RESUMO
Basal bodies (BBs) are conserved eukaryotic structures that organize cilia. They are comprised of nine, cylindrically arranged, triplet microtubules (TMTs) connected to each other by inter-TMT linkages which stabilize the structure. Poc1 is a conserved protein important for BB structural integrity in the face of ciliary forces transmitted to BBs. To understand how Poc1 confers BB stability, we identified the precise position of Poc1 in the Tetrahymena BB and the effect of Poc1 loss on BB structure. Poc1 binds at the TMT inner junctions, stabilizing TMTs directly. From this location, Poc1 also stabilizes inter-TMT linkages throughout the BB, including the cartwheel pinhead and the inner scaffold. The full localization of the inner scaffold protein Fam161A requires Poc1. As ciliary forces are increased, Fam161A is reduced, indicative of a force-dependent molecular remodeling of the inner scaffold. Thus, while not essential for BB assembly, Poc1 promotes BB interconnections that establish an architecture competent to resist ciliary forces.
Assuntos
Corpos Basais , Cílios , Microtúbulos , Proteínas de Protozoários , Tetrahymena thermophila , Corpos Basais/metabolismo , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Ligação Proteica , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Tetrahymena thermophila/metabolismo , Tetrahymena thermophila/genéticaRESUMO
Vertebrate photoreceptors are highly specialized retinal neurons that have cilium-derived membrane organelles called outer segments, which function as platforms for phototransduction. Male germ cell-associated kinase (MAK) is a cilium-associated serine/threonine kinase, and its genetic mutation causes photoreceptor degeneration in mice and retinitis pigmentosa in humans. However, the role of MAK in photoreceptors is not fully understood. Here, we report that zebrafish mak mutants show rapid photoreceptor degeneration during embryonic development. In mak mutants, both cone and rod photoreceptors completely lacked outer segments and underwent apoptosis. Interestingly, zebrafish mak mutants failed to generate axonemes during photoreceptor ciliogenesis, whereas basal bodies were specified. These data suggest that Mak contributes to axoneme development in zebrafish, in contrast to mouse Mak mutants, which have elongated photoreceptor axonemes. Furthermore, the kinase activity of Mak was found to be critical in ciliary axoneme development and photoreceptor survival. Thus, Mak is required for ciliogenesis and outer segment formation in zebrafish photoreceptors to ensure intracellular protein transport and photoreceptor survival.
Assuntos
Axonema , Cílios , Mutação , Proteínas Serina-Treonina Quinases , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/embriologia , Axonema/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Cílios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Mutação/genética , Apoptose , Masculino , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Sobrevivência Celular , Corpos Basais/metabolismo , Serina-Treonina Quinase 3RESUMO
Transition fibres and distal appendages surround the distal end of mature basal bodies and are essential for ciliogenesis, but only a few of the proteins involved have been identified and functionally characterised. Here, through genome-wide analysis, we have identified 30 transition fibre proteins (TFPs) and mapped their arrangement in the flagellated eukaryote Trypanosoma brucei. We discovered that TFPs are recruited to the mature basal body before and after basal body duplication, with differential expression of five TFPs observed at the assembling new flagellum compared to the existing fixed-length old flagellum. RNAi-mediated depletion of 17 TFPs revealed six TFPs that are necessary for ciliogenesis and a further three TFPs that are necessary for normal flagellum length. We identified nine TFPs that had a detectable orthologue in at least one basal body-forming eukaryotic organism outside of the kinetoplastid parasites. Our work has tripled the number of known transition fibre components, demonstrating that transition fibres are complex and dynamic in their composition throughout the cell cycle, which relates to their essential roles in ciliogenesis and flagellum length regulation.
