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1.
Structure ; 2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33651974

RESUMO

Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 65 left-handed spiral filament in the crystal structure at a resolution of 2.7 Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.

2.
Int J Mol Sci ; 21(22)2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33207549

RESUMO

Plasmodium falciparum parasitophorous vacuolar protein 1 (PfPV1), a protein unique to malaria parasites, is localized in the parasitophorous vacuolar (PV) and is essential for parasite growth. Previous studies suggested that PfPV1 cooperates with the Plasmodium translocon of exported proteins (PTEX) complex to export various proteins from the PV. However, the structure and function of PfPV1 have not been determined in detail. In this study, we undertook the expression, purification, and characterization of PfPV1. The tetramer appears to be the structural unit of PfPV1. The activity of PfPV1 appears to be similar to that of molecular chaperones, and it may interact with various proteins. PfPV1 could substitute CtHsp40 in the CtHsp104, CtHsp70, and CtHsp40 protein disaggregation systems. Based on these results, we propose a model in which PfPV1 captures various PV proteins and delivers them to PTEX through a specific interaction with HSP101.

3.
PLoS Genet ; 16(3): e1008664, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203505

RESUMO

Motile cilia/flagella are essential for swimming and generating extracellular fluid flow in eukaryotes. Motile cilia harbor a 9+2 arrangement consisting of nine doublet microtubules with dynein arms at the periphery and a pair of singlet microtubules at the center (central pair). In the central system, the radial spoke has a T-shaped architecture and regulates the motility and motion pattern of cilia. Recent cryoelectron tomography data reveal three types of radial spokes (RS1, RS2, and RS3) in the 96 nm axoneme repeat unit; however, the molecular composition of the third radial spoke, RS3 is unknown. In human pathology, it is well known mutation of the radial spoke head-related genes causes primary ciliary dyskinesia (PCD) including respiratory defect and infertility. Here, we describe the role of the primary ciliary dyskinesia protein Rsph4a in the mouse motile cilia. Cryoelectron tomography reveals that the mouse trachea cilia harbor three types of radial spoke as with the other vertebrates and that all triplet spoke heads are lacking in the trachea cilia of Rsph4a-deficient mice. Furthermore, observation of ciliary movement and immunofluorescence analysis indicates that Rsph4a contributes to the generation of the planar beating of motile cilia by building the distal architecture of radial spokes in the trachea, the ependymal tissues, and the oviduct. Although detailed mechanism of RSs assembly remains unknown, our results suggest Rsph4a is a generic component of radial spoke heads, and could explain the severe phenotype of human PCD patients with RSPH4A mutation.


Assuntos
Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Axonema/genética , Axonema/metabolismo , Cílios/genética , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Proteínas do Citoesqueleto/genética , Dineínas/metabolismo , Feminino , Flagelos/genética , Flagelos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética
4.
Cell Physiol Biochem ; 51(6): 2843-2857, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30562762

RESUMO

BACKGROUND/AIMS: Nodal cilia that rotate in the ventral node play an important role in establishing left-right asymmetry during embryogenesis; however, inv mutant cilia present abnormal movement and induce laterality defects. The mechanism of their motility, which is regulated by dynein activation and microtubule arrangement, has not been fully understood. This study analyzed the dynein-triggered ciliary motion in the abnormal ultrastructure of the inv mutant, aiming to quantitatively evaluate the influence of microtubule mislocalization on the movement of the cilium. METHODS: We established a realistic 3-D model of an inv mutant cilium with an ultrastructure based on tomographic datasets generated by ultra-high voltage electron microscopy. The time-variant activation of the axonemal dynein force was simulated by pairs of point loads and embedded at dynein-mounted positions between adjacent microtubule doublets in this mathematical model. Utilizing the finite element method and deformable grid, the motility of the mutant cilium that is induced by various dynein activation hypotheses was investigated and compared to experimental observation. RESULTS: The results indicate that for the inv mutant, simulations of the ciliary movement with the engagement of dyneins based on the distance-controlled pattern in the partially activation scenario are broadly consistent with the observation; the shortening of the microtubules induces smaller movement amplitudes, while the angles of the mislocalized microtubules affect the pattern of the ciliary movement, and during the ciliary movement, the microtubules swing and twist in the mutant ciliary body. CONCLUSION: More generally, this study implies that dynein engagement is sensitive to subtle geometric changes in the axoneme, and thus, this geometry greatly influences the integrity of a well-formed ciliary rotation.


