RESUMEN

The phospholipid-anchored membrane glycoprotein (gp)-80 mediates cell-cell adhesion through a homophilic trans-interaction mechanism during Dictyostelium development and is enriched in a Triton X-100-insoluble floating fraction. To elucidate how gp80 adhesion complexes assemble in the plasma membrane, gp80-gp80 and gp80-raft interactions were investigated. A low density raft-like membrane fraction was isolated using a detergent-free method. It was enriched in sterols, the phospholipid-anchored proteins gp80, gp138, and ponticulin, as well as DdCD36 and actin, corresponding to components found in the Triton X-100-insoluble floating fraction. Chemical cross-linking revealed that gp80 oligomers were enriched in the raft-like membrane fraction, implicating stable oligomer-raft interactions. However, gp80 oligomers resisted sterol sequestration and were partially dissociated with Triton X-100, suggesting that compartmentalization in rafts was not solely responsible for their formation. The trans-dimer known to mediate adhesion was identified, but cis-oligomerization predominated and displayed greater accumulation during development. In fact, oligomerization was dependent on the level of gp80 expression and occurred among isolated gp80 extracellular domains, indicating that it was mediated by direct gp80-gp80 interactions. Rafts existed in gp80-null cells and such pre-existent membrane domains may provide optimal microenvironments for gp80 cis-oligomerization and the assembly of adhesion complexes.

Asunto(s)

Adhesión Celular/fisiología , Dictyostelium/metabolismo , Glicoproteínas de Membrana/metabolismo , Microdominios de Membrana/metabolismo , Animales , Fraccionamiento Celular , Reactivos de Enlaces Cruzados/farmacología , Detergentes/química , Dictyostelium/citología , Dictyostelium/crecimiento & desarrollo , Metabolismo de los Lípidos , Lípidos/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Microdominios de Membrana/química , Microdominios de Membrana/efectos de los fármacos , Modelos Biológicos , Octoxinol/química , Polímeros/química , Polímeros/metabolismo , Esteroles/metabolismo

RESUMEN

Tumor metastasis involves many stage-specific adhesive interactions. The expression of several cell adhesion molecules, notably the integrin alpha(v)beta(3), has been associated with the metastatic potential of tumor cells. In this study, we used a novel in vitro assay to examine the role of alpha(v)beta(3) in the transmigration of melanoma cells through a monolayer of human lung microvascular endothelial cells. Confocal microscopy revealed the presence of the integrin alpha(v)beta(3) on melanoma membrane protrusions and pseudopods penetrating the endothelial junction. alpha(v)beta(3) was also enriched in heterotypic contacts between endothelial cells and melanoma cells. Transendothelial migration of melanoma cells was inhibited by either a cyclic Arg-Gly-Asp peptide or the anti-alpha(v)beta(3) monoclonal antibody LM609. Although both platelet endothelial cell adhesion molecule-1 and L1 are known to bind integrin alpha(v)beta(3), only L1 serves as a potential ligand for alpha(v)beta(3) during melanoma transendothelial migration. Also, polyclonal antibodies against L1 partially inhibited the transendothelial migration of melanoma cells. However, addition of both L1 and alpha(v)beta(3) antibodies did not show additive effects, suggesting that they are components of the same adhesion system. Together, the data suggest that interactions between the integrin alpha(v)beta(3) on melanoma cells and L1 on endothelial cells play an important role in the transendothelial migration of melanoma cells.

Asunto(s)

Movimiento Celular , Endotelio Vascular/metabolismo , Melanoma/metabolismo , Melanoma/patología , Glicoproteínas de Membrana/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Receptores de Vitronectina/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Eliminación de Gen , Humanos , Complejo de Antígeno L1 de Leucocito , Pulmón/irrigación sanguínea , Melanoma/genética , Microscopía Confocal , Metástasis de la Neoplasia , Oligopéptidos/farmacología , Seudópodos/metabolismo , Receptores de Vitronectina/genética , Factores de Tiempo , Células Tumorales Cultivadas

