RESUMEN
Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/fisiología , Dictyostelium/fisiología , Endocitosis , Neuropéptidos/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Actinas/inmunología , Animales , Adhesión Celular , División Celular , Membrana Celular/metabolismo , Movimiento Celular , Células Cultivadas , Quimiotaxis , Dictyostelium/citología , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Humanos , Mutagénesis Sitio-Dirigida , Neuropéptidos/genética , Proteínas Recombinantes de Fusión , Homología de Secuencia , Transducción de Señal , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1RESUMEN
A growing number of actin-associated membrane proteins have been implicated in motile processes, adhesive interactions, and signal transduction to the cell nucleus. We report here that supervillin, an F-actin binding protein originally isolated from bovine neutrophil plasma membranes, contains functional nuclear targeting signals and localizes at or near vinculin-containing focal adhesion plaques in COS7-2 and CV1 cells. Overexpression of full-length supervillin in these cells disrupts the integrity of focal adhesion plaques and results in increased levels of F-actin and vinculin. Localization studies of chimeric proteins containing supervillin sequences fused with the enhanced green fluorescent protein indicate that: (1) the amino terminus promotes F-actin binding, targeting to focal adhesions, and limited nuclear localization; (2) the dominant nuclear targeting signal is in the center of the protein; and (3) the carboxy-terminal villin/gelsolin homology domain of supervillin does not, by itself, bind tightly to the actin cytoskeleton in vivo. Overexpression of chimeras containing both the amino-terminal F-actin binding site(s) and the dominant nuclear targeting signal results in the formation of large nuclear bundles containing F-actin, supervillin, and lamin. These results suggest that supervillin may contribute to cytoarchitecture in the nucleus, as well as at the plasma membrane.
Asunto(s)
Actinas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Bovinos , Adhesión Celular , Línea Celular , Citoesqueleto/metabolismo , Cartilla de ADN/genética , Expresión Génica , Proteínas Fluorescentes Verdes , Laminas , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Señales de Localización Nuclear , Proteínas Nucleares/metabolismo , Fenotipo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Vinculina/metabolismoRESUMEN
Supervillin is a 205-kDa F-actin binding protein originally isolated from bovine neutrophils. This protein is tightly associated with both actin filaments and plasma membranes, suggesting that it forms a high-affinity link between the actin cytoskeleton and the membrane. Human supervillin cDNAs cloned from normal human kidney and from the cervical carcinoma HeLa S3 predict a bipartite structure with three potential nuclear localization signals in the NH2-terminus and three potential actin-binding sequences in the COOH-terminus. In fact, throughout its length, the COOH-terminal half of supervillin is similar to segments 2-6 plus the COOH-terminal "headpiece" of villin, an actin-binding protein in intestinal microvilli. A comparison of the bovine and human sequences indicates that supervillin is highly conserved at the amino acid level, with 79.2% identity of the NH2-terminus and conservation of three of the four nuclear localization signals found in bovine supervillin. The COOH-terminus is even more conserved, with 95.1% amino acid identity overall and 100% conservation of the villin-like headpiece. Supervillin mRNAs are expressed in all human tissue tested, bu are most abundant in muscle, bone marrow, thyroid gland, and salivary gland; comparatively little message is found in brain. Human supervillin mRNA is approximately 7.5 kb; this message is especially abundant in HeLa S3 cervical carcinoma, SW480 adenocarcinoma, and A549 lung carcinoma cell lines. The human supervillin gene (SVIL) is localized to a single chromosomal locus at 10p11.2, a region that is deleted in some prostate tumors.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de Microfilamentos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 10/genética , Clonación Molecular , Secuencia Conservada , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Señales de Localización Nuclear/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.
Asunto(s)
Actinas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Familia de Multigenes , Citoesqueleto de Actina/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Bovinos , Fraccionamiento Celular , Clonación Molecular , Perros , Células Epiteliales/química , Células Epiteliales/ultraestructura , Gelsolina/genética , Uniones Intercelulares , Riñón/química , Riñón/citología , Riñón/ultraestructura , Datos de Secuencia Molecular , Neutrófilos/química , Neutrófilos/ultraestructura , Señales de Localización Nuclear , Unión Proteica , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Microorganisms, including bacteria, fungi, protozoa, and microalgae, are composed predominantly of water which prohibits direct observation in a traditional scanning electron microscope (SEM). Preparation for SEM requires that microorganisms be fixed, frozen or dehydrated, and coated with a conductive film before observation in a high vacuum environment. Sample preparation may mechanically disturb delicate samples, compromise morphological information, and introduce other artifacts. The environmental scanning electron microscope (ESEM) provides a technology for imaging hydrated or dehydrated biological samples with minimal manipulation and without the need for conductive coatings. Sporulating cultures of three fungi, Aspergillus sp., Cunninghamella sp., and Mucor sp., were imaged in the ESEM to assess usefulness of the instrument in the direct observation of delicate, uncoated, biological specimens. Asexual sporophores showed no evidence of conidial displacement or disruption of sporangia. Uncoated algal cells of Euglena gracilis and Spirogyra sp. were examined using the backscatter electron detector (BSE) and the environmental secondary electron detector (ESD) of the ESEM. BSE images had more clearly defined intracellular structures, whereas ESD gave a clearer view of the surface E. gracilis cells fixed with potassium permanganate, Spirogyra sp. stained with Lugol's solution, and Saprolegnia sp. fixed with osmium tetroxide were compared using BSE and ESD to demonstrate that cellular details could be enhanced by the introduction of heavy metals. The effect of cellular water on signal quality was evaluated by comparing hydrated to critical point dried specimens.