Adseverin modulates morphology and invasive function of MCF7 cells

Adseverin (Ads) is a Ca²⁺-dependent actin-capping and severing protein that is highly expressed in gastric, prostate and bladder cancer cells. Currently it is unknown whether Ads contributes to the subcortical actin remodeling associated with the formation of cell extensions and matrix invasion in cancer. We compared cell extension formation and matrix degradation in Ads wildtype and Ads-null MCF7 breast cancer cells generated by CRISPR/Cas9. Compared with wildtype, Ads-null cells plated on fibronectin or collagen exhibited a more circular morphology with shorter cell extensions (37% reduction on fibronectin; p < 0.001). Reconstitution of Ads in Ads-null cells restored the formation of cell extensions (p < 0.05). While cell migration on two-dimensional matrices was unchanged by Ads deletion, the formation of cell extensions across Transwell membranes was reduced (~40% reduction, p < 0.05). When plated on fibrillar collagen, compared with wildtype, Ads-null cells showed reduced expression of MT1-MMP, collagen degradation (p < 0.05) and phagocytosis of collagen-coated beads (25% reduction; p = 0.001). We conclude that Ads is involved in the formation of cell extensions and collagen degradation in MCF7 cells, which may in turn affect matrix invasion and metastasis.     

Flightless I interacts with NMMIIA to promote cell extension formation,which enables collagen remodeling

We examined the role of the actin-capping protein flightless I (FliI) in collagen remodeling by mouse fibroblasts. FliI-overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions that stained for myosin10 and fascin and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI-knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI-overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with nonmuscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1–6 capped actin filaments efficiently, whereas FliI GLD 2–6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1–6) with the rod domain of NMMIIA (k_D = 0.146 μM), whereas FliI GLD 2–6 showed lower binding affinity (k_D = 0.8584 μM). Cells expressing FliI GLD 2–6 exhibited fewer cell extensions, did not colocalize with NMMIIA, and showed reduced collagen uptake compared with cells expressing FliI GLD 1–6. We conclude that FliI interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling in fibroblasts.     

Matrix-dependent Tiam1/Rac Signaling in Epithelial Cells Promotes Either Cell–Cell Adhesion or Cell Migration and Is Regulated by Phosphatidylinositol 3-Kinase

We previously demonstrated that both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin–mediated cell–cell adhesion in epithelial Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring E-cadherin–mediated cell–cell adhesion. Here we show that the Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin 1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin–mediated cell– cell adhesion. On different collagens, however, expression of Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase– PAK protein, is similar on fibronectin or collagen I, suggesting that rather the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin–mediated cell– cell adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac.     

Fibronectin–collagen binding and requirement during cellular adhesion

Fibronectin isolated from human plasma and from the extracellular matrices of cell monolayers mediates the attachment in vitro and spreading of trypsin-treated cells on a collagen substratum. Fibronectin-dependent kinetics of cellular attachment to collagen were studied for several adherent cell types. It was shown that trypsin-treated human umbilical-cord cells, mouse sarcoma CMT81 cells, endothelial cells, and human fibroblasts from a patient with Glanzmann's disease were completely dependent on fibronectin for their attachment to collagen, whereas guinea-pig and monkey smooth-muscle cells and chick-embryo secondary fibroblasts displayed varying degrees of dependence on fibronectin for their attachment. Radiolabelled human plasma fibronectin possessed similar affinity for collagen types I, II and III from a variety of sources. The fibronectin bound equally well to the collagens with or without prior urea treatment. However, in the fibronectin-mediated adhesion assay using PyBHK fibroblasts, a greater number of cells adhered and more spreading was observed on urea-treated collagen. Fibronectin extracted from the extracellular matrix of chick-embryo fibroblasts and that purified from human plasma demonstrated very similar kinetics of complexing to collagencoated tissue-culture dishes. Fibronectin from both sources bound to collagen in the presence of 0.05–4.0m-NaCl and over the pH range 2.6–10.6. The binding was inhibited when fibronectin was incubated with 40–80% ethylene glycol, the ionic detergents sodium dodecyl sulphate and deoxycholate, and the non-ionic detergents Nonidet P-40, Tween 80 and Triton X-100, all at a concentration of 0.1%. From these results we proposed that fibronectin–collagen complexing is mainly attributable to hydrophobic interactions.     

