RESUMEN
To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.
Asunto(s)
Proteoma , Espermatozoides , Animales , Masculino , Ratones , Axonema/química , Microscopía por Crioelectrón/métodos , Flagelos/metabolismo , Microtúbulos/metabolismo , Semen , Espermatozoides/química , Proteoma/análisisRESUMEN
Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm are powered by a microtubule-based molecular machine-the axoneme-but it is unclear how axonemal microtubules are ornamented to support motility in diverse fertilization environments. Here, we present high-resolution structures of native axonemal doublet microtubules (DMTs) from sea urchin and bovine sperm, representing external and internal fertilizers. We identify >60 proteins decorating sperm DMTs; at least 15 are sperm associated and 16 are linked to infertility. By comparing DMTs across species and cell types, we define core microtubule inner proteins (MIPs) and analyze evolution of the tektin bundle. We identify conserved axonemal microtubule-associated proteins (MAPs) with unique tubulin-binding modes. Additionally, we identify a testis-specific serine/threonine kinase that links DMTs to outer dense fibers in mammalian sperm. Our study provides structural foundations for understanding sperm evolution, motility, and dysfunction at a molecular level.
Asunto(s)
Motilidad Espermática , Cola del Espermatozoide , Masculino , Animales , Bovinos , Cola del Espermatozoide/química , Cola del Espermatozoide/metabolismo , Semen , Microtúbulos/metabolismo , Axonema/química , Espermatozoides , MamíferosRESUMEN
Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.
Asunto(s)
Axonema , Cilios , Trastornos de la Motilidad Ciliar , Flagelos , Mecanotransducción Celular , Humanos , Masculino , Inteligencia Artificial , Dineínas Axonemales/química , Dineínas Axonemales/metabolismo , Dineínas Axonemales/ultraestructura , Axonema/química , Axonema/metabolismo , Axonema/ultraestructura , Cilios/química , Cilios/metabolismo , Cilios/ultraestructura , Microscopía por Crioelectrón , Flagelos/química , Flagelos/metabolismo , Flagelos/ultraestructura , Microtúbulos/metabolismo , Chlamydomonas reinhardtii , Trastornos de la Motilidad Ciliar/metabolismo , Trastornos de la Motilidad Ciliar/patología , Trastornos de la Motilidad Ciliar/fisiopatología , Movimiento , Conformación ProteicaRESUMEN
Cilium formation and regeneration requires new protein synthesis, but the underlying cytosolic translational reprogramming remains largely unknown. Using ribosome footprinting, we performed global translatome profiling during cilia regeneration in Chlamydomonas and uncovered that flagellar genes undergo an early transcriptional activation but late translational repression. This pattern guided our identification of sphingolipid metabolism enzymes, including serine palmitoyltransferase (SPT), as essential regulators for ciliogenesis. Cryo-electron tomography showed that ceramide loss abnormally increased the membrane-axoneme distance and generated bulged cilia. We found that ceramides interact with intraflagellar transport (IFT) particle proteins that IFT motors transport along axoneme microtubules (MTs), suggesting that ceramide-IFT particle-IFT motor-MT interactions connect the ciliary membrane with the axoneme to form rod-shaped cilia. SPT-deficient vertebrate cells were defective in ciliogenesis, and SPT mutations from patients with hereditary sensory neuropathy disrupted cilia, which could be restored by sphingolipid supplementation. These results reveal a conserved role of sphingolipid in cilium formation and link compromised sphingolipid production with ciliopathies.
