Substratum-attached Material Research Articles

Amphoterin, the 30-kDa protein in a family of HMG1-type polypeptides. Enhanced expression in transformed cells, leading edge localization, and interactions with plasminogen activation.

Amphoterin is a heparin-binding protein that is developmentally regulated in brain and functionally involved in neurite outgrowth. Unexpectedly, amphoterin has a high mobility group 1 (HMG1)-type sequence. In the present study we have expressed amphoterin cDNA in a baculovirus vector and produced antibodies against the recombinant protein and several synthetic peptides. It was found that the amphoterin cDNA encodes the 30-kDa form of the protein isolated from tissues, whereas the co-purifying 28- and 29-kDa proteins (p28 and p29) have closely related but distinct primary structures. Partial amino acid sequencing shows several local changes in the sequences of p28 and p29 compared with amphoterin, suggesting the occurrence of a multigene family that encodes at least three different HMG1-type sequences in the rat. Studies using the probes that discern amphoterin from the other HMG1-type proteins indicate a high level expression in various transformed cell lines. Immunostaining of cells with the amphoterin-specific antibodies indicates a cytoplasmic localization that becomes remarkably enriched at the leading edges in spreading and motile cells. An extracellular localization is suggested by immunostaining of nonpermeabilized cells and by a plasminogen-dependent degradation of amphoterin in the substratum-attached material of cells. Tissue-derived and recombinant amphoterins strongly enhance the rate of plasminogen activation and promote the generation of surface-bound plasmin both by tissue-type and urokinase-type plasminogen activators. The results suggest an extracellular function for amphoterin in the leading edge of various invasive cells.

Glycosaminoglycan distribution in substratum adhesion sites of aging human skin fibroblasts, including papillary and reticular subpopulations

Glycosaminoglycan (GAG) distribution has been analyzed in the adhesion sites, left substratum-bound after EGTA-mediated detachment, of various human skin fibroblast populations grown in vitro in the presence (asc +) or absence (asc −) of ascorbate. Examination of these skin fibroblasts during the EGTA treatment by scanning electron microscopy reveals that (a) asc + cells detach much more rapidly than asc − cells, but (b)asc − or asc + cells leave the same two types of structures in longterm culture-generated substratum-attached material (L-SAM) — long linear restraction fibers and “footpat-like” structures. Most of the [ 3H]glucosamine-radiolabeled polysaccharides in L-SAM were shown to be GAGs. Fibroblasts from a full-thickness skin sample from a very young patient (AG4449) have similar distributions of the GAGs in both the EGTA-suspended cell and L-SAM fractions; however, asc + cell and L-SAM fractions contain relatively more heparan sulfate than the asc − fractions. In contrast, full-thickness skin fibroblasts from an elderly patient (AG2261) generate GAG distributions in their L-SAMs (with greatly elevated levels of hyaluronate and chondroitin sulfate) that are very different from those of the cell fractions and from those of AG44449: furthermore these distributions in AG2261 fractions do not change when shifted from asc − to asc + medium. These studies led to analyses of teh two major fibroblast subsets — papillary (PAP) or reticular (RET) — that can be isolated from the dermis of a newborn infant (patient 5). The GAG distributions in the RET fractions were different from those in PAP fractions; of special note was the greater length of heparan sulfate chains from all RET fractions examined when compared to PAP fractions. There was a remarkable similarity in the GAG distributions of asc + RET fractions when compared to the full-thickness AG2261 cell fractions. In summary, these studies demonstrate that asc − or asc + “young” cells generate different GAG distributions in their substratum adhesion sites, whereas “old” cells from a full thickness skin sample do not alter their distribution when shifted from asc − to asc + (this distribution is different from that of “young” cells). Furthermore, analyses of GAGs in papillary and reticular cell fractions reveal significant differences between the two, with considerable similarity of asc + reticular fractions of the full-thickness AG2261 fibroblasts, which is consistent with the enrichment of reticular fibroblasts in the skin of aging individuals.

Localization of the gangliosides GD2 and GD3 in adhesion plaques and on the surface of human melanoma cells.

The predominant gangliosides produced by two cultured human melanoma cell lines are GD3 and/or GD2. These gangliosides were found to be cell associated and present in substratum-attached material after cell removal by EDTA. Monoclonal antibodies directed to GD2 and GD3 specified the cell-surface distribution of these gangliosides and localized them in focal adhesion plaques at the interface of cells and their substratum. These attachment sites did not represent indiscriminant membrane fragments remaining after removal of cells with EDTA, because neither melanoma-associated proteoglycan nor class I histocompatibility antigens were detected by their respective antibodies. Our data suggest that the disialogangliosides GD2 and GD3 may be involved in the interaction between human melanoma cells and solid substrata.

