Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin - PubMed (original) (raw)
Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin
M Zhao et al. Mol Biol Cell. 1999 Apr.
Free PMC article
Abstract
Wounding corneal epithelium establishes a laterally oriented, DC electric field (EF). Corneal epithelial cells (CECs) cultured in similar physiological EFs migrate cathodally, but this requires serum growth factors. Migration depends also on the substrate. On fibronectin (FN) or laminin (LAM) substrates in EF, cells migrated faster and more directly cathodally. This also was serum dependent. Epidermal growth factor (EGF) restored cathodal-directed migration in serum-free medium. Therefore, the hypothesis that EGF is a serum constituent underlying both field-directed migration and enhanced migration on ECM molecules was tested. We used immunofluorescence, flow cytometry, and confocal microscopy and report that 1) EF exposure up-regulated the EGF receptor (EGFR); so also did growing cells on substrates of FN or LAM; and 2) EGFRs and actin accumulated in the cathodal-directed half of CECs, within 10 min in EF. The cathodal asymmetry of EGFR and actin staining was correlated, being most marked at the cell-substrate interface and showing similar patterns of asymmetry at various levels through a cell. At the cell-substrate interface, EGFRs and actin frequently colocalized as interdigitated, punctate spots resembling tank tracks. Cathodal accumulation of EGFR and actin did not occur in the absence of serum but were restored by adding ligand to serum-free medium. Inhibition of MAPK, one second messenger engaged by EGF, significantly reduced EF-directed cell migration. Transforming growth factor beta and fibroblast growth factor also restored cathodal-directed cell migration in serum-free medium. However, longer EF exposure was needed to show clear asymmetric distribution of the receptors for transforming growth factor beta and fibroblast growth factor. We propose that up-regulated expression and redistribution of EGFRs underlie cathodal-directed migration of CECs and directed migration induced by EF on FN and LAM.
Figures
Figure 1
Typical translocation of bovine CECs over 5 h in EF (150 mV/mm). Substrate and serum dependency are illustrated. Each point represents a single cell, located initially (0 h) at the center of the circular graph with final location plotted after 5 h in EF. The radius of each circle is 200 μm, and average cell length is ∼20 μm. The average cosine (directedness) of the distribution ± SEM and the number of cells plotted are indicated at upper right of each distribution. (for distributions for unstimulated cells and cells exposed to EF alone, see Zhao et al., 1996a).
Figure 2
FN or LAM significantly increased cathodal directedness of migration. The enhancement was concentration dependent. **, p < 0.01; *, p < 0.05 compared with that on the noncoated substratum in same EFs (shown at right). Directedness of 0 indicates random migration; directedness of 1 indicates all cells migrated directly cathodally; and −1 indicates direct anodal migration of all cells along the field vector. For methods of determining directedness, see Zhao et al. (1996a). Number of cells for each point, 43∼221.
Figure 3
Migration rate of CECs cultured in: serum-free DMEM (A) without EF (□) and with EF at 150 mV/mm (▨) and DMEM plus 10% FBS (B) without EF (□) and with EFs at 150 mV/mm (▨). **, p < 0.01 compared with that on the noncoated substratum without EF. (C) Coating concentration dependency of migration rate in serum-containing DMEM at 150 mV/mm. *, p < 0.05 compared with noncoated substratum in same EFs (shown at right). Number of cells for each point, 38∼221.
Figure 4
Representative fluorescent intensity flow cytometric histograms show the effect of LAM or FN with or without EF on membrane expression of EGFR on CECs. Isotype-matched negative control, OX21 as primary mAb, background fluorescence was similar with secondary Ab as negative control. M1 (positively stained cells) was set where the expression of gated events was <1% in isotype-matched negative control. (A) Control (without substratum coating, no EF) cells showed a population of positively stained cells, region M1, which can be further divided into a low peak (region M2) representing a subpopulation of low EGFR expression (EGFRLow) and a higher peak (region M3) representing a subpopulation of high EGFR expression (EGFRHigh) expression. (B) After 12 h in EF (100 mV/mm), positively stained cells increased, as shown by gated events in M1 as total, M2 as EGFRLow expression cells (p < 0.01), and M3 as EGFRHigh expression cells. The cells were cultured on plastic without substratum coating. (C) Withdrawal of serum from culture medium and exposed to EF (100 mV/mm) for 13 h shows a significant decrease in EGFR expression (p < 0.05 for EGFRHigh). The cells were cultured on plastic without substratum coating. (D) Addition of EGF (25 ng/ml) in serum-free medium and EF (100 mV/mm) for 13 h; EGFRLow expression was significantly up-regulated; compare with C. Also see text for detailed numerical data. (E) Significant increases in total EGFR expression and more dramatically in EGFRHigh were evident for CECs cultured on substratum of LAM (1 μg/ml) followed by 3 h of EF exposure at 100 mV/mm.