Assuntos
Cílios/fisiologia , Dineínas/metabolismo , Microtúbulos/metabolismo , Animais , Cílios/ultraestrutura , Simulação por Computador , Dineínas/ultraestrutura , Módulo de Elasticidade , Desenvolvimento Embrionário , Camundongos Endogâmicos ICR , Microtúbulos/ultraestrutura , Modelos Biológicos , Movimento
5.
R Soc Open Sci ; 5(8): 180601, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30225054

RESUMO

Left-right (L-R) asymmetry in the body plan is determined by nodal flow in vertebrate embryos. Shinohara et al. (Shinohara K et al. 2012 Nat. Commun.3, 622 (doi:10.1038/ncomms1624)) used Dpcd and Rfx3 mutant mouse embryos and showed that only a few cilia were sufficient to achieve L-R asymmetry. However, the mechanism underlying the breaking of symmetry by such weak ciliary flow is unclear. Flow-mediated signals associated with the L-R asymmetric organogenesis have not been clarified, and two different hypotheses-vesicle transport and mechanosensing-are now debated in the research field of developmental biology. In this study, we developed a computational model of the node system reported by Shinohara et al. and examined the feasibilities of the two hypotheses with a small number of cilia. With the small number of rotating cilia, flow was induced locally and global strong flow was not observed in the node. Particles were then effectively transported only when they were close to the cilia, and particle transport was strongly dependent on the ciliary positions. Although the maximum wall shear rate was also influenced by ciliary position, the mean wall shear rate at the perinodal wall increased monotonically with the number of cilia. We also investigated the membrane tension of immotile cilia, which is relevant to the regulation of mechanotransduction. The results indicated that tension of about 0.1 µN m-1 was exerted at the base even when the fluid shear rate was applied at about 0.1 s-1. The area of high tension was also localized at the upstream side, and negative tension appeared at the downstream side. Such localization may be useful to sense the flow direction at the periphery, as time-averaged anticlockwise circulation was induced in the node by rotation of a few cilia. Our numerical results support the mechanosensing hypothesis, and we expect that our study will stimulate further experimental investigations of mechanotransduction in the near future.

6.
Int J Mol Sci ; 19(8)2018 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-30126249

RESUMO

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits, and has the appearance of a jellyfish: Its body consists of a double ß-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. Based on structural information from archaeal prefoldins, mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the structure and mechanisms of eukaryotic prefoldins remain unknown. In this study, we succeeded in obtaining recombinant prefoldin from a thermophilic fungus, Chaetomium thermophilum (CtPFD). The recombinant CtPFD could not protect citrate synthase from thermal aggregation. However, CtPFD formed a complex with actin from chicken muscle and tubulin from porcine brain, suggesting substrate specificity. We succeeded in observing the complex formation of CtPFD and the group II chaperonin of C. thermophilum (CtCCT) by atomic force microscopy and electron microscopy. These interaction kinetics were analyzed by surface plasmon resonance using Biacore. Finally, we have shown the transfer of actin from CtPFD to CtCCT. The study of the folding pathway formed by CtPFD and CtCCT should provide important information on mechanisms of the eukaryotic prefoldin⁻chaperonin system.


Assuntos
Chaetomium/metabolismo , Proteínas Fúngicas/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Chaetomium/química , Chaetomium/genética , Galinhas , Clonagem Molecular , Cristalização , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expressão Gênica , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Agregados Proteicos , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Suínos
7.
Dev Cell ; 40(5): 439-452.e4, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28292423

RESUMO

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a-/-Wnt5b-/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry.


Assuntos
Padronização Corporal , Polaridade Celular , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteína Wnt-5a/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Comunicação Celular , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Domínio LIM/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Modelos Biológicos , Proteínas/metabolismo
8.
Artigo em Inglês | MEDLINE | ID: mdl-28213464

RESUMO

Visceral organs of vertebrates show left-right (L-R) asymmetry with regard to their position and morphology. Cilia play essential role in generating L-R asymmetry. A number of genes required for L-R asymmetry have now been identified in vertebrates, including human, many of which contribute to the formation and motility of cilia. In the mouse embryo, breaking of L-R symmetry occurs in the ventral node, where two types of cilia (motile and immotile) are present. Motile cilia are located at the central region of the node, and generate a leftward fluid flow. These motile cilia at the node are unique in that they rotate in the clockwise direction, unlike other immotile cilia such as airway cilia that show planar beating. The second type of cilia essential for L-R asymmetry is immotile cilia that are peripherally located immotile cilia. They sense a flow-dependent signal, which is either chemical or mechanical in nature. Although Ca2+ signaling is implicated in flow sensing, the precise mechanism remains unknown.