Asunto(s)
Axonema , Chlamydomonas , Cilios , Flagelos , Regeneración , Esfingolípidos , Axonema/química , Axonema/metabolismo , Ceramidas/metabolismo , Chlamydomonas/fisiología , Cilios/fisiología , Flagelos/fisiología , Transporte de Proteínas , Esfingolípidos/metabolismoRESUMEN
Applications in biotechnology and synthetic biology often make use of soluble proteins, but there are many potential advantages of anchoring enzymes to a stable substrate, including stability and the possibility for substrate channeling. To avoid the necessity of protein purification and chemical immobilization, there has been growing interest in bio-assembly of protein-containing nanoparticles, exploiting the self-assembly of viral capsid proteins or other proteins that form polyhedral structures. However, these nanoparticles are limited in size, which constrains the packaging and the accessibility of the proteins. An axoneme, the insoluble protein core of the eukaryotic flagellum or cilium, is a highly ordered protein structure that can be several microns in length, orders of magnitude larger than other types of nanoparticles. We show that when proteins of interest are fused to specific axonemal proteins and expressed in living Chlamydomonas reinhardtii cells, they become incorporated into linear arrays, which have the advantages of high protein loading capacity and single-step purification with retention of biomass. The arrays can be isolated as membrane-enclosed vesicles or as exposed protein arrays. The approach is demonstrated for both a fluorescent protein and an enzyme (beta-lactamase), showing that incorporation into axonemes retains protein function in a stable, easily isolated array form.
Asunto(s)
Axonema , Chlamydomonas reinhardtii , Axonema/química , Axonema/metabolismo , Chlamydomonas reinhardtii/metabolismo , Flagelos/química , Flagelos/metabolismoRESUMEN
The radial spoke (RS) heads of motile cilia and flagella contact projections of the central pair (CP) apparatus to coordinate motility, but the morphology is distinct for protozoa and metazoa. Here we show the murine RS head is compositionally distinct from that of Chlamydomonas Our reconstituted murine RS head core complex consists of Rsph1, Rsph3b, Rsph4a, and Rsph9, lacking Rsph6a and Rsph10b, whose orthologs exist in the protozoan RS head. We resolve its cryo-electron microscopy (cryo-EM) structure at 3.2-Å resolution. Our atomic model further reveals a twofold symmetric brake pad-shaped structure, in which Rsph4a and Rsph9 form a compact body extended laterally with two long arms of twisted Rsph1 ß-sheets and potentially connected dorsally via Rsph3b to the RS stalk. Furthermore, our modeling suggests that the core complex contacts the periodic CP projections either rigidly through its tooth-shaped Rsph4a regions or elastically through both arms for optimized RS-CP interactions and mechanosignal transduction.
Asunto(s)
Axonema/química , Axonema/metabolismo , Microscopía por Crioelectrón/métodos , Animales , Antígenos de Superficie , Chlamydomonas , Cilios , Proteínas del Citoesqueleto/química , Proteínas de Unión al ADN/química , Epítopos , Flagelos , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Mutación , Conformación Proteica , Proteínas RecombinantesRESUMEN
Asthenozoospermia is the most common cause of male infertility. Dynein protein arms play a crucial role in the motility of both the cilia and flagella, and defects in these proteins generally impair the axoneme structure and cause primary ciliary dyskinesia. But relatively little is known about the influence of dynein protein arm defects on sperm flagella function. Here, we recruited 85 infertile patients with idiopathic asthenozoospermia and identified bi-allelic mutations in DNAH7 (NM_018897.3) from three patients using whole-exome sequencing. These variants are rare, highly pathogenic, and very conserved. The spermatozoa from the patients with DNAH7 bi-allelic mutations showed specific losses in the inner dynein arms. The expression of DNAH7 in the spermatozoa from the DNAH7-defective patients was significantly decreased, but these patients were able to have their children via intra-cytoplasmic sperm injection treatment. Our study is the first to demonstrate that bi-allelic mutations in DNAH7 may impair the integrality of axoneme structure, affect sperm motility, and cause asthenozoospermia in humans. These findings may extend the spectrum of etiological genes and provide new clues for the diagnosis and treatment of patients with asthenozoospermia.
