Dendritic fibroblasts in three-dimensional collagen matrices - PubMed (original) (raw)

Dendritic fibroblasts in three-dimensional collagen matrices

Frederick Grinnell et al. Mol Biol Cell. 2003 Feb.

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.

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Figures

Figure 1

Figure 1

Human fibroblasts project a dendritic network of extensions in collagen matrices but not on collagen-coated coverslips. Fibroblasts were incubated 5 h on collagen-coated surfaces (A–C) or in collagen matrices (D–F). After 1 h, 50 ng/ml PDGF (B and E) or 10 μM LPA (C and F) was added to the incubations. At the end of the incubations, samples were fixed and stained for actin. Bar, 80 μm.

Figure 2

Figure 2

Extensions of the dendritic network contain a core of microtubules with actin concentrated at their tips. Fibroblasts were incubated 4 h in collagen matrices. PDGF at 50 ng/ml (A–C) or 10 μM LPA (D–F) was added to the incubations after 1 h. At the end of the incubations, samples were fixed and stained for actin and tubulin. (A and D) Actin; (B and E) tubulin; (C and F) overlay. Bar, 40 μm.

Figure 3

Figure 3

The dendritic network depends on actin and tubulin for formation and motility. Fibroblasts were incubated in collagen matrices for 15 min (A), 30 min (B), or 60 min (C). In some samples (D–K), 50 ng/ml PDGF was added to the incubations after 60 min, which were continued for an additional 4 h. Cytochalasin D (10 μM) was added after 15 min (E) or 4 h (G and I–K). Nocodazole (5 μM) was added after 15 min (F) or 4 h (H). At the end of the 5-h incubations, samples were fixed and stained for actin (A–H) or actin and tubulin (I, actin; J, tubulin; K, overlay). Bar, 80 μm for A–H and 40 μM for I–K.

Figure 4

Figure 4

LPA stimulates retraction of the fibroblast dendritic network. Fibroblasts were incubated in collagen matrices for 60 min at which time LPA (10 μM) was added and the incubations were continued for an additional 5 min (A), 15 min (B), or 60 min (C). In some samples (D–F), fibroblasts were incubated in collagen matrices for 60 min at which time PDGF (50 ng/ml) was added. The latter samples were incubated for an additional 4 h, at which time LPA (10 μM) was added for 60 min (E and F). In F, cytochalasin D (10 μM) was added 15 min before LPA. At the end of the incubations, samples were fixed and stained for actin. Bar, 80 μm for A–C and 40 μM for D–F.

Figure 5

Figure 5

Rho activation occurs in fibroblasts in collagen matrices after LPA stimulation. Fibroblasts were incubated in collagen matrices for 60 min (ATT) followed by 5 min in basal medium (DMEM/5% BSA) (BSA) containing LPA (10 μM) or PDGF (50 ng/ml) as indicated. Samples for each lane represented four matrices containing 4 × 105 cells each. At the end of the incubations, samples were lysed, and aliquots were used to determine GTP-loaded and total levels of Rac and Rho as shown.

Figure 6

Figure 6

In LPA stimulated cells, exotransferase C3 inhibits retraction of the fibroblast dendritic network but causes distortion cell extensions on coverslips. Fibroblasts loaded with FITC-rabbit IgG (A, B, E, and F) or with FITC-rabbit IgG and exotransferase C3 (C, D, G, and H) were incubated on collagen-coated coverslips (E–H) or in collagen matrices (A–D) for 4 h. LPA (10 μM) was added after 1 h. At the end of the incubations, samples were fixed and stained for actin (A, C, E, and G) and with FITC-conjugated goat anti-rabbit IgG to intensify the IgG signal (B, D, F, and H). Bar, 80 μm.

Figure 7

Figure 7

In PDGF stimulated cells, exotransferase C3 does not inhibit the fibroblast dendritic network but causes distortion of fibroblast extensions on coverslips. Fibroblasts loaded with FITC-rabbit IgG (A and B) or with FITC-rabbit IgG and exotransferase C3 (C–F) were incubated on collagen-coated coverslips (E and F) or in collagen matrices (A–D) for 4 h. PDGF (50 ng/ml) was added after 1 h. At the end of the incubations, samples were fixed and stained for actin (A, C, and E) and with FITC-conjugated goat anti-rabbit IgG to intensify the IgG signal (B, D, and F). Bar, 80 μm.

Figure 8

Figure 8

Rho kinase inhibitor blocks LPA-stimulated retraction of the fibroblast dendritic network. Fibroblasts were incubated 4 h in collagen matrices. LPA (10 μM) or nocodazole (5 μM) was added to the incubations after 3 h. In some samples (B, D, and F), Rho kinase inhibitor Y-27632 (10 μM) was added for 15 min before adding LPA or nocodazole. At the end of the incubations, samples were fixed and stained for actin. Bar, 40 μm.

Figure 9

Figure 9

Metabolic coupling of cells through the fibroblast dendritic network. Donor fibroblasts (labeled with DiI and Calcein AM) and unlabeled cells were incubated 2 h in collagen matrices. After 1 h, 50 ng/ml PDGF (C and D) or 10 μM LPA (E and F) was added. At the end of the incubations, samples were visualized without fixation for DiI (donor cells; A, C, and E) or Calcein AM (donor and recipient cells; B, D, and F). Asterisks indicate recipient cells. Bar, 80 μm.

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