RESUMEN

Treatment of PC12 cells with nerve growth factor induces their differentiation into sympathetic neuron-like cells and the concomitant expression of the neural cell adhesion molecule L1, a member of the Ig superfamily. To investigate the mechanism of L1-stimulated neurite outgrowth in PC12 cells, substrate-immobilized fusion proteins containing different extracellular domains of L1 were assayed for their neuritogenic activity. Surprisingly, domain Ig2 of L1, which was previously found to contain both homophilic binding and neuritogenic activities, failed to promote neurite outgrowth. In contrast, L1-Ig6 stimulated neurite outgrowth from PC12 cells. Despite this, homotypic binding of PC12 cells was significantly inhibited by antibodies against L1-Ig2, indicating that L1-L1 binding contributed to the intercellular adhesiveness of PC12 cells, but L1-stimulated neurite outgrowth depends on heterophilic interactions. Thus, PC12 cells provide a valuable model for the study of these two distinct functions of L1. Mutagenesis of L1-Ig6 highlighted the importance of the Arg-Gly-Asp motif in this domain for neuritogenesis. Inhibition studies using cyclic Arg-Gly-Asp-containing peptide and anti-integrin antibodies suggested the involvement of alphavbeta3 integrin. Furthermore, neurite outgrowth stimulated by L1-Ig6 was inhibited by lavendustin A and the MEK inhibitor PD98059, suggesting a signaling pathway that involves tyrosine kinase activation and the mitogen-activated protein kinase cascade.

Asunto(s)

Adhesión Celular/fisiología , Glicoproteínas de Membrana/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Neuritas/fisiología , Receptores de Vitronectina/fisiología , Neoplasias de las Glándulas Suprarrenales , Animales , Adhesión Celular/efectos de los fármacos , Agregación Celular , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Cinética , Complejo de Antígeno L1 de Leucocito , Glicoproteínas de Membrana/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/farmacología , Moléculas de Adhesión de Célula Nerviosa/genética , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Células PC12 , Fenoles/farmacología , Feocromocitoma , Ratas , Receptores de Vitronectina/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección

RESUMEN

We have isolated and characterized a Triton-insoluble floating fraction (TIFF) from Dictyostelium. Ten major proteins were consistently detected in TIFF, and six species were identified by mass spectrometry as actin, porin, comitin, regulatory myosin light chain, a novel member of the CD36 family, and the phospholipid-anchored cell adhesion molecule gp80. TIFF was enriched with many acylated proteins. Also, the sterol/phospholipid ratio of TIFF was 10-fold higher than that of the bulk plasma membrane. Immunoelectron microscopy showed that TIFF has vesicular morphology and confirmed the association of gp80 and comitin with TIFF membranes. Several TIFF properties were similar to those of Dictyostelium contact regions, which were isolated as a cytoskeleton-associated membrane fraction. Mass spectrometry demonstrated that TIFF and contact regions shared the same major proteins. During development, gp80 colocalized with F-actin, porin, and comitin at cell-cell contacts. These proteins were also recruited to gp80 caps induced by antibody cross-linking. Filipin staining revealed high sterol levels in both gp80-enriched cell-cell contacts and gp80 caps. Moreover, sterol sequestration by filipin and digitonin inhibited gp80-mediated cell-cell adhesion. This study reveals that Dictyostelium TIFF has structural properties previously attributed to vertebrate TIFF and establishes a role for Dictyostelium TIFF in cell-cell adhesion during development.

Asunto(s)

Vesículas Citoplasmáticas/metabolismo , Dictyostelium/citología , Dictyostelium/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Adhesión Celular , Octoxinol

RESUMEN

Extracellular EDTA suppressed in a dose-dependent manner the phagocytosis of yeast particles by Dictyostelium discoideum cells. Activity was restored fully by the addition of Ca(2+), and partially by the addition of Mn(2+)or Zn(2+), but Mg(2+)was ineffective. The pH-sensitive, Ca(2+)-specific chelator EGTA also inhibited phagocytosis at pH 7.5, but not at pH 5, and Ca(2+)restored the inhibited phagocytosis. In contrast, pinocytosis was unaffected by EDTA. Consistent with the idea that Ca(2+)was required for phagocytosis, D. discoideum growth on bacteria was inhibited by EDTA, which was then restored by the addition of Ca(2+). It is concluded that Ca(2+)was needed for efficient phagocytosis by D. discoideum amoebae. A search for Ca(2+)-dependent membrane proteins enriched in phagosomes revealed the presence of p24, a Ca(2+)-dependent cell-cell adhesion molecule-1 (DdCAD-1) that could be the target of the observed EDTA and EGTA inhibition. DdCAD-1-minus cells, however, had normal phagocytic activity. Furthermore, phagocytosis was inhibited by EDTA and rescued by Ca(2+)in the mutant just as in wild type. Thus, DdCAD-1 was not responsible for the observed Ca(2+)-dependence of phagocytosis, indicating that one or more different Ca(2+)-dependent molecule(s) was involved in the process.

