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1.
Nat Commun ; 13(1): 2056, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35440631

RESUMO

Several tissues contain cells with multiple motile cilia that generate a fluid or particle flow to support development and organ functions; defective motility causes human disease. Developmental cues orient motile cilia, but how cilia are locked into their final position to maintain a directional flow is not understood. Here we find that the actin cytoskeleton is highly dynamic during early development of multiciliated cells (MCCs). While apical actin bundles become increasingly more static, subapical actin filaments are nucleated from the distal tip of ciliary rootlets. Anchorage of these subapical actin filaments requires the presence of microridge-like structures formed during MCC development, and the activity of Nonmuscle Myosin II. Optogenetic manipulation of Ezrin, a core component of the microridge actin-anchoring complex, or inhibition of Myosin Light Chain Kinase interfere with rootlet anchorage and orientation. These observations identify microridge-like structures as an essential component of basal body rootlet anchoring in MCCs.


Assuntos
Actinas , Cílios , Citoesqueleto de Actina , Corpos Basais , Cílios/fisiologia , Citoesqueleto , Humanos
2.
J Cell Biol ; 211(5): 963-73, 2015 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-26644512

RESUMO

Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.


Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cílios/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas/metabolismo , Proteínas de Xenopus/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Corpos Basais/metabolismo , Drosophila melanogaster , Epiderme/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Xenopus laevis/metabolismo , Proteínas rho de Ligação ao GTP
4.
Kidney Int ; 87(6): 1191-200, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25671767

RESUMO

Nephronophthisis (NPH) is a heterogenetic autosomal recessive disorder associated with kidney cysts and multiple extrarenal manifestations. The disease-associated gene products (NPHPs) typically contain domains involved in protein-protein interactions, and appear to exert their tissue-specific functions in large protein complexes. Most NPHPs localize to the cilium and/or basal body; however, their precise molecular functions remain largely unknown. We have recently identified the SAM-domain containing protein Anks3 as a potential ANKS6/NPHP16-interacting protein, and report now that Anks3 interacts with several NPHPs as well as with Bicc1 and the oxygen-sensitive asparaginyl hydroxylase HIF1AN. Knockdown of anks3 in zebrafish embryos was associated with NPH-typical manifestations, including ciliary abnormalities, cyst formation, and laterality defects. In multi-ciliated epidermal cells, GFP-tagged Anks3 localizes to the cilium, but forms large aggregates in the absence of NPHP1, indicating that the negatively charged NPHP1 curtails the polymerization of Anks3. Collectively, these findings suggest that Anks3 is a cilia-associated molecule that partners with the ANKS6- and via NPHP1 to the NPHP1-4-8 module. Thus, developmental defects associated with Anks3 depletion in zebrafish suggest that ANKS3 mutations may cause NPH or NPH-like disease in humans.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Rim/embriologia , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cílios/metabolismo , Proteínas do Citoesqueleto , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Rim/anormalidades , Doenças Renais Císticas/metabolismo , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/metabolismo , Quinases Relacionadas a NIMA , Proteínas Nucleares/metabolismo , Polimerização , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Situs Inversus/genética , Xenopus , Proteínas de Xenopus/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
5.
Development ; 142(1): 174-84, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25516973

