RESUMEN

Caveolin-1 (Cav1) is an integral plasma membrane protein and a complex regulator of signal transduction. The Signal Transducer and Activator of Transcription-3 (Stat3) is activated by a number of receptor and non-receptor tyrosine kinases and is positively implicated in cancer. Despite extensive efforts, the relationship between Cav1 and Stat3 has been a matter of controversy. We previously demonstrated that engagement of E- or N-cadherin or cadherin-11 cell to cell adhesion molecules, as occurs with confluence of cultured cells, triggers a dramatic increase in the levels of tyr705 phosphorylated i.e. activated Stat3, by a mechanism requiring the cRac1 small GTPase. Since confluence was not taken into account in previous studies, we revisited the question of the relationship between Cav1 and Stat3-ptyr705 in non-transformed mouse fibroblasts and in human lung carcinoma cells, by examining their effect at different cell densities. Our results unequivocally demonstrate that Cav1 downregulates cadherin-11, by a mechanism which requires the Cav1 scaffolding domain. This cadherin-11 downregulation, in turn, leads to a reduction in cRac1 and Stat3 activity levels. Furthermore, in a feedback loop possibly through p53 upregulation, Stat3 downregulation increases Cav1 levels. Our data reveal the presence of a potent, negative regulatory loop between Cav1 and cadherin-11/Stat3, leading to Stat3 inhibition and apoptosis.

Asunto(s)

Cadherinas/genética , Caveolina 1/genética , Factor de Transcripción STAT3/genética , Proteína de Unión al GTP rac1/genética , Células A549 , Animales , Apoptosis/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Ratones , Fosforilación , Transducción de Señal , Proteína p53 Supresora de Tumor/genética

RESUMEN

Carbonic anhydrase IX (CAIX) is a hypoxia inducible factor 1-induced, cell surface pH regulating enzyme with an established role in tumor progression and clinical outcome. However, the molecular basis of CAIX-mediated tumor progression remains unclear. Here, we have utilized proximity dependent biotinylation (BioID) to map the CAIX 'interactome' in breast cancer cells in order to identify physiologically relevant CAIX-associating proteins with potential roles in tumor progression. High confidence proteins identified include metabolic transporters, ß1 integrins, integrin-associated protein CD98hc and matrix metalloprotease 14 (MMP14). Biochemical studies validate the association of CAIX with α2ß1 integrin, CD98hc and MMP14, and immunofluorescence microscopy demonstrates colocalization of CAIX with α2ß1 integrin and MMP14 in F-actin/cofilin-positive lamellipodia/pseudopodia, and with MMP14 to cortactin/Tks5-positive invadopodia. Modulation of CAIX expression and activity results in significant changes in cell migration, collagen degradation and invasion. Mechanistically, we demonstrate that CAIX associates with MMP14 through potential phosphorylation residues within its intracellular domain, and that CAIX enhances MMP14-mediated collagen degradation by directly contributing hydrogen ions required for MMP14 catalytic activity. These findings establish hypoxia-induced CAIX as a novel metabolic component of cellular migration and invasion structures, and provide new mechanistic insights into its role in tumor cell biology.

Asunto(s)

Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/enzimología , Anhidrasa Carbónica IX/metabolismo , Movimiento Celular/fisiología , Neoplasias Mamarias Experimentales/enzimología , Metaloproteinasa 14 de la Matriz/metabolismo , Animales , Antígenos de Neoplasias/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Anhidrasa Carbónica IX/genética , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Células MCF-7 , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Metaloproteinasa 14 de la Matriz/genética , Ratones , Podosomas/enzimología , Podosomas/genética , Podosomas/patología , Transfección