Asunto(s)

Calcio/química , Calcio/metabolismo , Dictyostelium/metabolismo , Fagocitosis/fisiología , Animales , Ácido Edético/química , Ácido Egtácico/química , Manganeso/química , Manganeso/metabolismo , Fagosomas/metabolismo , Fagosomas/ultraestructura , Pinocitosis/fisiología , Levaduras , Zinc/química , Zinc/metabolismo

RESUMEN

gp150 is a membrane glycoprotein which has been implicated in cell-cell adhesion in the postaggregation stages of Dictyostelium development. An analysis of its tryptic peptides by mass spectrometry has identified gp150 as the product of the lagC gene, which was previously shown to play a role in morphogenesis and cell-type specification. Antibodies raised against the GST-LagC fusion protein specifically recognized gp150 in wild-type cells and showed that it is missing in lagC-null cells. Immunolocalization studies have confirmed its enrichment in cell-cell contact regions. In mutant cells that lack the aggregation stage-specific cell adhesion molecule gp80, gp150 is expressed precociously. Moreover, these cells acquire EDTA-resistant cell-cell binding during aggregation, suggesting a role for gp150 in this process. Cells in which the genes encoding gp80 and gp150 are both inactivated do not acquire EDTA-resistant cell adhesion during aggregation. Strains transformed with an actin 15::lagC construct express gp150 precociously, but do not show EDTA-resistant adhesion during early development. However, vegetative cells expressing gp150 can be recruited into aggregates of 16-h lagC-null cells. These results, together with those obtained with the cell-to-substratum binding assay, indicate that gp150 mediates cell-cell adhesion via heterophilic interactions with another component that accumulates during the aggregation stage.

Asunto(s)

Dictyostelium/crecimiento & desarrollo , Dictyostelium/genética , Genes Protozoarios , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Animales , Anticuerpos Antiprotozoarios , Secuencia de Bases , Adhesión Celular/genética , Adhesión Celular/fisiología , Cartilla de ADN/genética , Dictyostelium/citología , Proteínas de la Membrana/inmunología , Mutación , Proteínas Protozoarias/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

We have examined the role of platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) during the transendothelial migration of melanoma cells using a novel in vitro system. Comparable studies have suggested the involvement of PECAM-1 in leukocyte transendothelial migration. Such studies have been confirmed using in vivo models of inflammation. These studies prompted us to examine the role of PECAM-1 in tumor cell transendothelial migration. Anti-PECAM-1 monoclonal antibodies, known to block leukocyte transendothelial migration, were tested in co-cultures of human melanoma cells seeded on a monolayer of human lung microvascular endothelial cells. None of these antibodies inhibited the transmigration of melanoma cells. Moreover, confocal microscopy revealed the dissolution of the PECAM-1 adhesion complexes in the endothelial junctions associated with melanoma cells and the lack of PECAM-1 in heterotypic contacts between transmigrating melanoma cells and adjacent endothelial cells. These data, therefore, indicate that PECAM-1 is not required for the transendothelial migration of melanoma cells.

Asunto(s)

Uniones Adherentes/inmunología , Movimiento Celular/fisiología , Endotelio Vascular/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/fisiología , Humanos , Microscopía Confocal , Células Tumorales Cultivadas

RESUMEN

A key event in cancer metastasis is the transendothelial migration of tumor cells. This process involves multiple adhesive interactions between tumor cells and the endothelium. After adhering to the surface of endothelial cells, tumor cells must penetrate the endothelial junction, which contains high concentrations of the cell adhesion molecules VE-cadherin and PECAM-1. Studies using an in vitro model system, consisting of melanoma cells which are seeded onto a monolayer of endothelial cells cultured on Matrigel, have revealed reorganization of the cytoskeleton and dynamic changes in the cell shape of both tumor and endothelial cells. The initial stages of transmigration are characterized by numerous membrane blebs protruding from the basolateral surfaces of the melanoma cells. Contact regions also show an abundance of microfilaments arising from the underlying endothelial cells. These adhesive interactions lead to the redistribution of both VE-cadherin and PECAM-1 and, consequently, a localized dissolution of the endothelial junction. The penetration of the endothelial junction is initiated by melanoma pseudopods. Despite the disappearance of VE-cadherin from the retracting endothelial junction, heterotypic contacts between the tumor cell and its surrounding endothelial cells show a high concentration of pan-cadherin staining, suggesting that transmigration of melanoma cells might yet be facilitated by interactions with another member of the cadherin family. Upon adhesion to the Matrigel, melanoma cells begin to spread and invade the matrix material, while the endothelial cells extend processes over the melanoma cells to reform the monolayer. Interestingly, the leading margins of these endothelial processes contain a high concentration ofN-cadherin. VE-cadherin and PECAM-1 reappear only when the advancing endothelial processes meet to reform the endothelial junction. Together, these observations suggest that endothelial cells actively participate in the transmigration of tumor cells and specific cadherins are involved in different steps of this complex process.

