Fibronectin filaments and actin microfilaments are organized into a fibronexus in Dupuytren's diseased tissue.код для вставкиСкачать
THE ANATOMICAL RECORD 230:175-182 (1991) Fibronectin Filaments and Actin Microfilaments Are Organized into a Fibronexus in Dupuytren’s Diseased Tissue JAMES J . TOMASEK AND CAROL J . HAAKSMA Department of Anatomy, New York Medical College, Valhalla, New York ABSTRACT The fibronexus is a close transmembrane association between fibronectin filaments and actin microfilaments. It has been found at the surfaces of fibroblasts in tissue culture, as well as within contracting granulation tissue. This specialized connection has been proposed to play an important role in the adhesive properties of fibroblasts. The purpose of this study is to determine whether the fibronexus is present in other contracting tissues besides granulation tissue, specifically in Dupuytren’s diseased tissue. Dupuytren’s disease is a pathologic condition in which the palmar aponeurosis becomes shortened leading to irreversible flexion of the digits. Shortening of the aponeurosis is believed to be a n active cellular process. Extracellular filaments and actin microfilaments form close transmembrane associations a t the surfaces of actin-rich fibroblasts in Dupuytren’s disease. Extracellular filaments extend from the cell surface into the surrounding tissue connecting fibroblasts with collagen fibrils and adjacent cells. In this study we have used immunoelectron microscopy to demonstrate that the extracellular filaments that participate in these close transmembrane associations contain fibronectin. High voltage electron microscopy has been used to examine the three-dimensional relationships between the cytoskeleton and fibronectin filaments in Dupuytren’s diseased tissue. We propose that the fibronexus is a dominant adhesive structure at the surface of fibroblasts in Dupuytren’s diseased tissue. The fibronexus, by mediating cell-to-cell and cell-to-matrix attachments, may serve to transmit contractile forces generated by actin microfilaments in these cells throughout the diseased tissue. The extracellular glycoprotein fibronectin plays a n onstrated that fibronectin filaments and actin microimportant role in the adhesion and spread of fibroblasts filaments form a fibronexus at the surfaces of fibroin vitro (Yamada et al., 1976; Yamada and Olden, blasts in 7-9 day old granulation tissue in guinea pigs 1978; Grinnell and Feld, 1979). The interaction of fi- (Singer et al., 1984). Based upon these results, Singer bronectin with the cell surface also promotes the for- et al. (1984) postulated that the fibronexus serves to mation of bundles of actin microfilaments (stress fi- transmit contractile forces generated by fibroblasts bers) in well-spread stationary fibroblasts (Singer and throughout the granulation tissue, and thereby affects Paradiso, 1981; Hynes et al. 1982; Woods and Couch- wound contraction. man, 1988). Fibronectin and actin are interrelated a s The purpose of this study is to determine whether the demonstrated by the codistribution of extracellular fi- fibronexus is present in other contracting tissues bebronectin filaments with intracellular bundles of actin sides granulation tissue, specifically the Dupuytren’s microfilaments (Hynes and Destree, 1978). Recent diseased tissue. The presence of fibronexus-like transstudies have demonstrated the existence of a trans- membrane associations in Dupuytren’s diseased tissue membrane receptor for fibronectin (Chen et al., 1985, would suggest that this association may play a n impor1986; Tamkun et al., 1986). This receptor is a member tant role in a variety of contracting tissues. In addition, of the p l integrin family of extracellular matrix recep- its presence might help in understanding the cellular tors (Hynes, 1987). It codistributes with fibronectin filaments and actin microfilaments, suggesting that i t may indirectly link these two filament systems together across the cell membrane (Chen et al., 1985, Received March 12, 1990; accepted November 14, 1990. 