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Biomedical subjects

A F Horwitz

Publications and source records attributed to A F Horwitz.

At least 19 recordsLinked to original sources
PubMed1992

LBL, a novel, developmentally regulated, laminin-binding lectin.

A 190/220-kDa complex found in integrin preparations was purified, and monoclonal antibodies were raised against it. The immunoaffinity-purified complex appears to be a trimer of very similar or identical 70-kDa subunits. It is a novel extracellular matrix molecule as determined by its subunit composition, N-terminal amino acid sequence, and in vivo localization. It is distributed widely in basement membranes including those from muscle, nerve, and kidney. It is also present in connective tissue regions such as perineurium and perimysium. It has the unusual property that it is initially expressed very late in avian development near the time of hatching. This protein is found to copurified with integrin because it binds to the carbohydrate support in Sepharose. Hemagglutination assays with mono- and disaccharides show that it functions as a lectin with galactoside-binding specificity. This protein is also found to bind strongly and specifically to laminin at a site distinct from its lectin activity, but does not bind to fibronectin or type IV collagen. The protein appears to be conserved and is a common contaminant of many laminin preparations. We call this novel protein "LBL" for laminin-binding lectin.

Amino Acids↗
PubMed1992

The alpha 5 beta 1 integrin associates with a dystrophin-containing lattice during muscle development.

The organization of the alpha 5 beta 1 integrin on skeletal muscle was studied in culture and in sections from adult and embryonic tissue using monoclonal antibodies specific for the alpha 5 subunit. The alpha 5 beta 1 integrin showed changes in organization and in the molecules with which it colocalizes. On early myoblasts, possessing a fibroblast-like morphology, the alpha 5 integrin organization was indistinguishable from that on fibroblasts; it was expressed prominently and localized in numerous focal contacts around the cell periphery. In bipolar myoblasts and early myotubes, the alpha 5 integrin was expressed only weakly and localized in a small number of focal contact-like structures. As myogenesis proceeded there was an apparent increase in integrin expression and a change in organization. In addition to the focal contact-like structures that persist throughout myogenesis in vitro, a dense lattice-like structure of integrin appeared. Fibrillar fibronectin, talin, and non-muscle alpha-actinin did not colocalize with the alpha 5 beta 1 integrin in the lattice structure as they did in the focal contact-like structures. However, dystrophin, which displayed a diffuse distribution earlier, now colocalized with the alpha 5 beta 1 integrin in the punctate lattice. Coincident with the registration of myofibrils into visible sarcomeres, the prominent dense, lattice structure disappeared leaving the focal contact-like structures as the only regions of organized alpha 5 beta 1 integrin. Despite the presence of the beta 1 integrin in neuromuscular or myotendinous junctions in vivo and on myotubes in vitro, the alpha 5 beta 1 integrin was not present in either junction. These observations suggest that the alpha 5 beta 1 integrin is involved in the adhesion of muscle to the extracellular matrix, the organization of the dystrophin-containing lattice, and the organization of nascent myofibrils which emanate from the focal contact- and stress fiber-like structures in muscle. Other integrins appear to anchor myofibrils at the myotendinous and neuromuscular junctions.

Animals↗
PubMed1992

Retrovirally introduced antisense integrin RNA inhibits neuroblast migration in vivo.

We used retrovirus-mediated gene transfer to ask whether integrins are involved in the development of neuroblasts in the chicken optic tectum. Vectors were constructed with the E. coli lacZ gene in the sense orientation and beta 1 integrin sequences in the antisense orientation. Tests in culture showed that the progeny of cells infected by these vectors were identifiable by expression of LacZ and had reduced levels of beta 1 integrins on their surfaces. We then injected these vectors into optic tecta on E3, at the height of neuronal production. Clones of LacZ-positive cells were analyzed 3-9 days later, as they migrated along radial glia to form the tectal plate. Antisense sequences had little effect on the proliferation of progenitors, or on the radial stacking of their progeny in the ventricular zone (E6). However, many antisense-bearing cells accumulated in the ventricular zone and failed to migrate into the tectal plate (E7.5 and E9). At later stages (E12), few antisense-bearing cells could be found. Thus, integrin appears to be required in the migratory process, and cells that fail to engage in integrin-mediated interactions may die.