Asunto(s)

Comunicación Celular/fisiología , Endotelio Vascular/fisiología , Melanoma/fisiopatología , Melanoma/ultraestructura , Animales , Cadherinas/fisiología , Moléculas de Adhesión Celular/fisiología , Movimiento Celular/fisiología , Endotelio Vascular/ultraestructura , Humanos , Microscopía Confocal , Células Tumorales Cultivadas

RESUMEN

The cell adhesion molecule L1 plays an important role in neural development, and mutations in human L1 have been implicated in X-linked hydrocephalus and related neurological diseases. We have previously demonstrated that recombinant proteins containing the second immunoglobulin-like domain (Ig2) of L1 contain both homophilic binding and neuritogenic activities. In this report, the involvement of L1 Ig2 in cell-cell adhesion and neuritogenesis was further evaluated in cell transfection studies. Transfectants expressing intact L1 were capable of undergoing L1-dependent self-aggregation and promoting neurite outgrowth from neural retinal cells. However, both activities were abolished in transfectants expressing L1delta2, a mutant L1 with Ig2 deleted. In competition experiments, the wild-type Ig2 fusion protein inhibited L1-dependent cell aggregation, whereas an Ig2 fusion protein containing the hydrocephalus mutation R184Q did not. Oligopeptides flanking Arg184 were therefore synthesized and assayed for their effects on L1-mediated cell-cell binding and neuritogenesis. The peptide L1-A, spanning the residues His178 and Gly191, inhibited both L1- and Ig2 fusion protein-mediated homophilic binding. When neural retinal cells were cultured on substrate-coated Ig2 fusion protein, peptide L1-A also abolished L1-dependent neurite outgrowth. Substitutions of several charged residues and hydrophobic residues with alanine in peptide analogues led to the loss of inhibitory effects, suggesting that multiple amino acids might be involved in L1-L1 binding. Taken together, these results identify an L1 homophilic binding site within the sequence HIKQDERVTMGQNG of Ig2 and demonstrate the requirement of L1 homophilic binding in the promotion of neurite outgrowth.

Asunto(s)

Inmunoglobulinas/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Animales , Sitios de Unión/fisiología , Agregación Celular/efectos de los fármacos , Agregación Celular/fisiología , Línea Celular Transformada , Cricetinae , Cricetulus , Femenino , Humanos , Inmunoglobulinas/fisiología , Complejo de Antígeno L1 de Leucocito , Mutación , Neuritas/efectos de los fármacos , Neuritas/fisiología , Ovario/citología , Péptidos/síntesis química , Péptidos/farmacología , Distribución Tisular , Transfección

RESUMEN

The cell adhesion molecule L1 is a potent inducer of neurite outgrowth and it has been implicated in X-linked hydrocephalus and related neurological disorders. To investigate the mechanisms of neurite outgrowth stimulated by L1, attempts were made to identify the neuritogenic sites in L1. Fusion proteins containing different segments of the extracellular region of L1 were prepared and different neuronal cells were assayed on substrate-coated fusion proteins. Interestingly, both immunoglobulin (Ig)-like domains 2 and 6 (Ig2, Ig6) promoted neurite outgrowth from dorsal root ganglion cells, whereas neural retinal cells responded only to Ig2. L1 Ig2 contains a previously identified homophilic binding site, whereas L1 Ig6 contains an Arg-Gly-Asp (RGD) sequence. The neuritogenic activity of Ig6 was abrogated by mutations in the RGD site. The addition of RGD-containing peptides also inhibited the promotion of neurite outgrowth from dorsal root ganglion cells by glutathione S-transferase-Ig6, implicating the involvement of an integrin. The monoclonal antibody LM609 against alphavbeta3 integrin, but not an anti-beta1 antibody, inhibited the neuritogenic effects of Ig6. These data thus provide the first evidence that the RGD motif in L1 Ig6 is capable of promoting neurite outgrowth via interaction with the alphavbeta3 integrin on neuronal cells.

