RESUMO
α-catenin is a key protein of adherens junctions (AJs) with mechanosensory properties. It also acts as a tumor suppressor that limits tissue growth. Here we analyzed the function of Drosophila α-Catenin (α-Cat) in growth regulation of the wing epithelium. We found that different α-Cat levels led to a differential activation of Hippo/Yorkie or JNK signaling causing tissue overgrowth or degeneration, respectively. α-Cat can modulate Yorkie-dependent tissue growth through recruitment of Ajuba, a negative regulator of Hippo signaling to AJs but also through a mechanism independent of Ajuba recruitment to AJs. Both mechanosensory regions of α-Cat, the M region and the actin-binding domain (ABD), contribute to growth regulation. Whereas M is dispensable for α-Cat function in the wing, individual M domains (M1, M2, M3) have opposing effects on growth regulation. In particular, M1 limits Ajuba recruitment. Loss of M1 causes Ajuba hyper-recruitment to AJs, promoting tissue-tension independent overgrowth. Although M1 binds Vinculin, Vinculin is not responsible for this effect. Moreover, disruption of mechanosensing of the α-Cat ABD affects tissue growth, with enhanced actin interactions stabilizing junctions and leading to tissue overgrowth. Together, our findings indicate that α-Cat acts through multiple mechanisms to control tissue growth, including regulation of AJ stability, mechanosensitive Ajuba recruitment, and dynamic direct F-actin interactions.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas com Domínio LIM/genética , Asas de Animais/crescimento & desenvolvimento , alfa Catenina/genética , Citoesqueleto de Actina/genética , Actinas/genética , Junções Aderentes/genética , Animais , Morte Celular/genética , Citoesqueleto/genética , Drosophila melanogaster/crescimento & desenvolvimento , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases/genética , Mecanotransdução Celular/genética , Proteínas Nucleares/genética , Domínios Proteicos/genética , Proteínas Serina-Treonina Quinases/genética , Transativadores/genética , Vinculina/genética , Asas de Animais/metabolismo , Proteínas de Sinalização YAPRESUMO
α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis.
Assuntos
Junções Aderentes/metabolismo , alfa Catenina/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Caderinas/química , Caderinas/metabolismo , Humanos , Estrutura Secundária de Proteína , alfa Catenina/químicaRESUMO
The cadherin-catenin adhesion complex is a key contributor to epithelial tissue stability and dynamic cell movements during development and tissue renewal. How this complex is regulated to accomplish these functions is not fully understood. We identified several phosphorylation sites in mammalian αE-catenin (also known as catenin α-1) and Drosophila α-Catenin within a flexible linker located between the middle (M)-region and the carboxy-terminal actin-binding domain. We show that this phospho-linker (P-linker) is the main phosphorylated region of α-catenin in cells and is sequentially modified at casein kinase 2 and 1 consensus sites. In Drosophila, the P-linker is required for normal α-catenin function during development and collective cell migration, although no obvious defects were found in cadherin-catenin complex assembly or adherens junction formation. In mammalian cells, non-phosphorylatable forms of α-catenin showed defects in intercellular adhesion using a mechanical dispersion assay. Epithelial sheets expressing phosphomimetic forms of α-catenin showed faster and more coordinated migrations after scratch wounding. These findings suggest that phosphorylation and dephosphorylation of the α-catenin P-linker are required for normal cadherin-catenin complex function in Drosophila and mammalian cells.