RESUMEN

Autocrine motility factor/ phosphoglucose isomerase (AMF/PGI) promotes cell survival by the pAkt survival pathway. Its receptor, gp78/AMFR, is an E3 ubiquitin ligase implicated in endoplasmic reticulum (ER)-associated protein degradation. We demonstrate here that AMF/PGI also protects against thapsigargin (TG)- and tunicamycin (TUN)-induced ER stress and apoptosis. AMF/PGI protection against the ER stress response is receptor mediated as it is not observed in gp78/AMFR-knockdown HEK293 cells. However, AMF/PGI protection against the ER stress response by TG and TUN was mediated only partially through PI3K/Akt activation. AMF/PGI reduction of the elevation of cytosolic calcium in response to either TG or inositol 1,4,5-trisphosphate receptor activation with ATP was gp78/AMFR-dependent, independent of mitochondrial depolarization and not associated with changes in ER calcium content. These results implicate regulation of ER calcium release in AMF/PGI protection against ER stress and apoptosis. Indeed, sequestration of cytosolic calcium with BAPTA-AM limited the ER stress response. Importantly, elevation of cytosolic calcium upon treatment with the calcium ionophore ionomycin, while not inducing an ER stress response, did prevent AMF/PGI protection against ER stress. By regulating ER calcium release, AMF/PGI interaction with gp78/AMFR therefore protects against ER stress identifying novel roles for these cancer-associated proteins in promoting tumor cell survival.

Asunto(s)

Señalización del Calcio/fisiología , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Glucosa-6-Fosfato Isomerasa/metabolismo , Receptores de Citocinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Respuesta de Proteína Desplegada/fisiología , Animales , Antibacterianos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células COS , Señalización del Calcio/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Chlorocebus aethiops , Retículo Endoplásmico/genética , Inhibidores Enzimáticos/farmacología , Glucosa-6-Fosfato Isomerasa/genética , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/genética , Tapsigargina/farmacología , Tunicamicina/farmacología , Ubiquitina-Proteína Ligasas/genética , Respuesta de Proteína Desplegada/efectos de los fármacos

RESUMEN

Raft-dependent endocytosis is in large part defined as the cholesterol-sensitive, clathrin-independent internalization of ligands and receptors from the plasma membrane. It encompasses the endocytosis of caveolae, smooth plasmalemmal vesicles that form a subdomain of cholesterol and sphingolipid-rich lipid rafts and that are enriched for caveolin-1. While sharing common mechanisms, like cholesterol sensitivity, raft endocytic routes show differential regulation by various cellular components including caveolin-1, dynamin-2 and regulators of the actin cytoskeleton. Dynamin-dependent raft pathways, mediated by caveolae and morphologically equivalent non-caveolin vesicular intermediates, are referred to as caveolae/raft-dependent endocytosis. In contrast, dynamin-independent raft pathways are mediated by non-caveolar intermediates. Raft-dependent endocytosis is regulated by tyrosine kinase inhibitors and, through the regulation of the internalization of various ligands, receptors and effectors, is also a determinant of cellular signaling. In this review, we characterize and discuss the regulation of raft-dependent endocytic pathways and the role of key regulators such as caveolin-1.

Asunto(s)

Endocitosis , Microdominios de Membrana/metabolismo , Animales , Caveolina 1/metabolismo , Humanos , Transducción de Señal

RESUMEN

The ER (endoplasmic reticulum) is composed of multiple domains including the nuclear envelope, ribosome-studded rough ER and the SER (smooth ER). The SER can also be functionally segregated into domains that regulate ER-Golgi traffic (transitional ER), ERAD (ER-associated degradation), sterol and lipid biosynthesis and calcium sequestration. The last two, as well as apoptosis, are critically regulated by the close association of the SER with mitochondria. Studies with AMFR (autocrine motility factor receptor) have defined an SER domain whose integrity and mitochondrial association can be modulated by ilimaquinone as well as by free cytosolic calcium levels in the normal physiological range. AMFR is an E3 ubiquitin ligase that targets its ligand directly to the SER via a caveolae/raft-dependent pathway. In the present review, we will address the relationship between the calcium-dependent morphology and mitochondrial association of the SER and its various functional roles in the cell.

Asunto(s)

Retículo Endoplásmico Liso/fisiología , Mitocondrias/fisiología , Animales , Células Cultivadas , Retículo Endoplásmico Liso/ultraestructura , Humanos , Mitocondrias/ultraestructura

RESUMEN

Sequence analysis revealed phospholipase A2 (PLA2) motifs in capsid proteins of parvoviruses. Although PLA2 activity is not known to exist in viruses, putative PLA2s from divergent parvoviruses, human B19, porcine parvovirus, and insect GmDNV (densovirus from Galleria mellonella), can emulate catalytic properties of secreted PLA2. Mutations of critical amino acids strongly reduce both PLA2 activity and, proportionally, viral infectivity, but cell surface attachment, entry, and endocytosis by PLA2-deficient virions are not affected. PLA2 activity is critical for efficient transfer of the viral genome from late endosomes/lysosomes to the nucleus to initiate replication. These findings offer the prospect of developing PLA2 inhibitors as a new class of antiviral drugs against parvovirus infections and associated diseases.

