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1.
Methods Mol Biol ; 2152: 207-224, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32524555

RESUMO

Our knowledge of the structure, localization, and interaction partners of cerebral cavernous malformations (CCM) proteins is mainly based on cell culture studies that lack the physiology of a three-dimensional multi-tissue environment. Uncovering the subcellular localization and the dynamic behavior of CCM proteins is an important aspect of characterizing the endothelial cell biology of CCM scaffold formation and for describing interactions with other protein complexes. However, the generation of specific antibodies to locate CCM scaffolds within cells has been challenging. To overcome the lack of functional antibodies, here, we describe the methodology involved in the generation of a construct for the expression of a fluorescently labeled CCM fusion construct and in the establishment of a transgenic zebrafish reporter line. The transgenic expression of fluorescently labeled CCM proteins within the developing zebrafish vasculature makes it possible to study the detailed subcellular localization and the dynamics of CCM proteins in vivo.


Assuntos
Animais Geneticamente Modificados , Expressão Gênica , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Biomarcadores , Clonagem Molecular , Endocárdio/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Genes Reporter , Humanos , Camundongos Knockout , Mutação , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Peixe-Zebra
2.
Circ Res ; 125(10): e43-e54, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31495257

RESUMO

RATIONALE: Pathological biomechanical signaling induces vascular anomalies including cerebral cavernous malformations (CCM), which are caused by a clonal loss of CCM1/KRIT1 (Krev interaction trapped protein 1), CCM2/MGC4607, or CCM3/PDCD10. Why patients typically experience lesions only in lowly perfused venous capillaries of the cerebrovasculature is completely unknown. OBJECTIVE: In contrast, animal models with a complete loss of CCM proteins lack a functional heart and blood flow and exhibit vascular anomalies within major blood vessels as well. This finding raises the possibility that hemodynamics may play a role in the context of this vascular pathology. METHODS AND RESULTS: Here, we used a genetic approach to restore cardiac function and blood flow in a zebrafish model of CCM1. We find that blood flow prevents cardiovascular anomalies including a hyperplastic expansion within a large Ccm1-deficient vascular bed, the lateral dorsal aorta. CONCLUSIONS: This study identifies blood flow as an important physiological factor that is protective in the cause of this devastating vascular pathology.


Assuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Neoplasias do Sistema Nervoso Central/diagnóstico por imagem , Modelos Animais de Doenças , Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico por imagem , Animais , Animais Geneticamente Modificados , Neoplasias do Sistema Nervoso Central/fisiopatologia , Angiografia Cerebral/métodos , Hemangioma Cavernoso do Sistema Nervoso Central/fisiopatologia , Peixe-Zebra
3.
Nat Commun ; 9(1): 2161, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29867082

RESUMO

Development of a multiple-chambered heart from the linear heart tube is inherently linked to cardiac looping. Although many molecular factors regulating the process of cardiac chamber ballooning have been identified, the cellular mechanisms underlying the chamber formation remain unclear. Here, we demonstrate that cardiac chambers remodel by cell neighbour exchange of cardiomyocytes guided by the planar cell polarity (PCP) pathway triggered by two non-canonical Wnt ligands, Wnt5b and Wnt11. We find that PCP signalling coordinates the localisation of actomyosin activity, and thus the efficiency of cell neighbour exchange. On a tissue-scale, PCP signalling planar-polarises tissue tension by restricting the actomyosin contractility to the apical membranes of outflow tract cells. The tissue-scale polarisation of actomyosin contractility is required for cardiac looping that occurs concurrently with chamber ballooning. Taken together, our data reveal that instructive PCP signals couple cardiac chamber expansion with cardiac looping through the organ-scale polarisation of actomyosin-based tissue tension.


Assuntos
Actomiosina/metabolismo , Polaridade Celular/fisiologia , Miocárdio/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Actomiosina/genética , Animais , Animais Geneticamente Modificados , Remodelamento Atrial/genética , Polaridade Celular/genética , Desenvolvimento Embrionário/genética , Coração/embriologia , Mutação , Miocárdio/citologia , Remodelação Ventricular/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
4.
Elife ; 72018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29364115

RESUMO

Endothelial cells respond to different levels of fluid shear stress through adaptations of their mechanosensitivity. Currently, we lack a good understanding of how this contributes to sculpting of the cardiovascular system. Cerebral cavernous malformation (CCM) is an inherited vascular disease that occurs when a second somatic mutation causes a loss of CCM1/KRIT1, CCM2, or CCM3 proteins. Here, we demonstrate that zebrafish Krit1 regulates the formation of cardiac valves. Expression of heg1, which encodes a binding partner of Krit1, is positively regulated by blood-flow. In turn, Heg1 stabilizes levels of Krit1 protein, and both Heg1 and Krit1 dampen expression levels of klf2a, a major mechanosensitive gene. Conversely, loss of Krit1 results in increased expression of klf2a and notch1b throughout the endocardium and prevents cardiac valve leaflet formation. Hence, the correct balance of blood-flow-dependent induction and Krit1 protein-mediated repression of klf2a and notch1b ultimately shapes cardiac valve leaflet morphology.


Assuntos
Células Endoteliais/fisiologia , Valvas Cardíacas/embriologia , Fatores de Transcrição Kruppel-Like/metabolismo , Mecanotransdução Celular , Glicoproteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteínas Musculares
5.
PLoS One ; 7(6): e40000, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22768194

RESUMO

Protein kinase C iota is required for various cell biological processes including epithelial tissue polarity and organ morphogenesis. To gain mechanistic insight into different roles of this kinase, it is essential to identify specific substrate proteins in their cellular context. The analog-sensitive kinase method provides a powerful tool for the identification of kinase substrates under in vivo conditions. However, it has remained a major challenge to establish screens based on this method in multicellular model organisms. Here, we report the methodology for in vivo conditions using the analog-sensitive kinase method in a genetically-tractable vertebrate model organism, the zebrafish. With this approach, kinase substrates can uniquely be labeled in the developing zebrafish embryo using bulky ATPγS analogs which results in the thiophosphorylation of substrates. The labeling of kinase substrates with a thiophosphoester epitope differs from phosphoesters that are generated by all other kinases and allows for an enrichment of thiophosphopeptides by immunoaffinity purification. This study provides the foundation for using the analog-sensitive kinase method in the context of complex vertebrate development, physiology, or disease.


Assuntos
Ensaios Enzimáticos/métodos , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Peixe-Zebra/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Embrião não Mamífero/enzimologia , Isoenzimas/química , Dados de Sequência Molecular , Proteínas Mutantes/química , Fosforilação , Proteína Quinase C/química , Especificidade por Substrato , Compostos de Sulfidrila/metabolismo , Peixe-Zebra/embriologia
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