RESUMEN
The mechanisms that regulate actin filament polymerization resulting in the morphogenesis of the brush border microvilli in epithelial cells remain unknown. Eps8, the prototype of a family of proteins capable of capping and bundling actin filaments, has been shown to bundle the microvillar actin filaments. We report that Eps8L1a, a member of the Eps8 family and a novel ezrin-interacting partner, controls microvillus length through its capping activity. Depletion of Eps8L1a leads to the formation of long microvilli, whereas its overexpression has the opposite effect. We demonstrate that ezrin differentially modulates the actin-capping and -bundling activities of Eps8 and Eps8L1a during microvillus assembly. Coexpression of ezrin with Eps8 promotes the formation of membrane ruffles and tufts of microvilli, whereas expression of ezrin and Eps8L1a induces the clustering of actin-containing structures at the cell surface. These distinct morphological changes are neither observed when a mutant of ezrin defective in its binding to Eps8/Eps8L1a is coexpressed with Eps8 or Eps8L1a nor observed when ezrin is expressed with mutants of Eps8 or Eps8L1a defective in the actin-bundling or -capping activities, respectively. Our data show a synergistic effect of ezrin and Eps8 proteins in the assembly and organization of actin microvillar filaments.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Túbulos Renales Proximales/citología , Microvellosidades/metabolismo , Actinas/metabolismo , Animales , Células Epiteliales/metabolismo , Células LLC-PK1 , Dominios y Motivos de Interacción de Proteínas , Sus scrofa , PorcinosRESUMEN
The highly related ERM (Ezrin, Radixin, Moesin) proteins provide a regulated linkage between the membrane and the underlying actin cytoskeleton. They also provide a platform for the transmission of signals in responses to extracellular cues. Studies in different model organisms and in cultured cells have highlighted the importance of ERM proteins in the generation and maintenance of specific domains of the plasma membrane. A central question is how do ERM proteins coordinate actin filament organization and membrane protein transport/stability with signal transduction pathways to build up complex structures? Through their interaction with numerous partners including membrane proteins, actin cytoskeleton and signaling molecules, ERM proteins have the ability to organize multiprotein complexes in specific cellular compartments. Likewise, ERM proteins participate in diverse functions including cell morphogenesis, endocytosis/exocytosis, adhesion and migration. This review focuses on aspects still poorly understood related to the function of ERM proteins in epithelial cell adhesion and migration.
Asunto(s)
Movimiento Celular , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Actinas/metabolismo , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Caenorhabditis elegans , Adhesión Celular , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/genética , Drosophila melanogaster , Células Epiteliales/citología , Células Epiteliales/metabolismo , Expresión Génica , Humanos , Uniones Intercelulares/fisiología , Proteínas de la Membrana/genética , Ratones , Proteínas de Microfilamentos/genética , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transducción de SeñalRESUMEN
Using newly generated monoclonal antibodies, we have compared the distribution of beta- and gamma-cytoplasmic actin in fibroblastic and epithelial cells, in which they play crucial roles during various key cellular processes. Whereas beta-actin is preferentially localized in stress fibers, circular bundles and at cell-cell contacts, suggesting a role in cell attachment and contraction, gamma-actin displays a more versatile organization, according to cell activities. In moving cells, gamma-actin is mainly organized as a meshwork in cortical and lamellipodial structures, suggesting a role in cell motility; in stationary cells, gamma-actin is also recruited into stress fibers. beta-actin-depleted cells become highly spread, display broad protrusions and reduce their stress-fiber content; by contrast, gamma-actin-depleted cells acquire a contractile phenotype with thick actin bundles and shrinked lamellar and lamellipodial structures. Moreover, beta- and gamma-actin depleted fibroblasts exhibit distinct changes in motility compared with their controls, suggesting a specific role for each isoform in cell locomotion. Our results reveal new aspects of beta- and gamma-actin organization that support their functional diversity.
Asunto(s)
Actinas/metabolismo , Citoplasma/metabolismo , Actinas/química , Actomiosina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Compartimento Celular , Movimiento Celular , Forma de la Célula , Células Epiteliales/citología , Células Epiteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Silenciador del Gen , Humanos , Mitosis , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/antagonistas & inhibidores , Transporte de Proteínas , Seudópodos/metabolismo , Ratas , Fracciones Subcelulares/metabolismo , Sus scrofaRESUMEN
Defects in myosin VIIa lead to developmental anomalies of the auditory and visual sensory cells. We sought proteins interacting with the myosin VIIa tail by using the yeast two-hybrid system. Here, we report on shroom2, a submembranous PDZ domain-containing protein that is associated with the tight junctions in multiple embryonic and adult epithelia. Shroom2 directly interacts with the C-terminal MyTH4-FERM domain of myosin VIIa and with F-actin. In addition, a shroom2 fragment containing the region of interaction with F-actin was able to protect actin filaments from cytochalasin-D-induced disruption in MDCK cells. Transfection experiments in MDCK and LE (L fibroblasts that express E-cadherin) cells led us to conclude that shroom2 is targeted to the cell-cell junctions in the presence of tight junctions only. In Ca(2+)-switch experiments on MDCK cells, ZO-1 (also known as TJP1) preceded GFP-tagged shroom2 at the differentiating tight junctions. ZO-1 directly interacts with the serine- and proline-rich region of shroom2 in vitro. Moreover, the two proteins colocalize in vivo at mature tight junctions, and could be coimmunoprecipitated from brain and cochlear extracts. We suggest that shroom2 and ZO-1 form a tight-junction-associated scaffolding complex, possibly linked to myosin VIIa, that bridges the junctional membrane to the underlying cytoskeleton, thereby contributing to the stabilization of these junctions.