Asunto(s)

Parvovirus/enzimología , Parvovirus/fisiología , Fosfolipasas A/metabolismo , Fosfolipasas A/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Calcio/metabolismo , Cápside/metabolismo , Núcleo Celular/metabolismo , Cromatografía en Capa Delgada , ADN/metabolismo , Endosomas/metabolismo , Vectores Genéticos , Hibridación in Situ , Lisosomas/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosfolipasas A2 , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Tiorredoxinas/metabolismo , Transfección

RESUMEN

Over 155 mutations within the V2 vasopressin receptor (AVPR2) gene are responsible for nephrogenic diabetes insipidus (NDI). The expression and subcellular distribution of four of these was investigated in transfected cells. These include a point mutation in the seventh transmembrane domain (S315R), a frameshift mutation in the third intracellular loop (804delG), and two nonsense mutations that code for AVPR2 truncated within the first cytoplasmic loop (W71X) and in the proximal portion of the carboxyl tail (R337X). RT-PCR revealed that mRNA was produced for all mutant receptor constructs. However, no receptor protein, as assessed by Western blot analysis, was detected for 804delG. The S315R was properly processed through the Golgi and targeted to the plasma membrane but lacked any detectable AVP binding or signaling. Thus, this mutation induces a conformational change that is compatible with endoplasmic reticulum (ER) export but dramatically affects hormone recognition. In contrast, the W71X and R337X AVPR2 were retained inside the cell as determined by immunofluorescence. Confocal microscopy revealed that they were both retained in the ER. To determine if calnexin could be involved, its interaction with the AVPR2 was assessed. Sequential coimmunoprecipitation demonstrated that calnexin associated with the precursor forms of both wild-type (WT) and mutant receptors in agreement with its general role in protein folding. Moreover, its association with the ER-retained R337X mutant was found to be longer than with the WT receptor suggesting that this molecular chaperone also plays a role in quality control and ER retention of misfolded G protein-coupled receptors.

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Diabetes Insípida Nefrogénica/genética , Diabetes Insípida Nefrogénica/metabolismo , Mutación , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Calnexina , Línea Celular , Permeabilidad de la Membrana Celular/genética , Diabetes Insípida Nefrogénica/etiología , Marcación de Gen , Humanos , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica/genética , Biosíntesis de Proteínas , Pliegue de Proteína , Ensayo de Unión Radioligante , Receptores de Vasopresinas/fisiología , Fracciones Subcelulares/metabolismo , Transcripción Genética , Transfección

RESUMEN

The Na(+)/H(+) exchanger NHE1 is involved in intracellular pH homeostasis and cell volume regulation and accumulates with actin in the lamellipodia of fibroblasts. In order to determine the role of NHE1 following epithelial transformation and the acquisition of motile and invasive properties, we studied NHE1 expression in polarized MDCK cells, Moloney Sarcoma virus (MSV) transformed MDCK cells and an invasive MSV-MDCK cell variant (MSV-MDCK-INV). Expression of NHE1 was significantly increased in MSV-MDCK-INV cells relative to MSV-MDCK and MDCK cells. NHE1 was localized with b-actin to the tips of MSV-MDCK-INV cell pseudopodia by immunofluorescence. Sensitivity of NHE1-mediated (22)Na uptake to ethylisopropylamiloride, a specific inhibitor of NHE1, was increased in MSV-MDCK cells relative to MDCK cells. Changes in intracellular pH induced upon EIPA treatment were also of higher magnitude in MSV-MDCK and MSV-MDCK-INV cells compared to wild-type MDCK cells, especially in Hepes-buffered DMEM medium. Inhibition of NHE1 by 50 microM ethylisopropylamiloride induced the disassembly of actin stress fibers and redistribution of the actin cytoskeleton in all cell types. However, in MSV-MDCK-INV cells, the effect of ethylisopropylamiloride treatment was more pronounced and associated with the increased reversible detachment of the cells from the substrate. Videomicroscopy of MSV-MDCK-INV cells revealed that within 20 minutes of addition, ethylisopropylamiloride induced pseudopodial retraction and inhibited cell motility. The ability of ethylisopropylamiloride to prevent nocodazole-induced formation of actin stress fibers in MSV-MDCK cells was more pronounced in Hepes medium relative to NaHCO(3) medium, showing that NHE1 can regulate actin stress fiber assembly in transformed MSV-MDCK cells via its intracellular pH regulatory effect. These results implicate NHE1 in the regulation of the actin cytoskeleton dynamics necessary for the adhesion and pseudopodial protrusion of motile, invasive tumor cells.