Assuntos
Caderinas/metabolismo , Caseína Quinase II/metabolismo , Caseína Quinase I/metabolismo , Adesão Celular , Drosophila melanogaster/metabolismo , alfa Catenina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Western Blotting , Caderinas/genética , Caseína Quinase I/genética , Caseína Quinase II/genética , Membrana Celular/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cães , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Imunofluorescência , Humanos , Imunoprecipitação , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Ovário/citologia , Ovário/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , alfa Catenina/química , alfa Catenina/genéticaRESUMO
The linkage of adherens junctions to the actin cytoskeleton is essential for cell adhesion. The contribution of the cadherin-catenin complex to the interaction between actin and the adherens junction remains an intensely investigated subject that centres on the function of α-catenin, which binds to cadherin through ß-catenin and can bind F-actin directly or indirectly. Here, we delineate regions within Drosophila α-Catenin (α-Cat) that are important for adherens junction performance in static epithelia and dynamic morphogenetic processes. Moreover, we address whether persistent α-catenin-mediated physical linkage between cadherin and F-actin is crucial for cell adhesion and characterize the functions of α-catenin monomers and dimers at adherens junctions. Our data support the view that monomeric α-catenin acts as an essential physical linker between the cadherin-ß-catenin complex and the actin cytoskeleton, whereas α-catenin dimers are cytoplasmic and form an equilibrium with monomeric junctional α-catenin.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Caderinas/metabolismo , Adesão Celular/fisiologia , Drosophila melanogaster/metabolismo , alfa Catenina/metabolismo , beta Catenina/metabolismo , Citoesqueleto de Actina/genética , Actinas/genética , Junções Aderentes/fisiologia , Regulação Alostérica , Animais , Caderinas/genética , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Processamento de Imagem Assistida por Computador , Immunoblotting , Técnicas Imunoenzimáticas , Imunoprecipitação , Mutação/genética , Multimerização Proteica , alfa Catenina/genética , beta Catenina/genéticaRESUMO
α-catenin associates the cadherin-catenin complex with the actin cytoskeleton. α-catenin binds to ß-catenin, which links it to the cadherin cytoplasmic tail, and F-actin, but also to a multitude of actin-associated proteins. These interactions suggest a highly complex cadherin-actin interface. Moreover, mammalian αE-catenin has been implicated in a cadherin-independent cytoplasmic function in Arp2/3-dependent actin regulation, and in cell signaling. The function and regulation of individual molecular interactions of α-catenin, in particular during development, are not well understood. We have generated mutations in Drosophila α-Catenin (α-Cat) to investigate α-Catenin function in this model, and to establish a setup for testing α-Catenin-related constructs in α-Cat-null mutant cells in vivo. Our analysis of α-Cat mutants in embryogenesis, imaginal discs and oogenesis reveals defects consistent with a loss of cadherin function. Compromising components of the Arp2/3 complex or its regulator SCAR ameliorate the α-Cat loss-of-function phenotype in embryos but not in ovaries, suggesting negative regulatory interactions between α-Catenin and the Arp2/3 complex in some tissues. We also show that the α-Cat mutant phenotype can be rescued by the expression of a DE-cadherin::α-Catenin fusion protein, which argues against an essential cytosolic, cadherin-independent role of Drosophila α-Catenin.
Assuntos
Junções Aderentes/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Deleção de Genes , alfa Catenina/genética , alfa Catenina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Junções Aderentes/genética , Animais , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Caderinas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Cabeça/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Larva/crescimento & desenvolvimento , Masculino , Mutagênese , Oogênese/genética , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Fenótipo , Espectrina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zigoto/metabolismo , alfa Catenina/deficiênciaRESUMO
The scaffolding protein anillin is required for completion of cytokinesis. Anillin binds filamentous (F) actin, nonmuscle myosin II, and septins and in cell culture models has been shown to restrict actomyosin contractility to the cleavage furrow. Whether anillin also serves this function during the incomplete cytokinesis that occurs in developing germ cells has remained unclear. Here, we show that anillin is required for cytokinesis in dividing Drosophila melanogaster spermatocytes and that anillin, septins, and myosin II stably associate with the cleavage furrow in wild-type cells. Anillin is necessary for recruitment of septins to the cleavage furrow and for maintenance of F-actin and myosin II at the equator in late stages of cytokinesis. Remarkably, expression of DE-cadherin suppresses the cytokinesis defect of anillin-depleted spermatocytes. DE-cadherin recruits beta-catenin (armadillo) and alpha-catenin to the cleavage furrow and stabilizes F-actin at the equator. Similarly, E-cadherin expression suppresses the cytokinesis defect caused by anillin knockdown in mouse L-fibroblast cells. Our results show that the anillin-septin and cadherin-catenin complexes can serve as alternative cassettes to promote tight physical coupling of F-actin and myosin II to the cleavage furrow and successful completion of cytokinesis.
