Cell-substrate Attachment Research Articles

Src, ras, and rac mediate the migratory response elicited by NGF and PMA in PC12 cells.

The combination of nerve growth factor (NGF) and phorbol 12-myristate 13-acetate (PMA) rapidly induced the locomotion of PC12 cells by sequentially stimulating lamellar spreading, ruffling with pinocytosis, and polarization by retraction from the substratum. During migration, cells acquired long processes as a result of several undisrupted cell-substratum attachment points. The effect of NGF on PC12 migration was blocked by K-252a, a selective inhibitor of the trk family of receptor tyrosine kinases. When PMA was added to cells expressing pp60v-src, the cells displayed the same morphological behavior as they did with NGF and PMA addition. Activated ras only partially substituted for the effects of NGF; but, when ras was inhibited, the number of migrating cells decreased significantly due to a defect in spreading and retraction. Expression of an activated form of rac stimulated spontaneous growth of lamellipodia and enhanced cell migration in response to PMA. Expression of a dominant negative form of rac inhibited cell spreading and motility. Also, as a later effect, rac-inhibited cells extended much shorter neurites than wild type cells in response to NGF alone. These results indicate that the cytoarchitectural changes induced by NGF and PMA in PC12 cells are mediated by src, ras, and rac. Whereas ras and rac activation affect lamellipodia extension and retraction but not pinocytotic ruffling, src activation is involved in all three events.

Localization of cranin (dystroglycan) at sites of cell-matrix and cell-cell contact: recruitment to focal adhesions is dependent upon extracellular ligands.

We report that cranin (dystroglycan) can become recruited to focal adhesions of cultured rat REF 52 fibroblasts and human aortic smooth muscle cells. Within mature focal adhesions, cranin was present within the plaque region defined by beta 1 integrin, vinculin and phosphotyrosine staining, but occupied a larger domain corresponding to the terminal segments of stress fibers that was more precisely co-extensive with the cytoskeletal proteins alpha-actinin, utrophin and aciculin. When REF 52 fibroblasts were plated on different substrata in the absence of protein synthesis and secretion in serum-free medium, focal clusters of cranin readily formed within 2 hours on matrix proteins that bind cranin directly (laminin or agrin) which were maintained as the focal adhesions became mature. In contrast, cranin failed to become targeted to cell-substratum attachment sites, either at early or later times, when cells were plated on a variety of other substrata that elicit formation of focal adhesions but do not bind cranin directly (fibronectin, vitronectin, collagen type IV, or anti-beta 1 integrin antibody TS2/16). These data strongly suggest that targeting of cranin to focal adhesions was dependent upon the presence of an extracellular ligand capable of binding cranin directly. However, some cultured nonmuscle cell lines (e.g., human umbilical vein endothelial cells, NIH 3T3 and CHO cells) failed to localize cranin to focal adhesions, even when plated on laminin. Cranin was also enriched at cell-cell adherens-type junctions of human normal breast MCF-10 epithelial cells, and at growth cones of E17 rat hippocampal axons. That cranin can become targeted to sites of cell-cell and cell-substratum contact in diverse cell types supports the hypothesis that cranin may be involved in mediating or regulating cell adhesion. The absence of muscle-specific and synapse-specific proteins within fibroblasts and epithelial cells provides a different context for thinking about cranin (dystroglycan) that may aid in discerning general principles of its structure and function.