Turnover of heparan sulfate proteoglycans from substratum adhesion sites of murine fibroblasts.

Substratum adhesion sites from murine Balb/c SVT2 fibroblasts are enriched in heparan sulfate proteoglycans which have been implicated in mediating adhesion of these cells to a fibronectin-adsorbed tissue culture substratum. Most of the heparan sulfate isolated from newly formed adhesion sites is found covalently attached to protein as proteoglycan while a significant portion of heparan sulfate from older sites has been identified as a single-chain species. This observation suggests that there may be catabolism of the heparan sulfate proteoglycan during the "maturation" of these adhesion sites at the cell's undersurface. Zwittergent 3-12 selectively extracts the single-chain class of heparan sulfate from either newly formed or "mature" adhesion sites while leaving the proteoglycan firmly bound in these sites. In an effort to further characterize the metabolism of these proteoglycans, substratum adhesion sites were isolated at various times after the cells had been pulse-radiolabeled using radioactive sulfate and subsequently chased. Greater than 80% of the sulfate-radiolabeled material is lost from the substratum-attached material within 24-48 h. Characterization of both the Zwittergent-soluble and -resistant heparan sulfate indicated that there was an initial accumulation followed by a rapid loss of a portion of the radiolabeled heparan sulfate as the single-chain Zwittergent-soluble class. However, most of the heparan sulfate proteoglycan was lost from the adhesion sites following approximately a 4-h time lag during the chase period without going through a smaller molecular weight intermediate. The turnover properties of the heparan sulfate proteoglycan in the EGTA-detachable cells were different from those in the substratum-attached fraction of the cell. The significance of these two different mechanisms of turnover of heparan sulfate proteoglycan in adhesion sites is discussed in relation to the role of this proteoglycan in mediating adhesion processes.

Modulation of cell-associated plasminogen activator activity by cocultivation of a stem cell and its tumorigenic descendant.

The effect of the presence of one cell type on the plasminogen activator activity of another cell type was studied. The cell types, AC and D, were isolated from a rat neuroblastoma (I. Imada and N. Sueoka, Dev. Biol. 66:97-108, 1978). AC cells are stem cells capable of multipotential differentiation in vitro and have little or no cell-associated plasminogen activator activity. D cells are tumorigenic and have high levels of cell-associated plasminogen activator activity. When AC cells were cocultivated with D cells, the plasminogen activator activity of the D cells was dramatically inhibited. The presence of as few as 1,250 AC cells inhibited 70% of the plasminogen activator activity of 20,000 D cells, as determined by a highly quantitative assay. The amount of inhibition by AC cells was proportional to the number of AC cells present. At increasing numbers of AC cells and a constant number of D cells, the Vmax for the activation of plasminogen proportionately decreased and the Km remained constant, implying that AC cells did not alter the structure or concentration of plasminogen. Inhibition was not mediated by a soluble inhibitor secreted by AC cells. Rather, attachment of AC cells adjacent to D cells, i.e., cell-to-cell contact, seemed to be required for inhibition. The substratum-attached material of AC cells, that which remained on the microwell surface after removal of AC cells with EDTA, inhibited D cell plasminogen activator activity. If plasminogen activator activity is involved in metastasis, then regulation of the plasminogen activator activity of one cell type by another cell type may be involved in determining which cells in a tumor can metastasize and where secondary tumors can arise.

Adhesive responses of fibroblast and neuroblastoma cells to substrata coated with polyvalent or monoclonal antibody to fibronectin