Asunto(s)
Astenozoospermia/genética , Axonema/química , Dineínas/genética , Adulto , Alelos , Simulación por Computador , Regulación hacia Abajo/genética , Desarrollo Embrionario/genética , Flagelos/genética , Humanos , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación , Inyecciones de Esperma Intracitoplasmáticas , Motilidad Espermática/genética , Cola del Espermatozoide/química , Espermatozoides/citología , Espermatozoides/ultraestructura , Secuenciación del ExomaRESUMEN
TomoAlign is a software package that integrates tools to mitigate two important resolution limiting factors in cryoET, namely the beam-induced sample motion and the contrast transfer function (CTF) of the microscope. The package is especially focused on cryoET of thick specimens where fiducial markers are required for accurate tilt-series alignment and sample motion estimation. TomoAlign models the beam-induced sample motion undergone during the tilt-series acquisition. The motion models are used to produce motion-corrected subtilt-series centered on the particles of interest. In addition, the defocus of each particle at each tilt image is determined and can be corrected, resulting in motion-corrected and CTF-corrected subtilt-series from which the subtomograms can be computed. Alternatively, the CTF information can be passed on so that CTF correction can be carried out entirely within external packages like Relion. TomoAlign serves as a versatile tool that can streamline the cryoET workflow from initial alignment of tilt-series to final subtomogram averaging during in situ structure determination.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Programas Informáticos , Proteínas Arqueales/química , Proteínas Arqueales/ultraestructura , Axonema/química , Axonema/ultraestructura , Endopeptidasas/química , Endopeptidasas/ultraestructura , Movimiento (Física) , Reproducibilidad de los Resultados , Tetrahymena thermophila/ultraestructuraRESUMEN
The ultrastructural features of axoneme organization within the cytoplasm and exflagellation were investigated in detail in microgametes of a malaria parasite, Plasmodium berghei, by electron and fluorescence microscopy. The kinetosomes (basal bodies) of the microgamete were characterized by an electron dense mass in which singlet microtubules (MTs) were embedded. Around the kinetosomes, several singlet and doublet MTs were recognized in transverse sections. Incomplete doublets with growing B-tubule were also observed. As precursors of the axoneme, arrays of over three doublets showed a tendency to encircle the central pair MTs. Some of the doublet MTs were already equipped with inner and outer dynein arms. In the microgamete, which lacks an intraflagellar transport (IFT) system, self-assembly of microtubular and associated components appeared to proceed stepwise from singlet MTs through arrays of one to nine doublet MTs, surrounding the central pair, to form the complete axoneme in a quite short time. At exflagellation, some extra doublets were occasionally included between the axoneme and the flagellar membrane. At high magnification, the outer dynein arm of the Plasmodium microgamete had a pistol-like shape representing a three-headed dynein molecule like that of other Alveolata.
Asunto(s)
Axonema/ultraestructura , Gametogénesis , Células Germinativas , Plasmodium berghei , Animales , Axonema/química , Dineínas/ultraestructura , Femenino , Células Germinativas/química , Células Germinativas/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Microscopía Fluorescente , Plasmodium berghei/fisiología , Plasmodium berghei/ultraestructuraRESUMEN
Flagellar dyneins are the molecular motors responsible for producing the propagating bending motions of cilia and flagella. They are located within a densely packed and highly organised super-macromolecular cytoskeletal structure known as the axoneme. Using the mesoscale simulation technique Fluctuating Finite Element Analysis (FFEA), which represents proteins as viscoelastic continuum objects subject to explicit thermal noise, we have quantified the constraints on the range of molecular conformations that can be explored by dynein-c within the crowded architecture of the axoneme. We subsequently assess the influence of crowding on the 3D exploration of microtubule-binding sites, and specifically on the axial step length. Our calculations combine experimental information on the shape, flexibility and environment of dynein-c from three distinct sources; negative stain electron microscopy, cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET). Our FFEA simulations show that the super-macromolecular organisation of multiple protein complexes into higher-order structures can have a significant influence on the effective flexibility of the individual molecular components, and may, therefore, play an important role in the physical mechanisms underlying their biological function.
Asunto(s)
Axonema/química , Dineínas/química , Flagelos/metabolismo , Sustancias Macromoleculares/química , Sitios de Unión , Cilios/metabolismo , Simulación por Computador , Microscopía por Crioelectrón , Citoesqueleto/metabolismo , Módulo de Elasticidad , Análisis de Elementos Finitos , Hidrólisis , Cinética , Microtúbulos/metabolismo , Movimiento (Física) , Probabilidad , Unión Proteica , Conformación Proteica , TermodinámicaRESUMEN
In eukaryotic cilia and flagella, various types of axonemal dyneins orchestrate their distinct functions to generate oscillatory bending of axonemes. The force-generating mechanism of dyneins has recently been well elucidated, mainly in cytoplasmic dyneins, thanks to progress in single-molecule measurements, X-ray crystallography, and advanced electron microscopy. These techniques have shed light on several important questions concerning what conformational changes accompany ATP hydrolysis and whether multiple motor domains are coordinated in the movements of dynein. However, due to the lack of a proper expression system for axonemal dyneins, no atomic coordinates of the entire motor domain of axonemal dynein have been reported. Therefore, a substantial amount of knowledge on the molecular architecture of axonemal dynein has been derived from electron microscopic observations on dynein arms in axonemes or on isolated axonemal dynein molecules. This review describes our current knowledge and perspectives of the force-generating mechanism of axonemal dyneins in solo and in ensemble.
