RESUMEN
BACKGROUND: Mutations in the fibrillin -1 gene (FBN1) cause Marfan syndrome (MFS), an autosomal dominant multi-system connective tissue disorder. The 200 different mutations reported in the 235 kb, 65 exon-containing gene include only one family with a genomic multi-exon deletion. METHODS: We used long-range RT-PCR for mutation detection and long-range genomic PCR and DNA sequencing for identification of deletion breakpoints, allele-specific transcript analyses to determine stability of the mutant RNA, and pulse-chase studies to quantitate fibrillin synthesis and extracellular matrix deposition in cultured fibroblasts. Southern blots of genomic DNA were probed with three overlapping fragments covering the FBN1 coding exons RESULTS: Two novel multi-exon FBN1 deletions were discovered. Identical nucleotide pentamers were found at or near the intronic breakpoints. In a Case with classic MFS, an in-frame deletion of exons 42 and 43 removed the C-terminal 24 amino acids of the 5th LTBP (8-cysteine) domain and the adjacent 25th calcium-binding EGF-like (6-cysteine) domain. The mutant mRNA was stable, but fibrillin synthesis and matrix deposition were significantly reduced. A Case with severe childhood-onset MFS has a de novo deletion of exons 44-46 that removed three EGF-like domains. Fibrillin protein synthesis was normal, but matrix deposition was strikingly reduced. No genomic rearrangements were detected by Southern analysis of 18 unrelated MFS samples negative for FBN1 mutation screening. CONCLUSIONS: Two novel deletion cases expand knowledge of mutational mechanisms and genotype/phenotype correlations of fibrillinopathies. Deletions or mutations affecting an LTBP domain may result in unstable mutant protein cleavage products that interfere with microfibril assembly.
RESUMEN
The NADPH oxidase of phagocytes is a membrane-bound heterodimeric flavocytochrome which catalyses the transfer of electrons from NADPH in the cytoplasm to oxygen in the phagosome. A number of cytosolic proteins are involved in its activation/deactivation: p47phox, p67phox, p40phox and the small GTP-binding protein, rac. The cytosolic phox proteins interact with the cytoskeleton in human neutrophils and, in particular, an interaction with coronin has been reported (Grogan A., Reeves, E., Keep, N. H., Wientjes, F., Totty, N., Burlingame, N. L., Hsuan, J., and Segal, A. W. (1997) J. Cell Sci. 110, 3071-3081). Here, we report on the interaction of another cytoskeletal protein, moesin, with the phox proteins. Moesin belongs to the ezrin-radixin-moesin family of F-actin-binding proteins and we show that it binds to p47phox and p40phox in a phosphoinositide-dependent manner. Furthermore, we show that its N-terminal part binds to the PX domain of p47phox and p40phox.
Asunto(s)
Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Membrana Celular/enzimología , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Inmunohistoquímica , Espectrometría de Masas , Proteínas de Microfilamentos/química , NADPH Oxidasas , Neutrófilos/metabolismo , Oxígeno/metabolismo , Fagocitos/enzimología , Fosfoproteínas/química , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de TiempoRESUMEN
The hepatitis B virus X protein (HBx) of the hepatitis B virus (HBV) has been involved in the development of hepatocellular carcinoma (HCC). However, its possible contribution to the metastatic spreading of liver tumors has not been explored so far. We report here the ability of HBx to enhance cell motility, both alone and in synergy with growth factors, and to induce a migratory phenotype in transformed cells. HBx altered the cellular morphology by inducing the formation of pseudopodial protrusions and cytoskeletal rearrangements, which was accompanied by the polarization of cell-surface adhesion molecules, including the hyaluronan (HA) receptor, CD44. Furthermore, HBx induced the redistribution to the pseudopodial tips of F-actin-binding proteins of the ezrin/radixin/moesin (ERM) family in a Rho- and Rac-dependent manner and increased the association of CD44 with moesin. The migration of HBx-bearing cells in response to HA and growth factors was impaired by a blocking anti-CD44 monoclonal antibody (mAb), suggesting that the HBx-induced cell motility is partially mediated by CD44. Interestingly, HBx-bearing cells showed increased HA-interaction efficiency as assessed under laminar flow conditions, which was the result, at least in part, of an enhanced binding affinity of CD44. HBx may therefore contribute to the acquisition of metastatic properties by modifying the migratory behavior of transformed hepatocytes and by increasing their ability to bind HA in the outer margin of the tumors or in secondary target organs.