Asunto(s)

Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuritas , Oligopéptidos/metabolismo , Receptores de Vitronectina/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Embrión de Pollo , Ganglios Espinales/citología , Humanos , Inmunoglobulinas/química , Complejo de Antígeno L1 de Leucocito , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/genética , Neuronas/citología , Oligopéptidos/farmacología , Proteínas Recombinantes de Fusión , Retina/citología

RESUMEN

In Dictyostelium discoideum, the cadA gene encodes the cell adhesion molecule DdCAD-1, a protein of M(r) 24,000, which mediates Ca(2+)-dependent cell-cell adhesion during development. We have examined the effects of cAMP, cell-cell contact, and growth conditions on cadA expression. cadA has a unique pattern of expression, which appears to be a combination of the expression patterns of early genes and aggregation-stage genes. Expression of the cadA gene in bacterially grown cells is activated at the beginning of the developmental cycle, followed by a period of rapid DdCAD-1 accumulation. The mRNA level reaches its maximum at 9 h of development and then declines to the basal level at approximately 18 h, while the protein level remains constant after reaching its maximum at 12 h. Pulse-chase experiments have demonstrated that DdCAD-1 has a significantly longer half-life than the average cellular protein. Transcription of the cadA gene is stimulated by exogenous cAMP pulses, leading to a 3- to 5-fold increase in the transcription rate. In the fgdA mutant, which lacks a functional G alpha 2, cAMP fails to enhance cadA expression, suggesting that cAMP stimulates cadA transcription via a G protein-dependent pathway. However, inhibition of cell-cell contact has no effect on the synthesis of DdCAD-1. Growth conditions also have a major influence on cadA expression. Axenically grown cells produce a high level of cadA transcripts during vegetative growth. The mRNA level shows a steady decrease during development and is reduced to the basal level by 12 h. In contrast, the level of DdCAD-1 remains relatively high throughout development, suggesting that axenic growth affects the accumulation of cadA mRNA but not the stability of the protein. These results indicate that multiple mechanisms are involved to maintain a high level of DdCAD-1 during development.

Asunto(s)

Proteínas de Unión al Calcio/biosíntesis , Calcio/fisiología , Moléculas de Adhesión Celular/biosíntesis , Dictyostelium/metabolismo , Proteínas Fúngicas/biosíntesis , Animales , Proteínas de Unión al Calcio/genética , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/genética , Comunicación Celular/fisiología , División Celular/fisiología , Medios de Cultivo , AMP Cíclico/metabolismo , Dictyostelium/crecimiento & desarrollo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/fisiología , Mutación

RESUMEN

DdCAD-1 is a 24-kD Ca2+-dependent cell- cell adhesion molecule that is expressed soon after the initiation of development in Dictyostelium cells. DdCAD-1 is present on the cell surface as well as in the cytosol. However, the deduced amino acid sequence of DdCAD-1 lacks a hydrophobic signal peptide or any predicted transmembrane domain, suggesting that it may be presented on the cell surface via a nonclassical transport mechanism. Here we report that DdCAD-1 is transported to the cell surface via contractile vacuoles, which are normally involved in osmoregulation. Immunofluorescence microscopy and subcellular fractionation revealed a preferential association of DdCAD-1 with contractile vacuoles. Proteolytic treatment of isolated contractile vacuoles degraded vacuole-associated calmodulin but not DdCAD-1, demonstrating that DdCAD-1 was present in the lumen. The use of hyperosmotic conditions that suppress contractile vacuole activity led to a dramatic decrease in DdCAD-1 accumulation on the cell surface and the absence of cell cohesiveness. Shifting cells back to a hypotonic condition after hypertonic treatments induced a rapid increase in DdCAD-1-positive contractile vacuoles, followed by the accumulation of DdCAD-1 on the cell membrane. 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole, a specific inhibitor of vacuolar-type H+-ATPase and thus of the activity of contractile vacuoles, also inhibited the accumulation of DdCAD-1 on the cell surface. Furthermore, an in vitro reconstitution system was established, and isolated contractile vacuoles were shown to import soluble DdCAD-1 into their lumen in an ATP-stimulated manner. Taken together, these data provide the first evidence for a nonclassical protein transport mechanism that uses contractile vacuoles to target a soluble cytosolic protein to the cell surface.

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Dictyostelium/metabolismo , Vacuolas/metabolismo , 4-Cloro-7-nitrobenzofurazano/farmacología , Animales , Transporte Biológico , Proteínas de Unión al Calcio/efectos de los fármacos , Proteínas de Unión al Calcio/aislamiento & purificación , Moléculas de Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/aislamiento & purificación , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Soluciones Hipertónicas , Soluciones Hipotónicas , Vacuolas/química , Vacuolas/efectos de los fármacos

