Dendritic Fibroblasts in Three-dimensional Collagen Matrices

Abstract

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...

Microtubule regulation of corneal fibroblast morphology and mechanical activity in 3-D culture

“…However, cells reside within 3-D extracellular matrices in vivo, and ECM geometry has also been shown to effect cell morphology, mechanical activity, and adhesion organization and composition (Abbott, 2003;Bard and Hay, 1975;Cukierman et al, 2001;Cukierman et al, 2002;Doane and Birk, 1991;Friedl and Brocker, 2000;Grinnell et al, 2003;Tomasek et al, 1982) While it is known that microtubules play a role in cell spreading and contractility within 3-D collagen matrices, these processes have not been assessed dynamically in individual cells. Furthermore, their influence on the subcellular pattern of force generation and local cell-induced matrix reorganization has not been reported previously.…”

Fibroblast mechanics in 3D collagen matrices☆

“…Fibroblast dendritic extensions have microtubule cores and actin rich tips [44]. Recently, we found that interfering with cytoplasmic microtubules prevents fibroblasts in relaxed collagen matrices from forming dendritic extensions.…”

“…Figure 2 shows that in the high tension state quadrants, differences between the morphology of human fibroblasts under pro-migratory (more ruffling) vs. pro-contractile (more stress fibers) conditions are subtle compared to the more profound changes that occur in the low tension state quadrants. In the later, cells protrude dendritic extensions under pro-migratory conditions, whereas dendritic extensions undergo transient retraction under pro-contractile conditions [44]. Cells in a pro-contractile, low tension state environment eventually re-protrude their extensions but in a more bipolar morphology [45].…”

“…That either pro-migratory or pro-contractile agonists can stimulate floating collagen matrix remodeling leads us to speculate that this process may represent a more general feature of tissue homeostasis. Fibroblasts in floating collagen matrices develop a dendritic network interconnected by gap junctions [44] analogous to the interconnected dendritic network of osteocytes -another connective tissue cell. The osteocyte dendritic network functions as the regulatory sensor to detect changes in tissue mechanics in bone [76,77].…”

Different Molecular Motors Mediate Platelet-derived Growth Factor and Lysophosphatidic Acid-stimulated Floating Collagen Matrix Contraction

“…As described previously, fibroblasts in collagen matrices project and retract a dendritic network of extensions. PDGF stimulates the size and complexity of this network, whereas LPA stimulates its retraction in a Rho kinase-dependent manner (9). Retraction occurs independently of and is not required for matrix contraction (10).…”