RESUMO
For left-right symmetry breaking in the mouse embryo, the basal body must become positioned at the posterior side of node cells, but the precise mechanism for this has remained unknown. Here, we examined the role of microtubules (MTs) and actomyosin in this basal body positioning. Exposure of mouse embryos to agents that stabilize or destabilize MTs or F-actin impaired such positioning. Active myosin II was detected at the anterior side of node cells before the posterior shift of the basal body, and this asymmetric activation was lost in Prickle and dachsous mutant embryos. The organization of basal-body associated MTs (baMTs) was asymmetric between the anterior and posterior sides of node cells, with anterior baMTs extending horizontally and posterior baMTs extending vertically. This asymmetry became evident after polarization of the PCP core protein Vangl1 and before the posterior positioning of the basal body, and it also required the PCP core proteins Prickle and dachsous. Our results suggest that the asymmetry in baMT organization may play a role in correct positioning of the basal body for left-right symmetry breaking.
Assuntos
Corpos Basais , Polaridade Celular , Actinas/metabolismo , Animais , Polaridade Celular/fisiologia , Cílios/metabolismo , Camundongos , Microtúbulos/metabolismoRESUMO
Cluap1/IFT38 is a ciliary protein that belongs to the IFT-B complex and is required for ciliogenesis. In this study, we have examined the behaviors of Cluap1 protein in nonciliated and ciliated cells. In proliferating cells, Cluap1 is located at the distal appendage of the mother centriole. When cells are induced to form cilia, Cluap1 is found in a novel noncentriolar compartment, the cytoplasmic IFT spot, which mainly exists once in a cell. Other IFT-B proteins such as IFT46 and IFT88 are colocalized in this spot. The cytoplasmic IFT spot is present in mouse embryonic fibroblasts (MEFs) but is absent in ciliogenesis-defective MEFs lacking Cluap1, Kif3a or Odf2. The cytoplasmic IFT spot is also found in mouse embryos but is absent in the Cluap1 mutant embryo. When MEFs are induced to form cilia, the cytoplasmic IFT spot appears at an early step of ciliogenesis but starts to disappear when ciliogenesis is mostly completed. These results suggest that IFT-B proteins such as Cluap1 accumulate in a previously undescribed cytoplasmic compartment during ciliogenesis.
Assuntos
Cílios/metabolismo , Citoplasma/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Cílios/ultraestrutura , Citoplasma/ultraestrutura , Fibroblastos , Proteínas de Choque Térmico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesinas , Camundongos , Camundongos Knockout , Proteínas Supressoras de TumorRESUMO
Sperm motility is driven by motile cytoskeletal elements in the tail, called axonemes. The structure of axonemes consists of 9 + 2 microtubules, molecular motors (dyneins), and their regulatory structures. Axonemes are well conserved in motile cilia and flagella through eukaryotic evolution. Deficiency in the axonemal structure causes defects in sperm motility, and often leads to male infertility. It has been known since the 1970s that, in some cases, male infertility is linked with other symptoms or diseases such as Kartagener syndrome. Given that these links are mostly caused by deficiencies in the common components of cilia and flagella, they are called "immotile cilia syndrome" or "primary ciliary dyskinesia," or more recently, "ciliopathy," which includes deficiencies in primary and sensory cilia. Here, we review the structure of the sperm flagellum and epithelial cilia in the human body, and discuss how male fertility is linked to ciliopathy.
RESUMO
Sperm chemotaxis occurs widely in animals and plants and plays an important role in the success of fertilization. Several studies have recently demonstrated that Ca(2+) influx through specific Ca(2+) channels is a prerequisite for sperm chemotactic movement. However, the regulator that modulates flagellar movement in response to Ca(2+) is unknown. Here we show that a neuronal calcium sensor, calaxin, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis. Calaxin inhibition resulted in significant loss of sperm chemotactic movement, despite normal increases in intracellular calcium concentration. Using a demembranated sperm model, we demonstrate that calaxin is essential for generation and propagation of Ca(2+)-induced asymmetric flagellar bending. An in vitro motility assay revealed that calaxin directly suppressed the velocity of microtubule sliding by outer-arm dynein at high Ca(2+) concentrations. This study describes the missing link between chemoattractant-mediated Ca(2+) signaling and motor-driven microtubule sliding during sperm chemotaxis.