RESUMO

Cilia are microtubule-based organelles that are present on most cells and are required for normal tissue development and function. Defective cilia cause complex syndromes with multiple organ manifestations termed ciliopathies. A crucial step during ciliogenesis in multiciliated cells (MCCs) is the association of future basal bodies with the apical plasma membrane, followed by their correct spacing and planar orientation. Here, we report a novel role for ELMO-DOCK1, which is a bipartite guanine nucleotide exchange factor complex for the small GTPase Rac1, and for the membrane-cytoskeletal linker Ezrin, in regulating centriole/basal body migration, docking and spacing. Downregulation of each component results in ciliopathy-related phenotypes in zebrafish and disrupted ciliogenesis in Xenopus epidermal MCCs. Subcellular analysis revealed a striking impairment of basal body docking and spacing, which is likely to account for the observed phenotypes. These results are substantiated by showing a genetic interaction between elmo1 and ezrin b. Finally, we provide biochemical evidence that the ELMO-DOCK1-Rac1 complex influences Ezrin phosphorylation and thereby probably serves as an important molecular switch. Collectively, we demonstrate that the ELMO-Ezrin complex orchestrates ciliary basal body migration, docking and positioning in vivo.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Corpos Basais/metabolismo , Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Axonema/metabolismo , Axonema/ultraestrutura , Membrana Celular/metabolismo , Cílios/ultraestrutura , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Fosforilação , Ligação Proteica , Xenopus laevis , Peixe-Zebra/embriologia , Proteínas rac de Ligação ao GTP
6.
Nat Genet ; 45(8): 951-6, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23793029

RESUMO

Nephronophthisis is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. Here we identify ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVS (NPHP2) and NPHP3. We show that ANKS6 localizes to the proximal cilium and confirm its role in renal development through knockdown experiments in zebrafish and Xenopus laevis. We also identify six families with ANKS6 mutations affected by nephronophthisis, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN hydroxylates ANKS6 and INVS and alters the composition of the ANKS6-INVS-NPHP3 module. Knockdown of Hif1an in Xenopus results in a phenotype that resembles loss of other NPHP proteins. Network analyses uncovered additional putative NPHP proteins and placed ANKS6 at the center of this NPHP module, explaining the overlapping disease manifestation caused by mutation in ANKS6, NEK8, INVS or NPHP3.


Assuntos
Doenças Renais Císticas/genética , Cinesinas/genética , Proteínas Nucleares/genética , Proteínas Quinases/genética , Fatores de Transcrição/genética , Animais , Cílios/metabolismo , Consanguinidade , Éxons , Técnicas de Silenciamento de Genes , Humanos , Íntrons , Doenças Renais Císticas/metabolismo , Cinesinas/metabolismo , Camundongos , Mutação , Quinases Relacionadas a NIMA , Proteínas Nucleares/metabolismo , Fenótipo , Doenças Renais Policísticas/genética , Ligação Proteica , Mapas de Interação de Proteínas , Proteínas Quinases/metabolismo , Transporte Proteico , Fatores de Transcrição/metabolismo , Xenopus/embriologia , Xenopus/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
7.
Cell ; 150(3): 533-48, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22863007

RESUMO

Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Exoma , Doenças Renais Císticas/genética , Proteínas dos Microtúbulos/metabolismo , Animais , Cílios/metabolismo , Técnicas de Silenciamento de Genes , Genes Recessivos , Humanos , Proteína Homóloga a MRE11 , Camundongos , Proteínas , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo
8.
Mech Dev ; 128(7-10): 376-86, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21843637

RESUMO

The centrosome is essential for the formation of the cilia and has been implicated in cell polarization and signaling during early embryonic development. A number of Wnt pathway components were found to localize at the centrosome, but how this localization relates to their signaling functions is unclear. In this study, we assessed a role for Diversin, a putative Wnt pathway mediator, in developmental processes that involve cilia. We find that Diversin is specifically localized to the basal body compartment near the base of the cilium in Xenopus multi-ciliated skin cells. Overexpression of Diversin RNA disrupted basal body polarization in these cells, suggesting that tightly regulated control of Diversin levels is crucial for this process. In cells depleted of endogenous Diversin, basal body structure appeared abnormal and this was accompanied by disrupted polarity, shortened or absent cilia and defective ciliary flow. These results are consistent with the involvement of Diversin in processes that are related to the acquisition of cell polarity and require ciliary functions.