Animals↗
PubMed1992

Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal association.

Wild-type and mutant chicken integrin beta 1 subunit (beta 1c) cDNAs were expressed in NIH 3T3 cells and assayed for localization in focal adhesions of cells plated on fibronectin substrates. Focal adhesion localization in stable transfected cells was assayed by indirect immunofluorescent staining with chicken-specific anti-beta 1c antibodies. Mutant beta 1c integrins containing internal deletions of 13 amino acids adjacent to the membrane, delta 759-771, and 20 centrally located amino acids, delta 771-790, localized in focal adhesions demonstrating that sequences required for direction to focal adhesion structures were not limited to one region of the cytoplasmic domain. Point mutations revealed three clusters of amino acids which contribute to localization in focal adhesions. These three clusters or signals are: cyto-1 (764-774), cyto-2 (785-788), and cyto-3 (797-800). The 11-residue cyto-1 signal is only found on integrin beta subunit sequences, except beta 4. Four residues within this region, D764, F768, F771, and E774, could not be altered without reducing focal adhesion staining intensities, and likely form a signal that occupies one side of an alpha helix. Mutations involving two cyto-1 residues, K770 and F771, also appeared to affect heterodimer affinity and specificity. Cyto-2 (785-788,), NPIY, is an NPXY signal that forms a tight turn motif. Cyto-2 provides a structural conformation, which when perturbed by proline removal or addition, inhibits integrin localization in focal adhesions. Cyto-3 (797-800), NPKY, resembles cyto-2, however, the nonconserved proline residue can be replaced without alteration of the localization phenotype. Cyto-3, therefore, constitutes a unique integrin signal, NXXY. Both serine and tyrosine residues at positions 790 and 788, respectively, which have been implicated in integrin phosphorylation/regulation, were conservatively replaced without detectable effect on focal adhesion localization. However, acidic replacements for these amino acids reduced focal adhesion staining intensities, suggesting that phosphorylation at these sites may negatively regulate integrin function.

3T3 Cells↗
PubMed1992

Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts.

Motile chick skeletal fibroblasts adhere to a laminin substrate by means of clustered beta 1 integrins. These integrin "macroaggregates" are similar to classic focal contacts but do not appear dark under interference-reflection microscopy. They contain alpha 5 integrin and are associated with extracellular fibronectin. To study their behavior during cell movement, time-lapse, low-light video microscopy was used to image integrins on living cells tagged with a fluorescent anti-beta 1 integrin antibody. Integrin macroaggregates remain fixed with respect to the substratum, despite the fact that they fluctuate in size, density, and shape over a period of minutes. Upon detachment of the cell rear, as much as 85% of the beta 1 integrin density of a macroaggregate remains behind on the substrate, along with both alpha 5 integrin and fibronectin. Release of the cell rear does not involve cleavage of the beta 1 integrin cytoplasmic domain from the remainder of the protein. These results indicate that cell motility does not require regulated detachment of integrin receptors from the substrate. On the other hand, cytoskeletal components and a variable fraction of the integrins are carried forward with the cell during detachment, suggesting that some type of cortical disassembly process does occur. Integrin macroaggregate structures are not recycled intact after detachment of the cell rear from the substrate. They do not persist on the cell surface, nor can they be seen to be engulfed by vesicles; yet, some of the individual integrins that make up these macroaggregates are eventually transported forward by both vesicular and cell-surface routes. Antibody-tagged integrins accumulate in dense patches at the lateral edges and dorsal surface of the cell, and move forward on the cell surface. The tagged integrins also enter cytoplasmic vesicles, which move forward within the cytoplasm. Macroaggregates generally form and grow at the cell front; however, application of fluorescent antibody causes integrins to disappear from the leading edge. Therefore, it has not been possible to directly visualize the recycling of the forward moving tagged integrins into new macroaggregates at the cell front. Surprisingly, under these conditions cells move normally despite the absence of any delivery of tagged integrin to the leading edge, indicating that recycling of integrins to the lamella is not required for apparently normal motility.