The major outer sheath protein of Treponema denticola inhibits the binding step of collagen phagocytosis in fibroblasts

Bacterial infections contribfute to the chronicity of connective tissue lesions in part by perturbing extracellular matrix remodelling processes. We examined a novel mechanism by which the major outer sheath protein (Msp) of the spirochaete Treponema denticola disrupts matrix remodelling mediated by intracellular digestion of collagen. The initial collagen-binding step of phagocytosis was examined in human gingival fibroblasts and Rat-2 fibroblasts. Cells were pretreated with Msp or vehicle, and binding of collagen-coated beads was measured by flow cytometry. Exposure to Msp induced a dose- and time-dependent decrease in cells that bound collagen beads; the inhibition of binding was reversed by absorption with anti-Msp antibodies. Msp-treated fibroblasts remained viable but underwent actin reorganization, including the assembly of a dense meshwork of subcortical actin filaments. Shear force assays showed that Msp abrogated collagen-binding interactions in the minimal affinity range required for stable adhesion. Fluorescence microscopy and immunoblotting showed equivalent amounts of beta1 integrin associated with collagen beads bound to Msp- and vehicle-treated cells. Photobleaching experiments found a similar percentage mobile fraction of beta1 integrins recovered in bleached areas of the plasma membrane. In contrast, Msp-induced inhibition of collagen binding was reversed by beta1 integrin affinity-activating antibodies and by latrunculin B, which prevented subcortical actin assembly. We conclude that native Msp of T. denticola inhibits the binding step of collagen phagocytosis in fibroblasts by inducing subcortical actin filament assembly and restricting affinity modulation of beta1 integrins. We suggest that, like Msp, bacterial toxins that target the cytoskeleton may also perturb the signalling networks required for cellular engagement of matrix ligands.     

Collagen remodeling by phagocytosis is determined by collagen substrate topology and calcium-dependent interactions of gelsolin with nonmuscle myosin IIA in cell adhesions

We examine how collagen substrate topography, free intracellular calcium ion concentration ([Ca²⁺]_i, and the association of gelsolin with nonmuscle myosin IIA (NMMIIA) at collagen adhesions are regulated to enable collagen phagocytosis. Fibroblasts plated on planar, collagen-coated substrates show minimal increase of [Ca²⁺]_i, minimal colocalization of gelsolin and NMMIIA in focal adhesions, and minimal intracellular collagen degradation. In fibroblasts plated on collagen-coated latex beads there are large increases of [Ca²⁺]_i, time- and Ca²⁺-dependent enrichment of NMMIIA and gelsolin at collagen adhesions, and abundant intracellular collagen degradation. NMMIIA knockdown retards gelsolin recruitment to adhesions and blocks collagen phagocytosis. Gelsolin exhibits tight, Ca²⁺-dependent binding to full-length NMMIIA. Gelsolin domains G4–G6 selectively require Ca²⁺ to interact with NMMIIA, which is restricted to residues 1339–1899 of NMMIIA. We conclude that cell adhesion to collagen presented on beads activates Ca²⁺ entry and promotes the formation of phagosomes enriched with NMMIIA and gelsolin. The Ca²⁺ -dependent interaction of gelsolin and NMMIIA in turn enables actin remodeling and enhances collagen degradation by phagocytosis.     

Differential binding to dorsal and ventral cell surfaces of fibroblasts: effect on collagen phagocytosis

Matrix remodeling by phagocytic fibroblasts is essential for growth and development but the regulatory processes are undefined. We evaluated the impact of spreading on the binding step of collagen phagocytosis with a novel culture system that more closely replicates phagocytosis in vivo than previous models. 3T3 cells were plated on collagen-coated beads, thereby loading only ventral surfaces (adhesion with spreading), or were allowed to spread on collagen films and then loaded with beads on their dorsal surfaces (adhesion without spreading). Ventral surfaces bound three-fold more beads than dorsal surfaces which was accompanied by accelerated phagosomal maturation. Arp3 and cortactin, markers of the actin-associated spreading machinery, strongly accumulated around ventrally but not dorsally loaded beads, suggesting that spreading contributes to enhanced binding of ventral surfaces. Further, ventral surfaces exhibited two-fold more free alpha2beta1 integrins, the major collagen receptors. Notably, compared to cells spread on collagen substrates, spreading cells exhibited a three-fold higher alpha2beta1 mobile fraction which was correlated with limited engagement of ventral receptors by actin filaments. Thus integrin ligation by actin filaments regulates the mobility of collagen receptors which in turn mediates the enhanced binding of collagen beads on spreading surfaces.     

