CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module.
ABSTRACT: Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.
Project description:Axonemal dynein ATPases direct eukaryotic ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here we describe a deficiency of CFAP45 in both humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar human ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is partially rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.
Project description:Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here we show using cryoelectron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the f β head perpendicular to the microtubule-sliding direction. In the absence of the tether complex, however, addition of ATP and vanadate caused a large conformational change in the IDA f head domains, suggesting that the movement of IDA f is mechanically restricted by the tether complex. Motility defects in flagella missing the tether demonstrates the importance of the IDA f-tether interaction in the regulation of ciliary/flagellar beating.
Project description:The physical basis of flagellar and ciliary beating is a major problem in biology which is still far from completely understood. The fundamental cytoskeleton structure of cilia and flagella is the axoneme, a cylindrical array of microtubule doublets connected by passive cross-linkers and dynein motor proteins. The complex interplay of these elements leads to the generation of self-organized bending waves. Although many mathematical models have been proposed to understand this process, few attempts have been made to assess the role of dyneins on the nonlinear nature of the axoneme. Here, we investigate the nonlinear dynamics of flagella by considering an axonemal sliding control mechanism for dynein activity. This approach unveils the nonlinear selection of the oscillation amplitudes, which are typically either missed or prescribed in mathematical models. The explicit set of nonlinear equations are derived and solved numerically. Our analysis reveals the spatio-temporal dynamics of dynein populations and flagellum shape for different regimes of motor activity, medium viscosity and flagellum elasticity. Unstable modes saturate via the coupling of dynein kinetics and flagellum shape without the need of invoking a nonlinear axonemal response. Hence, our work reveals a novel mechanism for the saturation of unstable modes in axonemal beating.
Project description:The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural, and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. In this study, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axoneme's ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ?2.3 × 10(5) ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights, to our knowledge, into the beating mechanism of flagella and a powerful tool for future studies.
Project description:The motility of cilia and flagella is powered by dynein ATPases associated with outer doublet microtubules. However, a flagellar kinesin-like protein that may function as a motor associates with the central pair complex. We determined that Chlamydomonas reinhardtii central pair kinesin Klp1 is a phosphoprotein and, like conventional kinesins, binds to microtubules in vitro in the presence of adenosine 5'-[beta,gamma-imido]triphosphate, but not ATP. To characterize the function of Klp1, we generated RNA interference expression constructs that reduce in vivo flagellar Klp1 levels. Klp1 knockdown cells have flagella that either beat very slowly or are paralyzed. EM image averages show disruption of two structures associated with the C2 central pair microtubule, C2b and C2c. Greatest density is lost from part of projection C2c, which is in a position to interact with doublet-associated radial spokes. Klp1 therefore retains properties of a motor protein and is essential for normal flagellar motility. We hypothesize that Klp1 acts as a conformational switch to signal spoke-dependent control of dynein activity.
Project description:The outer dynein arms (ODAs) of the flagellar axoneme generate forces needed for flagellar beating. Elucidation of the mechanisms underlying the chemomechanical energy conversion by the dynein arms and their orchestrated movement in cilia/flagella is of great importance, but the nucleotide-dependent three-dimensional (3D) movement of dynein has not yet been observed. In this study, we establish a new method for reconstructing the 3D structure of the in vitro reconstituted ODA-microtubule complex and visualize nucleotide-dependent conformational changes using cryoelectron microscopy and image analysis. As the complex went from the rigor state to the relaxed state, the head domain of the beta heavy chain shifted by 3.7 nm toward the B tubule and inclined 44 degrees inwards. These observations suggest that there is a mechanism that converts head movement into the axonemal sliding motion.
Project description:Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei.
Project description:Motile ciliopathies are characterized by specific defects in cilia beating that result in chronic airway disease, subfertility, ectopic pregnancy, and hydrocephalus. While many patients harbor mutations in the dynein motors that drive cilia beating, the disease also results from mutations in so-called dynein axonemal assembly factors (DNAAFs) that act in the cytoplasm. The mechanisms of DNAAF action remain poorly defined. Here, we show that DNAAFs concentrate together with axonemal dyneins and chaperones into organelles that form specifically in multiciliated cells, which we term DynAPs, for dynein axonemal particles. These organelles display hallmarks of biomolecular condensates, and remarkably, DynAPs are enriched for the stress granule protein G3bp1, but not for other stress granule proteins or P-body proteins. Finally, we show that both the formation and the liquid-like behaviors of DynAPs are disrupted in a model of motile ciliopathy. These findings provide a unifying cell biological framework for a poorly understood class of human disease genes and add motile ciliopathy to the growing roster of human diseases associated with disrupted biological phase separation.
Project description:Calaxin is a Ca2+-binding dynein-associated protein that regulates flagellar and ciliary movement. In ascidians, calaxin plays essential roles in chemotaxis of sperm. However, nothing has been known for the function of calaxin in vertebrates. Here we show that the mice with a null mutation in Efcab1, which encodes calaxin, display typical phenotypes of primary ciliary dyskinesia, including hydrocephalus, situs inversus, and abnormal motility of trachea cilia and sperm flagella. Strikingly, both males and females are viable and fertile, indicating that calaxin is not essential for fertilization in mice. The 9 + 2 axonemal structures of epithelial multicilia and sperm flagella are normal, but the formation of 9 + 0 nodal cilia is significantly disrupted. Knockout of calaxin in zebrafish also causes situs inversus due to the irregular ciliary beating of Kupffer's vesicle cilia, although the 9 + 2 axonemal structure appears to remain normal.
Project description:The Trypanosoma brucei flagellum controls motility and is crucial for cell polarity and division. Unique features of trypanosome motility suggest that flagellar beat regulation in this organism is unusual and worthy of study. The flagellar axoneme, required for motility, has a structure that is highly conserved among eukaryotes. Of the several dyneins in the axonemal inner arm complex, dynein f is thought to control flagellar waveform shape. A T. brucei gene predicted to encode the dynein f alpha heavy chain, TbDNAH10, was silenced using RNA interference in procyclic T. brucei cells. This resulted in immotile flagella, showing no movement except for occasional slight twitches at the tips. Cell growth slowed dramatically and cells were found in large clusters. Microscopic analysis of silenced cultures showed many cells with detached flagella, sometimes entangled between multiple cells. DAPI staining showed an increased frequency of mis-positioned kinetoplasts and multinucleate cells, suggesting that these cells experience disruption at an early cell cycle stage, probably secondary to the motility defect. TEM images showed apparently normal axonemes and no discernable defects in inner arm structure. This study demonstrates the use of RNAi as an effective method to study very large genes such as dynein heavy chains (HCs), and the immotility phenotype of these dynein knockdowns suggests that an intact inner arm is necessary for flagellar beating in T. brucei. Since analogous mutants in Chlamydomonas reinhardtii retain motility, this phenotype likely reflects differences in requirements for motility and/or dynein assembly between the two organisms and these comparative studies will help elucidate the mechanisms of flagellar beat regulation.