Assuntos
Cílios/fisiologia , Animais , Polaridade Celular , Camundongos
9.
Am J Hum Genet ; 99(2): 460-9, 2016 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-27486780

RESUMO

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.


Assuntos
Axonema/genética , Axonema/metabolismo , Proteínas de Transporte/genética , Cílios/patologia , Dineínas/química , Dineínas/metabolismo , Síndrome de Kartagener/genética , Síndrome de Kartagener/patologia , Mutação , Animais , Axonema/patologia , Axonema/ultraestrutura , Cílios/metabolismo , Cílios/ultraestrutura , Dineínas/genética , Dineínas/ultraestrutura , Exoma/genética , Éxons/genética , Imunofluorescência , Genes Recessivos , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Ligação Proteica , Xenopus , Proteínas de Xenopus/deficiência , Proteínas de Xenopus/genética
10.
PLoS One ; 11(7): e0159917, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27472658

RESUMO

Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming.


Assuntos
Caenorhabditis elegans/embriologia , Corrente Citoplasmática , Oócitos/metabolismo , Animais , Teorema de Bayes , Hidrodinâmica , Funções Verossimilhança , Camundongos , Modelos Biológicos , Estresse Mecânico
11.
Genes Cells ; 21(7): 728-39, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27353389

RESUMO

Lrrc6 encodes a cytoplasmic protein that is expressed specifically in cells with motile cilia including the node, trachea and testes of the mice. A mutation of Lrrc6 has been identified in human patients with primary ciliary dyskinesia (PCD). Mutant mice lacking Lrrc6 show typical PCD defects such as hydrocephalus and laterality defects. We found that in the absence of Lrrc6, the morphology of motile cilia remained normal, but their motility was completely lost. The 9 + 2 arrangement of microtubules remained normal in Lrrc6(-/-) mice, but the outer dynein arms (ODAs), the structures essential for the ciliary beating, were absent from the cilia. In the absence of Lrrc6, ODA proteins such as DNAH5, DNAH9 and IC2, which are assembled in the cytoplasm and transported to the ciliary axoneme, remained in the cytoplasm and were not transported to the ciliary axoneme. The IC2-IC1 interaction, which is the first step of ODA assembly, was normal in Lrrc6(-/-) mice testes. Our results suggest that ODA proteins may be transported from the cytoplasm to the cilia by an Lrrc6-dependent mechanism.


Assuntos
Cílios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animais , Dineínas do Axonema/genética , Axonema/genética , Axonema/patologia , Cílios/patologia , Citoplasma/genética , Citoplasma/metabolismo , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Dineínas/genética , Humanos , Síndrome de Kartagener/patologia , Camundongos , Camundongos Transgênicos , Mutação
12.
Dev Cell ; 35(2): 236-46, 2015 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-26506310

RESUMO

Determination of left-right asymmetry in mouse embryos is established by a leftward fluid flow that is generated by clockwise rotation of node cilia. How node cilia achieve stable unidirectional rotation has remained unknown, however. Here we show that brief exposure to the microtubule-stabilizing drug paclitaxel (Taxol) induces randomly directed rotation and changes the ultrastructure of node cilia. In vivo observations and a computer simulation revealed that a regular 9+0 arrangement of doublet microtubules is essential for stable unidirectional rotation of node cilia. The 9+2 motile cilia of the airway, which manifest planar beating, are resistant to Taxol treatment. However, the airway cilia of mice lacking the radial spoke head protein Rsph4a undergo rotational movement instead of planar beating, are prone to microtubule rearrangement, and are sensitive to Taxol. Our results suggest that the absence of radial spokes allows node cilia to rotate unidirectionally but, as a trade-off, renders them ultrastructurally fragile.