Asunto(s)
Actinas/metabolismo , Dineínas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Miosinas/metabolismo , Fosfoproteínas/metabolismo , Uniones Estrechas/metabolismo , Animales , Señalización del Calcio , Línea Celular , Membrana Celular/metabolismo , Perros , Estructuras Embrionarias/citología , Estructuras Embrionarias/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Ratones , Miosina VIIa , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Retina/citología , Retina/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
A subtracted library prepared from vestibular sensory areas [Nat. Genet. 26 (2000) 51] was used to identify a 960bp murine transcript preferentially expressed in the inner ear and testis. The cDNA predicts a basic 124aa protein that does not share any significant sequence homology with known proteins. Immunofluorescence and immunoelectron microscopy revealed that the protein is located mainly in the kinocilium of sensory cells in the inner ear. The protein was thus named kinocilin. In the mouse, kinocilin is first detected in the kinocilia of vestibular and auditory hair cells at embryonic days 14.5, and 18.5, respectively. In the mature vestibular hair cells, kinocilin is still present in the kinocilium. As the auditory hair cells begin to lose the kinocilium during postnatal development, kinocilin becomes distributed in an annular pattern at the apex of these cells, where it co-localizes with the tubulin belt [Hear. Res. 42 (1989) 1]. In mature auditory hair cells, kinocilin is also present at the level of the cuticular plate, at the base of each stereocilium. In addition, as the kinocilium regresses from developing auditory hair cells, kinocilin begins to be expressed by the pillar cells and Deiters cells, that both contain prominent transcellular and apical bundles of microtubules. By contrast, kinocilin was not detected in the supporting cells in the vestibular end organs. The protein is also present in the manchette of the spermatids, a transient structure enriched in interconnected microtubules. We propose that kinocilin has a role in stabilizing dense microtubular networks or in vesicular trafficking.
Asunto(s)
Células Ciliadas Auditivas/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Envejecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Diferenciación Celular/fisiología , Senescencia Celular/fisiología , Cilios/metabolismo , Cóclea/citología , Cóclea/metabolismo , ADN Complementario/metabolismo , Oído Interno/metabolismo , Embrión de Mamíferos/metabolismo , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/embriología , Células Ciliadas Auditivas/fisiología , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Testículo/metabolismo , Distribución TisularRESUMEN
A 3,673-bp murine cDNA predicted to encode a glycosylphosphatidylinositol-anchored protein of 1,088 amino acids was isolated during a study aimed at identifying transcripts specifically expressed in the inner ear. This inner ear-specific protein, otoancorin, shares weak homology with megakaryocyte potentiating factor/mesothelin precursor. Otoancorin is located at the interface between the apical surface of the inner ear sensory epithelia and their overlying acellular gels. In the cochlea, otoancorin is detected at two attachment zones of the tectorial membrane, a permanent one along the top of the spiral limbus and a transient one on the surface of the developing greater epithelial ridge. In the vestibule, otoancorin is present on the apical surface of nonsensory cells, where they contact the otoconial membranes and cupulae. The identification of the mutation (IVS12+2T>C) in the corresponding gene OTOA in one consanguineous Palestinian family affected by nonsyndromic recessive deafness DFNB22 assigns an essential function to otoancorin. We propose that otoancorin ensures the attachment of the inner ear acellular gels to the apical surface of the underlying nonsensory cells.
Asunto(s)
Oído Interno/metabolismo , Epitelio/metabolismo , Trastornos de la Audición/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/fisiología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Análisis Mutacional de ADN , ADN Complementario/metabolismo , Exones , Femenino , Proteínas Ligadas a GPI , Ligamiento Genético , Genotipo , Humanos , Masculino , Mesotelina , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Linaje , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Deafness is a common sensory defect in human. Our understanding of the molecular bases of this pathology comes from the study of a few genes that have been identified in human and/or in mice. Indeed, deaf mouse mutants are good models for studying and identifying genes involved in human hereditary hearing loss. Among these mouse mutants, twister was initially reported to have abnormal behavior and thereafter to be deaf. The recessive twister (twt) mutation has been mapped on mouse Chromosome (Chr) 7, homologous to the long arm of human Chr 15 (15q11). Otog, the gene encoding otogelin, a glycoprotein specific to all the acellular membranes of the inner ear, is also localized to mouse Chr 7, but in a region more proximal to the twister mutation, homologous to the short arm of human Chr 11 (11p15) carrying the two deafness loci, DFNB18 and USH1C. Mutant mice resulting from the knock-out of Otog, the Otog(tm1Prs) mice, present deafness and severe imbalance. Although twt had been mapped distally to Otog, these data prompted us to test whether twt could be due to a mutation in the Otog locus. Here, we demonstrate by genetic analysis that twt is actually allelic to Otog(tm1Prs). We further extend the phenotypical analysis of twister mice, documenting the association of a severe vestibular phenotype and moderate to severe form of deafness. Molecular analysis of the Otog gene revealed the absence of detectable expression of Otog in the twister mutant. The molecular and phenotypical description of the twt mouse mutation, Otog(twt), reported herein, highlights the importance of the acellular membranes in the inner ear mechanotransduction process.