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1.
PLoS Genet ; 16(12): e1009232, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33347437

RESUMO

Motile cilia can beat with distinct patterns, but how motility variations are regulated remain obscure. Here, we have studied the role of the coiled-coil protein CFAP53 in the motility of different cilia-types in the mouse. While node (9+0) cilia of Cfap53 mutants were immotile, tracheal and ependymal (9+2) cilia retained motility, albeit with an altered beat pattern. In node cilia, CFAP53 mainly localized at the base (centriolar satellites), whereas it was also present along the entire axoneme in tracheal cilia. CFAP53 associated tightly with microtubules and interacted with axonemal dyneins and TTC25, a dynein docking complex component. TTC25 and outer dynein arms (ODAs) were lost from node cilia, but were largely maintained in tracheal cilia of Cfap53-/- mice. Thus, CFAP53 at the base of node cilia facilitates axonemal transport of TTC25 and dyneins, while axonemal CFAP53 in 9+2 cilia stabilizes dynein binding to microtubules. Our study establishes how differential localization and function of CFAP53 contributes to the unique motion patterns of two important mammalian cilia-types.

2.
Nat Commun ; 11(1): 5520, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139725

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 Espermática/genética , Tomografia Computadorizada por Raios X , Sequenciamento Completo do Exoma
3.
Sci Adv ; 6(30): eaba1195, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32743070

RESUMO

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.

4.
Nat Ecol Evol ; 4(2): 261-269, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907383

RESUMO

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-Zebra
5.
Genes Cells ; 24(11): 731-745, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31554018

RESUMO

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 , Cinesina , Camundongos , Camundongos Knockout , Proteínas Supressoras de Tumor
6.
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
7.
Dev Biol ; 395(2): 331-41, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25224222

RESUMO

The transcription factor Pitx2c is expressed in primordial visceral organs in a left-right (L-R) asymmetric manner and executes situs-specific morphogenesis. Here we show that Pitx2c is also L-R asymmetrically expressed in the developing mouse limb. Human PITX2c exhibits the same transcriptional activity in the mouse limb. The asymmetric expression of Pitx2c in the limb also exhibits dorsal-ventral and anterior-posterior polarities, being confined to the posterior-dorsal region of the left limb. Left-sided Pitx2c expression in the limb is regulated by Nodal signaling through a Nodal-responsive enhancer. Pitx2c is expressed in lateral plate mesoderm (LPM)-derived cells in the left limb that contribute to various limb connective tissues. The number of Pitx2c(+) cells in the left limb was found to be negatively regulated by Pitx2c itself. Although obvious defects were not apparent in the limb of mice lacking asymmetric Pitx2c expression, Pitx2c may regulate functional L-R asymmetry of the limb.


Assuntos
Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/metabolismo , Morfogênese/fisiologia , Fatores de Transcrição/metabolismo , Animais , Primers do DNA/genética , Imunofluorescência , Galactosídeos , Técnicas de Introdução de Genes , Hibridização In Situ , Indóis , Camundongos , Camundongos Transgênicos , Tamoxifeno
8.
Development ; 130(9): 1725-34, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12642479

RESUMO

Inversin (Inv), a protein that contains ankyrin repeats, plays a key role in left-right determination during mammalian embryonic development, but its precise function remains unknown. Transgenic mice expressing an Inv and green fluorescent protein (GFP) fusion construct (Inv::GFP) were established to facilitate characterization of the subcellular localization of Inv. The Inv::GFP transgene rescued the laterality defects and polycystic kidney disease of Inv/Inv mice, indicating that the fusion protein is functional. In transgenic embryos, Inv::GFP protein was detected in the node monocilia. The fusion protein was also present in other 9+0 monocilia, including those of kidney epithelial cells and the pituitary gland, but it was not localized to 9+2 cilia. The N-terminal region of Inv (InvDeltaC) including the ankyrin repeats also localized to the node cilia and rescued the left-right defects of Inv/Inv mutants. Although no obvious abnormalities were detected in the node monocilia of Inv/Inv embryos, the laterality defects of such embryos were corrected by an artificial leftward flow of fluid in the node, suggesting that nodal flow is impaired by the Inv mutation. These results suggest that the Inv protein contributes to left-right determination as a component of monocilia in the node and is essential for the generation of normal nodal flow.


Assuntos
Cílios/metabolismo , Proteínas/metabolismo , Fatores de Transcrição , Animais , Repetição de Anquirina/genética , Fibroblastos/metabolismo , Genes Reporter/fisiologia , Rim/embriologia , Camundongos/embriologia , Camundongos Transgênicos , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
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