Asunto(s)
Proteínas Portadoras/farmacología , Receptores de Hialuranos/fisiología , Proteínas no Estructurales Virales/farmacología , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Citoesqueleto/ultraestructura , Células HeLa/citología , Células HeLa/ultraestructura , Humanos , Ácido Hialurónico/metabolismo , Invasividad Neoplásica/fisiopatología , Metástasis de la Neoplasia/fisiopatología , Fenotipo , Seudópodos/metabolismo , Distribución Tisular/efectos de los fármacos , Proteínas no Estructurales Virales/fisiología , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
BACKGROUND: The cell surface undergoes continuous change during cell movement. This is characterized by transient protrusion and partial or complete retraction of microspikes, filopodia, and lamellipodia. This requires a dynamic actin cytoskeleton, moesin, components of Rho-mediated signal pathways, rearrangement of membrane constituents and the formation of focal adhesion sites. While the immunofluorescence distribution of endogenous moesin is that of a membrane-bound molecule with marked enhancement in some but not all microextensions, the C-terminal fragment of moesin co-distributes with filamentous actin consistent with its actin-binding activity. By taking advantage of this property we studied the spontaneous protrusive activity of live NIH3T3 cells, expressing a fusion of GFP and the C-terminal domain of moesin. RESULTS: C-moesin-GFP localized to stress fibers and was enriched in actively protruding cellular regions such as filopodia or lamellipodia. This localization was reversibly affected by cytochalasin D. Multiple types of cytoskeletal rearrangements were observed that occurred independent of each other in adjacent regions of the cell surface. Assembly and disassembly of actin filaments occurred repeatedly within the same space and was correlated with either membrane protrusion and retraction, or no change in shape when microextensions were adherent. CONCLUSIONS: Shape alone provided an inadequate criterion for distinguishing between retraction fibers and advancing, retracting or stable filopodia. Fluorescence imaging of C-moesin-GFP, however, paralleled the rapid and dynamic changes of the actin cytoskeleton in microextensions. Regional regulatory control is implicated because opposite changes occurred in close proximity and presumably independent of each other. This new and sensitive tool should be useful for investigating mechanisms of localized actin dynamics in the cell cortex.
Asunto(s)
Células 3T3/química , Actinas/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Microscopía por Video , Proteínas Recombinantes de Fusión/metabolismo , Procesamiento de Señales Asistido por Computador , Células 3T3/efectos de los fármacos , Células 3T3/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Citocalasina D/farmacología , Citoesqueleto/metabolismo , Proteínas Fluorescentes Verdes , Ratones , Microscopía por Video/métodos , Estructura Secundaria de Proteína , Seudópodos/químicaRESUMEN
G(13) protein, one of the heterotrimeric guanine nucleotide-binding proteins (G proteins), regulates diverse and complex cellular responses by transducing signals from the cell surface presumably involving more than one pathway. Yeast two-hybrid screening of a mouse brain cDNA library identified radixin, a member of the ERM family of three closely related proteins (ezrin, radixin, and moesin), as a protein that interacted with Galpha(13). Interaction between radixin and Galpha(13) was confirmed by in vitro binding assay and by co-immunoprecipitation technique. Activated Galpha(13) induced conformational activation of radixin, as determined by binding of radixin to polymerized F-actin and by immunofluorescence in intact cells. Finally, two dominant negative mutants of radixin inhibited Galpha(13)-induced focus formation of Rat-1 fibroblasts but did not affect Ras-induced focus formation. Our results identifying a new signaling pathway for Galpha(13) indicate that ERM proteins can be activated by and serve as effectors of heterotrimeric G proteins.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de la Membrana/metabolismo , Células 3T3 , Animales , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Ratones , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Fosfoproteínas/metabolismo , Unión Proteica , Conformación Proteica , Transducción de Señal , LevadurasRESUMEN