Asunto(s)

Amilorida/análogos & derivados , Movimiento Celular , Transformación Celular Neoplásica , Seudópodos/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Amilorida/farmacología , Animales , Adhesión Celular , Línea Celular , Línea Celular Transformada , Tamaño de la Célula , Perros , Técnica del Anticuerpo Fluorescente Indirecta , Concentración de Iones de Hidrógeno , Microscopía por Video , Nocodazol/farmacología , Fenotipo , Seudópodos/fisiología , Seudópodos/ultraestructura , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Fibras de Estrés/metabolismo , Fibras de Estrés/ultraestructura

RESUMEN

Association between the ER and mitochondria has long been observed, and the formation of close contacts between ER and mitochondria is necessary for the ER-mediated sequestration of cytosolic calcium by mitochondria. Autocrine motility factor receptor (AMF-R) is a marker for a smooth subdomain of the ER, shown here by confocal microscopy to be distinct from, yet closely associated with the calnexin- or calreticulin-labeled ER. By EM, smooth ER AMF-R tubules exhibit direct interactions with mitochondria, identifying them as a mitochondria-associated smooth ER subdomain. In digitonin-permeabilized MDCK cells, the addition of rat liver cytosol stimulates the dissociation of smooth ER and mitochondria under conditions of low calcium. Using BAPTA chelators of various affinities and CaEGTA buffers of defined free Ca(2+) concentrations and quantitative confocal microscopy, we show that free calcium concentrations <100 nM favor dissociation, whereas those >1 microM favor close association between these two organelles. Therefore, we describe a cellular mechanism that facilitates the close association of this smooth ER subdomain and mitochondria when cytosolic free calcium rises above physiological levels.

Asunto(s)

Calcio/metabolismo , Retículo Endoplásmico Liso/metabolismo , Mitocondrias/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Biomarcadores , Calcio/análisis , Línea Celular , Quelantes/farmacología , Citosol/metabolismo , Citosol/ultraestructura , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Retículo Endoplásmico Liso/química , Retículo Endoplásmico Liso/ultraestructura , Técnica del Anticuerpo Fluorescente , Riñón/citología , Hígado/metabolismo , Microscopía Electrónica , Mitocondrias/ultraestructura , Ratas , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/análisis , Ubiquitina-Proteína Ligasas

RESUMEN

Autocrine motility factor receptor (AMF-R) is internalized via a clathrin-independent pathway to smooth endoplasmic reticulum tubules. This endocytic pathway is shown here to be inhibited by methyl-(beta)-cyclodextrin (m(beta)CD) implicating caveolae or caveolae-like structures in AMF internalization to smooth ER. AMF-R is also internalized via a clathrin-dependent pathway to a transferrin receptor-negative, LAMP-1/lgpA-negative endocytic compartment identified by electron microscopy as a multivesicular body (MVB). Endocytosed AMF recycles to cell surface fibrillar structures which colocalize with fibronectin; AMF-R recycling is inhibited at 20 degrees C, which blocks endocytosis past the early endosome, but not by m(beta)CD demonstrating that AMF-R recycling to fibronectin fibrils is mediated by clathrin-dependent endocytosis to MVBs. Microtubule disruption with nocodazole did not affect delivery of bAMF to cell surface fibrils indicating that recycling bAMF traverses the MVB but not a later endocytic compartment. Plating NIH-3T3 cells on an AMF coated substrate did not specifically affect cell adhesion but prevented bAMF delivery to cell surface fibronectin fibrils and reduced cell motility. AMF-R internalization and recycling via the clathrin-mediated pathway are therefore rate-limiting for cell motility. This recycling pathway to the site of deposition of fibronectin may be implicated in the de novo formation of cellular attachments or the remodeling of the extracellular matrix during cell movement.