RESUMEN

Transmigration of cancer cells through the vascular endothelium (diapedesis) is a key event in tumor metastasis. To investigate mechanisms involved in diapedesis, we used laser scanning confocal microscopy to examine the distribution of cadherins of WM239 melanoma cells as they migrated through a monolayer of activated human umbilical vein endothelial cells (EC) cultured on matrigel. Cadherins, including VE-cadherin, but not N-cadherin, were enriched in contacts between EC, whereas N-cadherin, but not VE-cadherin, was found in contacts between melanoma cells. During the early stages of diapedesis, EC located below the attached melanoma cells decreased in height and VE-cadherin disappeared from the EC contact located underneath the melanoma cell. Transendothelial migration began with small melanoma cell processes penetrating the VE-cadherin-negative regions between the EC. Subsequently, melanoma cells became intercalated between EC. Despite the absence of both VE-cadherin and N-cadherin, other members of the cadherin family were present in the heterotypic contacts between EC and melanoma cells. EC surrounding the intercalated melanoma cell subsequently extended processes and spread over the melanoma cell to re-form the endothelial monolayer. Interestingly, the leading margins of these EC processes contained high levels of N-cadherin, but not VE-cadherin. VE-cadherin-rich cell-cell contacts, however, reformed between advancing endothelial processes when they met above the melanoma cell. As the melanoma cells came into contact with the underlying matrigel, they spread out and adopted a fibroblast-like morphology. Addition of anti-N-cadherin antibodies to the assay resulted in a delay in the transendothelial migration of melanoma cells. Together, these results suggest that EC actively participate in diapedesis by disassembling and reassembling VE-cadherin-rich adherens junctions, and that N-cadherin plays an important role in the transmigration of melanoma cells and the reclosure of the endothelium.

Asunto(s)

Cadherinas/fisiología , Movimiento Celular , Endotelio Vascular/citología , Anticuerpos Monoclonales/inmunología , Antígenos CD , Adhesión Celular , Comunicación Celular , Humanos , Uniones Intercelulares , Melanoma , Células Tumorales Cultivadas

RESUMEN

To determine events that lead to the formation of intercellular contacts, we examined the spatial and temporal distribution of NCAM, cadherins, and F-actin in TM4 cells by immunofluorescence and laser scanning confocal microscopy. TM4 cells exhibited epithelioid characteristics and formed large overlapping lamella-like cell-cell contacts that contained a high concentration of NCAM. NCAM-rich lamellae formed from smaller NCAM patches at the ends of filopodia-like contacts between adjacent cells. Cadherins, as visualized by a pan-cadherin antibody, were present in a pattern distinctly different from that of NCAM. Although in filopodia-like contacts, both cadherins and NCAM were often concentrated at filopodial tips, in the larger lamella-like contacts that developed later, cadherins were located in an irregular punctate pattern only at the distal and more apical margins of the slanted NCAM-rich contact regions. Patterns of NCAM and microfilament (MF) bundle distribution were distinctly different, suggesting that the ends of these MF bundles were not physically linked to NCAM. By contrast, cadherins were concentrated at the ends of MF bundles at all stages of contact formation examined. Interestingly, this association of cadherins with MF bundles was mostly seen at the edge of the overlapping processes. In the lower cell process, MF bundles at the contact site were often arranged in random fashion, indicating an asymmetric distribution of MF in the junctional region. However, N-cadherin was enriched only at sites where MF bundles from both the upper and lower cell processes were aligned and terminated at the junctional membrane. Thus the organization of the actin cytoskeleton at cell-cell contact sites is influenced by the differential localization of different cadherins. These data also suggest that different mechanisms are involved in the accumulation of NCAM and cadherins in cell-cell contact regions.

Asunto(s)

Citoesqueleto de Actina/fisiología , Cadherinas/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Células de Sertoli/fisiología , Animales , Comunicación Celular , Línea Celular , Masculino , Ratones , Vinculina/metabolismo

RESUMEN

The neural cell adhesion molecule L1 has been shown to function as a homophilic ligand in a variety of dynamic neurological processes. Here we demonstrate that the sixth immunoglobulin-like domain of human L1 (L1-Ig6) can function as a heterophilic ligand for multiple members of the integrin superfamily including alphavbeta3, alphavbeta1, alpha5beta1, and alphaIIbbeta3. The interaction between L1-Ig6 and alphaIIbbeta3 was found to support the rapid attachment of activated human platelets, whereas a corresponding interaction with alphavbeta3 and alphavbeta1 supported the adhesion of umbilical vein endothelial cells. Mutation of the single Arg-Gly-Asp (RGD) motif in human L1-Ig6 effectively abrogated binding by the aforementioned integrins. A L1 peptide containing this RGD motif and corresponding flanking amino acids (PSITWRGDGRDLQEL) effectively blocked L1 integrin interactions and, as an immobilized ligand, supported adhesion via alphavbeta3, alphavbeta1, alpha5beta1, and alphaIIbbeta3. Whereas beta3 integrin binding to L1-Ig6 was evident in the presence of either Ca2+, Mg2+, or Mn2+, a corresponding interaction with the beta1 integrins was only observed in the presence of Mn2+. Furthermore, such Mn2+-dependent binding by alpha5beta1 and alphavbeta1 was significantly inhibited by exogenous Ca2+. Our findings suggest that physiological levels of calcium will impose a hierarchy of integrin binding to L1 such that alphavbeta3 or active alphaIIbbeta3 > alphavbeta1 > alpha5beta1. Given that L1 can interact with multiple vascular or platelet integrins it is significant that we also present evidence for de novo L1 expression on blood vessels associated with certain neoplastic or inflammatory diseases. Together these findings suggest an expanded and novel role for L1 in vascular and thrombogenic processes.

