Cell motility determines form and function of multicellular organisms. Most studies on fibroblast motility have been carried out using cells on the surfaces of culture dishes. In situ, however, the environment for fibroblasts is the three-dimensional extracellular matrix. In the current research, we studied the morphology and motility of human fibroblasts embedded in floating collagen matrices at a cell density below that required for global matrix remodeling (i.e., contraction). Under these conditions, cells were observed to project and retract a dendritic network of extensions. These extensions contained microtubule cores with actin concentrated at the tips resembling growth cones. Platelet-derived growth factor promoted formation of the network; lysophosphatidic acid stimulated its retraction in a Rho and Rho kinase-dependent manner. The dendritic network also supported metabolic coupling between cells. We suggest that the dendritic network provides a mechanism by which fibroblasts explore and become interconnected to each other in three-dimensional space. INTRODUCTIONForm and function of multicellular organisms depend on tissue-specific programs of cell motility (Trinkaus, 1984). Motility has been studied extensively using fibroblasts cultured on planar surfaces. Cells migrate over these surfaces using their flattened, ruffling lamellipodia (Lauffenburger and Horwitz, 1996;Mitchison and Cramer, 1996). Tractional force necessary for migration is exerted at newly formed cell-substratum adhesions (Galbraith and Sheetz, 1997;Oliver et al., 1999;Beningo et al., 2001). Formation and release of these adhesions along with regulation of cell protrusive and contractile activity requires complex molecular interactions between many adhesion, motor, and regulatory molecules (Schoenwaelder and Burridge, 1999;Borisy and Svitkina, 2000;Schwartz and Shattil, 2000;Geiger et al., 2001). Small G proteins are particularly important in the process because of their diverse effects on the actin cytoskeleton (Hall, 1998;Kaibuchi et al., 1999). Activation of Rac (e.g., by platelet-derived growth factor [PDGF]) stimulates cell protrusion, whereas activation of Rho (e.g., by lysophosphatidic acid) inhibits cell protrusion and stimulates cell contraction (Clark et al., 1998;Rottner et al., 1999).The flattened, lamellar appearance characteristic of fibroblasts on planar surfaces differs markedly from the in situ appearance of mesenchymal cells and connective tissue fibroblasts, which tend to be stellate or dendritic in shape, often with long, slender extensions (Breathnach, 1978;Trinkaus, 1984;Van Exan and Hardy, 1984;Omagari and Ogawa, 1990;Beertsen et al., 2000). In part, the differences in appearance of fibroblasts on planar surfaces compared with tissue may be a reflection of topographic responsiveness (Trinkaus, 1984); cells can detect nanometric substratum surface features (Curtis and Wilkinson, 1999). In addition, however, differences in substratum stiffness likely are important. Cells can modulate the strength of their adhesive i...