In response to the chemoattractants interleukin 8, C5a, N-formyl-methionyl-leucyl-phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1), intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are redistributed to a newly formed uropod in human neutrophils. The adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the cytoskeletal protein moesin in the uropod of stimulated neutrophils. Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by isolating a complex with glutathione S-transferase fusions of the cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as moesin and ezrin by Western blot analysis. ICAM-3 and moesin also coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity assay. Direct interaction of the cytoplasmic domains of ICAM-3 and PSGL-1 with the amino-terminal domain of recombinant moesin was demonstrated by protein-protein binding assays. These results suggest that the redistribution of PSGL-1 and its association with intracellular molecules, including the ezrin-radixin-moesin actin-binding proteins, regulate functions mediated by PSGL-1 in leukocytes stimulated by chemoattractants.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación , Moléculas de Adhesión Celular/sangre , Glicoproteínas de Membrana/sangre , Proteínas de Microfilamentos/sangre , Neutrófilos/química , Fosfoproteínas/sangre , Anticuerpos Monoclonales , Western Blotting , Membrana Celular/química , Cromatografía de Afinidad , Complemento C5a/farmacología , Citoplasma/química , Proteínas del Citoesqueleto , Humanos , Inmunoensayo , Interleucina-8/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/ultraestructura , Proteínas Recombinantes/sangreRESUMEN
The association of actin filaments with the plasma membrane maintains cell shape and adhesion. Here, we show that the plasma membrane ion exchanger NHE1 acts as an anchor for actin filaments to control the integrity of the cortical cytoskeleton. This occurs through a previously unrecognized structural link between NHE1 and the actin binding proteins ezrin, radixin, and moesin (ERM). NHE1 and ERM proteins associate directly and colocalize in lamellipodia. Fibroblasts expressing NHE1 with mutations that disrupt ERM binding, but not ion translocation, have impaired organization of focal adhesions and actin stress fibers, and an irregular cell shape. We propose a structural role for NHE1 in regulating the cortical cytoskeleton that is independent of its function as an ion exchanger.
Asunto(s)
Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Hidrógeno/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Sanguíneas/metabolismo , División Celular , Línea Celular , Tamaño de la Célula , Proteínas del Citoesqueleto/metabolismo , Adhesiones Focales/metabolismo , Humanos , Transporte Iónico , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Fosfoproteínas/metabolismo , Pruebas de Precipitina , Unión Proteica , Protones , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genética , Fibras de Estrés/metabolismoRESUMEN
Fibrillin-1 (FBN1) contains 47 epidermal growth factor (EGF)-like domains characterized by six conserved cysteine residues. Cysteine substitutions that disrupt one of the three disulfide bonds are frequent causes of Marfan syndrome (MFS). We identified 19 new substitutions involving cysteine residues in each of the six positions of EGF-like domains. Allele-specific mRNA assays revealed equal abundance of mutant and normal FBN1 transcripts in all 10 individuals studied. Quantitative pulse-chase analysis of fibrillin protein was performed on 25 mutant fibroblast strains with substitutions of 22 different cysteine residues in 18 different EGF-like domains spanning the entire gene. Normal synthesis and stability of mutant fibrillin molecules was seen in 20/25 individuals, 11 of whom showed delayed intracellular processing and/or secretion. In the remaining five cases, the mutant protein was apparently unstable. In four of these five cases, the second or third disulfide bond of EGF-like domains immediately preceding an 8-cysteine or hybrid domain was affected. All but two mutations caused severe reduction of matrix deposition, which was attributed to a dominant-negative effect of mutant molecules. For genotype/phenotype comparisons, clinical data on 25 probands and 19 mutation-positive family members were analyzed. Ocular manifestations were among the most consistent features (ectopia lentis in 86%, myopia in 80%). Nine mutations encoded by exons 26-32 resulted in early-onset classic MFS and, in one case, neonatal-lethal MFS. Mutations outside this region were associated with variable clinical phenotypes, including individuals with fibrillinopathies not meeting diagnostic criteria for MFS.