Fibronectin production by cultured human lung fibroblasts in three-dimensional collagen gel culture

Summary In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater retention of fibronectin in the vicinity of the producing cells.     

The effects of coating culture dishes with collagen on fibroblast cell shape and swirling pattern formation

Motile human-skin fibroblasts form macroscopic swirling patterns when grown to confluence on a culture dish. In this paper, we investigate the effect of coating the culture-dish surface with collagen on the resulting pattern, using human-skin fibroblast NB1RGB cells as the model system. The presence of the collagen coating is expected to enhance the adherence of the fibroblasts to the dish surface, and thereby also enhance the traction that the fibroblasts have as they move. We find that, contrary to our initial expectation, the coating does not significantly affect the motility of the fibroblasts. Their eventual number density at confluence is also unaffected. However, the coherence length of cell orientation in the swirling pattern is diminished. We also find that the fibroblasts cultured in collagen-coated dishes are rounder in shape and shorter in perimeter, compared with those cultured in uncoated polystyrene or glass culture dishes. We hypothesise that the rounder cell-shape which weakens the cell–cell nematic contact interaction is responsible for the change in coherence length. A simple mathematical model of the migrating fibroblasts is constructed, which demonstrates that constant motility with weaker nematic interaction strength does indeed lead to the shortening of the coherence length.Electronic supplementary materialThe online version of this article (10.1007/s10867-020-09556-3) contains supplementary material, which is available to authorized users.     

RhoA is down-regulated at cell–cell contacts via p190RhoGAP-B in response to tensional homeostasis

Breast epithelial cells cultured in three-dimensional (3D) collagen gels undergo ductal morphogenesis when the gel is compliant and they can achieve tensional homeostasis. We previously showed that this process requires down-regulation of Rho in compliant collagen gels, but the mechanism remains undefined. In this study, we find that p190RhoGAP-B, but not p190RhoGAP-A, mediates down-regulation of RhoA activity and ductal morphogenesis in T47D cells cultured in compliant 3D collagen gels. In addition, both RhoA and p190RhoGAP-B colocalize with p120-catenin at sites of cell–cell contact. The association between p190RhoGAP-B and p120-catenin is regulated by matrix compliance such that it increases in compliant vs. rigid collagen gels. Furthermore, knockdown of p120-catenin disrupts ductal morphogenesis, disregulates RhoA activity, and results in loss of p190B at cell–cell contacts. Consistent with these findings, using a RhoA-specific FRET biosensor (RhoA-FLARE.sc), we determined spatial RhoA activity to be significantly decreased at cell–cell contacts versus cell–ECM adhesions, and, of importance, spatial RhoA activity is regulated by p190B. This finding suggests that RhoA exists as an inactive pool at cell–cell contacts and is recruited to cell–ECM contacts within stiff matrices. Overall, these results demonstrate that RhoA is down-regulated at cell–cell contacts through p190RhoGAP-B, which is localized to cell–cell contacts by association with p120-catenin that is regulated by tensional homeostasis.     

Fibronectin binding site in type I collagen regulates fibronectin fibril formation

Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin- sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogenesis could be rescued by adding either type I collagen or a peptide fragment (CB.7) which contained the wild type fibronectin binding region of the alpha 1(I) chain to the cell culture. These results suggest that fibronectin fibrillogenesis in tissue culture is dependent on type I collagen synthesis, and define an important role for the fibronectin binding site in this process.     