Assuntos
Padronização Corporal/genética , Cílios/genética , Desenvolvimento Embrionário/genética , Animais , Cílios/fisiologia , Cílios/ultraestrutura , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Paclitaxel/administração & dosagem
13.
Biomed Mater Eng ; 24(6): 2495-501, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25226950

RESUMO

The cilia, presenting a rotational movement in the embryonic nodes, play a crucial role in the left-right specification during embryogenesis. The characteristic architecture of these cilia is based on a cylindrical arrangement of 9 doublet microtubules and the motion of the cilia is triggered by the dynein motors located between adjacent doublets by converting the chemical energy into mechanical work. Restricted by the inherent difficulties of experiments, the dynein activation patterns in moving cilia cannot be directly observed. Thus, the mechanism of nodal ciliary movement is still unclear. In this study, we present computational models of the nodal ciliary ultrastructure based on tomographic images of the ciliary body. By employing time accurate three-dimensional solid mechanics analysis, we investigate the dynein-triggered sliding between adjacent doublet microtubules and simulate the induced ciliary bending. As an exploratory study, two dynein activation patterns are proposed and their rationality is discussed. The mathematical model presented by this paper provides a platform to investigate various assumptions of dynein activity, facilitating us to propose the most possible dynein activation pattern and therefore improving our understandings regarding the protein-beating problems of cilia.


Assuntos
Cílios/fisiologia , Dineínas/fisiologia , Desenvolvimento Embrionário/fisiologia , Indução Embrionária/fisiologia , Modelos Biológicos , Proteínas Motores Moleculares/fisiologia , Movimento/fisiologia , Animais , Simulação por Computador , Humanos , Mecanotransdução Celular/fisiologia , Neurulação
14.
J Cell Biol ; 204(2): 203-13, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-24421334

RESUMO

Axonemal dynein complexes are preassembled in the cytoplasm before their transport to cilia, but the mechanism of this process remains unclear. We now show that mice lacking Pih1d3, a PIH1 domain-containing protein, develop normally but manifest male sterility. Pih1d3(-/-) sperm were immotile and fragile, with the axoneme of the flagellum lacking outer dynein arms (ODAs) and inner dynein arms (IDAs) and showing a disturbed 9+2 microtubule organization. Pih1d3 was expressed specifically in spermatogenic cells, with the mRNA being most abundant in pachytene spermatocytes. Pih1d3 localized to the cytoplasm of spermatogenic cells but was not detected in spermatids or mature sperm. The levels of ODA and IDA proteins were reduced in the mutant testis and sperm, and Pih1d3 was found to interact with an intermediate chain of ODA as well as with Hsp70 and Hsp90. Our results suggest that Pih1d3 contributes to cytoplasmic preassembly of dynein complexes in spermatogenic cells by stabilizing and promoting complex formation by ODA and IDA proteins.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Dineínas do Axonema/metabolismo , Espermatozoides/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Axonema/metabolismo , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Fertilidade/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , RNA Mensageiro/metabolismo , Espermatozoides/ultraestrutura , Testículo/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-23767475

RESUMO

Rotational movement of isolated single cilia in mice embryo was investigated, which generates leftward fluid flow in the node cavity and plays an important role in left-right determination. The leftward unidirectional flow results from tilting of the rotational axis of the cilium to the posterior side. By combining computational fluid dynamics with experimental observations, we demonstrate that the leftward stroke can be more effective than expected for cases in which cilia tilting alone is considered with the no-slip condition under constant driving torque. Our results suggest that the driving torque is asymmetric.


Assuntos
Padronização Corporal/fisiologia , Cílios/fisiologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/fisiologia , Lateralidade Funcional/fisiologia , Modelos Biológicos , Animais , Movimento Celular/fisiologia , Cílios/ultraestrutura , Simulação por Computador , Embrião de Mamíferos/ultraestrutura , Camundongos , Rotação , Estresse Mecânico
16.
Dev Biol ; 381(1): 203-12, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23742838

RESUMO

Qilin is one of several genes in zebrafish whose mutation results in cystic kidney. We have now studied the role of its mouse ortholog, Cluap1, in embryonic development by generating Cluap1 knockout (Cluap1-/-) mice. Cluap1-/- embryos died mid-gestation manifesting impairment of ciliogenesis in various regions including the node and neural tube. The basal body was found to be properly docked to the apical membrane of cells in the mutant, but the axoneme failed to grow. Cluap1 is a ciliary protein and is preferentially localized at the base and tip of cilia. Hedgehog signaling, as revealed with a Pacthed1-lacZ reporter gene, was lost in Cluap1-/- embryos at embryonic day (E) 8.5 but was ectopically expanded at E9.0. The Cluap1 knockout embryos also failed to manifest left-right asymmetric expression of Nodal in the lateral plate, most likely as a result of the loss of Hedgehog signaling in node crown cells that in turn leads to pronounced down-regulation of Gdf1 expression in these cells. Crown cell-specific restoration of Cluap1 expression rescued Gdf1 expression in crown cells and left-sided Nodal expression in the lateral plate of mutant embryos. Our results suggest that Cluap1 contributes to ciliogenesis by regulating the intraflagellar transport (IFT) cycle at the base and tip of the cilium.