Assuntos
Proteínas de Protozoários , Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sequência Conservada , Corpos Basais/metabolismo , Transporte Proteico , Fatores de Tempo , Flagelos/genética , Flagelos/metabolismo , Regulação da Expressão Gênica , Cílios/genética , Cílios/metabolismoRESUMO
The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Flagelos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Flagelos/metabolismo , Flagelos/química , Flagelos/ultraestrutura , Corpos Basais/metabolismo , Corpos Basais/química , Modelos Moleculares , Rotação , Conformação Proteica , Salmonella/metabolismo , Salmonella/química , Salmonella typhimurium/metabolismo , Salmonella typhimurium/químicaRESUMO
Cilia play critical roles in cell signal transduction and organ development. Defects in cilia function result in a variety of genetic disorders. Cep290 is an evolutionarily conserved ciliopathy protein that bridges the ciliary membrane and axoneme at the basal body (BB) and plays critical roles in the initiation of ciliogenesis and TZ assembly. How Cep290 is maintained at BB and whether axonemal and ciliary membrane localized cues converge to determine the localization of Cep290 remain unknown. Here, we report that the Cep131-Cep162 module near the axoneme and the Cby-Fam92 module close to the membrane synergistically control the BB localization of Cep290 and the subsequent initiation of ciliogenesis in Drosophila. Concurrent deletion of any protein of the Cep131-Cep162 module and of the Cby-Fam92 module leads to a complete loss of Cep290 from BB and blocks ciliogenesis at its initiation stage. Our results reveal that the first step of ciliogenesis strictly depends on cooperative and retroactive interactions between Cep131-Cep162, Cby-Fam92 and Cep290, which may contribute to the complex pathogenesis of Cep290-related ciliopathies.
Assuntos
Corpos Basais , Cognição , Animais , Sinais (Psicologia) , Axonema , Cílios/genética , Drosophila/genéticaRESUMO
Two mother centrioles in an animal cell are linked by intercentriolar fibers that have CROCC/rootletin as their main building block. Here, we investigated the regulatory role of intercentriolar/rootlet fibers in cilia assembly. The cilia formation rates were significantly reduced in the CEP250/C-NAP1 and CROCC/rootletin knockout (KO) cells, irrespective of the departure of the young mother centrioles from the basal bodies. In addition, centriolar satellites were dispersed throughout the cytoplasm in the CEP250 and CROCC KO cells. We observed that PCM1 directly binds to CROCC. Their interaction is critical not only for the accumulation of centriolar satellites near the centrosomes/basal bodies but also for cilia formation. Finally, we observed that the centriolar satellite proteins are localized at the intercentriolar/rootlet fibers in the kidney epithelial cells. Based on these findings, we propose that the intercentriolar/rootlet fibers function as docking sites for centriolar satellites near the centrosomes/basal bodies and facilitate the cilia assembly process.
Assuntos
Centríolos , Cílios , Corpos Basais , Centríolos/genética , Centrossomo , Grânulos Citoplasmáticos , Humanos , Células Epiteliais/citologiaRESUMO
For mucociliary clearance of pathogens, tracheal multiciliated epithelial cells (MCCs) organize coordinated beating of cilia, which originate from basal bodies (BBs) with basal feet (BFs) on one side. To clarify the self-organizing mechanism of coordinated intracellular BB-arrays composed of a well-ordered BB-alignment and unidirectional BB-orientation, determined by the direction of BB to BF, we generated double transgenic mice with GFP-centrin2-labeled BBs and mRuby3-Cep128-labeled BFs for long-term, high-resolution, dual-color live-cell imaging in primary-cultured tracheal MCCs. At early timepoints of MCC differentiation, BB-orientation and BB-local alignment antecedently coordinated in an apical microtubule-dependent manner. Later during MCC differentiation, fluctuations in BB-orientation were restricted, and locally aligned BB-arrays were further coordinated to align across the entire cell (BB-global alignment), mainly in an apical intermediate-sized filament-lattice-dependent manner. Thus, the high coordination of the BB-array was established for efficient mucociliary clearance as the primary defense against pathogen infection, identifying apical cytoskeletons as potential therapeutic targets.