Asunto(s)
Adenosina Trifosfato/metabolismo , Dineínas Axonemales/química , Flagelos/metabolismo , Microtúbulos/metabolismo , Animales , Dineínas Axonemales/metabolismo , Dineínas Axonemales/ultraestructura , Axonema/química , Axonema/metabolismo , Cilios/metabolismo , Cristalografía por Rayos X , Dineínas Citoplasmáticas/metabolismo , Flagelos/ultraestructuraRESUMEN
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous chronic destructive airway disease. PCD is traditionally diagnosed by nasal nitric oxide measurement, analysis of ciliary beating, transmission electron microscopy (TEM), and/or genetic testing. In most genetic PCD variants, laterality defects can occur. However, it is difficult to establish a diagnosis in individuals with PCD and central pair (CP) defects, and alternative strategies are required because of very subtle ciliary beating abnormalities, a normal ciliary ultrastructure, and normal situs composition. Mutations in HYDIN are known to cause CP defects, but the genetic analysis of HYDIN variants is confounded by the pseudogene HYDIN2, which is almost identical in terms of intron/exon structure. We have previously shown that several types of PCD can be diagnosed via immunofluorescence (IF) microscopy analyses. Here, using IF microscopy, we demonstrated that in individuals with PCD and CP defects, the CP-associated protein SPEF2 is absent in HYDIN-mutant cells, revealing its dependence on functional HYDIN. Next, we performed IF analyses of SPEF2 in respiratory cells from 189 individuals with suspected PCD and situs solitus. Forty-one of the 189 individuals had undetectable SPEF2 and were subjected to a genetic analysis, which revealed one novel loss-of-function mutation in SPEF2 and three reported and 13 novel HYDIN mutations in 15 individuals. The remaining 25 individuals are good candidates for new, as-yet uncharacterized PCD variants that affect the CP apparatus. SPEF2 mutations have been associated with male infertility but have not previously been identified to cause PCD. We identified a mutation of SPEF2 that is causative for PCD with a CP defect. We conclude that SPEF2 IF analyses can facilitate the detection of CP defects and evaluation of the pathogenicity of HYDIN variants, thus aiding the molecular diagnosis of CP defects.
Asunto(s)
Proteínas de Ciclo Celular/deficiencia , Cilios/química , Trastornos de la Motilidad Ciliar/genética , Proteínas de Microfilamentos/genética , Axonema/química , Axonema/ultraestructura , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Trastornos de la Motilidad Ciliar/diagnóstico , Trastornos de la Motilidad Ciliar/patología , Codón sin Sentido , Estudios de Cohortes , Análisis Mutacional de ADN , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Heterogeneidad Genética , Homocigoto , Humanos , Mutación con Pérdida de Función , Masculino , Proteínas de Microfilamentos/fisiología , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Depuración Mucociliar/genética , Mutación , Mutación Missense , Linaje , Cultivo Primario de Células , Situs Inversus/diagnóstico , Situs Inversus/genética , Situs Inversus/patologíaRESUMEN
The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.
Asunto(s)
Cuerpos Basales/metabolismo , Microtúbulos/metabolismo , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genética , Animales , Axonema/química , Axonema/genética , Axonema/metabolismo , Cuerpos Basales/química , Cuerpos Basales/parasitología , Centriolos/química , Centriolos/genética , Centriolos/parasitología , Flagelos/química , Flagelos/genética , Flagelos/parasitología , Humanos , Microtúbulos/química , Microtúbulos/parasitología , Estabilidad Proteica , Proteínas Protozoarias/química , Interferencia de ARN , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/patogenicidadRESUMEN
Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild-type Chlamydomonas with CA mutants. We identified a large (>2 MD) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and 3D structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.
