RESUMEN
The loss of the epithelial architecture and cell polarity/differentiation is known to be important during the tumorigenic process. Here we demonstrate that the brush border protein Myosin Ia (MYO1A) is important for polarization and differentiation of colon cancer cells and is frequently inactivated in colorectal tumors by genetic and epigenetic mechanisms. MYO1A frame-shift mutations were observed in 32% (37 of 116) of the colorectal tumors with microsatellite instability analyzed, and evidence of promoter methylation was observed in a significant proportion of colon cancer cell lines and primary colorectal tumors. The loss of polarization/differentiation resulting from MYO1A inactivation is associated with higher tumor growth in soft agar and in a xenograft model. In addition, the progression of genetically and carcinogen-initiated intestinal tumors was significantly accelerated in Myo1a knockout mice compared with Myo1a wild-type animals. Moreover, MYO1A tumor expression was found to be an independent prognostic factor for colorectal cancer patients. Patients with low MYO1A tumor protein levels had significantly shorter disease-free and overall survival compared with patients with high tumoral MYO1A (logrank test P = 0.004 and P = 0.009, respectively). The median time-to-disease recurrence in patients with low MYO1A was 1 y, compared with >9 y in the group of patients with high MYO1A. These results identify MYO1A as a unique tumor-suppressor gene in colorectal cancer and demonstrate that the loss of structural brush border proteins involved in cell polarity are important for tumor development.
Asunto(s)
Genes Supresores de Tumor , Mucosa Intestinal/metabolismo , Microvellosidades/metabolismo , Miosina Tipo I/fisiología , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Metilación de ADN , Humanos , Mutación , Miosina Tipo I/genética , Regiones Promotoras GenéticasRESUMEN
In enterocytes of the small intestine, endocytic trafficking of CFTR channels from the brush border membrane (BBM) to the subapical endosomes requires the minus-end motor, myosin VI (Myo6). The subapical localization of Myo6 is dependent on myosin Ia (Myo1a) the major plus-end motor associated with the BBM, suggestive of functional synergy between these two motors. In villus enterocytes of the Myo1a KO mouse small intestine, CFTR accumulated in syntaxin-3 positive subapical endosomes, redistributed to the basolateral domain and was absent from the BBM. In colon, where villi are absent and Myo1a expression is low, CFTR exhibited normal localization to the BBM in the Myo1a KO similar to WT. cAMP-stimulated CFTR anion transport in the small intestine was reduced by 58% in the KO, while anion transport in the colon was comparable to WT. Co-immunoprecipitation confirmed the association of CFTR with Myo1a. These data indicate that Myo1a is an important regulator of CFTR traffic and anion transport in the BBM of villus enterocytes and suggest that Myo1a may power apical CFTR movement into the BBM from subapical endosomes. Alternatively, it may anchor CFTR channels in the BBM of villus enterocytes as was proposed for Myo1a's role in BBM localization of sucrase-isomaltase.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Enterocitos/metabolismo , Cadenas Pesadas de Miosina/genética , Animales , Células CACO-2 , Cloruros , Colon/citología , Estimulación Eléctrica , Endocitosis , Enterocitos/citología , Exocitosis , Humanos , Intestino Delgado/citología , Transporte Iónico , Ratones , Ratones Noqueados , Microscopía Confocal , Microvellosidades/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Especificidad de Órganos , Vesículas Transportadoras/metabolismoRESUMEN
Brush border Myosin Ia (MYO1A) has been shown to be frequently mutated in colorectal tumors with microsatellite instability (MSI) and to have tumor suppressor activity in intestinal tumors. Here, we investigated the frequency of frameshift mutations in the A8 microsatellite in exon 28 of MYO1A in MSI gastric and endometrial tumors and found a high mutation rate in gastric (22/47; 46.8%) but not endometrial (3/48; 6.2%) tumors. Using a regression model, we show that MYO1A mutations are likely to confer a growth advantage to gastric, but not endometrial tumors. The mutant MYO1A(7A) protein was shown to lose its membrane localization in gastric cancer cells and a cycloheximide-chase assay demonstrated that the mutant MYO1A(7A) protein has reduced stability compared to the wild type MYO1A. Frequent MYO1A promoter hypermethylation was also found in gastric tumors. Promoter methylation negatively correlates with MYO1A mRNA expression in a series of 58 non-MSI gastric primary tumors (Pearson's r = -0.46; p = 0.0003) but not in a cohort of 54 non-MSI endometrial tumors and treatment of gastric cancer cells showing high MYO1A promoter methylation with the demethylating agent 5-aza-2'-deoxycytidine, resulted in a significant increase of MYO1A mRNA levels. We found that normal gastric epithelial cells, but not normal endometrial cells, express high levels of MYO1A. Therefore, when considered together, our findings suggest that MYO1A has tumor suppressor activity in the normal gastric epithelium but not in the normal endometrium and inactivation of MYO1A either genetically or epigenetically may confer gastric epithelial cells a growth advantage.