1986; Tamkun et al., 1986; Singer e t al., 1988). In Address reprint requests to Dr. James J. Tomasek, Department of regions where fibronectin filaments and actin microfil- Anatomical Sciences, Biomedical Sciences Building, Rm 553, P.O. Box University of Oklahoma-Health Science Center, Oklahoma aments are codistributed they form a close transmem- 26901, City, Okla 73190. brane association, which has been termed the fibroCarol J. Haaksma’s current address is the Department of Otorhinexus (Singer, 1979, 1982). In addition to being a n nolaryngology, University of Oklahoma-Health Science Center, Oklaimportant adhesive structure in vitro, it has been dem- homa City, Okla 73190. 0 1991 WILEY-LISS, INC 176 J.J. TOMASEK AND C.J. HAAKSMA mechanism responsible for tissue contraction in this disease. Dupuytren’s disease is a pathologic condition in which the palmar aponeurosis becomes shortened leading to irreversible flexion of the digits (Luck, 1959; Chiu and McFarlane, 1978). The shortening of the palmar fascia has been proposed to be due to a n active cellular contraction of this tissue (Gabbiani and Majno, 1972; Chiu and McFarlane, 1978; Gelberman et al., 1980). Present in the contracting palmar fascia are actin-rich fibroblasts, also termed “myofibroblasts,” which contain large bundles of actin microfilaments (Gabbiani and Majno, 1972; Chiu and McFarlane, 1978; Gelberman et al., 1980). Associated with these fibroblasts is a fibronectin-rich extracellular matrix (Tomasek et al., 1986). Extracellular filaments and actin microfilaments form close transmembrane associations at the surfaces of actin-rich fibroblasts present in Dupuytren’s diseased tissue (Tomasek et al., 1987). Structurally, these close transmembrane associations resemble the fibronexus previously described in granulation tissue (Singer et al., 1984) and cultured fibroblasts (Singer, 1979). In this study we demonstrate by immunoelectron microscopy that these extracellular filaments label with anti-fibronectin antibody. We report the use of a simplified immunoelectron microscopic procedure that can maintain preservation and antigenicity and still allow good penetration of antibody into the tissue. We also used high voltage electron microscopy to examine the three-dimensional relationships of the extracellular filamentous material and actin microfilament bundles t h a t form transmembrane associations at the surfaces of actin-rich fibroblasts in Dupuytren’s diseased tissue. Extracellular filaments were observed to interconnect fibroblasts with surrounding collagen fibrils and adjacent fibroblasts confirming our earlier electron microscopic study of Dupuytren’s diseased tissue (Tomasek et al., 1987). The results from this study suggest that a fibronexus-like transmembrane association is present at the surfaces of actin-rich fibroblasts in Dupuytren’s diseased palmar fascia. Furthermore, we propose that this adhesive complex plays a n important role in transmitting contractile forces generated by fibroblasts to the surrounding tissue in Dupuytren’s disease. MATERIALS AND METHODS Tissue Palmar fascia from patients with Dupuytren’s disease was obtained at the time of operation and transported to the laboratory in sterile ice-cold balanced saline solution. The surrounding cord and morphologically normal palmar fascia were dissected away from the nodular tissue, which was defined a s a hard, fusiform thickening in the palmar fascia. Nodular tissue was cut into small pieces and processed for light microscopic immunocytochemistry (Tomasek e t al., 1986), electron mocroscopic immunocytochemistry, and high voltage electron microscopy. Because actin-rich fibroblasts are present only during the active stage of the disease (Chiu and McFarlane, 1978), we screened tissues for the presence of these cells. This was done by staining cryostat sections with anti-actin antibodies (Tomasek et al., 1986). Five tissue samples were chosen for this study based upon the presence of large numbers of actin positive cells. Electron Microscopic lmmunocytochemistry Pieces of tissue were fixed in freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 20 minutes at room temperature. The tissue was rinsed in phosphate buffer followed by 0.1 M Tris buffer, pH 7.4, to quench any free aldehydes, and then infiltrated with 30% sucrose in phosphate buffer. Tissue pieces were placed in a drop of OCT compound (LAB-TEK Products, Miles Laboratories, Naperville, IL) on a piece of aluminum foil, and