Animals↗
PubMed1992

Developmentally regulated expression of alpha 6 integrin in avian embryos.

The distribution pattern of the avian alpha 6 integrin subunit was examined during early stages of development. The results show that this subunit is prevalent in cells of the developing nervous system and muscle. alpha 6 is first observed on neuroepithelial cells of the cranial neural plate and trunk neural tube. With time, immunoreactivity becomes prominent near the lumen and ventrolateral portions of the neural tube, co-distributing with neurons and axons, particularly notable on commissural neurons. The alpha 6 expression pattern is dynamic in the neural tube, with immunoreactivity peaking by embryonic day 6 (stage 30) and decreasing thereafter. The ventral roots and retina exhibit high levels of immunoreactivity throughout development. In the peripheral nervous system, alpha 6 immunoreactivity first appears on a subpopulation of sympathoadrenal cells around the dorsal aorta and later in the dorsal root ganglia shortly after gangliogenesis. Immunoreactivity appears on prospective myotomal cells as the somites delaminate into the dermomyotome and sclerotome, remaining prominent on myoblasts and differentiated muscle at all stages. The mesonephros also has intense immunoreactivity. In the periphery, alpha 6 immunoreactive regions often in proximity to laminin, which is thought to be the ligand of alpha 6 beta 1 integrin.

Animals↗
PubMed1991

Down-regulation of the chicken alpha 5 beta 1 integrin fibronectin receptor during development.

We have characterized the diversity of the chicken beta 1 integrin family and studied the expression of individual receptors during development. The diversity of the beta 1 integrin family was investigated by affinity purifying the beta 1 integrins from a variety of adult and embryonic tissues. These purifications reveal the relative levels of expression and also the differential expression of the alpha subunits in those tissues. Monoclonal antibodies were generated against the prominent 'band 1' of the embryonic chicken integrins and used to characterize the expression of this alpha subunit in embryonic and adult tissues. This alpha subunit is shown to be the chicken homologue of human alpha 5 fibronectin receptor. The chicken alpha 5 beta 1 integrin is the most prominent beta 1 integrin in the embryo and is expressed on the majority of cell types through the day 17 stage. The distribution of this receptor in the embryo closely parallels the distribution of its ligand, fibronectin. In adult tissues, expression of this receptor is greatly diminished relative to the expression of other alpha subunits. The cell type distribution is highly restricted: limited primarily to the vasculature and to connective tissue regions. These studies reveal a prominent role for the alpha 5 beta 1 integrin in embryonic cell types and a down-regulation of this receptor on many cell types during development.

Animals↗
PubMed1991

Expression of chicken integrin beta 1 subunit in rat PC12 cells.

We transfected rat pheochromocytoma (PC12) cells with a cDNA encoding chicken integrin beta 1 subunit. The chicken integrin beta 1 subunit produced in stable transfectants associated with two major alpha subunits of rat integrins to form interspecific chimeric receptors. These receptors mediated cell spreading and initial neurite outgrowth on laminin as did corresponding endogenous integrins, although they were slightly less effective in inducing cell adhesion to laminin. These results indicate that chicken integrin beta 1 may functionally substitute for beta 1 subunit of rat integrins in PC12 cells. Apparently, the structure of the integrin beta 1 subunit is highly conserved in the evolution of these species.

Adrenal Gland Neoplasms↗
PubMed1991

Repression of integrin beta 1 subunit expression by antisense RNA.