Asunto(s)
Sustitución de Aminoácidos/genética , Cisteína/genética , Factor de Crecimiento Epidérmico/química , Síndrome de Marfan/genética , Proteínas de Microfilamentos/genética , Mutación/genética , Adolescente , Adulto , Edad de Inicio , Anciano , Alelos , Células Cultivadas , Niño , Preescolar , Cisteína/metabolismo , Análisis Mutacional de ADN , Disulfuros/metabolismo , Exones/genética , Fibrilina-1 , Fibrilinas , Fibroblastos , Genes Letales/genética , Genotipo , Humanos , Recién Nacido , Síndrome de Marfan/epidemiología , Síndrome de Marfan/metabolismo , Síndrome de Marfan/fisiopatología , Proteínas de Microfilamentos/química , Persona de Mediana Edad , Datos de Secuencia Molecular , Fenotipo , ARN Mensajero/análisis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Activation of human platelets with thrombin transiently increases phosphorylation at (558)threonine of moesin as determined with phosphorylation state-specific antibodies. This specific modification is completely inhibited by the kinase inhibitor staurosporine and maximally promoted by the phosphatase inhibitor calyculin A, making it possible to purify the two forms of moesin to homogeneity. Blot overlay assays with F-actin probes labeled with either [32P]ATP or 125I show that only phosphorylated moesin interacts with F-actin in total platelet lysates, in moesin antibody immunoprecipitates, and when purified. In the absence of detergents, both forms of the isolated protein are aggregated. Phosphorylated, purified moesin co-sediments with alpha- or beta/gamma-actin filaments in cationic, but not in anionic, nonionic, or amphoteric detergents. The interaction affinity is high (Kd, approximately 1.5 nM), and the maximal moesin:actin stoichiometry is 1:1. This interaction is also observed in platelets extracted with cationic but not with nonionic detergents. In 0.1% Triton X-100, F-actin interacts with phosphorylated moesin only in the presence of polyphosphatidylinositides. Thus, both polyphosphatidylinositides and phosphorylation can activate moesin's high-affinity F-actin binding site in vitro. Dual regulation by both mechanisms may be important for proper cellular control of moesin-mediated linkages between the actin cytoskeleton and the plasma membrane.
Asunto(s)
Actinas/metabolismo , Plaquetas/metabolismo , Proteínas de Microfilamentos/metabolismo , Secuencia de Aminoácidos , Bioquímica/métodos , Citoesqueleto/química , Citoesqueleto/metabolismo , Detergentes/química , Humanos , Proteínas de Microfilamentos/aislamiento & purificación , Datos de Secuencia Molecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositoles/metabolismo , Fosforilación , Compuestos de Amonio Cuaternario , Treonina/metabolismoRESUMEN
Point and deletion mutants of moesin were examined for F-actin binding by blot overlay and co-sedimentation, and for intra- and intermolecular interactions with N- and C-terminal domains with yeast two-hybrid and in vitro binding assays. Wild-type moesin molecules interact poorly with F-actin and each other, and bind neither C- nor N-terminal fragments. Interaction with F-actin is strongly enhanced by replacement of Thr558 with aspartate (T558D), by deletion of 11 N-terminal residues (DelN11), by deletion of the entire N-terminal membrane-binding domain of both wild type and T558D mutant molecules, and by exposure to phosphatidylinositol 4, 5-diphosphate. Activation of F-actin binding is accompanied by changes in inter- and intramolecular domain interactions. The T558D mutation renders moesin capable of binding wild type but not mutated (T558D) C-terminal or wild type N-terminal fragments. The interaction between the latter two is prevented. DelN11 truncation enables binding of wild type N and C domain fragments. These changes suggest that the T558D mutation, mimicking phosphorylation of Thr558, promotes F-actin binding by disruption of interdomain interactions between N and C domains and exposure of the high affinity F-actin binding site in the C-terminal domain. Oscillation between activated and resting state could thus provide the structural basis for transient interactions between moesin and the actin cytoskeleton in protruding and retracting microextensions.
Asunto(s)
Actinas/metabolismo , Ácido Aspártico/metabolismo , Proteínas de Microfilamentos/metabolismo , Treonina/metabolismo , Sustitución de Aminoácidos , Ácido Aspártico/química , Secuencia de Bases , Cartilla de ADN , Humanos , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Treonina/químicaRESUMEN
Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1-320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6-8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.