Asunto(s)

Plaquetas/fisiología , Adhesión Celular , Endotelio Vascular/fisiología , Inmunoglobulinas/química , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/fisiología , Adulto , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Endotelio Vascular/citología , Humanos , Cinética , Complejo de Antígeno L1 de Leucocito , Fusión de Membrana , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Oligopéptidos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/biosíntesis , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/biosíntesis , Transfección

RESUMEN

Dictyostelium discoideum cells express DdCAD-1, a Ca(2+)-dependent cell-cell adhesion molecule, soon after the initiation of development. DdCAD-1 is a soluble protein which shares a significant degree of sequence similarity with E-cadherin. Laser scanning confocal microscopy of the subcellular localization of DdCAD-1 has revealed a nonrandom pattern of DdCAD-1 distribution. DdCAD-1 is present mostly as diffusely stained material in the cytoplasm during the initial stage of development. However, a drastic redistribution takes place before the onset of cell aggregation, when DdCAD-1 become localized predominantly at the cell periphery and an enrichment of DdCAD-1 occurs on membrane ruffles. A high concentration of DdCAD-1 also becomes associated with lamellipodia and filopodia, which often appear to participate in cell contact formation. Although DdCAD-1 is present in high concentrations in contact regions during early development, it disappears rapidly from these areas during cell aggregation. This redistribution is accompanied by an accumulation of the Ca(2+)-independent cell adhesion molecule gp80 in contact regions. During chemotactic migration, DdCAD-1 is present primarily on cells at the tip and on the outer margin of cell streams. In contrast, gp80 is concentrated in contact regions among cells within well-developed streams. This dynamic redistribution suggests a unique role for DdCAD-1 in the recruitment of cells into streams and in the formation of initial contacts, but it may not be required to maintain stable contacts in the presence of gp80.

Asunto(s)

Proteínas de Unión al Calcio/biosíntesis , Calcio/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Dictyostelium/crecimiento & desarrollo , Animales , Proteínas de Unión al Calcio/análisis , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/metabolismo , Agregación Celular , Comunicación Celular/fisiología , Movimiento Celular/fisiología , Dictyostelium/citología , Dictyostelium/metabolismo , Interleucina-6/análisis , Interleucina-6/metabolismo , Proteínas de la Membrana/fisiología , Microscopía Confocal , Microscopía Fluorescente , Microscopía de Contraste de Fase , Seudópodos/metabolismo , Seudópodos/ultraestructura , Propiedades de Superficie

RESUMEN

Dictyostelium discoideum expresses EDTA-sensitive cell-cell adhesion sites soon after the initiation of development, and a Ca2+-binding protein of Mr 24,000 (designated DdCAD-1) has been implicated in this type of adhesiveness. We have previously purified DdCAD-1 to homogeneity and characterized its cell binding activity (Brar, S. K., and Siu, C.-H. (1993) J. Biol. Chem. 268, 24902-24909). In this report, we describe the cloning of DdCAD-1 cDNAs. DNA sequencing revealed a single open reading frame coding for a polypeptide containing 213 amino acids. The identity of the cDNA was confirmed by amino acid sequences of two cyanogen bromide peptides. The deduced amino acid sequence of DdCAD-1 exhibits a relatively high degree of sequence similarity with members of the cadherin family and protein S of Myxococcus xanthus. Unlike the other cadherins, the carboxyl-terminal region of DdCAD-1 contains a Ca2+-binding motif. Although analyses of the sequence suggest that the polypeptide lacks a signal peptide sequence and a transmembrane domain, immunofluorescence microscopy demonstrates the association of DdCAD-1 with the ecto-surface of the plasma membrane. To investigate the structure/function relationships of DdCAD-1, glutathione S-transferase fusion proteins containing different DdCAD-1 fragments were expressed and assayed for their 45Ca2+ and cell binding activities. These studies revealed that the cell binding activity is dependent on the amino-terminal segment and not the carboxyl-terminal Ca2+-binding domain and showed additional Ca2+-binding site(s) within the amino-terminal segment.