A quail cell line (QT6-c) was co-transfected with pTEX vector expressing RNA complementary to chicken integrin beta 1 subunit mRNA (Anti-Int) and pRSVneo vector by a calcium phosphate method. Transfectants showing reduced expression of quail integrin beta 1 subunit were selected with an immunoblot assay, and a few positive clones were examined in detail. Northern blot and immunoblot analyses revealed that the Anti-Int caused a clear reduction of the transcript encoding integrin beta 1 subunit depending on culture conditions. The number of cell surface integrins also decreased in proportion to the decrement of the total amount of integrin beta 1 subunits. When one transfectant (QA23) was cultured in a serum-free medium, cell shape changed from fibroblast-like to neuron-like morphology accompanied by a low growth rate, and the cells did not form focal contact on fibronectin. A similar morphological change occurred in QT6-c cells when the cells were infected with Rous Sarcoma virus, which could produce the Anti-Int. The QA23 cells did not attach to fibronectin as efficiently as did the original QT6-c cells. These data suggest that reduced expression of integrin beta 1 subunit affects cell growth as well as cell morphology by disordering the interaction between integrins and matrix proteins and/or cytoplasmic proteins.

Animals↗
PubMed1989

Integrin on developing and adult skeletal muscle.

Avian integrin is a complex of integral membrane glycoproteins that appears to function as a dual receptors for both intracellular cytoskeletal and extracellular matrix components. Antibodies were raised against this complex and used to (1) immunolocalize integrin on cryosections of developing and adult muscle tissue and on developing myotube cultures in vitro and (2) immunoaffinity purify integrin from various fiber-type specific muscles. Integrin localization was compared with that of its putative cytoskeletal-associated and extracellular matrix ligands, talin and vinculin and fibronectin and laminin, respectively. The goal was to identify putative sites of interaction between the muscle sarcolemma and the cytoskeleton and the extracellular matrix and to reveal any differences in the molecular composition at these sites. Integrin's distribution on the sarcolemma of early (Day 12) embryonic limb muscle was random and punctate. On late embryonic (Days 17-19) limb muscle tissue its distribution was generally uniform but with occasional increased densities at specific sites along the sarcolemma. Posthatch (greater than 3 weeks) fast twitch muscle showed a highly regionalized distribution. These regions of integrin concentration coincided with densities of acetylcholine receptors, revealed by TRITC alpha-bungarotoxin labeling, and regions of muscle-tendon interaction, identified by morphological criteria. Tissue culture studies also demonstrated integrin densities at analogous sites in vitro, e.g., acetylcholine receptor clusters and sites at which myofibrils terminate at the sarcolemma. These integrin-rich sites were also shown to be Triton X-100 insoluble and therefore presumably are linked to the cytoskeleton or extracellular matrix. The localization of integrin on developing and adult muscle tissue was compared with that of fibronectin, laminin, vinculin, and talin using double, immunofluorescently labeled cryosections. In general, integrin did not colocalize exclusively with any one of its putative ligands. In the embryo, discrete densities of both talin and vinculin were observed at the myotendinous junction, whereas integrin immunoreactivity was widely distributed on muscle, vasculature, nerve, and connective tissue with no discernible sites of increased density. Laminin was primarily associated with muscle and nerve whereas fibronectin was prominent on connective tissue. On posthatch tissue, the distributions of talin, vinculin, laminin, and fibronectin were similar to those in the embryo, whereas the distribution of integrin was restricted to specific sites. The distribution of integrin was also examined for fiber-type specific differences on adu

Animals↗
PubMed1988

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.

Animals↗
PubMed1987

Integrin, the cell surface receptor for fibronectin and laminin, expresses the L2/HNK-1 and L3 carbohydrate structures shared by adhesion molecules.

On the basis of recent evidence that the carbohydrate structures designated L2/HNK-1 and L3 are shared by several neural adhesion molecules including L1, N-CAM, the myelin-associated glycoprotein and J1, we have suggested that other members of the L2/HNK-1 and L3 families are adhesion molecules. Here we show by Western blot analysis that integrin, the cell surface receptor for the extracellular matrix constituents fibronectin and laminin in chicken, also expresses the L2/HNK-1 and L3 epitopes and thus is another family member proven to be involved in adhesion.