rapidly frozen in stirring liquid Freon 22 (E. I. DuPont, Co., Wilmington, DE) cooled with liquid nitrogen. Tissue pieces were mounted onto metal stubs and cryosectioned a t 2-4 pm in a n IEC cryostat (Model CTI, International Equipment Company, Needham, MA). Frozen sections were transferred to phosphate buffered saline (PBS) in a 64 microwell plate and then allowed to thaw. It was found that allowing the sections to thaw in PBS rather than in air prevented extensive shrinkage of the tissue. Immunocytochemical staining was performed on sections in the microwell plate. Sections were first incubated for 3 hours a t room temperature in the primary antibody, rabbit anti-human plasma fibronectin antiserum, diluted 1:25 in PBS. Sections were rinsed three times in PBS followed by incubation for 3 hours at room temperature in secondary antibody, goat anti-rabbit IgG antibody conjugated to 10 nm colloidal gold (Janssen Life Sciences Products, Beerse, Belgium), diluted 1:5 in PBS. Sections were then rinsed three times in PBS and prepared for electron microscopy (Tomasek et al., 1987). As a control, the primary antibody was replaced with preimmune rabbit antiserum at a 1:25 dilution or PBS. Antibody Rabbit anti-human plasma fibronectin antibody raised against human plasma fibronectin was a gift from Dr. H. Godfrey (New York Medical College, Valhalla, NY). This antibody is monospecific against purified human plasma fibronectin and normal human serum on immunoelectrophoresis; i t did not react with a,-macroglobulin. When used to develop immunoblots of human plasma and purified human plasma fibronectin, this antibody detected only M, 460 kDa molecules (unreduced) or a doublet of M, 230-250 kDa (reduced) in both preparations. This antibody also cross reacts with purified fibroblast and placental fibronectin a s evaluated by immunoblots. High Voltage Electron Microscopy Tissue pieces were fixed in half-strength Karnovsky fixative (Karnovsky, 1965), post-fixed in 1% osmium tetroxide, stained with uranyl acetate, dehydrated, and embedded in Polybed 812 (Polysciences, Warrington, PA). One-half to 1 pm thick sections were cut on glass knives and picked up onto grids coated with a pale gold formvar film stabilized by the evaporation of carbon. Sections were stained with 2% aqueous uranyl acetate for 1 hour a t 37°C followed by 30-40 minutes in 0.2% lead citrate at room temperature. Thick sections were examined a t a n accelerating voltage of 1 million elec- FIBRONEXUS I N DUPUYTREN’S DISEASE tron volts using the AEI EM 7 high voltage electron microscope (NIH Biotechnology HVEM Resource, Wadsworth Center for Laboratories and Research, New York State Department of Health). RESULTS lmmunoelectron Microscopy of Transmembrane Associations in Dupuytren’s Disease It has been previously demonstrated that extracellular filaments and actin microfilaments form close transmembrane associations a t the surface of actinrich fibroblasts in Dupuytren’s diseased tissue (Tomasek et al., 1987). To determine whether the extracellular filaments contain fibronectin a n immunoelectron microscopic study of fibronectin labelling was performed using colloidal gold. The extracellular filaments were found to be rich in fibronectin (Figs. 1-3). This method of immunoelectron microscopy provides excellent visualization of the cell membrane and intracellular actin microfilaments allowing for identification of the close transmembrane association. Extracellular filaments labelled with anti-fibronectin antiserum were observed a t the surfaces of fibroblasts in Dupuytren’s diseased tissue (Figs. 1-3). These labelled filaments were present at the cell surface forming close transmembrane associations with actin-rich fibroblasts. Labelled extracellular filaments were observed to extend from the cell surface into the surrounding collagen-rich extracellular matrix (Fig. 1).Labelled extracellular filaments may also run from cell to cell forming close transmembrane associations at the surfaces of both cells (Fig. 2). There was little labelling of banded collagen fibrils (Figs. 1, 2). Most of the fibronectin in the extracellular matrix of Dupuytren’s diseased tissue appeared to be associated with extracellular filaments (Figs. 1-3). Controls incubated with either PBS (not