Both polyvalent and hybridoma-produced antibodies to fibronectin (Fn) were used to ‘map’ the immunoaccessible subsets of cell surface fibronectin on virus-transformed murine fibroblast SVT2 and rat neuroblastoma B104 cells. As one approach to this end, attachment and spreading responses of cells were measured on tissue culture substrata coated with antibody or with plasma fibronectin to compare their adhesive responses. Both SVT2 and B104 cells adhere poorly to polyvalent anti-Fn-coated substrata over short time intervals, but within several hours changes occur which permit cells to attach and spread as well on anti-Fn as on Fn (post-adsorption of the anti-Fn with Fn also generates a maximal response). This adhesive response could be completely prevented by predigesting the cells with Flavobacterium heparanase, but not with chondroitinase ABC, indicating that the cell surface Fn responsible for antibody-mediated adhesion is associated with heparan sulfate proteoglycans on the cell surface. The compositions of the substratum-attached material (left bound after EGTA-mediated detachment of cells) from cells attaching to anti-Fn or Fn were analysed by SDS-PAGE and found to be identical within the same cell type for the two different substrata. Three hybridoma-produced antibodies, which recognize different determinants on Fn, generated different adhesive responses for SVT2 or B104 cells when adsorbed to the substratum. SVT2 cells adhered well to antibody no. 32-coated substrata but poorly to antibodies 92 or 136; on the other hand, B104 cells responded similarly to all three antibodies over short times of attachment but much better to no. 32 after a several hour incubation. These experiments indicate that (1) much of the cell surface fibronectin is complexed with heparan sulfate proteoglycan and is initially inaccessible to bind to polyvalent antibody on the substratum to promote adhesion; (2) the surface of neuroblastoma cells contains a fibronectin-like molecule which is important in their substratum adhesion; and (3) monoclonal antibodies are valuable tools in ‘mapping’ the orientation of cell surface molecules like fibronectin by measuring adhesive responses to antibody-coated substrata.

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4)

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca 2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cell's undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca 2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.

Isolation and characterization of a large, neurite-associated glycoconjugate from neuroblastoma cells.

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.

Proteoglycans from the substratum adhesion sites of MSV-transformed Balb/c 3T3 cells

Kirsten murine sarcoma virus-transformed Balb/c 3T3 cells (KiMSV) are highly tumorigenic and metastatic in the appropriate murine host, are loosely adherent to the tissue culture substratum, and can be readily detached from the substratum by ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid treatment leaving their adhesion sites as substratum-attached material. Both long-term culture-generated adhesion sites (L-SAM) of KiMSV cells and newly formed adhesion sites of reattaching cells (R-SAM) contain high levels of hyaluronate (HA) and chondroitin sulfate (CS) whereas the R-SAM of parental Balb/c 3T3 cells is enriched in heparan sulfate (HS). A sizable fraction of KiMSV L-SAM proteoglycans (PG) and a smaller fraction of R-SAM PG's aggregate into two size classes of supramolecular complexes, after extraction off the substratum with 4 m guanidine hydrochloride, as determined by chromatography on columns of Sepharose CL2B in several buffer systems. Isopycnic density gradient analyses under associative conditions of KiMSV L-SAM generated three classes of material—high-density G A1 which contained some HA but principally CS and HS; intermediate-density G A2 which contained only HA; and low-density G A3 which contained some HA and principally glycoprotein. R-SAM gradients contained no G A2 but a sizable amount of “low-density” HA in G A3. When centrifuged under dissociative conditions, most of G A1 and all of G A2 from L-SAM shifted to the top of the gradient, whereas most of the HS-PG in R-SAM remained at the bottom of dissociative gradients. Comparison of these analyses with previous analyses of Balb/c 3T3 extracts demonstrates that (a) KiMSV cells generate adhesion sites with different PG contents than 3T3 sites; (b) the PG's of KiMSV sites have a reduced potential to aggregate into high-molecular-weight complexes but do form intermediate-size complexes not apparent in material from 3T3 sites; (c) these data support the hypothesis that HA is important in detachment of cells from extracellular matrices; and (d) HS-PG's in newly formed adhesion sites of KiMSV cells are considerably different from sites which have “matured”, indicating that there is metabolic activity in these sites during prolonged adherence and movement of transformed cells.

Plasma fibronectin—Binding glycosaminoglycans in the substratum adhesion sites of neural tumor cells

Polysaccharide from the substratum adhesion sites of rat glioma or neuroblastoma cells was passed through plasma fibronectin-Sepharose columns to identify polysaccharides which may be potential receptors on the cell surface for binding to this adhesion-promoting factor in the substratum-bound serum layer. Substratum-attached material was enriched in polysaccharide which binds to plasma fibronectin. Biochemical analyses identified the binding polysaccharide as highly N-sulfated heparan sulfate. These results, along with other evidence on the biochemical composition of the adhesion sites of glioma and neuroblastoma cells ( L. A. Culp, R. Ansbacher, and C. Domen, 1980, Biochemistry 19, 5899–5907 ), indicate that the fibronectin-dependent adhesion of these neural tumor cells to serum-coated tissue culture substrata may be mediated by heparan proteoglycans on their surface.