Assuntos
Polaridade Celular/fisiologia , Cílios/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Frações Subcelulares/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Animais , Centrossomo/metabolismo , Microscopia Confocal , Transdução de Sinais , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia
9.
Genes Cells ; 10(4): 369-79, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15773899

RESUMO

It has been suggested that ILK (integrin-linked kinase) participates in integrin- and growth factor-mediated signaling pathways and also functions as a scaffold protein at cell-extracellular matrix (ECM) adhesion sites. As the recently reported ILK knockout mice were found to die at the peri-implantation stage, the stage specific to mammals, little is known about the function of ILK in early developmental processes common to every vertebrate. To address this, we isolated a Xenopus ortholog of ILK (XeILK) and characterized its role in early Xenopus embryogenesis. XeILK was expressed constitutively and ubiquitously throughout the early embryogenesis. Depletion of XeILK with morpholino oligonucleotides (XeILK MO) caused severe defects in blastopore closure and axis elongation without affecting the mesodermal specification. Furthermore, XeILK MO was found to interfere with cell-cell and cell-ECM adhesions in dorsal marginal zone explants and to result in a significant loss of cell-ECM adhesions in activin-treated dissociated animal cap cells. These results thus indicate that XeILK plays an essential role in morphogenetic movements during gastrulation.


Assuntos
Gástrula/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Sequência de Aminoácidos , Animais , Adesão Celular , Matriz Extracelular/fisiologia , Gástrula/metabolismo , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética
10.
J Biol Chem ; 279(22): 22992-5, 2004 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-15031289

RESUMO

Src homology 2-containing phosphotyrosine phosphatase (Shp2) functions as a positive effector in receptor tyrosine kinase (RTK) signaling immediately proximal to activated receptors. However, neither its physiological substrate(s) nor its mechanism of action in RTK signaling has been defined. In this study, we demonstrate that Sprouty (Spry) is a possible target of Shp2. Spry acts as a conserved inhibitor of RTK signaling, and tyrosine phosphorylation of Spry is indispensable for its inhibitory activity. Shp2 was able to dephosphorylate fibroblast growth factor receptor-induced phosphotyrosines on Spry both in vivo and in vitro. Shp2-mediated dephosphorylation of Spry resulted in dissociation of Spry from Grb2. Furthermore, Shp2 could reverse the inhibitory effect of Spry on FGF-induced neurite outgrowth and MAP kinase activation. These findings suggest that Shp2 acts as a positive regulator in RTK signaling by dephosphorylating and inactivating Spry.


Assuntos
Proteínas Tirosina Fosfatases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Ativação Enzimática , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Fosforilação , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Domínios de Homologia de src
11.
Nat Cell Biol ; 4(11): 850-8, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12402043

RESUMO

Sprouty (Spry) inhibits signalling by receptor tyrosine kinases; however, the molecular mechanism underlying this function has not been defined. Here we show that after stimulation by growth factors Spry1 and Spry2 translocate to the plasma membrane and become phosphorylated on a conserved tyrosine. Next, they bind to the adaptor protein Grb2 and inhibit the recruitment of the Grb2-Sos complex either to the fibroblast growth factor receptor (FGFR) docking adaptor protein FRS2 or to Shp2. Membrane translocation of Spry is necessary for its phosphorylation, which is essential for its inhibitor activity. A tyrosine-phosphorylated octapeptide derived from mouse Spry2 inhibits Grb2 from binding FRS2, Shp2 or mouse Spry2 in vitro and blocks activation of the extracellular-signal-regulated kinase (ERK) in cells stimulated by growth factor. A non-phosphorylated Spry mutant cannot bind Grb2 and acts as a dominant negative, inducing prolonged activation of ERK in response to FGF and promoting the FGF-induced outgrowth of neurites in PC12 cells. Our findings suggest that Spry functions in a negative feedback mechanism in which its inhibitor activity is controlled rapidly and reversibly by post-translational mechanisms.


Assuntos
Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fosfoproteínas/fisiologia , Proteínas ras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células COS , Bovinos , Linhagem Celular , Membrana Celular/metabolismo , DNA Complementar/metabolismo , Dimerização , Ativação Enzimática , Genes Dominantes , Células HeLa , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Células PC12 , Peptídeos/química , Fosfoproteínas/metabolismo , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/metabolismo , Ratos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Tirosina/química , Tirosina/metabolismo , Xenopus
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