Asunto(s)
Neoplasias Endometriales/genética , Microvellosidades/metabolismo , Cadenas Pesadas de Miosina/genética , Miosina Tipo I/genética , Neoplasias Gástricas/genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Secuencia de Bases , Western Blotting , Metilación de ADN , Cartilla de ADN , Decitabina , Neoplasias Endometriales/patología , Femenino , Humanos , Microscopía Confocal , Mutación , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/patologíaRESUMEN
The actin motor myosin VI regulates endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine, but the endocytic adaptor linking CFTR to myosin VI is unknown. Dab2 (Disabled 2) is the binding partner for myosin VI, clathrin, and alpha-AP-2 and directs endocytosis of low density lipoprotein receptor family members by recognizing a phosphotyrosine-binding domain. However, CFTR does not possess a phosphotyrosine-binding domain. We examined whether alpha-AP-2 and/or Dab2 were binding partners for CFTR and the role of myosin VI in localizing endocytic adaptors in the intestine. CFTR co-localized with alpha-AP-2, Dab2, and myosin VI and was identified in a complex with all three endocytic proteins in the intestine. Apical CFTR was increased in the intestines of Dab-2 KO mice, suggesting its involvement in regulating surface CFTR. Glutathione S-transferase pulldown assays revealed binding of CFTR to alpha-AP-2 (but not Dab2) in the intestine, whereas Dab-2 interacted with alpha-AP-2. siRNA silencing of alpha-AP-2 in cells significantly reduced CFTR endocytosis, further supporting alpha-AP-2 as the direct binding partner for CFTR. alpha-AP-2 and Dab2 localized to the terminal web regions of enterocytes, but Dab2 accumulated in this location in Snell's Waltzer myosin VI((sv/sv)) intestine. Ultrastructural examination revealed that the accumulation of Dab2 correlated with prominent involution and the loss of normal positioning of the intermicrovillar membranes that resulted in expansion of the terminal web region in myosin VI((sv/sv)) enterocytes. The findings support alpha-AP-2 in directing myosin VI-dependent endocytosis of CFTR and a requirement for myosin VI in membrane invagination and coated pit formation in enterocytes.
Asunto(s)
Canales de Cloruro/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mucosa Intestinal/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Factor de Transcripción AP-2/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Proteínas Reguladoras de la Apoptosis , Endocitosis , Enterocitos/metabolismo , Masculino , Ratones , Ratones Noqueados , Microvellosidades/metabolismo , Modelos Biológicos , Ratas , Ratas Sprague-DawleyRESUMEN
Myosin-IXb (Myo9b) is a single-headed, processive motor that contains a Rho-GTPase-activating protein (GAP) domain within its tail. Although tail-less myosin- IXb motor domain moves towards the minus end of the actin filament, we show here that full-length myosin-IXb is a plus-end-directed motor. This suggests that the tail domain of myosin-IXb regulates motor directionality.
