RESUMEN
Actin filament assembly is a tightly regulated process that functions in many aspects of cell physiology. Members of the Ena/VASP (Drosophila Enabled/vasodilator-stimulated phosphoprotein) family are key players in regulating actin filament assembly, in many cases through their association with binding partners that display a particular proline-rich motif, FPPPP. Ena/VASP proteins interact with these partners via the highly conserved Ena/VASP homology 1 (EVH1) domain. The diverse array of binding partners for EVH1 domains, including cytoskeletal proteins such as zyxin, transmembrane guidance receptors such as Roundabout, and the T-cell signaling protein Fyb/SLAP, shows that these interactions are likely to be important in a number of cellular processes that require regulated actin filament assembly.
Asunto(s)
Movimiento Celular , Polaridad Celular , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/fisiología , Secuencias de Aminoácidos , Animales , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/fisiología , Epitelio/crecimiento & desarrollo , Proteínas de Microfilamentos/metabolismo , Modelos Biológicos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiología , Prolina/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Adherens junctions, which are cadherin-mediated junctions between cells, and focal adhesions, which are integrin-mediated junctions between cells and the extracellular matrix, are protein complexes that link the actin cytoskeleton to the plasma membrane and, in turn, to the extracellular environment. Zyxin is a LIM domain protein that is found in vertebrate adherens junctions and focal adhesions. Zyxin's molecular architecture and binding partner repertoire suggest roles in actin assembly and dynamics, cell motility, and nuclear-cytoplasmic communication. In order to study the function of zyxin in development, we have identified a zyxin orthologue in Drosophila melanogaster that we have termed Zyx102. Like its vertebrate counterparts, Zyx102 displays three carboxy-terminal LIM domains, a potential nuclear export signal, and three proline-rich motifs, one of which matches the consensus for mediating an interaction with Ena/VASP (Drosophila Enabled/Vasodilator-stimulated phosphoprotein) proteins. Here we show that Zyx102 and Enabled (Ena), the Drosophila member of the Ena/VASP family, can interact specifically in vitro and that this interaction does not occur when a particular mutant form of Ena, encoded by the lethal ena210 allele, is used. Lastly, we show that the zyx102 gene and Drosophila Ena are co-expressed during oogenesis and early embryogenesis, indicating that the two proteins may be able to interact during the development of the Drosophila egg chamber and early embryo.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/genética , Proteínas de Homeodominio/genética , Filogenia , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Mutación , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , ZixinaRESUMEN
The zyxin family of proteins function as cytoskeletal regulators in adhesion, actin assembly, and cell motility. Though fibroblasts derived from zyxin-null mice show striking defects in motility and response to mechanical stimuli, the mice are viable and fertile. In Drosophila melanogaster, the family is represented by a single homologue, Zyx102. To study the role of zyxin during development, we generated a zyx102 RNA-interference transgenic line that allows for the conditional knockdown of Zyx102. When UAST-zyx102-dsRNAi expression is driven broadly by Actin5C-GAL4, loss of Zyx102 results in lethality during the pharate adult stage, a narrow developmental window during which the fly must molt, resorb molting fluid, fill adult trachea with air, and execute a behavioral program to eclose. Zyx102 knockdown animals attempt to emerge, but their adult trachea do not fill with air. If dissected from the pupal case, knockdown individuals appear morphologically normal, but remain inviable.