Asunto(s)
Membrana Celular/metabolismo , Proteínas del Citoesqueleto , Fibroblastos/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Células 3T3 , Citoesqueleto de Actina/metabolismo , Animales , Proteínas Sanguíneas/metabolismo , Adhesión Celular/fisiología , Membrana Celular/ultraestructura , Fibroblastos/ultraestructura , Proteínas Fluorescentes Verdes , Procesamiento de Imagen Asistido por Computador , Proteínas Luminiscentes/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Microfilamentos/genética , Microscopía Fluorescente , Microscopía de Interferencia , Fosfoproteínas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Fracciones Subcelulares/metabolismo , TransfecciónRESUMEN
BACKGROUND: Ezrin belongs to a family of plasma membrane-cytoskeleton linking, actin binding proteins (Ezrin-radixin-Moesin family) involved in signal transduction, growth control, cell-cell adhesion, and microvilli formation. METHODS: The expression of ezrin was examined in glomerular cells in culture, during kidney development, in the mature kidney, and in five different experimental kidney disease models in the rat. RESULTS: Ezrin was specifically expressed in glomerular epithelial cells in developing glomeruli in mature glomeruli and in glomerular epithelial cells in culture. Distinct from its other family members, moesin and radixin, which are predominantly expressed in glomerular endothelial and mesangial areas, ezrin protein (by immunohistochemistry) was specifically and exclusively modulated during podocyte injury and regeneration. Ezrin immunohistochemistry was able to visualize cell body attenuation, pseudocysts, and in particular vacuolation of injured podocytes, a feature that usually has to be identified at the ultrastructural level, and was strikingly increased in binucleated podocytes or podocytes that were partially or completely detached from the underlying GBM (frequently also binucleated). Infiltrating macrophages also express ezrin, but can easily be differentiated from podocytes by their round shape and higher level of expression. CONCLUSIONS: Ezrin likely has a role in the cytoskeletal organization, such as reassembling of acting filaments accompanying podocyte injury and regeneration. Since suitable light microscopic markers for the identification of glomerular epithelial cells are rare, ezrin may also be a useful marker for podocytes in normal and injured glomeruli.
Asunto(s)
Enfermedades Renales/metabolismo , Glomérulos Renales/crecimiento & desarrollo , Glomérulos Renales/metabolismo , Fosfoproteínas/metabolismo , Envejecimiento/metabolismo , Animales , Biomarcadores , Células Cultivadas , Proteínas del Citoesqueleto , Células Epiteliales/metabolismo , Enfermedades Renales/patología , Enfermedades Renales/fisiopatología , Glomérulos Renales/patología , Masculino , Mitosis/fisiología , Ratas , Ratas Sprague-Dawley , Valores de ReferenciaRESUMEN
BACKGROUND: Estrogen receptor (ER)-positive breast carcinomas possess a less aggressive phenotype than ER-negative breast carcinomas. We hypothesize that a set of genes exists that is expressed only in ER-negative breast carcinomas, which account for the more malignant phenotypic characteristics of these tumors. METHODS: We have used a new technique of polymerase chain reaction select suppression subtractive hybridization to identify genes that are expressed only in ER-negative carcinomas. RESULTS: Seventy-one cDNA clones generated by suppression subtractive hybridization were screened by Northern blot analysis with RNA from ER-positive MCF7 and ER-negative MDA-MB-231 breast carcinoma cell lines. Fifteen clones were differentially expressed in MDA-MB-231 cells. Five of these 15 clones were consistently found to be associated with the ER-negative phenotype in a panel of eight breast carcinoma cell lines. Sequence analysis demonstrated that three of these clones were derived from vimentin and two clones from moesin. Western blot analysis with antihuman moesin antibody confirmed that moesin protein was overexpressed in ER-negative breast carcinoma cell lines but absent from ER-positive breast carcinomas. Moesin mRNA was examined in a panel of 29 primary breast carcinomas with semi-quantitative reverse transcriptase-polymerase chain reaction. Moesin expression was found to be decreased significantly in ER-positive compared with ER-negative tumors (P < .01). CONCLUSIONS: Vimentin and moesin are differentially expressed in association with the ER-negative breast cancer phenotype. Moesin is a membrane/actin filament protein involved in dynamic restructuring of the cell surface and filopodia, a cell structure needed for cell adhesion and motility. Moesin may play a role in the invasiveness and pattern of metastasis characteristic of ER-negative breast cancers.
Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Microfilamentos , Proteínas/genética , Receptores de Estrógenos/genética , Northern Blotting , Western Blotting , Neoplasias de la Mama/patología , Femenino , Humanos , Fenotipo , Proteínas/análisis , ARN Mensajero/análisis , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/fisiología , Vimentina/genéticaRESUMEN
The neurofibromatosis type 2 (NF2) tumor suppressor gene encodes merlin, a protein with homology to the cell membrane/F-actin linking proteins, moesin, ezrin and radixin. Unlike these closely related proteins, merlin lacks a C-terminal F-actin binding site detectable by actin blot overlays, and the GFP-tagged merlin C-terminal domain co-distributes with neither stress fibers nor cortical actin in NIH3T3 cells. Merlin also differs from the other three proteins in its inter- and intramolecular domain interactions, as shown by in vitro binding and yeast two-hybrid assays. As is true for ezrin, moesin and radixin, the N- and C-terminal domains of merlin type 1 bind to each other. However, full-length merlin and its N- and C-terminal domains, as well as the C-terminal domain of ezrin, interact with other full-length merlin type 1 molecules, and its C-terminal domain interacts with itself. Merlin 1 function in cells may thus depend on intra- and intermolecular interactions and their modulation, which include interactions with other members of this protein family.
Asunto(s)
Actinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos , Proteínas/metabolismo , Células 3T3 , Actinas/química , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , Escherichia coli , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Neurofibromina 2 , Proteínas/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Chemokines as well as the signaling through the adhesion molecules intercellular adhesion molecule (ICAM)-3 and CD43 are able to induce in T lymphocytes their switching from a spherical to a polarized motile morphology, with the formation of a uropod at the rear of the cell. We investigated here the role of CD43 in the regulation of T-cell polarity, CD43-cytoskeletal interactions, and lymphocyte aggregation. Pro-activatory anti-CD43 monoclonal antibody (MoAb) induced polarization of T lymphocytes with redistribution of CD43 to the uropod and the CCR2 chemokine receptor to the leading edge of the cell. Immunofluorescence analysis showed that all three ezrin-radixin-moesin (ERM) actin-binding proteins localized in the uropod of both human T lymphoblasts stimulated with anti-CD43 MoAb and tumor-infiltrating T lymphocytes. Radixin localized at the uropod neck, whereas ezrin and moesin colocalized with CD43 in the uropod. Biochemical analyses showed that ezrin and moesin coimmunoprecipitated with CD43 in T lymphoblasts. Furthermore, in these cells, the CD43-associated moesin increased after stimulation through CD43. The interaction of moesin and ezrin with CD43 was specifically mediated by the cytoplasmic domain of CD43, as shown by precipitation of both ERM proteins with a GST-fusion protein containing the CD43 cytoplasmic tail. Videomicroscopy analysis of homotypic cell aggregation induced through CD43 showed that cellular uropods mediate cell-cell contacts and lymphocyte recruitment. Immunofluorescence microscopy performed in parallel showed that uropods enriched in CD43 and moesin localized at the cell-cell contact areas of cell aggregates. The polarization and homotypic cell aggregation induced through CD43 was prevented by butanedione monoxime, indicating the involvement of myosin cytoskeleton in these phenomena. Altogether, these data indicate that CD43 plays an important regulatory role in remodeling T-cell morphology, likely through its interaction with actin-binding proteins ezrin and moesin. In addition, the redistribution of CD43 to the uropod region of migrating lymphocytes and during the formation of cell aggregates together with the enhancing effect of anti-CD43 antibodies on lymphocyte cell recruitment suggest that CD43 plays a key role in the regulation of cell-cell interactions during lymphocyte traffic.