Assuntos
Dineínas/fisiologia , Proteínas Sensoras de Cálcio Intracelular/fisiologia , Espermatozoides/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Carbamatos/farmacologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Ciona intestinalis/citologia , Ciona intestinalis/fisiologia , Masculino , Microtúbulos/fisiologia , Modelos Biológicos , Proteínas Motores Moleculares/fisiologia , Piperidinas/farmacologia , Motilidade dos Espermatozoides/fisiologia , Cauda do Espermatozoide/fisiologia , Espermatozoides/efeitos dos fármacosRESUMO
Immotile cilia at the ventral node of mouse embryos are required for sensing leftward fluid flow that breaks left-right symmetry of the body. However, the flow-sensing mechanism has long remained elusive. In this work, we show that immotile cilia at the node undergo asymmetric deformation along the dorsoventral axis in response to the flow. Application of mechanical stimuli to immotile cilia by optical tweezers induced calcium ion transients and degradation of Dand5 messenger RNA (mRNA) in the targeted cells. The Pkd2 channel protein was preferentially localized to the dorsal side of immotile cilia, and calcium ion transients were preferentially induced by mechanical stimuli directed toward the ventral side. Our results uncover the biophysical mechanism by which immotile cilia at the node sense the direction of fluid flow.
Assuntos
Sinalização do Cálcio , Cálcio , Cílios , Mecanotransdução Celular , Animais , Camundongos , Cálcio/metabolismo , Cílios/fisiologia , Embrião de MamíferosRESUMO
Light-responsive regulation of ciliary motility is known to be conducted through modulation of dyneins, but the mechanism is not fully understood. Here, we report a novel subunit of the two-headed f/I1 inner arm dynein, named DYBLUP, in animal spermatozoa and a unicellular green alga. This subunit contains a BLUF (sensors of blue light using FAD) domain that appears to directly modulate dynein activity in response to light. DYBLUP (dynein-associated BLUF protein) mediates the connection between the f/I1 motor domain and the tether complex that links the motor to the doublet microtubule. Chlamydomonas lacking the DYBLUP ortholog shows both positive and negative phototaxis but becomes acclimated and attracted to high-intensity blue light. These results suggest a mechanism to avoid toxic strong light via direct photoregulation of dyneins.
RESUMO
BACKGROUND INFORMATION: Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca(2+) has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca(2+)-binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized. RESULTS: We identified a novel neuronal calcium sensor family protein, named calaxin (Ca(2+)-binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF-hand Ca(2+)-binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross-linking experiment showed that calaxin binds to beta-tubulin in situ. Overlay experiments further indicated that calaxin binds the beta-dynein heavy chain of outer arm dynein in the presence of Ca(2+). CONCLUSIONS: These results suggest that calaxin is a potential Ca(2+)-dependent modulator of outer arm dynein in metazoan cilia and flagella.
Assuntos
Chlamydomonas/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Proteínas de Protozoários/metabolismo , Espermatozoides/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Cálcio/metabolismo , Chlamydomonas/química , Chlamydomonas/classificação , Chlamydomonas/genética , Cílios/genética , Dineínas/genética , Flagelos/genética , Masculino , Dados de Sequência Molecular , Família Multigênica , Proteínas Sensoras de Cálcio Neuronal/química , Proteínas Sensoras de Cálcio Neuronal/genética , Filogenia , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Espermatozoides/químicaRESUMO
Unidirectional fluid flow generated by motile cilia at the left-right organizer (LRO) breaks left-right (L-R) symmetry during early embryogenesis in mouse, frog and zebrafish. The chick embryo, however, does not require motile cilia for L-R symmetry breaking. The diversity of mechanisms for L-R symmetry breaking among vertebrates and the trigger for such symmetry breaking in non-mammalian amniotes have remained unknown. Here we examined how L-R asymmetry is established in two reptiles, Madagascar ground gecko and Chinese softshell turtle. Both of these reptiles appear to lack motile cilia at the LRO. The expression of the Nodal gene at the LRO in the reptilian embryos was found to be asymmetric, in contrast to that in vertebrates such as mouse that are dependent on cilia for L-R patterning. Two paralogues of the Nodal gene derived from an ancient gene duplication are retained and expressed differentially in cilia-dependent and cilia-independent vertebrates. The expression of these two Nodal paralogues is similarly controlled in the lateral plate mesoderm but regulated differently at the LRO. Our in-depth analysis of reptilian embryos thus suggests that mammals and non-mammalian amniotes deploy distinct strategies dependent on different Nodal paralogues for rendering Nodal activity asymmetric at the LRO.