Kinetics of cell detachment: peeling of discrete receptor clusters

Clustering of cell surface adhesion receptors is an essential step in the development of focal contacts, specialized cell-substrate attachment sites where receptors are simultaneously linked to extracellular ligand and cytoskeletal proteins. Previously, we examined the effect of receptor clustering on attachment strength. Here, we employ the numerical methodology developed by Dembo and colleagues (Dembo, M., D.C. Torney, K. Saxman, and D. Hammer. 1988. Proc. R. Soc. Lond. B. 234:55-83) to investigate the kinetics of cell detachment when receptors are clustered into discrete patches. We show that the membrane peeling velocity decreases if receptors are clustered within a patch located inside the contact region. Peeling of clusters is influenced by the chemistry and mechanics of receptor-ligand bonds within the patch. Detachment is also prohibited if the applied tension equals the critical tension of the patch, unless the patch length is small compared with the boundary length over which membrane bending occurs, in which case the patch will peel. Peeling of these short patches only occurs when the mechanical stiffness of clustered bonds is within an optimal range. We compare our model predictions with experimental measurements of T lymphocyte detachment from ICAM-1 substrates. We demonstrate that if discrete patches of receptors are present, detachment occurs through intervals of slow and fast peeling, similar to the dynamics of T lymphocyte peeling, indicating that clustering of LFA-1 receptors is one possible explanation for the observed detachment kinetics in this system.

Expression and distribution of functional integrins in rat CNS glia.

In previous studies, we have reported on the expression of beta 1 integrins in type 1 astrocytes and their function in cell-substratum attachment (Tawil et al., J Cell Biol 120:261-271, 1993). Here we extend those findings by providing evidence that type 1 astrocytes express integrins of the beta 3 and possibly beta 4 subclasses and that the former (alpha v beta 3) functions in attachment by recognizing the peptide, Arg-Gly-Asp, in vitronectin. In addition, we have examined immunocytochemically the expression of beta 1 integrins on type 2 astrocytes and oligodendrocytes. The pattern of expression of integrins on these two cell types is distinct from type 1 astrocytes; most notably type 1 astrocytes but not oligodendrocytes or type 2 astrocytes express alpha 1 beta 1 heterodimers. Since type 2 astrocytes and oligodendrocytes originate from a common precursor (O-2A), the alpha 1 beta 1 heterodimer may be a functional marker which distinguishes O-2A-derived cells from those of the type 1 astrocyte lineage.

Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength

Although a biphasic dependence of cell migration speed on cell-substratum adhesiveness has been predicted theoretically, experimental data directly demonstrating a relationship between these two phenomena have been lacking. To determine whether an optimal strength of cell-substratum adhesive interactions exists for cell migration, we measured quantitatively both the initial attachment strength and migration speed of human smooth muscle cells (HSMCs) on a range of surface concentrations of fibronectin (Fn) and type IV collagen (CnIV). Initial attachment strength was measured in order to characterize short time-scale cell-substratum interactions, which may be representative of dynamic interactions involved in cell migration. The critical fluid shear stress for cell detachment, determined in a radial-flow detachment assay, increased linearly with the surface concentrations of adsorbed Fn and CnIV. The detachment stress required for cells on Fn, 3.6 +/- 0.2 x 10(-3) mu dynes/absorbed molecule, was much greater than that on CnIV, 5.0 +/- 1.4 x 10(-5) mu dynes/absorbed molecule. Time-lapse videomicroscopy of individual cell movement paths showed that the migration behavior of HSMCs on these substrates varied with the absorbed concentration of each matrix protein, exhibiting biphasic dependence. Cell speed reached a maximum at intermediate concentrations of both proteins, with optimal concentrations for migration at 1 x 10(3) molecules/micron2 and 1 x 10(4) molecules/micron2 on Fn and CnIV, respectively. These optimal protein concentrations represent optimal initial attachment strengths corresponding to detachment shear stresses of 3.8 mu dyne/micron2 on Fn and 1.5 mu dyne/micron2 on CnIV. Thus, while the optimal absorbed protein concentrations for migration on Fn and CnIV differed by an order of magnitude, the optimal initial attachment strengths for migration on these two proteins were very similar. Further, the same minimum strength of initial attachment, corresponding to a detachment shear stress of approximately 1 mu dyne/micron2, was required for movement on either protein. These results suggest that initial cell-substratum attachment strength is a central variable governing cell migration speed, able to correlate observations of motility on substrata differing in adhesiveness. They also demonstrate that migration speed depends in biphasic manner on attachment strength, with maximal migration at an intermediate level of cell-substratum adhesiveness.