Type XIII collagen: a novel cell adhesion component present in a range of cell–matrix adhesions and in the intercalated discs between cardiac muscle cells

Recent analysis of type XIII collagen surprisingly showed that it is anchored to the plasma membranes of cultured cells via a transmembrane segment near its amino terminus. Here we demonstrate that type XIII collagen is concentrated in cultured skin fibroblasts and several other human mesenchymal cell lines in the focal adhesions at the ends of actin stress fibers, co-localizing with the known focal adhesion components talin and vinculin. This co-occurrence was also observed in rapidly forming adhesive structures of spreading and moving fibroblasts and in disrupting focal adhesions following microinjection of the Rho-inhibitor C3 transferase into the cells, suggesting that type XIII collagen is an integral focal adhesion component. Moreover, it appears to have an adhesion-related function since cell-surface expression of type XIII collagen in cells with weak basic adhesiveness resulted in improved cell adhesion on selected culture substrata. In tissues type XIII collagen was found in a range of integrin-mediated adherens junctions including the myotendinous junctions and costameres of skeletal muscle as well as many cell–basement membrane interfaces. Some cell–cell adhesions were found to contain type XIII collagen, most notably the intercalated discs in the heart. Taken together, the results strongly suggest that type XIII collagen has a cell adhesion-associated function in a wide array of cell–matrix junctions.     

CELL POSITION-DEPENDENT RECIPROCAL FEEDBACK REGULATION OF TYPE I COLLAGEN GENE EXPRESSION IN CULTURED HUMAN SKIN FIBROBLASTS

In order to investigate possible cell positional effects on the gene expression of human dermal fibroblasts, the authors cultured the cells on non-coated polystyrene culture dishes, type I collagen-coated dishes, or collagen gels formed by type I collagen, or suspended them in type I collagen gels and measured collagen synthesis by the cells. The production rate of type I collagen was similar whether cells were cultured on non-coated polystyrene or on type I collagen-coated dishes, but it was suppressed significantly when the cells were placed within the collagen gel matrix. Time-dependent expression of genes for α1(I) and α2(I) collagen chains was measured by Northern blot analysis. A significant increase in mRNA levels for these chains was observed when the cells were cultured for three days on type I collagen-coated dishes or on collagen gels. On the other hand, a significant decrease in the mRNA levels was observed after 2 days and later, when the cells were cultured within type I collagen gel matrix. These results indicate that human dermal fibroblasts recognize their position on or in type I collagen (extracellular matrix) and respond by changing their expression patterns of type I collagen chain genes. The results of the kinetics of gene expression also suggest that upregulation and downregulation of type I collagen genes are controlled by different mechanisms.     

α2β1 Integrin Mediates Dermal Fibroblast Attachment to Type VII Collagen via a 158-Amino-Acid Segment of the NC1 Domain

Dermal fibroblasts are in apposition to type VII (anchoring fibril) collagen in both unwounded and wounded skin. The NC1 domain of type VII collagen contains multiple submodules with homology to known adhesive molecules, including fibronectin type III-like repeats and a potential RGD cell attachment site. We previously reported the structure and matrix binding properties of authentic and recombinant NC1. In this study, we examined the interaction between dermal fibroblasts and the NC1 domain of type VII collagen. We found that both recombinant and authentic NC1 vigorously promoted human fibroblast attachment. Adhesion of fibroblasts to NC1 was dose dependent, saturable, and abolished by both polyclonal and monoclonal antibodies to NC1. Cell adhesion to NC1 was divalent cation dependent and specifically inhibited by a monoclonal antibody directed against the α2 or β1 integrin subunits, but not by the presence of RGD peptides. Furthermore, the cell-binding activity of NC1 was not conformation dependent, since heat-denatured NC1 still promoted cell adhesion. Using a series of recombinant NC1 deletion mutant proteins, the cell binding site of NC1 was mapped to a 158-aa (residues 202–360) subdomain. We conclude that human dermal fibroblasts interact with the NC1 domain of type VII collagen and this cell–matrix interaction is mediated by the α2β1 integrin and is RGD independent.     