illustrated) or preimmune rabbit serum (Fig. 4) exhibited very little colloidal gold labelling. Ultrastructure of Close Transmembrane Associations at the Surfaces of Fibroblasts in Dupuytren ’s Disease High voltage electron microscopy of Dupuytren’s diseased tissue confirmed the presence of a population of fibroblasts containing large bundles of actin microfilaments with closely associated extracellular filamentous material (Figs. 5-8). The fibronectin-rich filaments and actin microfilaments were observed to lie in close association a t the fibroblast surface (Figs. 5-8). Transmembrane associations were observed to consist of a bundle of extracellular filaments which parallel both the plasmalemma and actin microfilaments (Figs. 5-8). The distance over which the filaments and plasmalemma are closely associated can be quite extensive, ranging up to 1.5 pm in length (Fig. 5). Occasional densities in the plasmalemma are present in regions of the transmembrane associations giving the appearance that the cell has spread and strongly attached onto the fibronectin filaments (Figs. 5, 6). Bundles of fibronectin-rich filaments were observed to extend from close transmembrane associations a t the cell surface into the surrounding tissue (Figs. 5-8). These filaments were observed to extend for distances up to 4 pm (Fig. 5). Extracellular filaments may extend 177 from cell to cell, forming close transmembrane associations a t the surfaces of both cells (Fig. 5). Collagen fibrils were frequently observed to lie in close association with extracellular filaments a t the cell surface, suggesting that collagen fibrils may be linked to the cell by this complex transmembrane association (Figs. 5-8). Collagen fibrils are also associated with fibronectin-rich filaments which have extended away from the cell surface (Fig. 8). Extracellular filaments were observed to wrap partially around a bundle of collagen fibrils (Fig. 5). These images give the impression that the extracellular filaments are interconnecting actinrich fibroblasts in the diseased tissue with each other as well as with the surrounding collagen fibrils. DISCUSSION This study has demonstrated that fibronectin-containing filaments and actin microfilaments form a fibronexus-like transmembrane association at the surfaces of fibroblasts in Dupuytren’s diseased palmar fascia. Previously we have demonstrated that extracellular filaments and actin microfilaments form a close transmembrane association with one another a t the surfaces of fibroblasts in this tissue (Tomasek et al., 1987). Structurally, this association resembles the fibronexus previously described in fibroblasts cultured in vitro (Singer, 1979) and in granulation tissue (Singer et al., 1984). To test the hypothesis that the close transmembrane association observed in Dupuytren’s diseased palmar fascia is similar to the fibronexus, we examined this tissue with immunoelectron microscopy. Extracellular filaments were found to label with anti-fibronectin antiserum. Moreover, the excellent preservation in our immunoelectron micrographs allowed us to establish that these labelled extracellular filaments participate in the close transmembrane associations present at the surfaces of actinrich fibroblasts. Using high voltage electron microscopy we demonstrated that these contacts can be quite extensive a t the cell surface extending up to at least 1.5 pm in length. In addition, fibronectin-rich extracellular filaments can extend from the cell surface for distances up to at least 4 pm connecting the cells with the surrounding collagen fibrils and other actin-rich fibroblasts. These results demonstrate that the fibronexus may be a n important adhesive structure in Dupuytren’s disease. The mechanism by which fibronectin-rich filaments and actin microfilaments are linked across the cell membrane is not known. Neither actin nor fibronectin are integral membrane proteins. Recently a fibronectin receptor of the p l integrin family has been described (Hynes, 1987). This receptor has been demonstrated to colocalize with fibronectin filaments and actin microfilament bundles in cultured fibroblasts (Chen et al., 1985, 1986; Tamkun et al., 1986; Singer e t al., 1988). The extracellular domain of this receptor can bind directly to fibronectin (Tamkun et al., 1986; Hynes, 1987). The intracellular domain may bind indirectly to actin through actin-binding proteins (Horwitz et al., 1986). These results suggest that the fibronectin receptor could be a n integral component of the fibronexus indirectly linking fibronectin and actin across the plasmalemma. It remains to be determined whether the fibronectin receptor is a part of the fibronexus present 178 J.J. TOMASEK AND C.J. HAAKSMA Figs. 1-4. Transmission electron micrographs of actin-rich fibroblasts in Dupuytren’s diseased palmar fascia stained by the immunogold procedure with anti-fibronectin antiserum. Fig. 1: Labelled extracellular filaments participate in a transmembrane association with a bundle of microfilaments (mf) at the cell surface. Collagen fibrils (cf) display little labelling. Fig. 2 Labelled extracellular filaments interconnect two actin-rich fibroblasts. cf, collagen fibrils; mf, microfilaments. Fig. 3: Labelled extracellular filaments form a transmembrane association with a bundle of actin microfilaments (mf) at the cell surface. Fig. 4: Preimmune serum control demonstrates little labelling of extracellular filaments participating in a transmembrane association. mf, microfilaments. (Fig. 1, ~65,000;Fig. 2, ~82,000; Fig. 3, x 50,000; Fig. 4, x 55,000.) in Dupuytren’s diseased tissue. It is noteworthy that cultured fibroblasts, obtained from Dupuytren’s diseased palmar fascia, will form a fibronexus to which the fibronectin receptor colocalizes (Tomasek, unpublished observations). It has been proposed that contraction in Dupuytren’s disease is a n active cellular process. The actin-rich fibroblasts, also termed “myofibroblasts,” are believed to be the cell type responsible for generating contractile force leading to tissue contraction (Gabbiani and FIBRONEXUS IN DUPUYTREN’S DISEASE 179 Figs. 5,6.Stereo pairs of high voltage electron micrographs of actinrich fibroblasts and associated extracellular filaments in Dupuytren’s diseased palmar fascia. Fig. 5 A bundle of extracellular filaments (arrow) forms close associations with two different actin-rich fibroblasts (curved arrow and arrowhead). Densities are present in the plasmalemma where it participates in the close transmembrane association between extracellular filaments and actin microfilaments (arrowhead).The same bundle of extracellular fibrils (arrow) appears to partially encircle a bundle of collagen fibrils. Fig. 6 A transmembrane association between a cell containing a bundle of actin microfilaments and extracellular filaments (arrowheads). Collagen fibrils are closely associated with extracellular filaments a t the cell surface (arrow). (Fig. 5, x 25,000; Fig. 6, x 25,000.) Majno, 1972; Chiu and McFarlane, 1978; Gelberman et al., 1980; Tomasek et al., 1986,1987). In order for these cells to participate in tissue contraction they must be able to generate and transmit contractile force. Stress fibers are capable of undergoing contraction (Kreis and Birchmeier, 1980; Drenckhahn and Wagner, 1986). Fi- 180 J.J. TOMASEK AND C.J. HAAKSMA Figs. 7,8.Stereo pairs of high voltage electron micrographs of actinrich fibroblasts and associated extracellular filaments in Dupuytren’s diseased palmar fascia. Fig.7 A transmembrane association between actin microfilaments and extracellular filaments is present at the cell surface (arrowhead).Fig. 8 Extracellular filaments (arrow) appear to extend from the cell surface to surrounding collagen fibrils. (Fig. 7, x 20,000; Fig. 8, x 20,000.) bronectin-containing extracellular filaments extending from the fibronexus at the cell surface could transmit this force to the surrounding collagen fibrils and fibroblasts. Cell contraction would result in displacement of collagen fibrils and adjacent fibroblasts leading to a shortening of the tissue. Fibronectin was localized primarily at the periphery of the extracellular filaments participating in the formation of the fibronexus. It is possible that these extracellular filaments contain, in addition to fibronectin, other extracellular matrix macromolecules. Studies on the composition of similar extracellular filaments formed by fibroblasts in tissue culture have been contradictory as to whether they contain collagen (Furcht et al., 1980) or are exclusively