Asunto(s)
Chlamydomonas/genética , Cilios/química , Microtúbulos/química , Mutación , Axonema/química , Movimiento Celular , Tomografía con Microscopio Electrónico , Flagelos/química , Genotipo , Conformación Molecular , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme assembly, structure, and motility require a radially arranged set of doublet microtubules, each decorated in repeating patterns with non-tubulin components. We use single-particle cryo-electron microscopy to visualize and build an atomic model of the repeating structure of a native axonemal doublet microtubule, which reveals the identities, positions, repeat lengths, and interactions of 38 associated proteins, including 33 microtubule inner proteins (MIPs). The structure demonstrates how these proteins establish the unique architecture of doublet microtubules, maintain coherent periodicities along the axoneme, and stabilize the microtubules against the repeated mechanical stress induced by ciliary motility. Our work elucidates the architectural principles that underpin the assembly of this large, repetitive eukaryotic structure and provides a molecular basis for understanding the etiology of human ciliopathies.
Asunto(s)
Axonema/ultraestructura , Cilios/ultraestructura , Ciliopatías/patología , Microtúbulos/ultraestructura , Axonema/química , Axonema/genética , Movimiento Celular/genética , Cilios/química , Cilios/genética , Ciliopatías/genética , Ciliopatías/metabolismo , Microscopía por Crioelectrón , Humanos , Proteínas de Microtúbulos/química , Proteínas de Microtúbulos/ultraestructura , Microtúbulos/química , Microtúbulos/genética , Estrés MecánicoRESUMEN
Primary cilia carry out numerous signaling and sensory functions, and defects in them, "ciliopathies," cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. We have combined cryoelectron tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening cytoplasm. Within this framework, we report spatial distributions of key proteins in wild-type (WT) mice and the effects on them of genetic deficiencies in 3 models of Bardet-Biedl syndrome.
Asunto(s)
Síndrome de Bardet-Biedl/patología , Cilios/ultraestructura , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Cilio Conector de los Fotorreceptores/ultraestructura , Segmento Externo de la Célula en Bastón/ultraestructura , Imagen Individual de Molécula/métodos , Animales , Axonema/química , Axonema/ultraestructura , Centriolos/ultraestructura , Modelos Animales de Enfermedad , Proteínas del Ojo/análisis , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/análisis , Microtúbulos/ultraestructura , Complejos Multiproteicos , Proteínas Musculares/análisis , Cilio Conector de los Fotorreceptores/química , Proteínas Qa-SNARE/análisis , Proteínas Supresoras de Tumor/análisisRESUMEN
Cilia, the hair-like protrusions that beat at high frequencies to propel a cell or move fluid around are composed of radially bundled doublet microtubules. In this study, we present a near-atomic resolution map of the Tetrahymena doublet microtubule by cryoelectron microscopy. The map demonstrates that the network of microtubule inner proteins weaves into the tubulin lattice and forms an inner sheath. From mass spectrometry data and de novo modeling, we identified Rib43a proteins as the filamentous microtubule inner proteins in the protofilament ribbon region. The Rib43a-tubulin interaction leads to an elongated tubulin dimer distance every 2 dimers. In addition, the tubulin lattice structure with missing microtubule inner proteins (MIPs) by sarkosyl treatment shows significant longitudinal compaction and lateral angle change between protofilaments. These results are evidence that the MIPs directly affect and stabilize the tubulin lattice. It suggests that the doublet microtubule is an intrinsically stressed filament and that this stress could be manipulated in the regulation of ciliary waveforms.