Assuntos
Corpos Basais , Citoesqueleto , Camundongos , Animais , Microtúbulos , Cílios , Células EpiteliaisRESUMO
Primary cilia mediate sensory signaling in multiple organisms and cell types but have structures adapted for specific roles. Structural defects in them lead to devastating diseases known as ciliopathies in humans. Key to their functions are structures at their base: the basal body, the transition zone, the "Y-shaped links," and the "ciliary necklace." We have used cryo-electron tomography with subtomogram averaging and conventional transmission electron microscopy to elucidate the structures associated with the basal region of the "connecting cilia" of rod outer segments in mouse retina. The longitudinal variations in microtubule (MT) structures and the lumenal scaffold complexes connecting them have been determined, as well as membrane-associated transition zone structures: Y-shaped links connecting MT to the membrane, and ciliary beads connected to them that protrude from the cell surface and form a necklace-like structure. These results represent a clearer structural scaffold onto which molecules identified by genetics, proteomics, and superresolution fluorescence can be placed in our emerging model of photoreceptor sensory cilia.
Assuntos
Centríolos , Cílios , Humanos , Animais , Camundongos , Tomografia com Microscopia Eletrônica , Microscopia Eletrônica de Transmissão , Corpos BasaisRESUMO
The unicellular protozoan Trypanosoma brucei has a single flagellum that is involved in cell motility, cell morphogenesis, and cell division. Inheritance of the newly assembled flagellum during the cell cycle requires its correct positioning, which depends on the faithful duplication or segregation of multiple flagellum-associated cytoskeletal structures, including the basal body, the flagellum attachment zone, and the hook complex. Along the flagellum attachment zone sites a set of four microtubules termed the microtubule quartet (MtQ), whose molecular function remains enigmatic. We recently reported that the MtQ-localized protein NHL1 interacts with the microtubule-binding protein TbSpef1 and regulates flagellum inheritance by promoting basal body rotation and segregation. Here, we identified a TbSpef1- and NHL1-associated protein named SNAP1, which co-localizes with NHL1 and TbSpef1 at the proximal portion of the MtQ, depends on TbSpef1 for localization and is required for NHL1 localization to the MtQ. Knockdown of SNAP1 impairs the rotation and segregation of the basal body, the elongation of the flagellum attachment zone filament, and the positioning of the newly assembled flagellum, thereby causing mis-placement of the cell division plane, a halt in cleavage furrow ingression, and an inhibition of cytokinesis completion. Together, these findings uncover a coordinating role of SNAP1 with TbSpef1 and NHL1 in facilitating flagellum positioning and cell division plane placement for the completion of cytokinesis.
Assuntos
Flagelos , Microtúbulos , Proteínas de Protozoários , Trypanosoma brucei brucei , Corpos Basais/metabolismo , Divisão Celular , Segregação de Cromossomos , Flagelos/metabolismo , Microtúbulos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMO
The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. Crb3 knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific Crb3 knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.
Assuntos
Carcinogênese , Centro Organizador dos Microtúbulos , Animais , Camundongos , Corpos Basais , Diferenciação Celular , Transformação Celular Neoplásica , HiperplasiaRESUMO
Primary cilia are essential sensory organelles that develop when an inhibitory cap consisting of CP110 and other proteins is eliminated. The degradation of CP110 by the ubiquitin-dependent proteasome pathway mediated by NEURL4 and HYLS1 removes the inhibitory cap. Here, we investigated the suitability of rapamycin-mediated dimerization for centriolar recruitment and asked whether the induced recruitment of NEURL4 or HYLS1 to the centriole promotes primary cilia development and CP110 degradation. We used rapamycin-mediated dimerization with ODF2 to induce their targeted recruitment to the centriole. We found decreased CP110 levels in the transfected cells, but independent of rapamycin-mediated dimerization. By knocking down ODF2, we showed that ODF2 controls CP110 levels. The overexpression of ODF2 is not sufficient to promote the formation of primary cilia, but the overexpression of NEURL4 or HYLS1 is. The co-expression of ODF2 and HYLS1 resulted in the formation of tube-like structures, indicating an interaction. Thus, ODF2 controls primary cilia formation by negatively regulating the concentration of CP110 levels. Our data suggest that ODF2 most likely acts as a scaffold for the binding of proteins such as NEURL4 or HYLS1 to mediate CP110 degradation.