Asunto(s)

Clatrina/metabolismo , Endocitosis/fisiología , Fibronectinas/metabolismo , Receptores de Citocinas/metabolismo , Células 3T3 , Animales , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Glucosa-6-Fosfato Isomerasa/metabolismo , Humanos , Ratones , Receptores del Factor Autocrino de Motilidad , Ubiquitina-Proteína Ligasas

RESUMEN

The MSV-MDCK-INV invasive variant of Moloney sarcoma virus (mos) transformed MDCK cells express multiple beta-actin-rich pseudopodia (P. U. Le et al., Cancer Res. 58, 1631-1635, 1998). We show here that the tips of these actively protruding cellular domains are morphologically distinct presenting numerous blebs and selectively pass through 1-microm-pore filters. The pseudopodia were purified from the underside of the filters and a major protein component was identified as the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). By confocal microscopy, GAPDH colocalized with actin in MSV-MDCK-INV pseudopodia localizing this glycolytic enzyme to this site of active actin polymerization. Inhibition of glycolysis with 2-deoxyglucose or oxamate induced a rapid transformation of beta-actin-rich pseudopodia into extended lamellipodia and prevented cell motility. A localized glycolytic supply of energy therefore regulates the formation of beta-actin-rich pseudopodial protrusions and thereby the motility of invasive tumor cells.

Asunto(s)

Actinas/análisis , Transformación Celular Neoplásica , Glucólisis , Virus del Sarcoma Murino de Moloney/fisiología , Seudópodos/ultraestructura , Animales , Fraccionamiento Celular , Línea Celular Transformada , Movimiento Celular , Perros , Gliceraldehído-3-Fosfato Deshidrogenasas/análisis , Riñón , Microscopía Confocal , Microscopía Electrónica de Rastreo , Mitocondrias/metabolismo , Consumo de Oxígeno , Seudópodos/patología

RESUMEN

Mouse Staufen (mStau) is a double-stranded RNA-binding protein associated with polysomes and the rough endoplasmic reticulum (RER). We describe a novel endogenous isoform of mStau (termed mStau(i)) which has an insertion of six amino acids within dsRBD3, the major double-stranded RNA (dsRNA)-binding domain. With a structural change of the RNA-binding domain, this conserved and widely distributed isoform showed strongly impaired dsRNA-binding ability. In transfected cells, mStau(i) exhibited the same tubulovesicular distribution (RER) as mStau when weakly expressed; however, when overexpressed, mStau(i) was found in large cytoplasmic granules. Markers of the RER colocalized with mStau(i)-containing granules, showing that overexpressed mStau(i) could still be associated with the RER. Cotransfection of mStau(i) with mStau relocalized overexpressed mStau(i) to the reticular RER, suggesting that they can form a complex on the RER and that a balance between these isoforms is important to achieve proper localization. Coimmunoprecipitation demonstrated that the two mStau isoforms are components of the same complex in vivo. Analysis of the immunoprecipitates showed that mStau is a component of an RNA-protein complex and that the association with mStau(i) drastically reduces the RNA content of the complex. We propose that this new isoform, by forming a multiple-isoform complex, regulates the amount of RNA in mStau complexes in mammalian cells.

Asunto(s)

Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Retículo Endoplásmico Rugoso/metabolismo , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fracciones Subcelulares

RESUMEN

Autocrine motility factor (AMF) is identical to the glycolytic enzyme phosphohexose isomerase (PHI) and overexpression of AMF/PHI is associated with tumor malignancy. In order to study the overexpression of AMF/PHI, an HA-tagged AMF construct was transiently transfected into Cos7 cells. Expression of a tagged AMF-HA allowed us to determine that over a period of 16 hours only a small amount (0.1-1%) of total cellular AMF-HA was secreted into the cell medium. Cell-associated AMF-HA was exclusively cytosolic as it could be completely extracted with Triton X-100 and concentrated within actin rich pseudopodial domains. Treatment of the cells with the glycolysis inhibitor oxamate disrupted the association of AMF-HA with actin concentrations demonstrating that glycolysis regulates the formation of these AMF/PHI-associated actin-rich protrusions. AMF/PHI is a well-characterized tumor cell secreted cytokine and we identify here an alternate intracellular function for this glycolytic enzyme/cytokine in cell motility.