The specificity of integrin-ligand interactions in cultured human renal epithelium.

Members of the beta 1 integrin family are present at the basolateral membrane of human renal tubular epithelium in vivo and at the ventral surfaces of cultured renal epithelial cells, at the sites appropriate for cell substratum adhesion. In this study we have proven that these molecules are indeed functional in mediating cell substratum attachment in normal human renal epithelium by using monoclonal antibodies to integrin alpha subunits to block initial cell attachment. The importance of arginine-glycine-aspartic acid (RGD) recognition by cell surface receptors in various extracellular ligands was also examined using synthetic peptides. RGDS peptide strongly inhibited attachment to plain plastic or fibronectin-coated substrata but had no effect on cell adhesion to laminin-coated coverslips.

A theoretical analysis for the effect of focal contact formation on cell-substrate attachment strength

For many cell types, growth, differentiation, and motility are dependent on receptor-mediated adhesion to ligand-coated surfaces. Focal contacts are strong, specialized, adhesive connections between cell and substrate in which receptors aggregate and connect extracellular ligand to intracellular cytoskeletal molecules. In this paper, we present a mathematical model to examine how focal contact formation affects cellular adhesive strength. To calculate adhesive strength with and without focal contacts, we use a one-dimensional tape peeling analysis to determine the critical tension necessary to peel the membrane. Receptor-ligand bonds are modeled as adhesive springs. In the absence of focal contacts, we derive analytic expressions for the critical tension at low and high ligand densities and show how membrane morphology affects adhesion. Then, focal contacts are modeled as cytoplasmic nucleation centers which bind adhesion receptors. The extent of adhesive strengthening upon focal contact formation depends on the elastic rigidity of the cytoskeletal connections, which determines the structural integrity of the focal contact itself. We consider two limits to this elasticity, very weak and rigid. Rigid cytoskeletal connections give much greater attachment strengths. The dependence of attachment strength on measurable model parameters is quite different in these two limits, which suggests focal contact structure might be deduced from properly performed adhesion experiments. Finally, we compare our model to the adhesive strengthening response reported for glioma cell adhesion to fibronectin (Lotz et al., 1989. J. Cell Biol. 109:1795-1805). Our model successfully predicts the observed detachment forces at 4 degrees C and yields values for the number of fibronectin receptors per glioma cell and the density of cytoskeletal connection molecules (talin) involved in receptor clusters which are consistent with measurements for other cell types. Comparison of the model with data at 37 degrees C suggests that while cytoskeletal cross-linking and clustering of fibronectin receptors significantly increases adhesion strength, specific glioma cell-substratum attachment sites possess little mechanical rigidity and detach through a peeling mechanism, consistent with the view that these sites of < or = 15 nm cell-substrate separation are precursors to fully formed, elastically rigid focal contacts.

Motion of cells sedimenting on a solid surface in a laminar shear flow

Cell adhesion often occurs under dynamic conditions, as in flowing blood. A quantitative understanding of this process requires accurate knowledge of the topographical relationships between the cell membrane and potentially adhesive surfaces. This report describes an experimental study made on both the translational and rotational velocities of leukocytes sedimenting of a flat surface under laminar shear flow. The main conclusions are as follows: (a) Cells move close to the wall with constant velocity for several tens of seconds. (b) The numerical values of translational and rotational velocities are inconsistent with Goldman's model of a neutrally buoyant sphere in a laminar shear flow, unless a drag force corresponding to contact friction between cells and the chamber floor is added. The phenomenological friction coefficient was 7.4 millinewton.s/m. (c) Using a modified Goldman's theory, the width of the gap separating cells (6 microns radius) from the chamber floor was estimated at 1.4 micron. (d) It is shown that a high value of the cell-to-substrate gap may be accounted for by the presence of cell surface protrusions of a few micrometer length, in accordance with electron microscope observations performed on the same cell population. (e) In association with previously reported data (Tissot, O., C. Foa, C. Capo, H. Brailly, M. Delaage, and P. Bongrand. 1991. Biocolloids and Biosurfaces. In press), these results are consistent with the possibility that cell-substrate attachment be initiated by the formation of a single molecular bond, which might be considered as the rate limiting step.