Assuntos
Corpos Basais , Centríolos , Cílios , Dimerização , SirolimoRESUMO
Tetrahymena thermophila possesses arrays of motile cilia that promote fluid flow for cell motility. These consist of intricately organized basal bodies (BBs) that nucleate and position cilia at the cell cortex. Tetrahymena cell geometry and spatial organization of BBs play important roles in cell size, swimming, feeding, and division. How cell geometry and BB organization are established and maintained remains poorly understood, and prior studies have been limited due to difficulties in accurate BB identification and small sample size. We therefore developed an automated image processing pipeline that segments single cells, distinguishes unique BB populations, assigns BBs into distinct ciliary rows, and distinguishes new from mature BBs. We identified unique features to describe the variation of cell shape and BB spatial organization in unsynchronized single-cell images. The results reveal asymmetries in BB distribution and ingression of the cytokinetic furrow within the cell. Moreover, we establish novel spatial and temporal waves in new BB assembly through the cell cycle. Finally, we used measurements from single cells across the cell cycle to construct a generative model that allows synthesis of movies depicting single cells progressing through the cell cycle. Our approach is expected to be of particular value for characterizing Tetrahymena mutants.
Assuntos
Tetrahymena thermophila , Tetrahymena , Tetrahymena thermophila/metabolismo , Corpos Basais/metabolismo , Ciclo Celular , Divisão Celular , Movimento Celular , Cílios/metabolismoRESUMO
Primary cilia are sensory organelles essential for embryonic and postnatal development, and tissue homeostasis in adulthood. They are generated in a cell cycle-dependent manner and found on most cells of the body. Although cilia formation is intensively investigated virtually nothing is known about the transcriptional regulation of primary ciliation. We used here Odf2/Cenexin, encoding a protein of the mother centriole and the basal body that is mandatory for primary cilia formation, as the target gene for the identification of transcriptional activators. We identified a consensus binding site for Fox transcription factors (TFs) in its promoter region and focused here on the Fox family. We found transcriptional activation of Odf2 neither by FOXO TFs nor by the core TF for multiciliation, FOXJ1. However, we identified FOXA1 as a transcriptional activator of Odf2 by reporter gene assays and qRT-PCR, and showed by qWB that Foxa1 knockdown caused a decrease in ODF2 and CP110 proteins. We verified the binding sequence of FOXA1 in the Odf2 promoter by ChIP. Finally, we demonstrated that knockdown of FOXA1 affected primary cilia formation. We, thus, showed for the first time, that FOXA1 regulates primary ciliation by transcriptional activation of ciliary genes.
Assuntos
Cílios , Proteínas de Choque Térmico , Cílios/genética , Cílios/metabolismo , Proteínas de Choque Térmico/metabolismo , Centríolos/metabolismo , Corpos Basais/metabolismo , Regulação da Expressão GênicaRESUMO
The Ciliary Adhesion (CA) complex forms in close association with the basal bodies of cilia during the early stages of ciliogenesis and is responsible for mediating complex interactions with the actin networks of multiciliated cells (MCCs). However, its precise localization with respect to basal body accessory structures and the interactions that lead to its establishment in MCCs are not well understood. Here, we studied the distribution of the CA proteins using super-resolution imaging and possible interactions with the microtubule network. The results of this study reveal that the apical CA complex forms at the distal end of the basal foot and depends on microtubules. Our data also raise the possibility that CAs may have additional roles in the regulation of the organization of the microtubule network of MCCs.