Adhesion sites of neural tumor cells: Morphogenesis of substratum-attached material

An analysis by scanning electron microscopy (SEM) has been performed of the attachment, neurite outgrowth, EGTA-mediated detachment, and morphological characteristics of substratum-attached material (SAM) for non-neurite- or neurite-containing rat neuroblastoma cells growing on serum-coated plastic coverslips. Attachment is initiated by filopodial contact with the substratum and with subsequent broad spreading of the surface membrane; footpad-type adhesion sites commonly observed in fibroblasts are not apparent at the periphery of these neuronal cells. During serum starvation, neurite extension occurs by elongation into bipolar cells, membrane ruffling and filopodial extension at these polar ends, and growth cone extension over the substratum. With time, some growth cones terminate membrane ruffling and spread extensively into a footpad-like morphology. EGTA-mediated detachment occurs by cell body rounding and pulling away from small focal areas of contact between the surface membrane and the substratum. After complete detachment, two morphologically different classes of SAM are identified. Non-neurite-containing neuroblastoma cells leave large membranous pools of SAM which are rigid and raised off the substratum, revealing small focal contact areas. A second morphological class of SAM is identified in neurite-containing cultures as small pools of membranous material tightly bound to the substratum and reminiscent of the footpad SAM deposited by fibroblasts. Along with the biochemical differences noted previously for the SAMs from non-neurite- or neurite-containing cultures, these studies indicate that the adhesion between the growth cone of neurites and the serum-coated substratum is significantly different from the adhesion processes occurring between the cell body and the substratum.

Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen.

Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substratum. The adhesion deficit in these cells was not corrected by raising the concentration of divalent cations or of serum to levels 10-fold greater than those normally utilized in cell culture. However, both WT and ADv clones could adhere, spread, and attain a normal CHO morphology on substrata coated with concanavalin A or poly-L-lysine. In addition, the adhesion variants could attach to substrata coated with "footpad" material (substratum-attached material) derived from monolayers of human diploid fibroblasts or WT CHO cells. These observations suggest that the variant clones may have a cell surface defect that prevents them from utilizing exogeneous fibronectin as an adhesion-promoting ligand; however the variants seem to have normal cytoskeletal and metabolic capacities that allow them to attach and spread on substrata coated with alternative ligands. These variants should be extremely useful in studying the molecular basis of cell adhesion.

The effect of fibronectin and substratum-attached material on the spreading of chick embryo mesoderm cells in vitro

Endoblast and hypoblast tissue, dissected from early chick embryos, was explanted and cultured on glass or plastic substrata. These tissues grew rapidly to form epithelial sheets. Under the same conditions, mesoderm, dissected without the aid of dissociating agents, grew poorly. After 24 h in culture, the mesoderm explants consisted of a sparse outgrowth of fibroblast-like cells. When pieces of mesoderm were seeded onto the dorsal surface of the epithelia, however, the cells penetrated the sheet and rapidly spread on the substratum within 4 h. If the epithelial sheet was detached from the substratum and the mesoderm then seeded onto areas of substratum previously occupied by epithelium, similar rapid spreading occurred. This effect could be produced in the absence of serum. The method used to remove the epithelium (EDTA, detergent or manual dissection) did not influence the result. When the substratum-attached material (SAM) was examined by scanning electron microscopy, 2 types of material were seen. One type appeared to be the remains of detached filopodia and cytoplasmic lamellae, while the other appeared to be of extracellular origin. Both these types reacted positively by immunofluorescence using anti-fibronectin serum. SAM derived from mesoderm reacted negatively. When mesoderm was cultured in the presence of plasma fibronectin on unmodified plastic or glass, spreading was complete in 4-5 h and thus was similar to mesoderm seeded onto SAM. The morphology of mesoderm explants on SAM or in the presence of plasma fibronectin was more epithelial than on untreated substratum in normal medium. Hypoblast and endoblast cultured in the presence of anti-fibronectin serum failed to spread normally, apparently being unable to attach to the substratum. Mesoderm did not spread rapidly on SAM in the presence of this antiserum. Cycloheximide reversibly inhibited the spreading of hypoblast and endoblast, and this effect could be eliminated, at least for hypoblast, by the addition of plasma fibronectin. Covering attachment sites on the substratum with bovine serum albumin, thereby preventing the attachment of SAM or fibronectin, also inhibited spreading. It is proposed that mesoderm cells have low levels of surface fibronectin in comparison with endoblast and hypoblast, and that this results in a comparatively low adhesiveness, which is important for its morphogenetic activity within the embryo.