Membrane-type 1 matrix metalloproteinase regulates fibronectin assembly and N-cadherin adhesion

Fibronectin matrix formation requires the increased cytoskeletal tension generated by cadherin adhesions, and is suppressed by membrane-type 1 matrix metalloproteinase (MT1-MMP). In a co-culture of Rat1 fibroblasts and MT1-MMP-silenced HT1080 cells, fibronectin fibrils extended from Rat1 to cell–matrix adhesions in HT1080 cells, and N-cadherin adhesions were formed between Rat1 and HT1080 cells. In control HT1080 cells contacting with Rat1 fibroblasts, cell–matrix adhesions were formed in the side away from Rat1 fibroblasts, and fibronectin assembly and N-cadherin adhesions were not formed. The role of N-cadherin adhesions in fibronectin matrix formation was studied using MT1-MMP-silenced HT1080 cells. MT1-MMP knockdown promoted fibronectin matrix assembly and N-cadherin adhesions in HT1080 cells, which was abrogated by double knockdown with either integrin β₁ or fibronectin. Conversely, inhibition of N-cadherin adhesions by its knockdown or treatment with its neutralizing antibody suppressed fibronectin matrix formation in MT1-MMP-silenced cells. These results demonstrate that fibronectin assembly initiated by MT1-MMP knockdown results in increase of N-cadherin adhesions, which are prerequisite for further fibronectin matrix formation.     

Fibroblast morphogenesis on 3D collagen matrices: The balance between cell clustering and cell migration

Fibroblast clusters have been observed in tissues under a variety of circumstances: in fibrosis and scar, in the formation of hair follicle dermal papilla, and as part of the general process of mesenchymal condensation that takes place during development. Cell clustering has been shown to depend on features of the extracellular matrix, growth factor environment, and mechanisms to stabilize cell–cell interactions. In vitro studies have shown that increasing the potential for cell–cell adhesion relative to cell–substrate adhesion promotes cell clustering. Experimental models to study fibroblast clustering have utilized centrifugation, hanging drops, and substrata with poorly adhesive, soft and mechanically unstable properties. In this review, we summarize work on a new, highly tractable, cell clustering research model in which human fibroblasts are incubated on the surfaces of collagen matrices. Fibroblast clustering occurs under procontractile growth factor conditions (e.g., serum or the serum lipid agonist lysophosphatidic acid) but not under promigratory growth factor conditions (e.g., platelet-derived growth factor) and can be reversed by switching growth factor environments. Cell contraction plays a dual role in clustering to bring cells closer together and to stimulate cells to organize fibronectin into a fibrillar matrix. Binding of fibroblasts to a shared fibronectin fibrillar matrix stabilizes clusters, and fragmentation of the fibrillar matrix occurs when growth factor conditions are switched to promote cell dispersal.     

Nested collagen matrices: a new model to study migration of human fibroblast populations in three dimensions

Fibroblast-3D collagen matrix culture provides a model system to analyze cell physiology under conditions that more closely resemble tissue than conventional 2D cell culture. Previous work has focused primarily on remodeling and contraction of collagen matrices by fibroblasts, and there has been little research on migration of cell populations within the matrix. Here, we introduce a nested collagen matrix model to analyze migration of fibroblasts in 3D collagen matrices. Nested collagen matrices were prepared by embedding contracted cell-containing matrices (also called dermal equivalents) inside cell-free matrices; migration occurred from the former to the latter. Control experiments with human dermal fragments in place of dermal equivalents confirmed the reliability of the model. Human fibroblast migration in nested collagen matrices occurred after a lag phase of 8-16 h, and cells migrating out of the inner matrices were bipolar with leading dendritic extensions. Migration was myosin II, Rho kinase and metalloproteinase-dependent but did not require plasma fibronectin. Platelet-derived growth factor but not lysophosphatidic acid or serum stimulated cell migration, although all three of these physiological agonists promote matrix remodeling and contraction. The nested collagen matrix model is a relatively easy, rapid and quantitative method to measure migration of cell populations. Our studies using this model demonstrate important differences between regulation of fibroblast migration and remodeling in collagen matrices.     