Assuntos
Padronização Corporal , Cílios , Animais , Embrião de Galinha , Madagáscar , Camundongos , Répteis , Peixe-ZebraRESUMO
Immotile cilia sense extracellular signals such as fluid flow, but whether Ca2+ plays a role in flow sensing has been unclear. Here, we examined the role of ciliary Ca2+ in the flow sensing that initiates the breaking of left-right (L-R) symmetry in the mouse embryo. Intraciliary and cytoplasmic Ca2+ transients were detected in the crown cells at the node. These Ca2+ transients showed L-R asymmetry, which was lost in the absence of fluid flow or the PKD2 channel. Further characterization allowed classification of the Ca2+ transients into two types: cilium-derived, L-R-asymmetric transients (type 1) and cilium-independent transients without an L-R bias (type 2). Type 1 intraciliary transients occurred preferentially at the left posterior region of the node, where L-R symmetry breaking takes place. Suppression of intraciliary Ca2+ transients delayed L-R symmetry breaking. Our results implicate cilium-derived Ca2+ transients in crown cells in initiation of L-R symmetry breaking in the mouse embryo.
RESUMO
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.
Assuntos
Nucleotídeos de Adenina/metabolismo , Astenozoospermia/genética , Proteínas do Citoesqueleto/deficiência , Situs Inversus/genética , Adolescente , Adulto , Animais , Astenozoospermia/patologia , Axonema/ultraestrutura , Sistemas CRISPR-Cas/genética , Cílios/metabolismo , Cílios/ultraestrutura , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Epididimo/patologia , Feminino , Flagelos/metabolismo , Flagelos/ultraestrutura , Humanos , Mutação com Perda de Função , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Planárias/citologia , Planárias/genética , Planárias/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Situs Inversus/diagnóstico por imagem , Situs Inversus/patologia , Motilidade dos Espermatozoides/genética , Tomografia Computadorizada por Raios X , Sequenciamento do ExomaRESUMO
Sperm chemotaxis toward a chemoattractant is very important for the success of fertilization. Calaxin, a member of the neuronal calcium sensor protein family, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis of ascidian, Ciona intestinalis. Here, we present the crystal structures of calaxin both in the open and closed states upon Ca2+ and Mg2+ binding. The crystal structures revealed that three of the four EF-hands of a calaxin molecule bound Ca2+ ions and that EF2 and EF3 played a critical role in the conformational transition between the open and closed states. The rotation of α7 and α8 helices induces a significant conformational change of a part of the α10 helix into the loop. The structural differences between the Ca2+- and Mg2+-bound forms indicates that EF3 in the closed state has a lower affinity for Mg2+, suggesting that calaxin tends to adopt the open state in Mg2+-bound form. SAXS data supports that Ca2+-binding causes the structural transition toward the closed state. The changes in the structural transition of the C-terminal domain may be required to bind outer-arm dynein. These results provide a novel mechanism for recognizing a target protein using a calcium sensor protein.
Assuntos
Proteínas Sensoras de Cálcio Intracelular/química , Simulação de Dinâmica Molecular , Animais , Sítios de Ligação , Cálcio/metabolismo , Ciona intestinalis/química , Flagelos/química , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Magnésio/metabolismo , Simulação de Acoplamento Molecular , Ligação ProteicaRESUMO
Changes in protein by posttranslational modifications comprise an important mechanism for the control of many cellular processes. Several flagellar proteins are methylated on arginine residues during flagellar resorption; however, the function is not understood. To learn more about the role of protein methylation during flagellar dynamics, we focused on protein arginine methyltransferases (PRMTs) 1, 3, 5, and 10. These PRMTs localize to the tip of flagella and in a punctate pattern along the length, very similar, but not identical, to that of intraflagellar transport (IFT) components. In addition, we found that PRMT 1 and 3 are also highly enriched at the base of the flagella, and the basal localization of these PRMTs changes during flagellar regeneration and resorption. Proteins with methyl arginine residues are also enriched at the tip and base of flagella, and their localization also changes during flagellar assembly and disassembly. PRMTs are lost from the flagella of fla10-1 cells, which carry a temperature-sensitive mutation in the anterograde motor for IFT. The data define the distribution of specific PRMTs and their target proteins in flagella and demonstrate that PRMTs are cargo for translocation within flagella by the process of IFT.