fibronectin (Chen et al., 1978). Type VI collagen has been described as a major collagen component of extracellular matrices (Bruns et al., 1986). However, i t is unlikely that type VI collagen is a major component of the extracellular filaments described in this paper. Similar extracellular filaments formed by cultured fibroblasts stain for fibronectin but not type VI collagen (Bruns et al., 1986). In addition, we have not observed any periodicity in the extracellular filaments that form the fibronexus in Dupuytren’s diseased tissue. Fibronectin does appear to be a major component of these extracellular filaments. The location of fibronectin a t the periphery of these extracellular filaments may be impor- FIBRONEXUS IN DUPUYTREN’S DISEASE 181 Chen, W.-T., J. Wang, T. Hasegawa, S.S. Yamada, and K.M. Yamada 1986 Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides. J . Cell Biol., 103.1649-1661. Chiu, H.F., and R.M. McFarlane 1978 Pathogenesis of Dupuytren’s contracture: A correlative clinical-pathological study. J . Hand Surg., 3:l-10. Drenckhahn, D., and J . Wagner 1986 Stress fibers in the splenic sinus endothelium in situ: Molecular structure, relationship to the extracellular matrix, and contractility. J. Cell Biol., 102; 1738-1747. Eddy, R.J., J.A. Petro, and J.J. Tomasek 1988 Evidence for the nonmuscle nature of the “myofibroblast” of granulation tissue and hypertrophic scar. Am. J. Pathol., 130t252-260. Furcht, L.T., D. Smith, G. Wendelschafer-Crabb, D.F. Mosher, and J.M. Foidart 1980 Fibronectin Presence in native collagen fibrils of human fibroblasts: immunoperoxidase and immunoferritin localization. J. Histochem. Cytochem., 28t1319-1333. Gabbiani, G., and G. Majno 1972 Dupuytren’s contracture: Fibroblast contraction? An ultrastructural study. Am. J. Pathol., 66:131146. Gelberman, R.H., D. Amiel, R.M. Rudolph, and R.M. Vance 1980 Dupuytren’s contracture. An electron microscopic, biochemical and clinical correlative study. J . Bone Joint Surg., 62A:425-432. Grinnell, F., R.E. Billingham, and L. Burgess 1981 Distribution of fibronectin during wound healing. J . Invest. Dermatol., 76r181189. Grinnell, F., and M.K. Feld 1979 Initial adhesion of human fibroblasts in serum-free medium: Possible role of secreted fibronectin. Cell, 17t117-129. Horwitz, A,, K. Duggan, C. Buck, M.C. Beckerle, and K. Burridge 1986 Interaction of plasma membrane fibronectin receptor with talin-A transmembrane linkage. Nature, 320:53 1-533. Hynes, R.O. 1987 Integrins: A family of cell surface receptors. Cell, 48:549-554. Hynes, R.O., and A.T. Destree 1978 Relationships between fibronectin (LETS protein) and actin. Cell, 15:875-886. Hynes, R.O., A.T. Destree, and D.D. Wagner 1982 Relationships between microfilaments, cell-substratum adhesion, and fibronectin. Cold Spring Harbor Symp. Quant. Biol., 46:659-670. Karnovsky, M.J. 1965 A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electro microscopy. J . Cell Biol., 27: 137a (Abstract). Kreis, T.E., and W. Birchmeier 1980 Stress fiber sarcomeres of fibroblasts are contractile. Cell, 22t555-561. Luck, J.V. 1959 Dupuytren’s contracture. A new concept of the pathogenesis correlated with surgical management. J . Bone Joint Surg., 41-A:635-642. Singer, 1.1. 1979 The fibronexus: A transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts. Cell, 16:675-685. Singer, 1.1.1982 Fibronexus formation is a n early event during fibronectin-induced restoration of more normal morphology and substrate adhesion patterns in transformed hamster fibroblasts. J . ACKNOWLEDGMENTS Cell Sci., 56r1-20. The authors would like to acknowledge Dr. Robert Singer, I.I., D.W. Kawka, D.M. Kazazis, and R.A.F. Clark 1984 In vivo codistribution of fibronectin and actin fibers in granulation tisSchultz and the many members of the New York Socisue: Immunofluorescence and electron microscopic studies of the ety for Surgery of the Hand for their contributions of fibronexus a t the myofibroblast surface. J. Cell Biol., 98t2091tissue. We thank Dr. Henry Godfrey for generously 2106. providing the antibody used in this study. We would Singer, I.I., and P.R. Paradiso 1981 A transmembrane relationship between fibronectin and vinculin (130Kd Protein): Serum modualso like to thank Dr. Min Song for help with high lation in normal