Asunto(s)

Actinas/metabolismo , Expresión Génica , Glucosa-6-Fosfato Isomerasa/metabolismo , Animales , Células COS , Movimiento Celular , Medios de Cultivo Condicionados/metabolismo , Citosol/efectos de los fármacos , Citosol/enzimología , Citosol/metabolismo , Dimerización , Técnica del Anticuerpo Fluorescente , Expresión Génica/efectos de los fármacos , Glucosa-6-Fosfato Isomerasa/química , Glucosa-6-Fosfato Isomerasa/genética , Glucólisis/efectos de los fármacos , L-Lactato Deshidrogenasa/antagonistas & inhibidores , L-Lactato Deshidrogenasa/metabolismo , Peso Molecular , Octoxinol/metabolismo , Ácido Oxámico/farmacología , Unión Proteica/efectos de los fármacos , Seudópodos/efectos de los fármacos , Seudópodos/enzimología , Seudópodos/metabolismo , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transfección

RESUMEN

The peptide sequence of autocrine motility factor (AMF), a tumor secreted cytokine that induces cell motility, corresponds to that of the previously identified cytokine/enzyme, neuroleukin/glucose-6-phosphate isomerase. Neuroleukin is a neurotrophic factor that promotes neuronal survival and sprouting at the neuromuscular junction. The AMF receptor (AMF-R) has been identified and shown to be highly expressed in malignant tumors with minimal expression in adjacent normal tissue. Neuroleukin mRNA is highly expressed in the cerebellum and we therefore undertook a developmental study of AMF-R expression in rat cerebellum. As determined by immunoblot, AMF-R is expressed at equivalent high levels in brain and cerebellum of postnatal day 5 (P5) and 12 (P12) rats and at significantly reduced levels in the adult. Coimmunofluorescence studies with MAP-2 and gamma-actin revealed that at P12, AMF-R was mainly localized to Purkinje and granule cells. Moreover, the premigratory cells of the external granular layer were also immunoreactive for AMF-R suggesting a role for AMF-R in granule cell migration during cerebellar development in the first two weeks after birth. In the adult, AMF-R distribution was similar to P12, although weaker, and was localized to Purkinje and granule cells. AMF-R labeling of GFAP positive glial processes could not be detected in cerebellar sections although in cerebellar primary cultures, both neurons and glial cells were labeled for AMF-R. In neurons, AMF-R labeling was present in the cell body, neurites and growth cones. These data indicate that regulation of the neurotrophic function of neuroleukin might be regulated spatially and temporally by expression of its receptor, AMF-R, in developing and adult cerebellum.

Asunto(s)

Cerebelo/embriología , Neuronas/metabolismo , Receptores de Citocinas/metabolismo , Actinas/metabolismo , Factores de Edad , Animales , Movimiento Celular/fisiología , Células Cultivadas , Cerebelo/citología , Regulación hacia Abajo/fisiología , Embrión de Mamíferos , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/metabolismo , Conos de Crecimiento/metabolismo , Conos de Crecimiento/ultraestructura , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/metabolismo , Neuritas/metabolismo , Neuritas/ultraestructura , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Células de Purkinje/citología , Células de Purkinje/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores del Factor Autocrino de Motilidad , Ubiquitina-Proteína Ligasas

RESUMEN

Transfection of Mv1Lu mink lung type II alveolar cells with beta1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the beta1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella.

Asunto(s)

Autofagia/fisiología , Orgánulos/fisiología , Animales , Línea Celular , Lisosomas/ultraestructura , Visón , N-Acetilglucosaminiltransferasas/genética , Orgánulos/ultraestructura , Transfección , Vacuolas/ultraestructura

RESUMEN

Polarization of the motile cell is associated with the formation of a distinct plasma membrane domain, the pseudopod, whose stabilization determines the directionality of cell movement. The rapid movement of cells over a substrate requires that an essential aspect of cell motility must be the supply of the necessary molecular machinery to the site of pseudopodial extension. Renewal of this pseudopodial domain requires the directed delivery to the site of pseudopodial protrusion of proteins which regulate actin cytoskeleton dynamics, cell-substrate adhesion, and localized degradation of the extracellular matrix. Polarized targeting mechanisms include the targeted delivery of beta-actin mRNA to the leading edge and microtubule-based vesicular traffic. The latter may include Golgi-derived vesicles of the biosynthetic pathway as well as clathrin-dependent and clathrin-independent endocytosis and recycling. Coordination of protrusive activities and supply mechanisms is critical for efficient cellular displacement and may implicate small GTPases of the Rho family. While the specific molecular mechanisms underlying pseudopodial protrusion of the motile cell are well-characterized, discussion of these diverse mechanisms in the context of cellular polarization has been limited.