Asunto(s)
Cilios/química , Proteínas de Microtúbulos/química , Tetrahymena/química , Tubulina (Proteína)/química , Axonema/química , Microscopía por Crioelectrón , Citoesqueleto/química , Espectrometría de Masas , Microtúbulos/química , Simulación de Dinámica Molecular , Paclitaxel/química , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Protozoarias/química , Estrés MecánicoRESUMEN
The 9 + 2 axoneme structure of the motile flagellum/cilium is an iconic, apparently symmetrical cellular structure. Recently, asymmetries along the length of motile flagella have been identified in a number of organisms, typically in the inner and outer dynein arms. Flagellum-beat waveforms are adapted for different functions. They may start either near the flagellar tip or near its base and may be symmetrical or asymmetrical. We hypothesized that proximal/distal asymmetry in the molecular composition of the axoneme may control the site of waveform initiation and the direction of waveform propagation. The unicellular eukaryotic pathogens Trypanosoma brucei and Leishmania mexicana often switch between tip-to-base and base-to-tip waveforms, making them ideal for analysis of this phenomenon. We show here that the proximal and distal portions of the flagellum contain distinct outer dynein arm docking-complex heterodimers. This proximal/distal asymmetry is produced and maintained through growth by a concentration gradient of the proximal docking complex, generated by intraflagellar transport. Furthermore, this asymmetry is involved in regulating whether a tip-to-base or base-to-tip beat occurs, which is linked to a calcium-dependent switch. Our data show that the mechanism for generating proximal/distal flagellar asymmetry can control waveform initiation and propagation direction.
Asunto(s)
Dineínas/química , Flagelos/fisiología , Axonema/química , Flagelos/química , Multimerización de ProteínaRESUMEN
The massive dynein motor complexes that drive ciliary and flagellar motility require cytoplasmic preassembly, a process requiring dedicated dynein assembly factors (DNAAFs). How DNAAFs interact with molecular chaperones to control dynein assembly is not clear. By analogy with the well-known multifunctional HSP90-associated cochaperone, R2TP, several DNAAFs have been suggested to perform novel R2TP-like functions. However, the involvement of R2TP itself (canonical R2TP) in dynein assembly remains unclear. Here we show that in Drosophila melanogaster, the R2TP-associated factor, Wdr92, is required exclusively for axonemal dynein assembly, likely in association with canonical R2TP. Proteomic analyses suggest that in addition to being a regulator of R2TP chaperoning activity, Wdr92 works with the DNAAF Spag1 at a distinct stage in dynein preassembly. Wdr92/R2TP function is likely distinct from that of the DNAAFs proposed to form dynein-specific R2TP-like complexes. Our findings thus establish a connection between dynein assembly and a core multifunctional cochaperone.
Asunto(s)
Dineínas Axonemales/química , Cilios/genética , Proteínas HSP90 de Choque Térmico/química , Proteómica , Animales , Dineínas Axonemales/genética , Axonema/química , Axonema/genética , Cilios/química , Drosophila melanogaster/química , Drosophila melanogaster/genética , Proteínas HSP90 de Choque Térmico/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Unión Proteica , Pliegue de ProteínaRESUMEN
Genomics and proteomics studies in Chlamydomonas have revealed that an axoneme is composed of 200-600 types of proteins, including uncharacterized proteins collectively named flagellar-associated proteins (FAPs). Nine FAPs contain the EF-hand motif; however, they have not yet been well characterized. To find components responsible for Chlamydomonas-specific waveform changes coupled with intracellular Ca2+ concentrations, we focused on FAP85, an EF-hand motif-containing FAP specific to Chlamydomonas and its relatives. We cloned the cDNA encoding FAP85, expressed it in Escherichia coli cells, and generated a polyclonal antibody against the expressed protein. Immunoblotting showed that FAP85 was present in every axoneme of several flagellar mutants lacking major axonemal components. Immuno-electron microscopy revealed that anti-FAP85 antibodies were found only on the inner wall of A-tubules of the doublets exposed by N-lauroylsarcosine (Sarkosyl) treatment. The zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) applied to 0.6 M KCl-extracted axonemes generated a 75-kDa complex containing ß-tubulin and FAP85. Further characterization of FAP85 and its effects on microtubule dynamics showed that FAP85 binds to tubulin and stabilized microtubules. According to these results, we conclude that FAP85 is a novel member of microtubule-binding proteins, localizing on the inner wall of the A-tubule and stabilizing microtubules.Key words: Chlamydomonas, flagella, doublet microtubule, microtubule inner proteins.