Assuntos
Corpos Basais , Cílios , Cílios/metabolismo , Corpos Basais/metabolismo , Microtúbulos/metabolismo , Actinas/metabolismoRESUMO
Motile cilia beat with an asymmetric waveform consisting of a power stroke that generates a propulsive force and a recovery stroke that returns the cilium back to the start. Cilia are anchored to the cell cortex by basal bodies (BBs) that are directly coupled to the ciliary doublet microtubules (MTs). We find that, consistent with ciliary forces imposing on BBs, bending patterns in BB triplet MTs are responsive to ciliary beating. BB bending varies as environmental conditions change the ciliary waveform. Bending occurs where striated fibers (SFs) attach to BBs and mutants with short SFs that fail to connect to adjacent BBs exhibit abnormal BB bending, supporting a model in which SFs couple ciliary forces between BBs. Finally, loss of the BB stability protein Poc1, which helps interconnect BB triplet MTs, prevents the normal distributed BB and ciliary bending patterns. Collectively, BBs experience ciliary forces and manage mechanical coupling of these forces to their surrounding cellular architecture for normal ciliary beating.
Assuntos
Corpos Basais , Cílios , Corpos Basais/metabolismo , Cílios/metabolismo , Microtúbulos/metabolismo , Fenômenos MecânicosRESUMO
The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body (BB) of the flagellum. Here, we studied the architecture of the exclusion zone filament (EZF) of the TAC, the only known component of which is p197, that connects the BB with the mitochondrial outer membrane (OM). We show that p197 has three domains that are all essential for mitochondrial DNA inheritance. The C terminus of p197 interacts with the mature and probasal body (pro-BB), whereas its N terminus binds to the peripheral OM protein TAC65. The large central region of p197 has a high α-helical content and likely acts as a flexible spacer. Ultrastructure expansion microscopy (U-ExM) of cell lines exclusively expressing p197 versions of different lengths that contain both N- and C-terminal epitope tags demonstrates that full-length p197 alone can bridge the â¼270-nm distance between the BB and the cytosolic face of the OM. Thus U-ExM allows the localization of distinct domains within the same molecules and suggests that p197 is the TAC subunit most proximal to the BB. In addition, U-ExM revealed that p197 acts as a spacer molecule, as two shorter versions of p197, with the repeat domain either removed or replaced by the central domain of the Trypanosoma cruzi p197 ortholog reduced the distance between the BB and the OM in proportion to their predicted molecular weight.
Assuntos
Replicação do DNA , DNA Mitocondrial , Genoma Mitocondrial , Membranas Mitocondriais , Proteínas de Protozoários , Trypanosoma brucei brucei , Corpos Basais/química , DNA Mitocondrial/genética , Epitopos/química , Flagelos/química , Membranas Mitocondriais/química , Proteínas de Protozoários/química , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genéticaRESUMO
Centrosomal protein of 164 kDa (CEP164) is located at distal appendages of primary cilia and is necessary for basal body (BB) docking to the apical membrane. To investigate the function of photoreceptor CEP164 before and after BB docking, we deleted CEP164 during retina embryonic development (Six3Cre), in postnatal rod photoreceptors (iCre75) and in mature retina using tamoxifen induction (Prom1-ETCre). BBs dock to the cell cortex during postnatal day 6 (P6) to extend a connecting cilium (CC) and an axoneme. P6 retina-specific knockouts (retCep164-/-) are unable to dock BBs, thereby preventing formation of CC or outer segments (OSs). In rod-specific knockouts (rodCep164-/-), Cre expression starts after P7 and CC/OS form. P16 rodCep164-/- rods have nearly normal OS lengths, and maintain OS attachment through P21 despite loss of CEP164. Intraflagellar transport components (IFT88, IFT57 and IFT140) were reduced at P16 rodCep164-/- BBs and CC tips and nearly absent at P21, indicating impaired intraflagellar transport. Nascent OS discs, labeled with a fluorescent dye on P14 and P18 and harvested on P19, showed continued rodCep164-/- disc morphogenesis but absence of P14 discs mid-distally, indicating OS instability. Tamoxifen induction with PROM1ETCre;Cep164F/F (tamCep164-/-) adult mice affected maintenance of both rod and cone OSs. The results suggest that CEP164 is key towards recruitment and stabilization of IFT-B particles at the BB/CC. IFT impairment may be the main driver of ciliary malfunction observed with hypomorphic CEP164 mutations.