Asunto(s)

Movimiento Celular/fisiología , Polaridad Celular/fisiología , Actinas/fisiología , Animales , Adhesión Celular/fisiología , Membrana Celular/fisiología , Citoesqueleto/fisiología , Matriz Extracelular/fisiología , Humanos , Microtúbulos/fisiología

RESUMEN

Staufen (Stau) is a double-stranded RNA (dsRNA)-binding protein involved in mRNA transport and localization in Drosophila. To understand the molecular mechanisms of mRNA transport in mammals, we cloned human (hStau) and mouse (mStau) staufen cDNAs. In humans, four transcripts arise by differential splicing of the Stau gene and code for two proteins with different N-terminal extremities. In vitro, hStau and mStau bind dsRNA via each of two full-length dsRNA-binding domains and tubulin via a region similar to the microtubule-binding domain of MAP-1B, suggesting that Stau cross-links cytoskeletal and RNA components. Immunofluorescent double labeling of transfected mammalian cells revealed that Stau is localized to the rough endoplasmic reticulum (RER), implicating this RNA-binding protein in mRNA targeting to the RER, perhaps via a multistep process involving microtubules. These results are the first demonstration of the association of an RNA-binding protein in addition to ribosomal proteins, with the RER, implicating this class of proteins in the transport of RNA to its site of translation.

Asunto(s)

Proteínas de Drosophila , Retículo Endoplásmico Rugoso/metabolismo , ARN Bicatenario/metabolismo , Tubulina (Proteína)/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Detergentes , Drosophila melanogaster , Humanos , Ratones , Datos de Secuencia Molecular , Octoxinol , ARN Bicatenario/genética , Proteínas de Unión al ARN , Homología de Secuencia de Aminoácido

RESUMEN

Autocrine motility factor receptor (AMF-R) is a cell surface receptor that is also localized to a smooth subdomain of the endoplasmic reticulum, the AMF-R tubule. By postembedding immunoelectron microscopy, AMF-R concentrates within smooth plasmalemmal vesicles or caveolae in both NIH-3T3 fibroblasts and HeLa cells. By confocal microscopy, cell surface AMF-R labeled by the addition of anti-AMF-R antibody to viable cells at 4 degreesC exhibits partial colocalization with caveolin, confirming the localization of cell surface AMF-R to caveolae. Labeling of cell surface AMF-R by either anti-AMF-R antibody or biotinylated AMF (bAMF) exhibits extensive colocalization and after a pulse of 1-2 h at 37 degreesC, bAMF accumulates in densely labeled perinuclear structures as well as fainter tubular structures that colocalize with AMF-R tubules. After a subsequent 2- to 4-h chase, bAMF is localized predominantly to AMF-R tubules. Cytoplasmic acidification, blocking clathrin-mediated endocytosis, results in the essentially exclusive distribution of internalized bAMF to AMF-R tubules. By confocal microscopy, the tubular structures labeled by internalized bAMF show complete colocalization with AMF-R tubules. bAMF internalized in the presence of a 10-fold excess of unlabeled AMF labels perinuclear punctate structures, which are therefore the product of fluid phase endocytosis, but does not label AMF-R tubules, demonstrating that bAMF targeting to AMF-R tubules occurs via a receptor-mediated pathway. By electron microscopy, bAMF internalized for 10 min is located to cell surface caveolae and after 30 min is present within smooth and rough endoplasmic reticulum tubules. AMF-R is therefore internalized via a receptor-mediated clathrin-independent pathway to smooth ER. The steady state localization of AMF-R to caveolae implicates these cell surface invaginations in AMF-R endocytosis.