Assuntos
Cílios/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Morfogênese/genética , Animais , Padronização Corporal , Regulação para Baixo , Fibroblastos/metabolismo , Genes Reporter , Genótipo , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Óperon Lac , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Transdução de Sinais
17.
PLoS One ; 8(3): e60406, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23544137

RESUMO

The determination of left-right body asymmetry in mouse embryos depends on the interplay of molecules in a highly sensitive structure, the node. Here, we show that the localization of Cerl2 protein does not correlate to its mRNA expression pattern, from 3-somite stage onwards. Instead, Cerl2 protein displays a nodal flow-dependent dynamic behavior that controls the activity of Nodal in the node, and the transmission of the laterality information to the left lateral plate mesoderm (LPM). Our results indicate that Cerl2 initially localizes and prevents the activation of Nodal genetic circuitry on the right side of the embryo, and later its right-to-left translocation shutdowns Nodal activity in the node. The consequent prolonged Nodal activity in the node by the absence of Cerl2 affects local Nodal expression and prolongs its expression in the LPM. Simultaneous genetic removal of both Nodal node inhibitors, Cerl2 and Lefty1, sustains even longer and bilateral this LPM expression.


Assuntos
Padronização Corporal , Embrião de Mamíferos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Nodal/metabolismo , Animais , Embrião de Mamíferos/citologia , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Nodal/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Somitos/citologia , Somitos/metabolismo , Fatores de Tempo
18.
Phys Rev Lett ; 110(24): 248107, 2013 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-25165968

RESUMO

Rotational movement of mouse node cilia generates leftward fluid flow in the node cavity, playing an important role in left-right determination in the embryo. Although rotation of numerous cilia was believed necessary to trigger the determination, recent reports indicate the action of two cilia to be sufficient. We examine cooperative cilia movement via hydrodynamic interaction. Results show cilia to be cooperative, having phases locked in a certain relation; a system with a pair of nonidentical cilia can achieve phase-locked states more easily than one with a pair of identical cilia.


Assuntos
Cílios/fisiologia , Modelos Biológicos , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário , Lateralidade Funcional , Hidrodinâmica , Camundongos , Rotação
19.
Nat Commun ; 3: 1322, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23271656

RESUMO

Breaking of left-right symmetry in mouse embryos requires fluid flow at the node, but the precise action of the flow has remained unknown. Here we show that the left-right asymmetry of Cerl2 expression around the node, a target of the flow, is determined post-transcriptionally by decay of Cerl2 mRNA in a manner dependent on its 3' untranslated region. Cerl2 mRNA is absent specifically from the apical region of crown cells on the left side of the node. Preferential decay of Cerl2 mRNA on the left is initiated by the leftward flow and further enhanced by the operation of Wnt-Cerl2 interlinked feedback loops, in which Wnt3 upregulates Wnt3 expression and promotes Cerl2 mRNA decay, whereas Cerl2 promotes Wnt degradation. Mathematical modelling and experimental data suggest that these feedback loops behave as a bistable switch that can amplify in a noise-resistant manner a small bias conferred by fluid flow.


Assuntos
Retroalimentação Fisiológica , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Animais , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Transgênicos , Conformação de Ácido Nucleico , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , Transdução de Sinais , Proteína Wnt3/genética , Proteína Wnt3/metabolismo
20.
Science ; 338(6104): 226-31, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-22983710

RESUMO

Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca(2+) channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.


Assuntos
Padronização Corporal , Embrião de Mamíferos/fisiologia , Fatores de Determinação Direita-Esquerda/metabolismo , Organizadores Embrionários/fisiologia , Canais de Cátion TRPP/metabolismo , Animais , Líquidos Corporais/fisiologia , Cálcio/metabolismo , Cílios/metabolismo , Cílios/fisiologia , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cinesina/genética , Fatores de Determinação Direita-Esquerda/genética , Camundongos , Camundongos Mutantes , Mutação , Organizadores Embrionários/citologia , Transdução de Sinais , Canais de Cátion TRPP/genética
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