Directional persistence of EGF-induced cell migration is associated with stabilization of lamellipodial protrusions - PubMed (original) (raw)

Directional persistence of EGF-induced cell migration is associated with stabilization of lamellipodial protrusions

Brian D Harms et al. Biophys J. 2005 Feb.

Abstract

Migrating cells can sustain a relatively constant direction of lamellipodial protrusion and locomotion over timescales ranging from minutes to hours. However, individual waves of lamellipodial extension occur over much shorter characteristic times. Little understanding exists regarding how cells might integrate biophysical processes across these disparate timescales to control the directional persistence of locomotion. We address this issue by examining the effects of epidermal growth factor (EGF) stimulation on long-timescale directional persistence and short-timescale lamellipodial dynamics of EGF receptor-transfected Chinese hamster ovary cells migrating on fibronectin-coated substrata. Addition of EGF increased persistence, with the magnitude of increase correlating with fibronectin coating concentration. Kymographic analysis of EGF-stimulated lamellipodial dynamics revealed that the temporal stability of lamellipodial protrusions similarly increased with fibronectin concentration. A soluble RGD peptide competitor reduced both the persistence of long-timescale cell paths and the stability of short-timescale membrane protrusions, indicating that cell-substratum adhesion concomitantly influences lamellipodial dynamics and directional persistence. These results reveal the importance of adhesion strength in regulating the directional motility of cells and suggest that the short-timescale kinetics of adhesion complex formation may play a key role in modulating directional persistence over much longer timescales.

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Figures

FIGURE 1

FIGURE 1

Migration behavior of EGFR-transfected CHO cells is affected by Fn and EGF. Time-lapse videomicroscopy was used to capture the motility responses of individual CHO K1 and CHO-EGFR cells. (A and B) Without EGFR-GFP transfection, EGF does not affect the migratory response of CHO K1 cells; (C_–_F) Transfected CHO-EGFR cells respond to EGF by upregulating their migration in a Fn-dependent manner. Digital images were taken every 15 min for a total of 6 h per experiment. Each wind-rose plot shows centroid tracks from 10 representative cells in a typical experiment, with the initial position of each track superimposed at 0,0 for clarity. Distance between hatch marks on both axes is 50 _μ_m.

FIGURE 2

FIGURE 2

Effects of EGF and Fn on speed and persistence for migrating CHO-EGFR cells. Transfection of EGFR-GFP into CHO K1 cells alters cell speed (A) and persistence (B) in the presence, but not the absence, of EGF. Data in A and B were obtained using a 10 _μ_g/ml Fn coating concentration. Asterisks denote inequality at 95% confidence for key comparison. (C) EGF stimulation, but not Fn coating concentration, modulates CHO-EGFR speed. Single-factor ANOVA: p = 0.54 (S, −EGF), p = 0.90 (S, +EGF). (D) Persistence time increases with Fn coating concentration in the presence of EGF. ANOVA: p = 0.88 (P, −EGF), p = 0.03 (P, +EGF). To derive the speed of a single cell, total cell path length was divided by the total time of observation. Single cell persistence times were derived from nonlinear least-squares regression using individual cell speed and root mean-square displacement data as inputs. Reported S and P data represent the mean ± 2 SE for three experiments per condition, with 80–100 cells per experiment.

FIGURE 3

FIGURE 3

Lamellipodial membrane dynamics of CHO-EGFR cells. Representative short-timescale kymographs from time-lapse movies of CHO-EGFR cells stimulated with EGF and migrating on 1 _μ_g/ml (A) and 30 _μ_g/ml (B) Fn. Kymographs depict variations in lamellipodial activity along a one-pixel-wide line drawn perpendicularly to the cell membrane in sequential phase contrast images. Ascending contours show lamellipodial protrusion, whereas descending contours show lamellipodial retraction. Dark regions indicate membrane ruffling.

FIGURE 4

FIGURE 4

Lamellipodial stability correlates with directional persistence in CHO-EGFR migration. (A) Fn coating concentration does not affect the average velocity of membrane protrusion. The average slope of ascending kymograph membrane waves defines protrusion velocity. ANOVA: p = 0.86. (B) Protrusion stability increases with Fn concentration in the presence of EGF. Stability represents the time elapsed between the beginning of a single protrusion wave and its spatial peak. Nonoverlapping 95% confidence intervals are indicated by asterisks. ANOVA: p = 0.03.

FIGURE 5

FIGURE 5

Soluble RGD peptide inhibits cell adhesion. The average area of CHO-EGFR cells migrating in the presence of EGF was determined for all Fn coating concentrations; cell adhesion as described by cell area increased in parallel with Fn. Addition of peptide inhibitor abrogated this Fn-dependent effect on adhesion in a dose-dependent fashion.

FIGURE 6

FIGURE 6

RGD peptide reduces both long-timescale directional persistence and short-timescale protrusion stability. (A) Wind-rose plots for migration experiments of CHO-EGFR cells plated on 30 _μ_g/ml Fn in the presence of EGF and varying amounts of GRGDSP, a soluble competitive inhibitor of Fn binding to _α_5_β_1 integrins. Distance between hatch marks on both axes is 50 _μ_m. (B) Cell speeds and (C) persistence times for the RGD migration experiments. Speed and persistence were derived as in Fig. 2. ANOVA: p = 0.71 for RGD-dependent cell speed, p = 0.002 for RGD-dependent persistence. Nonoverlapping 95% confidence intervals are indicated by asterisks. (D) Kymography was performed on CHO-EGFR cells plated on 30 _μ_g/ml Fn in the presence of EGF and either GRGDSP or control GRADSP peptide. Nonoverlapping 95% confidence intervals are indicated by asterisks (p = 0.01).

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