Asunto(s)

Clatrina/fisiología , Endocitosis/fisiología , Retículo Endoplásmico Liso/metabolismo , Receptores de Citocinas/metabolismo , Células 3T3 , Animales , Transporte Biológico , Membrana Celular/metabolismo , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Retículo Endoplásmico Liso/fisiología , Retículo Endoplásmico Liso/ultraestructura , Células HeLa , Humanos , Ligandos , Ratones , Microscopía Inmunoelectrónica , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/fisiología , Receptores de Citocinas/ultraestructura , Ubiquitina-Proteína Ligasas

RESUMEN

The increased polylactosamine glycosylation of LAMP-2 in MDCK cells cultured for 1 day relative to cells cultured for 3 days has been correlated with its slower rate of Golgi transit (Nabi and Rodriguez-Boulan, 1993, Mol. Biol. Cell., 4, 627-635). To determine if the differential polylactosamine glycosylation of LAMP-2 is a consequence of glycosyltransferase expression levels, the activities of beta1-6GlcNAc-TV, beta1-3GlcNAc-T(i), beta1-2GlcNAc-TI, beta1, 4Gal-T, alpha2-6sialyl-T, and alpha2-3sialyl-T were assayed and no significant differences in the activities of these enzymes in 1 and 3 day cell extracts were detected. During MDCK epithelial polarization, the Golgi apparatus undergoes morphological changes and apiconuclear Golgi networks were more evident in 3 day cells. Treatment with nocodazole disrupted Golgi networks and generated numerous Golgi clusters in both 1 day and 3 day cells. In the presence of nocodazole the differential migration of LAMP-2 in 1 and 3 day MDCK cells was maintained and could be eliminated by treatment with endo-beta-galactosidase, indicating that gross Golgi morphology did not influence the extent of LAMP-2 polylactosamine glycosylation. Nocodazole treatment did, however, result in the faster migration of LAMP-2 which was not due to modification of core N-glycans as the precursor form of the glycoprotein migrated with an identical molecular size. Following incubation at 20 degrees C, which prevents the exit of proteins from the trans-Golgi network, the molecular size of LAMP-2 increased to a similar extent in both 1 and 3 day MDCK cells. Extending the time of incubation at 20 degrees C did not influence the size of LAMP-2, demonstrating that its glycosylation is modified not by its retention within the Golgi but rather by its equivalent slower Golgi passage at the lower temperature in both 1 and 3 day cells. An identical effect was observed in nocodazole treated cells, demonstrating that Golgi residence time determines the extent of LAMP-2 polylactosamine glycosylation, even in isolated Golgi clusters.

Asunto(s)

Amino Azúcares/metabolismo , Antígenos CD/metabolismo , Aparato de Golgi/metabolismo , Glicoproteínas de Membrana/metabolismo , Polisacáridos/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Secuencia de Carbohidratos , Línea Celular , Perros , Glicosilación/efectos de los fármacos , Glicosiltransferasas/metabolismo , Aparato de Golgi/efectos de los fármacos , Riñón/citología , Proteínas de Membrana de los Lisosomas , Datos de Secuencia Molecular , Nocodazol/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Temperatura

RESUMEN

An invasive variant of Moloney sarcoma virus-transformed MDCK cells (MSV-MDCK-INV), which was isolated by the repeated selection of cells that successfully traversed a Matrigel-coated filter, exhibits increased motile ability and presents an elongated cell shape and numerous pseudopodia. Although stress fibers are present in both MDCK and MSV-MDCK cells, MSV-MDCK-INV cells contain no stress fibers and exhibit a dense concentration of actin at the tips of pseudopodia. Relative to both MDCK and MSV-MDCK cells, the MSV-MDCK-INV cells exhibit increased expression of beta-actin and redistribution of beta-actin to the tips of pseudopodia. These actin concentrations are enriched in both F- and G-actin and, thus, represent dynamic regions of actin cytoskeleton remodeling. The acquisition of invasive properties by epithelial transformants is, therefore, associated with the increased expression of beta-actin and its concentration in actin-rich domains, which may drive pseudopodial extension and facilitate tumor cell invasion.

Asunto(s)

Actinas/metabolismo , Transformación Celular Viral , Seudópodos/metabolismo , Actinas/fisiología , Animales , Línea Celular Transformada , Tamaño de la Célula , Perros , Immunoblotting , Riñón , Microscopía Fluorescente , Virus del Sarcoma Murino de Moloney , Invasividad Neoplásica