and transformed fibroblasts. Cell, 24t481-492. voltage electron microscopy a t the National Institutes Singer, I.I., S. Scott, D.W. Kawka, D.M. Kazazis, J . Gailit, and E. of Health Biotechnology Resources Laboratories in AlRouslahti 1988 Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extrabany, NY. This research was supported by a grant from cellular matrix accumulation. J. Cell Biol., 106r2171-2182. the Orthopedic Research and Education Foundation. Tamkun, J.W., D.W. DeSimone, D. Fonda, R.S. Patel, C. Buck, A.F. Horwitz, and R.O. Hynes 1986 Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin LITERATURE CITED and actin. Cell, 46r271-282. Bruns, R.R., W. Press, E. Engvall, R. Timple, and J. Gross 1986 Type Tomasek, J.J., R.J. Schultz, C.W. Episalla, and S.A. Newman 1986 The cytoskeleton and extracellular matrix of the Dupuytren’s disVI collagen in extracellular, 100-nm periodic filaments and ease “myofibroblast”: An immunofluorescence study of a nonfibrils: Identification by immunoelectron microscopy. J. Cell muscle cell type. J. Hand Surg., IIr365-371. Biol., 103:393-404. Chen, L.B., A. Murray, R.A. Segal, A. Bushnell, and M.L. Walsh 1978 Tomasek, J.J., R.J. Schultz, and C.J. Haaksma 1987 Extracellular matrix-cytoskeletal connections at the surface of the specialized Studies on intercellular LETS glycoprotein matrices. Cell, 14: contractile fibroblast (myofibroblast) in Dupuytren’s disease. J. 377-391. Bone Joint Surg., 69-At1400-1407. Chen, W.-T., E. Hasegawa, C. Weinstock, and K.M. Yamada 1985 Development of cell surface linkage complexes in cultured fibro- Woods, A,, and J.R. Couchman 1988 Focal adhesions and cell-matrix . interactions. Collagen Rel. Res., 8:155-182 blasts. J . Cell Biol., 100:1103-lli4. tant. In a peripheral location, fibronectin can interact with the fibronectin receptor a t the cell surface and with other extracellular components, such as the type I collagen fibrils. This study has demonstrated that a structure similar to the previously described fibronexus is present in Dupuytren’s diseased tissue. The fibronexus has previously been demonstrated to be a n important adhesive structure for fibroblasts in vitro (Singer, 1979, 1982) and is postulated to play a similar role in granulation tissue (Singer et al., 1984). The fibronexus may be a n important adhesive structure during active contraction in Dupuytren’s disease. These results also suggest that the fibronexus may play a n important adhesive role in other contractile tissues which contain fibronectin and actin-rich fibroblasts, such as hypertrophic scars (Eddy et al., 1988). In addition to playing a role in cell adhesion, fibronectin can stimulate actin bundle formation and induce fibronexus formation in transformed (Singer, 1982) and normal fibroblasts (Woods and Couchman, 1988). Singer et al. (1984) have postulated t h a t fibronectin may play a n important role in causing the formation of the fibronexus in fibroblasts in granulation tissue. Whether fibronectin plays such a role in Dupuytren’s disease is unclear. Fibronectin labelling, in Dupuytren’s diseased tissue, is found only in association with actin-rich fibroblasts and only during the active stage of the disease (Tomasek et al., 1986). However, unlike granulation tissue where the fibroblasts migrate into a fibronectin-rich clot (Grinnell et al., 1981), i t is not known in Dupuytren’s disease which appears first, the fibronectin matrix or the actin bundles. Once formed, the fibronexus is a dominant adhesive structure at the surfaces of actin-rich fibroblasts in Dupuytren’s diseased tissue. Fibronectin-rich filaments extend from the fibronexus at the cell surface to adjacent cells and collagen fibrils. Thus the fibronexus, by mediating cell-to-cell and cell-to-matrix attachments, may serve to transmit contractile forces generated by actin microfilaments in these cells throughout the diseased tissue. 182 J.J. TOMASEK AND C.J. HAAKSMA Yamada, K.M., and K. Olden 1978 Fibronectins-adhesive glycoproteins of cell surface and blood. Nature (London),275t179-184. Yamada, K.M., S. Yamada, and I. Pastan 1976 Cell surface protein partially restores morphology, adhesiveness, and contact inhibition of movement to transformed fibroblasts. Proc. Natl. Acad. Sci. USA, 73t1217-1221.