Animals↗
PubMed1987

Cell surface receptors for extracellular matrix molecules.

Table 2 lists most of the extracellular matrix and related receptors identified to date. The wide range of binding affinities of these receptors for their ligands may be important to their function. The affinity of integrins for fibronectin is moderate, with a dissociation constant in the micromolar range. This affinity level leads to relatively rapid dissociation and reformation of receptor-ligand complexes. Thus changes in component concentration can shift binding equilibria within minutes (the time scale of many biologic phenomena) and change the number or organization of adhesive complexes. This type of interaction would be useful in motile cells, in which adhesions must form and dissociate rapidly. In contrast, the affinity of the 68-kDa laminin receptor for its ligand is three orders of magnitude higher. Such levels of affinity would be useful in stabilizing tissue. Members of the integrin family appear to recognize an RGD sequence on the ligands to which they bind. Since there are many ligands containing the RGD sequence, the question of specificity arises. Avian integrin shows little specificity and appears to behave as a multifunctional, promiscuous receptor for extracellular matrix molecules. Figure 1 summarizes our current view of the structural and functional features of avian integrin. In contrast, the mammalian receptors for vitronectin and fibronectin are specific for their respective ligands. More than one of these receptors may be present simultaneously on a cell surface, e.g. fibroblasts express receptors for fibronectin, laminin, and vitronectin at the same time. This multiplicity of receptors provides potential mechanisms for generating the adhesive differences among cells believed to play a prominent role in morphogenesis. Further adhesive differences may stem from the formation of different combinations of various alpha- and beta-subunits expressed in the cell. The mechanism of regulation of adhesive interactions with the extracellular matrix is only beginning to be explored. There are several levels at which this regulation might occur. Integrin appears to be more regionalized in more developed cells that are integral parts of tissue structures. Changes in receptor distribution could alter the relative strength of adhesive interactions. In certain instances, avian integrin disappears, or its concentration is reduced, e.g. during the development of embryonic lung (Chen et al 1986) and erythroid cells (Patel & Lodish 1985). Post-translational modifications provide yet another mechanism for regulating integrin-ligand binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Integrin, a transmembrane glycoprotein complex mediating cell-substratum adhesion.

A monoclonal antibody, CSAT, which inhibits the adhesion of chick cells to substrata coated with fibronectin, laminin and vitronectin, has been used to identify a cell surface receptor required for cell-substratum adhesion. This receptor, termed integrin, is found on the ventral surface of cells in close contact adhesion sites, at the periphery of adhesion plaques and beneath stress fibres. It is a heterodimer consisting of non-covalently linked alpha and beta subunits. Integrin binds directly to laminin, fibronectin and vitronectin with dissociation constants in the micromolar range. The binding of integrin to matrix molecules is sensitive to peptides carrying the cell-binding sequence Arg-Gly-Asp and requires heteromeric integrity. Integrin also binds directly to the cytoskeleton-associated protein talin. Thus, integrin has the properties of a transmembrane molecule capable of bringing extracellular matrix and cytoskeleton-associated molecules in proper juxtaposition to form adhesion structures. The integrin beta subunit is phosphorylated following Rous sarcoma virus transformation. Phosphorylation alters the ability of the receptor to bind extracellular matrix molecules as well as talin, suggesting a mechanism for the alteration of cellular adhesive and morphological properties following malignant transformation. A major phosphorylation site is on the cytoplasmic domain of the beta subunit. Synthetic peptides homologous with this region of integrin inhibit integrin-talin binding. The gene for the beta subunit of integrin has been sequenced. Its structure is consistent with the membrane-spanning properties of the receptor. Integrin is structurally and serologically related to adhesion receptors from mammalian tumour cells, fibroblasts, platelets and lymphocytes. It appears to be a member of a supergene family of receptors involved in cellular adhesive interactions. Antibody and peptide inhibition experiments have suggested a role for integrin and integrin-like molecules in cell migration, neurite extension, neural differentiation, histogenesis and embryonic development in Drosophila. Thus, integrin appears representative of a set of evolutionarily conserved, biologically important adhesive molecules.