Assuntos
Flagelos/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Arginina/metabolismo , Axonema/metabolismo , Axonema/fisiologia , Transporte Biológico , Chlamydomonas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Flagelos/fisiologia , Metilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteína-Arginina N-Metiltransferases/fisiologiaRESUMO
Through their coordinated alignment and beating, motile cilia generate directional fluid flow and organismal movement. While the mechanisms used by multiciliated epithelial tissues to achieve this coordination have been widely studied, much less is known about regulation of monociliated tissues such as those found in the vertebrate node and swimming planktonic larvae. Here, we show that a calcium sensor protein associated with outer arm dynein, calaxin, is a critical regulator for the coordinated movements of monocilia. Knockdown of calaxin gene in sea urchin embryos results in uncoordinated ciliary beating and defective directional movement of the embryos, but no apparent abnormality in axoneme ultrastructure. Examination of the beating cycle of individual calaxin-deficient cilia revealed a marked effect on the waveform and spatial range of ciliary bending. These findings indicate that calaxin-mediated regulation of ciliary beating is responsible for proper basal body orientation and ciliary alignment in fields of monociliated cells.
Assuntos
Cílios/fisiologia , Dineínas/metabolismo , Ouriços-do-Mar/fisiologia , Animais , Axonema/ultraestrutura , Corpos Basais , Cílios/genética , Cílios/metabolismo , Dineínas/genética , Embrião não Mamífero/fisiologia , Movimento , Orientação Espacial , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genéticaRESUMO
The complex waveforms characteristic of motile eukaryotic cilia and flagella are produced by the temporally and spatially regulated action of multiple dynein subforms generating sliding between subsets of axonemal microtubules. Multiple protein complexes have been identified that are associated with the doublet microtubules and that mediate regulatory signals between key axonemal structures, such as the radial spokes and central apparatus, and the dynein arm motors; these complexes include the N-DRC, MIA, and CSC complexes. Previous studies have shown that PACRG (parkin co-regulated gene) forms a complex that is anchored to the axonemal doublet microtubules. Loss of PACRG causes defects in ciliary motility and cilia related diseases. Here, we use an in vitro microtubule sliding assay to demonstrate that PACRG and its interactors are part of a signaling pathway that includes the central apparatus, radial spokes and specific inner dynein arm subforms to control dynein-driven microtubule sliding. Using a biochemical approach, our studies also indicate that PACRG interacts with the radial spokes. © 2016 Wiley Periodicals, Inc.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Plantas/metabolismo , Chlamydomonas reinhardtii/genética , Cílios/genética , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Chaperonas Moleculares/genética , Proteínas de Plantas/genéticaRESUMO
Metazoan spermatozoa, especially those from marine invertebrates and fish, are excellent sources for isolating axonemal dyneins because of their cellular homogeneity and the large amounts that can be collected. Sperm flagella can be easily isolated by homogenization and subsequent centrifugation. Axonemes are obtained by demembranation of flagella with the nonionic detergent Triton X-100. The outer arm dyneins have been most widely studied because they are specifically extracted by a high-salt solution and can be isolated as a relatively pure fraction of ~20S two-headed dynein by sucrose density gradient centrifugation. Only a few reports have described the isolation of inner arm dyneins from sperm and the protocol has room for improvement. Sperm show clear changes in motility at fertilization, which are exerted through the regulation of axonemal dyneins by protein phosphorylation and Ca(2+) binding. Therefore dyneins from sperm flagella are an excellent biochemically tractable source for studying the regulation of axonemal dyneins. Here we describe protocols used for purification of flagellar dyneins from sperm of tunicates, sea urchins, and fish. The techniques described here could be applied to other species with appropriate modifications.
Assuntos
Bioquímica/métodos , Dineínas/isolamento & purificação , Cauda do Espermatozoide/metabolismo , Animais , Centrifugação com Gradiente de Concentração , Cromatografia por Troca Iônica , Ciona intestinalis/metabolismo , Peixes/metabolismo , Masculino , Ouriços-do-Mar/metabolismo , Cauda do Espermatozoide/químicaRESUMO
Germline transgenesis with a Tc1/mariner superfamily Minos transposon was achieved in the ascidian Ciona intestinalis. Transgenic lines that express transposases in germ cells are very useful for remobilizing transposon copies. In the present study, we created transposase-expressing lines of Minos in Ciona. A Ciona gene encoding protamine (Ci-prm) is expressed in the testes and sperm. Transgenic lines expressing Minos transposase in the testes and sperm were created with a cis-element of Ci-prm, and used for enhancer detection. Double-transgenic animals between transposase lines and a transgenic line with an enhancer detection vector passed on several independent enhancer detection events to subsequent progeny. This technique allowed us to isolate transgenic lines that express GFP in restricted tissues. This system provides an easy and efficient method for large-scale enhancer detection in Ciona intestinalis.