Asunto(s)
Proteínas Motoras Moleculares/metabolismo , Miosinas/metabolismo , Actinas/metabolismo , Anticuerpos/metabolismo , Miosina Tipo V/metabolismo , Estructura Terciaria de Proteína , Factores de TiempoRESUMEN
Myo2p is an essential class V myosin in budding yeast with several identified functions in organelle trafficking and spindle orientation. The present study demonstrates that Myo2p is a component of a large RNA-containing complex (Myo2p-RNP) that is distinct from polysomes based on sedimentation analysis and lack of ribosomal subunits in the Myo2p-RNP. Microarray analysis of RNAs that coimmunoprecipitate with Myo2p revealed the presence of a large number of mRNAs in this complex. The Myo2p-RNA complex is in part composed of the RNA processing body (P-body) based on coprecipitation with P-body protein subunits and partial colocalization of Myo2p with P-bodies. P-body disassembly is delayed in the motor mutant, myo2-66, indicating that Myo2p may facilitate the release of mRNAs from the P-body.
Asunto(s)
Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/química , Miosina Tipo V/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , Sustancias Macromoleculares , Cadenas Pesadas de Miosina/genética , Miosina Tipo V/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Orgánulos/metabolismo , Polirribosomas/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Hongos/química , ARN de Hongos/genética , ARN Mensajero/química , ARN Mensajero/genética , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vesículas Secretoras/metabolismo , Vacuolas/metabolismoRESUMEN
Myosin VI (Myo6) is an actin-based motor protein implicated in clathrin-mediated endocytosis in nonneuronal cells, though little is known about its function in the nervous system. Here, we find that Myo6 is highly expressed throughout the brain, localized to synapses, and enriched at the postsynaptic density. Myo6-deficient (Snell's waltzer; sv/sv) hippocampus exhibits a decrease in synapse number, abnormally short dendritic spines, and profound astrogliosis. Similarly, cultured sv/sv hippocampal neurons display decreased numbers of synapses and dendritic spines, and dominant-negative disruption of Myo6 in wild-type hippocampal neurons induces synapse loss. Importantly, we find that sv/sv hippocampal neurons display a significant deficit in the stimulation-induced internalization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPARs), and that Myo6 exists in a complex with the AMPAR, AP-2, and SAP97 in brain. These results suggest that Myo6 plays a role in the clathrin-mediated endocytosis of AMPARs, and that its loss leads to alterations in synaptic structure and astrogliosis.
Asunto(s)
Endocitosis/fisiología , Cadenas Pesadas de Miosina/fisiología , Receptores AMPA/metabolismo , Sinapsis/fisiología , Complejo 2 de Proteína Adaptadora/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfato/farmacología , Animales , Astrocitos/metabolismo , Astrocitos/ultraestructura , Encéfalo/metabolismo , Encéfalo/ultraestructura , Química Encefálica , Dendritas/metabolismo , Dendritas/ultraestructura , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Homólogo 1 de la Proteína Discs Large , Dineínas/genética , Dineínas/metabolismo , Endocitosis/efectos de los fármacos , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Guanilato-Quinasas , Insulina/farmacología , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/metabolismo , Microscopía Electrónica , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Neuronas/ultraestructura , Receptores AMPA/análisis , Receptores de Glutamato/metabolismo , Sacarosa/farmacología , Sinapsis/ultraestructura , Membranas Sinápticas/química , Membranas Sinápticas/efectos de los fármacos , Sinaptosomas/química , Sinaptosomas/efectos de los fármacos , Transferrina/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismoRESUMEN
Drosophila myosin IB (Myo1B) is one of two class I myosins in the Drosophila genome. In the larval and adult midgut enterocyte, Myo1B is present within the microvillus (MV) of the apical brush border (BB) where it forms lateral tethers between the MV membrane and underlying actin filament core. Expression of green fluorescent protein-Myo1B tail domain in the larval gut showed that the tail domain is sufficient for localization of Myo1B to the BB. A Myo1B deletion mutation exhibited normal larval gut physiology with respect to food uptake, clearance, and pH regulation. However, there is a threefold increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive enterocyte nuclei in the Myo1B mutant. Ultrastructural analysis of mutant midgut revealed many perturbations in the BB, including membrane tethering defects, MV vesiculation, and membrane shedding. The apical localization of both singed (fascin) and Dmoesin is impaired. BBs isolated from mutant and control midgut revealed that the loss of Myo1B causes the BB membrane and underlying cytoskeleton to become destabilized. Myo1B mutant larvae also exhibit enhanced sensitivity to oral infection by the bacterial pathogen Pseudomonas entomophila, and severe cytoskeletal defects are observed in the BB of proximal midgut epithelial cells soon after infection. Resistance to P. entomophila infection is restored in Myo1B mutant larvae expressing a Myo1B transgene. These results indicate that Myo1B may play a role in the local midgut response pathway of the Imd innate immune response to Gram-negative bacterial infection.