Animals↗
PubMed1986

Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin.

We describe the isolation, characterization, and sequence of cDNA clones encoding one subunit of the complex of membrane glycoproteins that forms part of the transmembrane connection between the extracellular matrix and the cytoskeleton. The cDNA sequence encodes a polypeptide of 89 kd that has features strongly suggesting the presence of a large N-terminal extracellular domain, a single transmembrane segment, and a small C-terminal cytoplasmic domain. The extracellular domain contains a threefold repeat of a novel 40 residue cysteine-rich segment, and the cytoplasmic domain contains a tyrosine residue that is a potential site for phosphorylation by tyrosine kinases. We propose the name integrin for this protein complex to denote its role as an integral membrane complex involved in the transmembrane association between the extracellular matrix and the cytoskeleton.

Actins↗
PubMed1986

Integrin (the CSAT antigen): functionality requires oligomeric integrity.

Integrin, the cell-substrate attachment (CSAT) antigen, is a complex of integral membrane glycoproteins whose apparent function is to mediate cell-substratum adhesion by serving as a transmembrane link between the extracellular matrix and elements of the cytoskeleton. Previous attempts to separate the members of this complex under nondenaturing conditions have been successful. We have now produced a monoclonal antibody "G" that is specific for the lower molecular mass cysteine-rich band 3 of the complex. Using an antibody affinity column containing this monoclonal antibody, it is possible to dissociate integrin into two fractions, one containing band 3, the other containing bands 1 plus 2. Neither fraction will by itself bind fibronectin, laminin, or talin. However, when the fractions are combined, the reconstituted integrin elutes from a gel filtration column in the same position as the native complex, and binding activity to these molecules returns. Further, it is shown by gel filtration that the recognition site for the adhesion-disrupting monoclonal antibodies CSAT and JG22 is on band 3, supporting the contention that integrin is an oligomer. The data presented here is consistent with integrin being either a mixture of heterodimers, each with a common subunit and reacting with a particular extracellular matrix molecule, or a single heterotrimer capable of binding to several different extracellular matrix molecules.

Animals↗
PubMed1986

The participation of a putative cell surface receptor for laminin and fibronectin in peripheral neurite extension.

We have used the CSAT (cell substrate attachment) monoclonal antibody (Mab), which is directed against a putative laminin and fibronectin receptor, to examine its role in the adhesive phenomena of neurons. This antibody was previously found to disturb the adhesion of several classes of fibroblasts and muscle. Here we report its effects upon neuronal-substrate adhesion. Two sources of neurons were investigated--the dorsal root and ciliary ganglia. Both responded similarly. Neurons plated in the presence of the CSAT Mab did not adhere to the substratum and process formation was inhibited completely for at least 24-48 hr. In explant cultures, when neurons were first allowed to extend processes prior to addition of the CSAT Mab, the results depended on the particular substrate. With some substrates, the neurites bundled and detached from the substratum; with others, they retracted and regrew to form large fascicles or bundles of processes. In dissociated cultures that already had extended processes, neurites fasciculated and cell bodies aggregated in response to the presence of the CSAT Mab. The magnitude of this response varied, depending upon the substrate. The antigen was localized, using immunofluorescence, on neuronal cell bodies, axons, and growth cones. This distribution correlated with its biological effects on all parts of the neuron. The antigen was isolated from neuronal cultures by immunoaffinity purification. It migrated in the molecular weight range of 140 kDa on reducing SDS-PAGE. This antigen is very similar to that isolated from fibroblasts, which is an integral membrane glycoprotein complex. The data presented implicate the participation of the CSAT antigen in neurite extension and fasciculation.

Animals↗