Assuntos
Corpos Basais , Corantes Fluorescentes , Animais , Corpos Basais/metabolismo , Cílios/metabolismo , Corantes Fluorescentes/metabolismo , Camundongos , Transporte Proteico/genética , Células Fotorreceptoras Retinianas Cones , TamoxifenoRESUMO
The gap junction complex functions as a transport channel across the membrane. Among gap junction subunits, gap junction protein α1 (GJA1) is the most commonly expressed subunit. A recent study showed that GJA1 is necessary for the maintenance of motile cilia; however, the molecular mechanism and function of GJA1 in ciliogenesis remain unknown. Here, we examined the functions of GJA1 during ciliogenesis in human retinal pigment epithelium-1 and Xenopus laevis embryonic multiciliated-cells. GJA1 localizes to the motile ciliary axonemes or pericentriolar regions beneath the primary cilium. GJA1 depletion caused malformation of both the primary cilium and motile cilia. Further study revealed that GJA1 depletion affected several ciliary proteins such as BBS4, CP110, and Rab11 in the pericentriolar region and basal body. Interestingly, CP110 removal from the mother centriole was significantly reduced by GJA1 depletion. Importantly, Rab11, a key regulator during ciliogenesis, was immunoprecipitated with GJA1 and GJA1 knockdown caused the mislocalization of Rab11. These findings suggest that GJA1 regulates ciliogenesis by interacting with the Rab11-Rab8 ciliary trafficking pathway.
Assuntos
Centríolos , Cílios , Animais , Corpos Basais , Centríolos/metabolismo , Centrossomo/metabolismo , Cílios/metabolismo , Conexina 43/metabolismo , Humanos , Xenopus laevisRESUMO
Land plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is a continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning spermatogenesis remain to be elucidated. We successfully identified candidate genes involved in spermatogenesis, deeply divergent BLD10s, by computational analyses combining multiple methods and omics data. We then examined the functions of BLD10s in the liverwort Marchantia polymorpha and the moss Physcomitrium patens. MpBLD10 and PpBLD10 are required for normal basal body (BB) and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, and thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. We identified orthologs of MpBLD10 and PpBLD10 in diverse Streptophyta and found that MpBLD10 and PpBLD10 are orthologous to BLD10/CEP135 family proteins, which function in BB assembly. However, BLD10s evolved especially quickly in land plants and MpBLD10 might have acquired additional functions in spermatozoid formation through rapid molecular evolution.
Assuntos
Bryopsida , Marchantia , Animais , Corpos Basais , Bryopsida/genética , Cromatina/metabolismo , Gametogênese Vegetal , Marchantia/genética , Marchantia/metabolismo , Filogenia , Espermatogênese/genéticaRESUMO
Hydrodynamic flow produced by multiciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cell's cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by focused ion beam scanning electron microscopy imaging that cilia are coupled both longitudinally and laterally in the ciliate Tetrahymena thermophila by the underlying BB and cortical cytoskeletal network. To visualize the behavior of individual cilia in live, immobilized Tetrahymena cells, we developed Delivered Iron Particle Ubiety Live Light (DIPULL) microscopy. Quantitative and computer analyses of ciliary dynamics reveal that BB connections control ciliary waveform and coordinate ciliary beating. Loss of BB connections reduces cilia-dependent fluid flow forces.