Asunto(s)
Drosophila melanogaster/metabolismo , Enterocitos/metabolismo , Miosina Tipo I/metabolismo , Pseudomonas/patogenicidad , Animales , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/ultraestructura , Epitelio/metabolismo , Tracto Gastrointestinal , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Larva/genética , Larva/metabolismo , Microscopía Electrónica de Transmisión , Mutación/genética , Miosina Tipo I/genética , Miosina Tipo I/ultraestructura , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/patología , Sensibilidad y Especificidad , Tasa de SupervivenciaRESUMEN
Myosin 1e (Myo1e) is one of two Src homology 3 domain-containing "long-tailed" type I myosins in vertebrates, whose functions in health and disease are incompletely understood. Here, we demonstrate that Myo1e localizes to podocytes in the kidney. We generated Myo1e-knockout mice and found that they exhibit proteinuria, signs of chronic renal injury, and kidney inflammation. At the ultrastructural level, renal tissue from Myo1e-null mice demonstrates changes characteristic of glomerular disease, including a thickened and disorganized glomerular basement membrane and flattened podocyte foot processes. These observations suggest that Myo1e plays an important role in podocyte function and normal glomerular filtration.
Asunto(s)
Miosinas/fisiología , Podocitos/patología , Animales , Nitrógeno de la Urea Sanguínea , Tasa de Filtración Glomerular , Enfermedades Renales/etiología , Glomérulos Renales/patología , Ratones , Ratones Noqueados , Miosina Tipo I , Miosinas/análisis , Podocitos/químicaRESUMEN
Myosin-Va, a processive actin-based motor thought to be involved in organelle transport, now stands ready to join myosin-II (from muscle) as one of the most highly characterized members of the myosin superfamily. Recent reports from the laboratories of Goldman and colleagues and Selvin and colleagues have provided unprecedented, high-resolution views of the structural changes that take place while this motor moves along its track. Taken together, the new results indicate that myosin-Va tilts its light chain binding domains to 'walk' along actin in a hand-over-hand fashion.
Asunto(s)
Proteínas Motoras Moleculares/metabolismo , Movimiento , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Animales , Modelos Moleculares , Conformación ProteicaRESUMEN
To gain insight regarding myosin-1A (M1A) function, we expressed a dominant negative fragment of this motor in the intestinal epithelial cell line, CACO-2BBE. Sucrase isomaltase (SI), a transmembrane disaccharidase found in microvillar lipid rafts, was missing from the brush border (BB) in cells expressing this fragment. Density gradient centrifugation, affinity purification, and immunopurification of detergent-resistant membranes isolated from CACO-2BBE cells and rat microvilli (MV) all indicate that M1A and SI reside on the same population of low density (approximately 1.12 g/ml) membranes. Chemical cross-linking of detergent-resistant membranes from rat MV indicates that SI and M1A may interact in a lipid raft complex. The functional significance of such a complex is highlighted by expression of the cytoplasmic domain of SI, which results in lower levels of M1A and a loss of SI from the BB. Together, these studies are the first to assign a specific role to M1A and suggest that this motor is involved in the retention of SI within the BB.