Figure 5
Confocal images of CECs double labeled for actin filaments (red) and EGFR (green). Colocalization is indicated by yellow. (a) Cell cultured without EF, no obvious asymmetric distribution of EGFR or F-actin. (b) Cell cultured in serum-free medium at 150 mV/mm, no obvious asymmetric distribution of EGFR or F-actin. (C) Cell cultured in EF in serum. Obvious accumulation of EGFR and F-actin cathodally was evident. (a′–c′) Fluorescence intensity in arbitrary units along a line drawn across the cells in a–c, respectively. Asymmetry of both actin and EGFR is evident both visually and digitally only for the cell in c. Figure 6. (A) Confocal images of a live CEC labeled with EGFR antibodies, showing dynamic accumulation of EGFR at the cathode facing side (arrows) when exposed to 300 mV/mm for a 20-min duration, cultured in DMEM with 10% FBS. Bar, 20 μm. (B) Asymmetry of EGFR and F-actin in a cell exposed to 150 mV/mm for 3 h. (B′) Representative vertical section (through the line drawn across the cell in B) shows actin and EGFR staining in an interdigitating pattern in punctate spots near the cell–substratum interface.
Figure 7
Marked cathodal accumulation of EGFR on live CECs revealed by fluorescein EGF binding. (A) Cells were exposed to EF for 3 h at 150 mV/mm and labeled as described in MATERIALS AND METHODS. (B) Ai of EGFR (in 10–20 cells for each group) as revealed by fluorescein EGF binding (see MATERIALS AND METHODS for details). An Ai value >0 indicates higher fluorescence intensity on the cathodal facing side; values close to 1 indicate extreme cathodal asymmetry of EGFR. *, p < 0.001 when compared with that of the cells in serum-free medium or cells not exposed to EF.
Figure 8
Ai of F-actin and EGFR from serial optical sections through a single cell, showing close correlation of the subcellular localization of F-actin (A) and EGFR (B). An Ai value >0 indicates higher fluorescence intensity on the cathodal facing side; therefore, values close to 1 indicate extreme cathodal asymmetry of F-actin or EGFR. Each section is 0.5 μm thick.
Figure 9
Confocal images of CECs exposed to EF of 150 mV/mm for 12–16 h and double labeled with rhodamine-phalloidin for F-actin (red) and antibodies against growth factor receptors (green): TGFR (top panel) or FGFR (lower panel). Colocalization is indicated by yellow. (A) Two single cells showing stronger staining at the cathodal side for both TGFR and F-actin. (V1 and V2) Vertical sections along the line V1 and V2 in A. (B) Grouped cells also show accumulation of TGFR staining at the cathodal side (arrows). (V3) Vertical section cut along line V3 in B shows asymmetric accumulation of TGFR at the cathodal facing side. (B′ and B") TGFR and F-actin asymmetry in the basal and middle sections of B. (C) Two control cells (no EF exposure) show F-actin and FGFR staining. (D) Striking asymmetric distribution of the FGFR (green) and actin (red) observed in two cells after 16 h in EF of 150 mV/mm.
Figure 10
Model illustrating potential role of lateral EF in directing corneal epithelial migration at a wound edge. Beyond ∼1 mm away from a wound, the transcorneal PD indicated at the left (+25 mV inside positive) is supported by the high resistance of the tight junctions in the upper sheet of epithelial cells. At the wound the PD collapses to zero, FN has replaced the normal basement membrane, and the upper sheet of epithelial cells rapidly slides down to make contact with the transient basement membrane within ∼1 h (Kuwabara et al., 1976). Direct measurements indicate that the lateral fields in the tear fluid are higher than those in the stroma. However (see DISCUSSION), the relevant subepithelial voltage drop immediately below the top sheet of corneal epithelium has not been measured and is likely to be substantial, given the tight packing of these differentiated and flattened cells. The lower surface of the cells of a sliding sheet therefore would be migrating toward a cathode. In corneal epithelial wounds, the tight junction–specific protein occludin is present just one cell back from the leading edge (Danjo and Gipson, 1998).
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