Cell surface oligosaccharides on Dictyostelium during development

ABSTRACT Developmental studies of the changes in protein glycosylation are useful in elucidating the role of oligosaccharides in biological events. We have used the chemical technique, hydrazinolysis, to release oligosaccharides from cell surface glycoproteins of Dictyostelium discoideum. Oligomannose type, xylose- and fucose-containing oligosaccharides were found to be present. The charged oligosaccharides contained sulphate and mannose 6-phosphate residues; no sialic acid was detected. The charged oligosaccharides also contained significant amounts of xylose, arabinose, fucose and galactose, as well as mannose and N-acetylglucosamine, which were the main constituents of the neutral glycans. By monitoring the chemical characteristics of the liberated oligosaccharides, dramatic changes in both the charge and size distribution of cell surface oligosaccharides were observed throughout the 24 h period of cell development. A comparison, however, between the neutral glycan structures of prestalk and prespore cells, over the same time frame showed no dramatic differences. Discoidin, a lectin present on the cell surface of 8 h cells, was found not to be glycosylated. Affinity chromatography using immobilised discoidin was used to probe a sugar library made from the cell surface glycoproteins of 8h cells. Discoidin was found to bind selectively an oligosaccharide with the structure Manα3(Manα6)(Xylβ2)Manβ4GlcNAc. This oligosaccharide lacks a conventional N,N′-diacetyl- chitobiose core and has only been previously observed in plant glycoproteins. Peptide-N-glycosidase F treatment of horseradish peroxidase released an identical structure, confirming that the oligosaccharide was not a degradation fragment of the hydrazine. The oligosaccharide was found to inhibit discoidin-mediated haemagglutination with a Ki of 0.75 mM, a concentration approximately 100 times lower than that for galactose. The correlation between changes in the amoebal plasma membrane oligosaccharide structures and the biological events occurring at different stages of development such as cell-cell adhesion and cell-substratum attachment suggest an important role for sugars in these processes.

Cadmium-induced multistep transformation of cultured Indian muntjac skin fibroblasts

During the past five years we have made a series of cadmium-transformed and resistant fibroblast cell lines by continuous low-level exposure to cadmium. In the present paper we describe the use of four of these lines with varying degrees of transformation to investigate the multistep nature of cadmium carcinogenesis. These include: (a) M cell, an immortal but nontransformed muntjac skin fibroblast line; (b) CCR5, a morphologically transformed and cadmium-resistant line derived from M cells after 20-months continuous exposure to small step-wise increases in cadmium; (c) SCR5, a tumorigenic line derived by selection (in the absence of cadmium) of rapidly growing CCR5 agar colonies; (d) T1, a line derived from an SCR5 tumour growing in a nude mouse. We have compared the morphological characteristics of the four cell lines using light and electron microscopy and evaluated their ability to grow in liquid culture, soft agar and nude mice. We have also examined the changes which have occurred in their cytoskeletons and extracellular matrices using fluorescent antibodies to actin, tubulin and fibronectin and related these to the strength of their cell-cell and cell-substrate attachments and to their levels of transformation and tumorigenesis. We have shown that, while some changes occur in a single step (e.g. intracellular cytoskeletal changes), others are gradual (e.g. changes in extracellular matrix, focus formation and ability to grow in soft agar). We conclude that continuous exposure to low levels of cadmium can initiate growth and structural changes which subsequently lead to cell transformation and tumorigenesis on the removal of cadmium. Though change with cadmium was slow, many of the transformed characteristics are similar to those reported for viral and chemically transformed cells.