Asunto(s)
Proteínas de Unión a Calmodulina/metabolismo , Membrana Celular/enzimología , Mucosa Intestinal/enzimología , Complejo Sacarasa-Isomaltasa/metabolismo , Animales , Células CACO-2 , Proteínas de Unión a Calmodulina/genética , Membrana Celular/ultraestructura , Digestión/fisiología , Disacáridos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Mucosa Intestinal/ultraestructura , Microdominios de Membrana/metabolismo , Microdominios de Membrana/ultraestructura , Microvellosidades/enzimología , Microvellosidades/ultraestructura , Cadenas Pesadas de Miosina , Miosina Tipo I , Estructura Terciaria de Proteína/fisiología , Ratas , Fracciones SubcelularesRESUMEN
Microtubules (MTs) help establish and maintain cell polarity by promoting actin-dependent membrane protrusion at the leading edge of the cell, but the molecular mechanisms that mediate cross-talk between actin and MTs during this process are unclear. We demonstrate that the Abl-related gene (Arg) nonreceptor tyrosine kinase is required for dynamic lamellipodial protrusions after adhesion to fibronectin. arg-/- fibroblasts exhibit reduced lamellipodial dynamics as compared with wild-type fibroblasts, and this defect can be rescued by reexpression of an Arg-yellow fluorescent protein fusion. We show that Arg can bind MTs with high affinity and cross-link filamentous actin (F-actin) bundles and MTs in vitro. MTs concentrate and insert into Arg-induced F-actin-rich cell protrusions. Arg requires both its F-actin-binding domains and its MT-binding domain to rescue the defects in lamellipodial dynamics of arg-/- fibroblasts. These findings demonstrate that Arg can mediate physical contact between F-actin and MTs at the cell periphery and that this cross-linking activity is required for Arg to regulate lamellipodial dynamics in fibroblasts.
Asunto(s)
Actinas/metabolismo , Fibroblastos/metabolismo , Microtúbulos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Seudópodos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/ultraestructura , Animales , Sitios de Unión/genética , Adhesión Celular/genética , Línea Celular Transformada , Movimiento Celular/genética , Fibroblastos/ultraestructura , Fibronectinas/metabolismo , Regulación de la Expresión Génica/genética , Ratones , Microtúbulos/ultraestructura , Dinámicas no Lineales , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Quinasas/genética , Seudópodos/ultraestructuraRESUMEN
Phagocytosis of invading pathogens or cellular debris requires a dramatic change in cell shape driven by actin polymerization. For antibody-covered targets, phagocytosis is thought to proceed through the sequential engagement of Fc-receptors on the phagocyte with antibodies on the target surface, leading to the extension and closure of the phagocytic cup around the target. We find that two actin-dependent molecular motors, class 1 myosins myosin 1e and myosin 1f, are specifically localized to Fc-receptor adhesions and required for efficient phagocytosis of antibody-opsonized targets. Using primary macrophages lacking both myosin 1e and myosin 1f, we find that without the actin-membrane linkage mediated by these myosins, the organization of individual adhesions is compromised, leading to excessive actin polymerization, slower adhesion turnover, and deficient phagocytic internalization. This work identifies a role for class 1 myosins in coordinated adhesion turnover during phagocytosis and supports a mechanism involving membrane-cytoskeletal crosstalk for phagocytic cup closure.
Asunto(s)
Actinas/metabolismo , Adhesión Celular/fisiología , Miosina Tipo I/metabolismo , Miosinas/metabolismo , Fagocitosis/fisiología , Actinas/ultraestructura , Animales , Células de la Médula Ósea , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Femenino , Microscopía Intravital , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Microscopía Fluorescente , Miosina Tipo I/genética , Miosinas/genética , Cultivo Primario de Células , Células RAW 264.7 , Receptores Fc/metabolismo , Receptores Fc/ultraestructura , Imagen de Lapso de TiempoRESUMEN
Cells have evolved multiple mechanisms to overcome the effects of entropy and diffusion to create a highly ordered environment. For cells to function properly, some components must be anchored to provide a framework or structure. Others must be rapidly transported over long distances to generate asymmetries in cell morphology and composition. To accomplish long-range transport, cells cannot rely on diffusion alone as many large organelles and macromolecular complexes are essentially immobilized by the dense meshwork of the cytosol. One strategy used by cells to overcome diffusion is to harness the free energy liberated by ATP hydrolysis through molecular motors. Myosins are a family of actin based molecular motors that have evolved a variety of ways to contribute to cellular organization through numerous modifications to the manner they convert that free energy into mechanical work.
