Fig 1.
The morphology of two sublines of Walker carcinosarcoma WC 256 cells migrating in an adhesive mode but forming different protrusions.
(A, C) Weakly adherent cells forming blebs spontaneously (BC); (B, D) Strongly adherent cells forming lamellipodia (LC); photographs obtained in DIC optics (A, B) or in SEM (C, D). Arrows indicate blebs at the leading edges of cells.
Table 1.
Parameters characterizing the morphology of blebbing and lamellipodia forming cell.
Fig 2.
Distribution of F-actin in migrating BC and LC cells.
Cells were transfected with LifeAct, a 17-amino-acid peptide, which stains F-actin in eukaryotic cells. (A) Sequence of frames showing bleb formation (indicated by arrow) in weakly adherent BC cells. (B) Strongly adherent lamellipodia forming LC cell.
Fig 3.
Comparison of motile activity of BC and LC cells.
(A) Composite trajectories of BC and LC cells migrating under isotropic conditions are shown as circular diagrams. In each diagram, the initial point of each trajectory was placed at the center of the circle. Each trajectory was constructed from 120 (BC) or 48 (LC) successive positions of cell centroids recorded at 15-sec (BC) or 150-sec (LC) time intervals, respectively. The movement of BC and LC was recorded for 30 minutes and 150 minutes, respectively. (B) Diagram showing the speed of cell movement and speed of cell displacement (n = 50); *Statistically significant (p<0.05).
Table 2.
Parameters characterizing BC cell migration under isotropic conditions and after the application of an electric field.
Table 3.
Parameters characterizing LC cell migration under isotropic conditions and after the application of an electric field.
Fig 4.
The effect of dcEF on the migration of BC and LC cells.
(A) Composite trajectories of BC and LC cells migrating in the absence and in the presence of dcEFs are shown as circular diagrams. In each diagram, the initial point of each trajectory was placed at the center of the circle. The x-axis corresponds to the direction of the electric field. The cathode was always placed at the right side of the diagram. Each trajectory was constructed from 120 (BC) or 48 (LC) successive positions of cell centroids recorded at 15-sec (BC) or 150-sec (LC) time intervals, immediately after the exposure of cells to dcEF. The movement of BC and LC was recorded for 30 minutes and 150 minutes, respectively. (B) Diagrams presenting the values of directional cosines γ for BC and LC cells depending on applied field strength; mean ± SEM; p<0.05 vs 0 V/cm (C) The dynamics of reversibility of the direction of cell movement. The directionality of cell movement was completely reversible upon reversing the field polarity (3V/cm). For both cell sublines the average directional cosines γ were analyzed every 5 minutes. Perpendicular lines indicate the time when the electric field polarity was reversed (n = 50). *Statistically significant vs. 0 V/cm (p<0.05).
Fig 5.
The effect of Rac, Cdc42, Rho and ROCK inhibitors on electrotaxis of BC and LC cells and the effect of dcEF on the activity of small Rho GTP-ases.
(A) Composite trajectories of BC and LC cells migrating in the presence of dcEF (3 V/cm) and 50 μM NSC23766 (Rac1 inhibitor), 50 μM ZCL278 (Cdc42 inhibitor III), 30 μM Rhosin (Rho inhibitor) or 10 μM Y-27632 (ROCK inhibitor) are shown as circular diagrams. In each diagram, the initial point of each trajectory was placed at the center of the circle. The x-axis corresponds to the direction of the electric field. The cathode was always placed at the right side of the diagram. Each trajectory was constructed from 120 (BC) or 48 (LC) successive positions of cell centroids recorded at 15-sec (BC) or 150-sec (LC) time intervals, immediately after exposure of cells to dcEF. The movement of BC and LC was recorded for 30 minutes and 150 minutes, respectively (n = 50). (B) The diagrams depict the values of directional cosines γ. (C) The activity of Rac1, Cdc42 and RhoA in BC and LC cells under isotropic condition and after exposition of cells to dcEF for 5 and 15 minutes determined by the G-Lisa assay. *Statistically significant vs. 3 V/cm (p<0.05).
Table 4.
Quantitative data showing the effects of Rac, Cdc42, Rho and ROCK inhibitors on electrotaxis of BC cells.
Table 5.
Quantitative data showing the effects of Rac, Cdc42, Rho and ROCK inhibitors on electrotaxis of LC cells.
Fig 6.
The role of intracellular Ca2+ and myosin II in electrotaxis of BC and LC cells.
(A) Composite trajectories of BC and LC cells migrating in the presence of dcEF (3 V/cm) and 30 μM BAPTA-AM (chelator of [Ca2+]i), 10 μM ML-7 (MLCK inhibitor) or 50 μM blebbistatin (myosin II inhibitor) shown as circular diagrams. In each diagram, the initial point of each trajectory was placed at the center of the circle. The x-axis corresponds to the direction of the electric field. The cathode was always placed at the right side of the diagram. Each trajectory was constructed from 120 (BC) or 48 (LC) successive positions of cell centroids recorded at 15-sec (BC) or 150-sec (LC) time intervals, immediately after exposure of cells to dcEF. The movement of BC and LC cells was recorded for 30 minutes and 150 minutes, respectively (n = 50). (B) The diagrams depict the values of directional cosines γ. *Statistically significant vs. 3 V/cm (p<0.05).
Table 6.
Quantitative data showing the effects of BAPTA, blebbistatin and ML-7 on the electrotaxis of BC cells.
Table 7.
Quantitative data showing the effects of BAPTA, blebbistatin and ML-7 on the electrotaxis of LC cells.