Analysis of wound healing in an in vitro model: Early appearance of laminin and a 125×103M r polypeptide during adhesion complex formation

The adhesion complex, which plays an important role in cell-substratum attachment, consists of a cellular hemidesmosomal plaque, anchoring filaments, the basement membrane zone and anchoring fibrils. An analysis of the temporal sequence of assembly of the adhesion complex was undertaken in an in vitro model of epithelial cell wound healing by immunofluorescence and electron microscopy. A monoclonal antibody directed against a 125K (K = 10(3) Mr) polypeptide (mAbHD), bullous pemphigoid (BP) autoantibodies, antibodies directed against collagen type VII and laminin antibodies were used as markers for anchoring filaments, the hemidesmosome, anchoring fibrils and the laminin component of the basement membrane zone, respectively. Fluorescence labeling could be detected with mAbHD before labeling with BP autoantibodies or collagen type VII antibodies. Laminin fluorescence was detected at the same time as mAbHD. Furthermore, the 125K polypeptide and laminin were located extracellularly prior to the appearance of BP antigen and collagen type VII. The appearance of the hemidesmosomal plaque at the electron microscope level succeeded the localization of BP antigen in basal cells detected by immunofluorescence microscopy. No evidence for the coordinated appearance of BP antigen, collagen type VII and laminin was observed in this model. We discuss the possibility that the 125K protein and laminin may play roles in the initiation of complex formation. Furthermore, although basement membrane zone components were detected early in adhesion complex re-formation, formation of the lamina densa region of the basement membrane zone followed the appearance of the hemidesmosomal plaque, indicating a role for the hemidesmosomal plaque in organizing the structure of the lamina densa.

Microtubule formation and neurite growth in cerebellar macroneurons which develop in vitro: evidence for the involvement of the microtubule-associated proteins, MAP-1a, HMW-MAP2 and Tau

The relationship between the expression of microtubule-associated proteins (MAPs) and microtubule formation was studied in embryonic cerebellar macroneurons maintained in culture. The results obtained suggest that in these neurons high molecular weight-MAP2 (HMW-MAP2) acts as a promoter of tubulin assembly since its induction and pattern of distribution are highly correlated with the increase in microtubule mass which parallels axonal and dendritic growth; MAP-1a may have a similar role but restricted to the assembly of dendritic microtubules. On the other hand, Tau expression and accumulation follows a time course identical to that of the induction of stable microtubules; besides, at all stages of neurite differentiation and growth this protein seems to be preferentially associated with this subset of microtubules as opposed to the other MAPs, observations which suggest an important role for this protein in determining microtubule stability during axonal and dendritic elongation. Finally, the present results show that environmental stimuli are capable of regulating the expression of these MAPs; the induction of each of them varies as a function of the type of signal. Thus, while diffusable substances are able to dramatically induce HMW-MAP2, MAP-1a and Tau inductions depend on cell substrate attachment and/or cell-cell interactions.

Sodium- N-butyrate induces secretion and substrate accumulation of p52 in kirsten sarcoma virus-transformed rat kidney fibroblasts

1. 1. A group of five transformation-responsive secreted proteins, ranging in molecular mass from 31 to 70 kDa, were identified in cultured normal rat kidney (NRK) fibroblasts. 2. 2. One such protein (p52) was found to be a major secreted and substrate-attached component of NRK cells. 3. 3. Kirsten sarcoma virus-transformed NRK cells failed to accumulate p52 in either the secreted or substrate-associated protein compartments; this protein was inducible, however, in transformed cells by culture in 2mM sodium- n-butyrate. 4. 4. Kinetics of p52 induction in transformed NRK cells, relative to the time course of increased cell spreading, and its enrichment in the substrate-associated protein fraction suggest that p52 might function in cell-substrate attachment.

Polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a renal epithelial cell line.