Asunto(s)
Fenómenos Fisiológicos Celulares , Miosinas/metabolismo , Actinas/química , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Drosophila , Cinética , Modelos Moleculares , Miosinas/química , Conformación ProteicaRESUMEN
Spa2p is a nonessential protein that regulates yeast cell polarity. It localizes early to the presumptive bud site and remains at sites of growth throughout the cell cycle. To understand how Spa2p localization is regulated and to gain insight into its molecular function in cell polarity, we used a coimmunoprecipitation strategy followed by tandem mass spectrometry analysis to identify proteins that associate with Spa2p in vivo. We identified Myo1p, Myo2p, Pan1p, and the protein encoded by YFR016c as proteins that interact with Spa2p. Strikingly, all of these proteins are involved in cell polarity and/or actin function. Here we focus on the functional significance of the interactions of Spa2p with Myo2p and Myo1p. We find that localization of Spa2GFP to sites of polarized growth depends on functional Myo2p but not on Myo1p. We also find that Spa2p, like Myo2p, cosediments with F-actin in an ATP-sensitive manner. We hypothesize that Spa2p associates with actin via a direct or indirect interaction with Myo2p and that Spa2p may be involved in mediating polarized localization of polarity proteins via Myo2p. In addition, we observe an enhanced cell-separation defect in a myo1spa2 strain at 37 degrees C. This provides further evidence that Spa2p is involved in cytokinesis and cell wall morphogenesis.
Asunto(s)
Actinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Polaridad Celular , Pared Celular/metabolismo , Citocinesis , Proteínas del Citoesqueleto , Proteínas Fúngicas/metabolismo , Proteínas de Microfilamentos , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Unión Proteica , Saccharomyces cerevisiae/citologíaRESUMEN
To develop our understanding of myosin-1a function in vivo, we have created a mouse line null for the myosin-1a gene. Myosin-1a knockout mice demonstrate no overt phenotypes at the whole animal level but exhibit significant perturbations and signs of stress at the cellular level. Among these are defects in microvillar membrane morphology, distinct changes in brush-border organization, loss of numerous cytoskeletal and membrane components from the brush border, and redistribution of intermediate filament proteins into the brush border. We also observed significant ectopic recruitment of another short-tailed class I motor, myosin-1c, into the brush border of knockout enterocytes. This latter finding, a clear demonstration of functional redundancy among vertebrate myosins-I, may account for the lack of a whole animal phenotype. Nevertheless, these results indicate that myosin-1a is a critical multifunctional component of the enterocyte, required for maintaining the normal composition and highly ordered structure of the brush border.
Asunto(s)
Enterocitos/metabolismo , Enterocitos/ultraestructura , Microvellosidades/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Animales , Colon/metabolismo , Colon/ultraestructura , Duodeno/metabolismo , Duodeno/ultraestructura , Íleon/metabolismo , Íleon/ultraestructura , Ratones , Ratones Noqueados , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Microvellosidades/ultraestructura , Cadenas Pesadas de Miosina/deficiencia , Cadenas Pesadas de Miosina/genética , FenotipoRESUMEN
Myosin 1E is one of two "long-tailed" human Class I myosins that contain an SH3 domain within the tail region. SH3 domains of yeast and amoeboid myosins I interact with activators of the Arp2/3 complex, an important regulator of actin polymerization. No binding partners for the SH3 domains of myosins I have been identified in higher eukaryotes. In the current study, we show that two proteins with prominent functions in endocytosis, synaptojanin-1 and dynamin, bind to the SH3 domain of human Myo1E. Myosin 1E co-localizes with clathrin- and dynamin-containing puncta at the plasma membrane and this co-localization requires an intact SH3 domain. Expression of Myo1E tail, which acts in a dominant-negative manner, inhibits endocytosis of transferrin. Our findings suggest that myosin 1E may contribute to receptor-mediated endocytosis.
