechanism of the oscillatory movement, we constructed a simple model system composed of MTs, outer-arm dyneins, and DNA origami that crosslinks the MTs. Electron microscopy (EM) showed patches of dynein molecules crossbridging two MTs in two opposite orientations; the oppositely oriented dyneins are expected to produce opposing forces. The optical trapping experiments showed that the dynein-MT-DNA-origami complex actually oscillate back and forth after photolysis of caged ATP. Intriguingly, the complex, when held at one end, showed repetitive bending motions. The results show that a simple system composed of ensembles of oppositely oriented dyneins, MTs, and inter-MT crosslinkers, without the additional regulatory structures, has an intrinsic ability to cause oscillation and repetitive bending motions.

Chlamydomonas ARMC2/PF27 is an obligate cargo adapter for IFT of radial spokes

Intraflagellar transport (IFT) carries proteins into flagella but how IFT trains interact with the large number of diverse proteins required to assemble flagella remains largely unknown. Here, we show that IFT of radial spokes in Chlamydomonas requires ARMC2/PF27, a conserved armadillo repeat protein associated with male infertility and reduced lung function. Chlamydomonas ARMC2 was highly enriched in growing flagella and tagged ARMC2 and the spoke protein RSP3 comigrated on anterograde trains. In contrast, a cargo and an adapter of inner and outer dynein arms moved independently of ARMC2, indicating that unrelated cargoes distribute stochastically onto the IFT trains. After concomitant unloading at the flagellar tip, RSP3 attached to the axoneme whereas ARMC2 diffused back to the cell body. In armc2/pf27 mutants, IFT of radial spokes was abolished and the presence of radial spokes was limited to the proximal region of flagella. We conclude that ARMC2 is a cargo adapter required for IFT of radial spokes to ensure their assembly along flagella. ARMC2 belongs to a growing class of cargo-specific adapters that enable flagellar transport of preassembled axonemal substructures by IFT.

LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction based on microtubules, and some attached projections. Radial spokes (RSs) facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 radial spoke proteins (RSP1-23), with the roles of some radial spoke proteins remained unknown. Recently, RSP15 has been reported to be located to the stalk of RS2, but its homolog in mammals has not been explored. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. We found that LRRC23 localizes to the RS complex within murine sperm flagella and interacts with RSPH3A/B. The knockout of Lrrc23 resulted in male infertility due to RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was unaffected significantly. These data indicate that LRRC23 is a key regulator underpinning the integrity of RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia.

Kif9 is an active kinesin motor required for ciliary beating and proximodistal patterning of motile axonemes

Most motile cilia have a stereotyped structure of nine microtubule outer doublets and a single central pair of microtubules. The central pair microtubules are surrounded by a set of proteins, termed the central pair apparatus. A specific kinesin, Klp1 projects from the central pair and contributes to ciliary motility in Chlamydomonas. The vertebrate orthologue, Kif9 is required for beating in mouse sperm flagella, but the mechanism of Kif9/Klp1 function remains poorly defined. Here, using Xenopus epidermal multiciliated cells, we show that Kif9 is necessary for ciliary motility as well as leads to defects in the distal localization of not only central pair proteins, but also radial spokes and dynein arms. In addition, single-molecule assays in vitro revealed that Xenopus Kif9 is a processive motor, though like axonemal dyneins it displays no processivity in ciliary axonemes in vivo. Thus, our data suggest that Kif9 plays both indirect and direct role in ciliary motility.

Structural organization of the C1b projection within the ciliary central apparatus

'9+2' motile cilia contain 9 doublet microtubules and a central apparatus (CA) composed of two singlet microtubules with associated projections. The CA plays crucial roles in regulating ciliary motility. Defects in CA assembly or function usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of most CA projections remain largely unknown. Here, we combined genetic approaches and quantitative proteomics with cryo-electron tomography and subtomogram averaging to compare the CA of wild-type Chlamydomonas with those of two CA mutants. Our results show that two conserved proteins, FAP42 and FAP246, are localized to the L-shaped C1b projection of the CA. We also identified another novel CA candidate protein, FAP413, which interacts with both FAP42 and FAP246. FAP42 is a large protein that forms the peripheral 'beam' of the C1b projection, and the FAP246-FAP413 subcomplex serves as the 'bracket' between the beam (FAP42) and the C1b 'pillar' that attaches the projection to the C1 microtubule. The FAP246-FAP413-FAP42 complex is essential for stable assembly of both the C1b and C1f projections, and loss of any of these proteins leads to ciliary motility defects. Our results provide insight into the subunit organization and 3D structure of the C1b projection, suggesting that the FAP246-FAP413-FAP42 subcomplex is part of a large interconnected CA-network that provides mechanical support and may play a role in mechano-signaling between the CA and radial spokes to regulate dynein activity and ciliary beating.

LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice

Cilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction of structures including dynein arms, radial spokes (RSs), microtubules, and the dynein regulatory complex (DRC). RSs facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 different proteins (RSP1-23), with the roles of RSP13, RSP15, RSP18, RSP19, and RSP21 remained poorly understood. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. Through immunoelectron microscopy, we demonstrate that LRRC23 localizes to the RS complex within murine sperm flagella. We further found that LRRC23 was able to interact with RSHP9 and RSPH3A/B. The knockout of Lrrc23 resulted in RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was unaffected by the loss of this protein. Spermatozoa lacking LRRC23 were unable to efficiently pass through the uterotubal junction and exhibited defective zona penetration. Together these data indicate that LRRC23 is a key regulator underpinning the integrity of RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia.

Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Protein 4 homolog A ( RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A-/- PCD remains elusive. Here, we present the native, three-dimensional structure of RSPH4A-/- human respiratory cilia using samples collected non-invasively from a PCD patient. Using cryo-electron tomography and subtomogram averaging, we compared the structures of control and RSPH4A-/- cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1-/- cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A-/- cilia. However, RSPH4A-/- cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A-/- PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination. [Media: see text]

Ccdc113/Ccdc96 complex, a novel regulator of ciliary beating that connects radial spoke 3 to dynein g and the nexin link

Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern.

Genipin-Cross-Linked Layer-by-Layer Chitosan/Hydroxyapatite Composite Rod for Bone Fracture Fixation

The synthesis and optimization of chitosan-based inorganic/organic composites have gained growing interest in the last years. In the current study, genipin crosslinked hydroxyapatite/Chitosan (HA/CHI) composite rods (GCHR), which had been prepared and reported in our prevenient studies, were further evaluated via their formation of layer-by-layer structure, cross-linking degree and so on. The cross-linking degree in the range of 28.73∼43.32% was moderate for the in situ periodic precipitation and the formation of homogeneous HA/CHI/Genipin composite rods. In this range the composite rods could obtain a complex structure combined with layer-by-layer structure and radial spokes structure. Benifiting from the special structure, the rods can obtain superior mechanical properties and become fresh device for internal fixation of fracture. Mouse bone marrow mesenchymal stem cells (mBMSCs) were co-cultured with extract of the GCHR100 rods to evaluate the cytocompatibility of the composite. The results showed that the leachate of the GCHR100 rods significantly up-regulated osteogenesis-related genes like ALP, Runx2 and OCN after 7 days compared with control (P< 0.05).