Polarized epithelial cell monolayers contain two distinct plasma membrane domains as delineated by the presence of tight junctions--i.e., an apical surface that faces the external environment and a basolateral surface that functions both in cell-cell contact and cell-substrate attachment. Central to the understanding of epithelial cell polarity is the question of how such cell-surface specializations are generated. A different class of membrane glycoproteins has recently emerged that may yield new insight into the mechanism underlying the biogenesis of this polarity. Members of this class contain a large extracellular protein domain linked to the membrane via glycosyl-phosphatidylinositol. Using a polarized renal epithelial cell line (Madin-Darby canine kidney), we identified endogenous glycosyl-phosphatidylinositol-anchored proteins through release by a phosphatidylinositol-specific phospholipase C. Six glycosyl-phosphatidylinositol-anchored proteins of 110, 85, 70, 55, 38, and 35 kDa were identified and appeared to be restricted to the apical surface. Our data are consistent with the notion that the glycosyl-phosphatidylinositol membrane anchor may contain the necessary information for "targeting" to the apical surface.

Myoblast migration specifically inhibited in the chick embryo by grafted CSAT hybridoma cells secreting an anti-integrin antibody.

We report a teratological method in which mouse hybridoma cells are grafted into a chick host. CSAT (Cell Substratum ATtachment) hybridoma was used. It produces an antibody directed against the avian integrin complex. The grafts were performed during the second and third days of incubation either at the level of the somites or in the coelom of the chick embryo. The anomalies were revealed by means of a monoclonal antibody that recognizes myogenic cells as soon as they become committed in the myotome. When embryos were grafted at the level of the somites, body wall muscles failed to develop on the side of the graft only. After coelomic grafting, total agenesis of abdominal muscles was induced. The anomalies were specific since the engraftment of three control hybridoma clones induced no change in muscle formation. These control hybridomas produce antibodies directed against the same molecular complex but not against the same epitope as CSAT. The injection of hybridoma cells in an embryo appears as a method of general interest to determine the long-term consequences of perturbing a specific developmental process.

The new basement membrane antigen recognized by the monoclonal antibody GB3 is a large size glycoprotein: modulation of its expression by retinoic acid

Further biochemical investigations on the hemidesmosone-associated epidermal basement membrane component recognized by the monoclonal antibody GB3 are presented in this study. We previously found that the expression of this constituent is impaired in a severe genedermatosis termed lethal junctional epidermolysis bullosa. We demonstrate now that this factor is a very large glycoprotein (apparent molecular weight, 600 kDa) made up of polypeptides in the range of 93.5 to 150 kDa, and containing N-linked oligosaccharide chains. Both endo- β- N-acetylglucosaminidases and neuraminidase hydrolysis, as well as concanavalin A binding experiments were performed on the GB3 radioimmunoprecipitated peptides from cultured human keratinocytes. They showed that the antigen subunits probably bear both ‘high-mannose’ and ‘complex’ type glycosidic chains. The chronic exposure of cultured human keratinocytes to retinoic acid (10 −8 to 10 −6 M) resulted in no apparent changes in the overall bulk of these glycosidic chains, but a dose-dependent increase of synthesis and secretion of the antigen was observed. A relative induction factor of 4 was obtained in cultures treated with 10 t-6 M retinoic acid. This induction was also observed morphologically by indirect immunofluorescence at the basement membrane zone from cultured human keratinocytes grown on dead de-epidermized dermis. These results further emphasize the influence of glycoprotiens in cell-cell and cell-substratum attachment. Furthermore, the ability to modulate this antigen must be relevant for the understanding of the molecular defect involved in lethal junctional epidermolysis bullosa.

Cell-Substrate Adhesion: Induction of Cell Spreading and Apical/Basal Plasma Membrane Polarity

This chapter discusses the observations and molecular mechanisms regulating the formation of transcellular polarity of the plasma membrane (PM) induced by cell–substrate adhesion. Observations suggest that the sequence of events leading to polarity began with cell–substrate attachment during which time extracellular matrix (ECM) receptors are moved to the basal PM domain by vesicle transport from the apical PM and an internal pool of PM. To a lesser extent, proteins are moved by lateral diffusion from the apical PM. Three types of ECM receptors are thought to be in the cell: those that initiate cell spreading, those that are docking proteins acting as receptors to target vesicles to the basal PM, and those that tracked the cytoskeleton used in the trafficking of vesicles. A mechanism that appeared to be responsible for cell spreading is the clustering of the ECM-receptors in the basal PM, which contacts the substrate. Clustering the receptors dramatically increased their binding to the cytoskeleton and thereby set in motion cell spreading and the beginning of transcellular polarization.