Asunto(s)
Antígenos CD , Comunicación Celular/fisiología , Proteínas de Microfilamentos , Fosfoproteínas/fisiología , Proteínas/fisiología , Sialoglicoproteínas/fisiología , Linfocitos T/citología , Linfocitos T/fisiología , Transporte Biológico , Células Cultivadas , Proteínas del Citoesqueleto , Humanos , Uniones Intercelulares/fisiología , Leucosialina , Unión ProteicaRESUMEN
During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane-cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the uropods, whereas moesin decorated the most distal part of the uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, beta-actin and alpha-tubulin, clustered at the cell leading edge and uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti-ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin-ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin-ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación , Moléculas de Adhesión Celular/metabolismo , Polaridad Celular/fisiología , Proteínas del Citoesqueleto , Proteínas de Microfilamentos , Proteínas/metabolismo , Linfocitos T/química , Linfocitos T/citología , Proteínas Sanguíneas/análisis , Western Blotting , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/química , Movimiento Celular/fisiología , Quimiocinas/farmacología , Citoplasma/química , Citoplasma/metabolismo , Humanos , Receptores de Hialuranos/análisis , Molécula 1 de Adhesión Intercelular/análisis , Proteínas de la Membrana/análisis , Fosfoproteínas/análisis , Pruebas de Precipitina , Estructura Terciaria de Proteína , Proteínas/análisis , Linfocitos T/efectos de los fármacosRESUMEN
The small GTPases Rho and Rac regulate actin filament assembly and the formation of integrin adhesion complexes to produce stress fibers and lamellipodia, respectively, in mammalian cells. Although numerous candidate effectors that might mediate these responses have been identified using the yeast two-hybrid and affinity purification techniques, their cellular roles remain unclear. We now describe a biological assay that allows components of the Rho and Rac signaling pathways to be identified. Permeabilization of serum-starved Swiss 3T3 cells with digitonin in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) induces both actin filament and focal adhesion complex assembly through activation of endogenous Rho and Rac. These responses are lost when GTPgammaS is added 6 min after permeabilization, but can be reconstituted using concentrated cytosolic extracts. We have achieved a 10,000-fold purification of the activity present in pig brain cytosol and protein sequence analysis shows it to contain moesin. Using recombinant proteins, we show that moesin and its close relatives ezrin and radixin can reconstitute stress fiber assembly, cortical actin polymerization and focal complex formation in response to activation of Rho and Rac.
Asunto(s)
Actinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Permeabilidad de la Membrana Celular , Proteínas del Citoesqueleto , Proteínas de Unión al GTP/fisiología , Proteínas de Microfilamentos , Células 3T3 , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiología , Actinas/fisiología , Animales , Sitios de Unión , Proteínas Sanguíneas/fisiología , Encéfalo/metabolismo , Moléculas de Adhesión Celular/fisiología , Citoesqueleto/efectos de los fármacos , Citosol/metabolismo , Interacciones Farmacológicas , GTP Fosfohidrolasas/metabolismo , Integrinas/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Fosfoproteínas/fisiología , Proteínas/fisiología , Porcinos , Proteínas de Unión al GTP rac , Proteínas de Unión al GTP rhoRESUMEN
More than 70 unique fibrillin-1 mutations have been identified in individuals with a variety of phenotypic changes. These range from severe neonatal lethal forms of Marfan syndrome to adult onset manifestations, mitral valve prolapse syndromes to isolated features such as ectopia lentis, Marfanoid body habitus and ascending aortic aneurysm and/or dissection. Fibrillin-1 mutations result in structurally and functionally defective fibrillin-1 molecules and microfibrils. Recent molecular genetic and fibrillin-1 biosynthesis studies suggest that individuals with fibrillin-1 abnormalities can be further subdivided into groups that are associated with distinct differences in severity and prognosis. In recognition of the expanding scope of related connective tissue disorders, we propose the terms microfibrillar disorder for disorders affecting fibrillin-containing microfibrils, and the more narrow concept of fibrillinopathy for clinical entities associated with abnormalities of fibrillin-1 or fibrillin-2. This latter category includes the previously defined disorders Marfan syndrome, congenital contractual arachnodactyly, and forms of ascending aortic aneurysm and/or dissection.