Assuntos
Actomiosina/metabolismo , Proteínas Contráteis/metabolismo , Citocinese/fisiologia , Drosophila melanogaster/fisiologia , Espermatócitos/fisiologia , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Caderinas/metabolismo , Cateninas/metabolismo , Proteínas Contráteis/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Recuperação de Fluorescência Após Fotodegradação , GTP Fosfo-Hidrolases/metabolismo , Células L , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Miosina Tipo II/metabolismo , Ligação Proteica , Interferência de RNA , Septinas , Espermatócitos/citologia , Espermatócitos/metabolismo , TransfecçãoRESUMO
BACKGROUND: Kinesin II-mediated anterograde intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia in various cell types. Kinesin associated protein (KAP) is identified as the non-motor accessory subunit of Kinesin II, but its role in the corresponding motor function is not understood. RESULTS: We show that mutations in the Drosophila KAP (DmKap) gene could eliminate the sensory cilia as well as the sound-evoked potentials of Johnston's organ (JO) neurons. Ultrastructure analysis of these mutants revealed that the ciliary axonemes are absent. Mutations in Klp64D, which codes for a Kinesin II motor subunit in Drosophila, show similar ciliary defects. All these defects are rescued by exclusive expression of DmKAP and KLP64D/KIF3A in the JO neurons of respective mutants. Furthermore, reduced copy number of the DmKap gene was found to enhance the defects of hypomorphic Klp64D alleles. Unexpectedly, however, both the DmKap and the Klp64D mutant adults produce vigorously motile sperm with normal axonemes. CONCLUSIONS: KAP plays an essential role in Kinesin II function, which is required for the axoneme growth and maintenance of the cilia in Drosophila type I sensory neurons. However, the flagellar assembly in Drosophila spermatids does not require Kinesin II and is independent of IFT.
Assuntos
Proteínas de Transporte/metabolismo , Cílios/metabolismo , Proteínas de Drosophila/metabolismo , Flagelos/metabolismo , Cinesinas/metabolismo , Neurônios Aferentes/metabolismo , Estimulação Acústica , Animais , Transporte Biológico , Proteínas de Transporte/genética , Cílios/ultraestrutura , Drosophila , Proteínas de Drosophila/genética , Potenciais Evocados Auditivos , Flagelos/ultraestrutura , Masculino , Microscopia Confocal , Microscopia Eletrônica , Mutagênese , Mutação/genética , Neurônios Aferentes/ultraestrutura , Espermatozoides/citologia , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Transgenes/genéticaRESUMO
We have identified the Drosophila homologue of the non-motor accessory subunit of kinesin-II motor complex. It is homologous to the SpKAP115 of the sea urchin, KAP3A and KAP3B of the mouse, and SMAP protein in humans. In situ hybridization using a DmKAP specific cRNA probe has revealed a dynamic pattern of expression in the developing nervous system. The staining first appears in a subset of cells in the embryonic central nervous system at stage 13 and continues till the first instar larva stage. At the third instar larva stage the staining gets restricted to a few cells in the optic lobe and in the ventral ganglion region. It has also stained a subset of sensory neurons from late stage 13 and till the first instar larva stage. The DmKAP expression pattern in the nervous system corresponds well with that of Klp64D and Klp68D as reported earlier. In addition, we have found that the DmKAP gene is constitutively expressed in the germline cells and in follicle cells during oogenesis. These cells are also stained using an antibody to KLP68D protein, but mRNA in situ hybridization using KLP64D specific probe has not stained these cells. Together these results proved a basis for further analysis of tissue specific function of DmKAP in future.