Asunto(s)

Proteínas de Unión al Calcio/biosíntesis , Moléculas de Adhesión Celular/biosíntesis , Dictyostelium/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Cadherinas/química , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Moléculas de Adhesión Celular/química , Pollos , Clonación Molecular , ADN Complementario , ADN de Plantas/química , ADN de Plantas/metabolismo , Glutatión Transferasa/biosíntesis , Datos de Secuencia Molecular , Myxococcus xanthus , Proteína S/química , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Homología de Secuencia de Aminoácido

RESUMEN

The cell adhesion molecule L1 plays an important role in neural development. We have previously demonstrated that the second immunoglobulin-like domain (Ig2) of L1 contains both homophilic binding and neuritogenic activities (Zhao, X., and Siu, C.-H. (1995) J. Biol. Chem. 270, 29413-29421). Recently, two mutations (R184Q and H210Q) within the Ig2 region of the human L1 gene have been shown to be responsible for X-linked hydrocephalus and the related MASA (mental retardation, aphasia, shuffling gait, and adducted thumbs) syndrome. Glutathione S-transferase-Ig2 fusion proteins containing these mutations were used to evaluate their effects on L1. The homophilic binding activity of fusion proteins and their ability to promote neurite outgrowth from retinal cells were examined. The R184Q mutation led to a complete loss of both homophilic binding and neuritogenic activities, while the H210Q mutation resulted only in a partial loss. These results provide, for the first time, direct demonstration of the deleterious effects of hydrocephalus/MASA mutations on two intrinsic properties of L1.

Asunto(s)

Hidrocefalia/genética , Mutación , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuritas/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células CHO , Cricetinae , Cartilla de ADN , ADN Complementario , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Complejo de Antígeno L1 de Leucocito , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Síndrome

RESUMEN

Integrin alpha v beta 3 is distinct in its capacity to recognize the sequence Arg-Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, alpha v beta 3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca(++)-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the alpha v-integrin subunit and could be significantly inhibited by an antibody to the alpha v beta 3 heterodimer. M21 cells also displayed some alpha v beta 3-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and alpha v beta 3 was also observed to promote significant haptotactic cell migration. To map the site of alpha v beta 3 ligation we used recombinant L1 fragments comprising the entire extracellular domain of human L1. Significant alpha v beta 3-dependent adhesion and spreading was evident on a L1 fragment containing Ig-like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Ig-like domain of L1 abrogated M21 cell adhesion. We conclude that alpha v beta 3-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, alpha v beta 3 may recognize L1 in a cell-cell or cell-substrate interaction.

Asunto(s)

Moléculas de Adhesión de Célula Nerviosa/metabolismo , Receptores de Vitronectina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos , Antígenos de Superficie/metabolismo , Secuencia de Bases , Sitios de Unión , Adhesión Celular , Línea Celular , Movimiento Celular , Cartilla de ADN , Humanos , Cinética , Complejo de Antígeno L1 de Leucocito , Ligandos , Melanoma , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Moléculas de Adhesión de Célula Nerviosa/aislamiento & purificación , Oligopéptidos , Reacción en Cadena de la Polimerasa , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales Cultivadas

RESUMEN

The neural cell adhesion molecule NCAM is a member of the immunoglobulin (Ig) superfamily. NCAM can undergo homophilic binding and heterophilic interactions with cell surface components and is often concentrated at sites of intercellular contact. To investigate the molecular basis of this biased surface distribution, we examined L cell transfectants expressing wild-type or mutant forms of chick NCAM-140 by laser scanning confocal microscopy. Mutant NCAMs that lacked Ig-like domains 1, 2, 4, or 5 were preferentially localized in contact regions. However, the relative concentration of these mutant NCAMs in contact sites was substantially reduced compared with wild-type NCAM. In contrast, NCAM redistribution to intercellular contacts was abolished in cells expressing mutant NCAMs that either lacked Ig-like domain 3 or contained mutations in the homophilic binding site in this domain. In heterotypic contacts between PC12 cells and L cell transfectants, colocalization of rat NCAM and chick NCAM was again dependent on the integrity of the homophilic binding site of the NCAM expressed on L cells. These results provide evidence that homophilic binding is the main mechanism by which NCAM becomes redistributed to intercellular contacts. They also implicate a role for other Ig-like domains in the accumulation of NCAM at cell-cell contacts.

Asunto(s)

Moléculas de Adhesión de Célula Nerviosa/metabolismo , Animales , Sitios de Unión , Comunicación Celular , Células Cultivadas , Embrión de Pollo , Células L , Ratones , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Células PC12 , Ratas , Transfección