Purification of two smooth muscle glycoproteins related to integrin. Distribution in cultured chicken embryo fibroblasts.

We have purified two membrane glycoproteins from chicken gizzard smooth muscle. In the presence of reducing agents, these proteins have molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 165,000 and 130,000, but they migrate at 165,000 and 110,000 without reduction. The two proteins can also be isolated as a complex in buffers containing physiologic salt concentrations. This complex has physical properties similar to two proteins of the integrin family of receptors for extracellular matrix proteins, the cell substratum attachment antigen from chicken embryos, and the glycoprotein IIb IIIa complex from mammalian platelets. When the smooth muscle complex is visualized by electron microscopy, it has a striking resemblance to both avian integrin and the glycoprotein IIb IIIa complex. Smooth muscle is a good source of the 165,000 and 130,000 proteins, and purification of both the individual subunits and the complex is achieved using conventional biochemical techniques. Antibodies directed against the 130,000 protein cross-react with integrin but do not cross-react with the 165,000 protein. Immunofluorescence microscopy using these antibodies reveals staining of fibroblast focal contacts and fibrillar streaks which coalign with fibronectin. Whereas monoclonal antibodies against integrin label the periphery of the focal contact more intensely than the center, the anti-130,000-protein serum stains the entire focal contact. Antibodies directed against the 165,000 protein also stain focal contacts and fibrillar streaks of fibroblasts in tissue culture. On the basis of similar physical properties, biochemical characteristics, and immunological cross-reactivity we conclude that the 165,000/130,000 complex is a smooth muscle integrin.

Cell attachment and chemotaxis can utilize the same peptide sequence of fibronectin

Fibronectin and fragments of the parent molecule are involved in cell-substrate attachment, cell migration and chemotaxis. The cell attachment sequence, Gly-Arg-Gly-Asp-Ser-Pro, was synthesized and tested for its ability to effect ligamentum nuchae fibroblast chemotaxis. This hexapeptide and fibronectin itself both caused directed cell migration, with maximal activity in the 10 −10 to 10 −9 M range. These data demonstrate that the fibronectin cell binding site and chemotaxis site share a common sequence.

Embryonic neural retinal cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT).

Cell attachment and neurite outgrowth by embryonic neural retinal cells were measured in separate quantitative assays to define differences in substrate preference and to demonstrate developmentally regulated changes in cellular response to different extracellular matrix glycoproteins. Cells attached to laminin, fibronectin, and collagen IV in a concentration-dependent fashion, though fibronectin was less effective for attachment than the other two substrates. Neurite outgrowth was much more extensive on laminin than on fibronectin or collagen IV. These results suggest that different substrates have distinct effects on neuronal differentiation. Neural retinal cell attachment and neurite outgrowth were inhibited on all three substrates by two antibodies, cell substratum attachment antibody (CSAT) and JG22, which recognize a cell surface glycoprotein complex required for cell interactions with several extracellular matrix constituents. In addition, retinal cells grew neurites on substrates coated with the CSAT antibodies. These results suggest that cell surface molecules recognized by this antibody are directly involved in cell attachment and neurite extension. Neural retinal cells from embryos of different ages varied in their capacity to interact with extracellular matrix substrates. Cells of all ages, embryonic day 6 (E6) to E12, attached to collagen IV and CSAT antibody substrates. In contrast, cell attachment to laminin and fibronectin diminished with increasing embryonic age. Age-dependent differences were found in the profile of proteins precipitated by the CSAT antibody, raising the possibility that modifications of these proteins are responsible for the dramatic changes in substrate preference of retinal cells between E6 and E12.