Asunto(s)
Dinaminas/metabolismo , Endocitosis , Miosina Tipo I/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Células 3T3 , Animales , Células COS , Chlorocebus aethiops , Vesículas Cubiertas por Clatrina/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Ratones , Miosina Tipo I/química , Unión Proteica , Transporte de Proteínas , Ratas , Sinapsis/metabolismo , Extractos de Tejidos , Transferrina/metabolismo , Técnicas del Sistema de Dos Híbridos , Dominios Homologos srcRESUMEN
Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Pollos/metabolismo , Microtúbulos/metabolismo , Contracción Muscular/fisiología , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Encéfalo/metabolismo , Calcio/metabolismo , Células Cultivadas , Insectos/virología , Microscopía Electrónica , Microscopía Fluorescente , Unión Proteica , Estructura Terciaria de Proteína/fisiologíaRESUMEN
Genetic studies have implicated MYO9B, which encodes myosin IXb (Myo9b), a motor protein with a Rho GTPase activating domain (RhoGAP), as a susceptibility gene for inflammatory bowel disease (IBD). Moreover, we have recently shown that knockdown of Myo9b in an intestinal epithelial cell line impairs wound healing and barrier function. Here, we investigated whether mice lacking Myo9b have impaired intestinal barrier function and features of IBD. Myo9b knock out (KO) mice exhibit impaired weight gain and fecal occult blood (indicator of gastrointestinal bleeding), and increased intestinal epithelial cell apoptosis could be detected along the entire intestinal axis. Histologic analysis revealed intestinal mucosal damage, most consistently observed in the ileum, which included superficial ulceration and neutrophil infiltration. Focal lesions contained neutrophils and ultrastructural examination confirmed epithelial discontinuity and the deposition of extracellular matrix. We also observed impaired mucosal barrier function in KO mice. Transepithelial electrical resistance of KO ileum is >3 fold less than WT ileum. The intestinal mucosa is also permeable to high molecular weight dextran, presumably due to the presence of mucosal surface ulcerations. There is loss of tight junction-associated ZO-1, decreased lateral membrane associated E-cadherin, and loss of terminal web associated cytokeratin filaments. Consistent with increased Rho activity in the KO, there is increased subapical expression of activated myosin II (Myo2) based on localization of phosphorylated Myo2 regulatory light chain. Except for a delay in disease onset in the KO, no difference in dextran sulfate sodium-induced colitis and lethality was observed between wild-type and Myo9b KO mice.
Asunto(s)
Íleon/patología , Enfermedades Inflamatorias del Intestino/genética , Intestinos/patología , Miosina Tipo II/metabolismo , Animales , Humanos , Enfermedades Inflamatorias del Intestino/patología , Ratones , Ratones NoqueadosRESUMEN
In mice and humans, loss of myosin VI (Myo6) function results in deafness, and certain Myo6 mutations also result in cardiomyopathies in humans. The current studies have utilized the Snell's waltzer (sv) mouse (a functional null mutation for Myo6) to determine if this mouse also exhibits cardiac defects and thus used to determine the cellular and molecular basis for Myo6-associated heart disease. Myo6 is expressed in mouse heart where it is predominantly expressed in vascular endothelial cells (VECs) based on co-localization with the VEC cell marker CD31. Sv/sv heart mass is significantly greater than that of sv/+ littermates, a result of left ventricle hypertrophy. The left ventricle of the sv/sv exhibits extensive fibrosis, both interstitial and perivascular, based on histologic staining, and immunolocalization of several markers for fibrosis including fibronectin, collagen IV, and the fibroblast marker vimentin. Myo6 is also expressed in lung VECs but not in VECs of intestine, kidney, or liver. Sv/sv lungs exhibit increased periaveolar fibrosis and enlarged air sacs. Electron microscopy of sv/sv cardiac and lung VECs revealed abnormal ultrastructure, including luminal protrusions and increased numbers of cytoplasmic vesicles. Previous studies have shown that loss of function of either Myo6 or its adaptor binding partner synectin/GIPC results in impaired arterial development due to defects in VEGF signaling. However, examination of synectin/GIPC-/- heart revealed no fibrosis or significantly altered VEC ultrastructure, suggesting that the cardiac and lung defects observed in the sv/sv mouse are not due to Myo6 function in arterial development.