Asunto(s)
Aneurisma de la Aorta/complicaciones , Proteínas de la Matriz Extracelular/genética , Síndrome de Marfan/complicaciones , Proteínas de Microfilamentos/genética , Adulto , Aneurisma de la Aorta/genética , Proteínas de Unión al Calcio/genética , Proteínas Contráctiles/genética , Tejido Elástico , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Humanos , Síndrome de Marfan/diagnóstico , Síndrome de Marfan/genética , Mutación , Fenotipo , Factores de Empalme de ARN , Análisis de Secuencia de ADNRESUMEN
Laminin-5 (previously known as kalinin, epiligrin, and nicein) is an adhesive protein localized to the anchoring filaments within the lamina lucida space of the basement membrane zone lying between the epidermis and dermis of human skin. Anchoring filaments are structures within the lamina lucida and lie immediately beneath the hemidesmosomes of the overlying basal keratinocytes apposed to the basement membrane zone. Human keratinocytes synthesize and deposit laminin-5. Laminin-5 is present at the wound edge during reepithelialization. In this study, we demonstrate that laminin-5, a powerful matrix attachment factor for keratinocytes, inhibits human keratinocyte migration. We found that the inhibitory effect of laminin-5 on keratinocyte motility can be reversed by blocking the alpha3 integrin receptor. Laminin-5 inhibits keratinocyte motility driven by a collagen matrix in a concentration-dependent fashion. Using antisense oligonucleotides to the alpha3 chain of laminin-5 and an antibody that inhibits the cell binding function of secreted laminin-5, we demonstrated that the endogenous laminin-5 secreted by the keratinocyte also inhibits the keratinocyte's own migration on matrix. These findings explain the hypermotility that characterizes keratinocytes from patients who have forms of junctional epidermolysis bullosa associated with defects in one of the genes encoding for laminin-5 chains, resulting in low expression and/or functional inadequacy of laminin-5 in these patients. These studies also suggest that during reepithelialization of human skin wounds, the secreted laminin-5 stabilizes the migrating keratinocyte to establish the new basement membrane zone.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Queratinocitos/citología , Antígenos CD/fisiología , Células Cultivadas , Colágeno/fisiología , Proteínas del Citoesqueleto , Humanos , Técnicas Inmunológicas , Integrina alfa3 , Integrinas/fisiología , Masculino , Oligonucleótidos Antisentido/farmacología , Fosfoproteínas/metabolismo , KalininaRESUMEN
Actin and associated proteins at the cytoskeleton-plasma membrane interface stabilize the membrane bilayer, control cell shape, and delimit specialized membrane domains. To identify membrane proteins that bind directly to F-actin, we have developed a blot overlay assay with 125I-labeled F-actin. In the soil amoebae, Dictyostelium discoideum, the major proteins reactive in this assay are p30a, a 34-kD peripheral membrane protein that is concentrated in filopodia and at sites of cell-cell adhesion, and ponticulin, a 17-kD transmembrane glycoprotein required for efficient chemotaxis and for control of pseudopod dynamics. Proteins with apparent molecular masses of approximately 34- and approximately 17-kD also are observed on F-actin blot overlays of many mammalian cell lines. However, in mammalian cells, the most prominent F-actin binding proteins in this assay exhibit apparent molecular masses of 78-, 80-, 81-, approximately 120-, and 205-kD. Bovine neutrophils contain the 78-, 81-, and 205-kD proteins, all of which co-isolate with a plasma membrane-enriched fraction. We have previously identified the 78-, 80-, and 81-kD proteins as moesin, radixin, and ezrin, respectively. These proteins, which are members of the protein 4.1 superfamily, colocalize with actin in cell surface extensions and have been implicated in the protrusion of microvilli, filopodia, and membrane ruffles. The 205-kD protein (p205) appears to be absent from current databases, and its characteristics are still under investigation. We here report that the 120-kD protein is drebrin, a submembranous actin-binding protein originally identified as a developmentally regulated brain protein. Thus, it appears that F-actin blot overlays provide an efficient assay for simultaneous monitoring of a subset of F-actin binding proteins, including p30a, ponticulin, moesin, radixin, ezrin, p205, and drebrin.