To learn more about the regulation of contraction of collagen matrices by fibroblasts, we compared the ability of lysophosphatidic acid (LPA) and platelet-derived growth factor (PDGF) to stimulate contraction of floating and stressed collagen matrices. In floating collagen matrices, PDGF and LPA stimulated contraction with similar kinetics, but appeared to utilize complementary signaling pathways since contraction obtained by the combination of growth factors exceeded that observed with saturating concentrations of either alone. The PDGF-simulated pathway was selectively inhibited by the protein kinase inhibitor KT5926. In stressed collagen matrices, PDGF and LPA stimulated contraction with different kinetics, with LPA acting rapidly and PDGF acting only after an ϳ1-h lag period. Pertussis toxin, known to block signaling through the G i class of heterotrimeric G-proteins, inhibited LPA-stimulated contraction of floating but not stressed matrices, suggesting that LPA-stimulated contraction depends on receptors coupled to different G-proteins in floating and stressed matrices. On the other hand, the Rho inhibitor C3 exotransferase blocked contraction of both floating and stressed collagen matrices. These results suggest the possibility that distinct signaling mechanisms regulate contraction of floating and stressed collagen matrices.Closure of cutaneous wounds involves three processes: epithelialization, connective tissue deposition, and contraction. Wound contraction, which brings the margins of open wounds together (1, 2), is believed to be mediated by specialized fibroblasts called myofibroblasts because of their content of actin stress fibers and ␣-smooth muscle actin (3). The myofibroblast phenotype can occur early or late during the wound contraction process depending on the mechanical resistance of surrounding tissue (4). Myofibroblasts have also been implicated in the pathology of wound contractures and fibrotic disease (5, 6).Using several different culture models, we and others have studied the ability of fibroblasts to reorganize and contract collagen matrices in vitro. In the "floating" model (7), a freshly polymerized collagen matrix containing fibroblasts is released from the culture dish and allowed to float in culture medium, and contraction occurs in the absence of external mechanical load and without appearance of actin stress fibers in the cells (8). In the "attached" model, a polymerized collagen matrix containing fibroblasts remains attached to the culture dish during contraction. In this case, mechanical load (i.e. isometric tension) develops during contraction, and cellular stress fibers assemble (9 -11). Finally, the two-step "stressed" model combines an initial period of attached matrix contraction leading to mechanical loading, followed by release of the matrices, resulting in mechanical unloading and further contraction as mechanical stress dissipates (i.e. stress-relaxation) (12).Contraction of collagen matrices depends on cell binding to collagen through ␣ 2 ␤ 1 integrins (13-15) and r...

Mechanical and physical features of the extracellular environment dramatically impact cell shape. Fibroblasts interacting with 3D relaxed collagen matrices appear much different from cells on 2D collagen-coated surfaces and form dendritic cell extensions that contain microtubule cores and actin-rich tips. We found that interfering with cellular microtubules caused cells in relaxed matrices to remain round and unable to form dendritic extensions, whereas fibroblasts on coverslips formed lamellipodial extensions and were spread completely without microtubules but were unable to become polarized. Fibroblasts in relaxed collagen matrices lack stress fibers, focal adhesions, and focal adhesion signaling. Fibroblasts on collagen-coated coverslips that were unable to develop stress fibers and focal adhesions, because of either adding blebbistatin to the cells or use of soft coverslips, also formed microtubule-dependent dendritic extensions. Conversely, fibroblasts interacting with precontracted collagen matrices developed stress fibers and lamellipodial extensions and required microtubules for polarization but not spreading. Our findings demonstrate an unexpected relationship between the role of microtubules in cell spreading and the tension state of cell-matrix interactions. At a low tension state (absence of stress fibers and focal adhesions) typical of fibroblasts in relaxed collagen matrices, cells spread with dendritic extensions whose formation requires microtubules; at a high tension state (stress fibers and focal adhesions) typical of cells on coverslips, cells spread with lamellipodial extensions and microtubules are required for cell polarization but not for spreading.adhesion ͉ cell plasticity ͉ cytoskeleton ͉ extracellular matrix ͉ mechanosignaling M echanistic explanations for the functions of hierarchical systems such as tissue cells require an understanding of cell shape and cell composition (1). Since the early days of cell culture, it has been clear that mechanical and physical features of the extracellular environment have a dramatic impact on cell shape. More than 80 years ago, Lewis and Lewis (2) observed mesenchymal cells on glass coverslips and reported that the cells were highly flattened with ''tension striae'' or stress fibers. Around the same time, Weiss (3) cultured mesenchymal cells in blood plasma clots and showed that cell shape varied from stellate to bipolar depending on the orientation of the fibrous network of the clot.Subsequent work has confirmed differences in the shape of cells interacting with 2D rigid surfaces (glass or plastic) vs. 3D flexible matrices (e.g., collagen or fibrin) (4-6). Ironically, most research concerning regulation of cell shape has been carried out with 2D rigid surfaces although stress fibers can rarely be seen in fibroblasts in tissues except under conditions of wound repair and fibrosis (7,8). On the other hand, the diversity of fibroblast shapes observed by Weiss (3) resembles the plasticity of cells in tissues (9-11).Recently, it has become clear that dif...

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