Collagen phagocytosis by fibroblasts is regulated by decorin

Decorin is a small, leucine-rich proteoglycan that binds to collagen and regulates fibrillogenesis. We hypothesized that decorin binding to collagen inhibits phagocytosis of collagen fibrils. To determine the effects of decorin on collagen degradation, we analyzed phagocytosis of collagen and collagen/decorin-coated fluorescent beads by Rat-2 and gingival fibroblasts. Collagen beads bound to gingival cells by alpha2beta1 integrins. Binding and internalization of decorin/collagen-coated beads decreased dose-dependently with increasing decorin concentration (p < 0.001). Inhibition of binding was sustained over 5 h (p < 0.001) and was attributed to interactions between decorin and collagen and not to decorin-collagen receptor interactions. Both the non-glycosylated decorin core protein and the thermally denatured decorin significantly inhibited collagen bead binding (approximately 50 and 89%, respectively; p < 0.05). Mimetic peptides corresponding to leucine-rich repeats 1-3, encompassed by a collagen-binding approximately 11-kDa cyanogen bromide fragment of decorin and leucine-rich repeats 4 and 5, previously shown to bind to collagen, were tested for their ability to inhibit collagen bead binding. Although the synthetic peptide 3 alone exhibited saturable binding to collagen, neither peptides 3 nor 1 and 2 markedly inhibited phagocytosis. Leucine-rich repeat 3 bound to a triple helical peptide containing the alpha2 integrin-binding site of collagen. When collagen beads were co-incubated with peptides 3 and 4, inhibition of collagen phagocytosis (55%) was equivalent to intact native/recombinant core protein. Thus a novel collagen binding domain in decorin acts cooperatively with leucine-rich repeat 4 to mask the alpha2beta1 integrin-binding site on collagen, an important sequence for the phagocytosis of collagen fibrils.     

Regulation of thrombospondin secretion by cells in culture

Thrombospondin (TS), a 450,000 molecular weight glycoprotein, is released from α-granules of thrombin-activated platelets and is secreted and incorporated into the extracellular matrix by several cell types in culture. We have examined the effects of cell density and transformation on the production of TS in cell culture. The levels of TS, per cell, in the culture medium of endothelial cells, smooth muscle cells, and fibroblasts were greater at lower cell densities; in fibroblasts the levels of two other extracellular matrix proteins, fibronectin and collagen, were unaffected by cell density. Our evidence indicates that the higher levels of TS in the culture medium, determined for lower-density cells, were achieved by an increased secretion of the protein rather than by a reduction in degradation or incorporation into the extracellular matrix. TS production by normal and transformed Wl-38 fibroblasts was the same, although the fibronectin level in the culture medium of the transformed cells was substantially decreased. These findings suggest that the production of TS by cells in culture is regulated in a different fashion from that of fibronectin or collagen.     

Cell-surface charge and cell-surface hydrophobicity of collagen-bindingAeromonas andVibrio strains

Partitioning in aqueous polymer two-phase systems of polyethylene glycol and dextran was used to detect and compare cell-surface charge and cell-surface hydrophobicity of Aeromonas hydrophila, A. caviae, A. sobria, Vibrio cholerae, and V. anguillarum strains. These strains have cell-surface components that bound either native or thermally denatured type I collagen (i.e., a mixture of the α1+α2 chains) and gelatin immobilized on latex beads. Our goals were: (1) to compare the possible relationship between the cell-surface charge/hydrophobicity and binding to collagen and (2) to evaluate the influence of the culture media on the expression of surface properties. There was no apparent relationship between cell-surface charge, cell-surface hydrophobicity, and binding to collagen. The expression of surface properties was dependent on the culture media. There was no relationship between binding to immobilized collagen and binding to soluble ¹²⁵I-labeled collagen. Particle-agglutination reactivity differed when using various collagen-coated microbead preparations. There were general differences in the particle-agglutination reactivity when collagen-coated latex beads were prepared using different coating procedures. The negative charge and hydrophobicity of the various collagen-coated microbead preparations were also studied by partitioning in the two-phase system of polyethylene glycol and dextran. Under these conditions, the α1+α2 collagen-chain mixture covalently immobilized on carboxy-modified latex beads was less hydrophobic and negatively charged than gelatin and native collagen immobilized on the same kind of latex beads. For latex beads passively coated with collagen preparations, the α1+α2 collagen-chain mixture was more hydrophobic than gelatin and native collagen. We suggest that for screening collagen-binding among Vibrio and Aeromonas strains, a reliable and sensitive particle-agglutination assay should consider the collagen preparation and the coating procedure for the immobilization of collagen onto the latex beads. In this regard, carboxy-modified latex beads coated with an α1+α2 collagen-chain mixture gave the best results. Received: 9 January 1995 / Accepted: 30 May 1995