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First published online 22 February 2005
doi: 10.1242/jcs.01646

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Golgi polarization in a strong electric field

Biomedical Sciences, School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK

View larger version (91K): [in a new window]   Fig. 1. An electric field of 300mV/mm directs CHO cell migration and Golgi polarization. (A-C) Time-lapse images showing morphological polarization and directional migration of CHO cells in a DC EF. (D) Outlines of the labelled cells from A-C highlight morphological polarization and cell migration. (E-F) Golgi (red) polarization and actin (green) distribution in CHO cells cultured in the absence (E) or presence (F) of an EF for 3 hours. The cells were fixed and triple-labelled with GM130 antibody (Golgi marker, red), FITC-phalloidin (F-actin, green) and DAPI (blue). (F') Golgi polarization in control experiments with cross-current medium flow where chemical gradients, ionic gradients and fluctuation in temperature and pH were eliminated. (G) Cells with the Golgi falling in the indicated quadrant were scored. (H) An EF of 300 mV/mm increased the percentage of cells with the Golgi polarizing towards the cathode with time. Results shown are the mean±s.e.m. For each time point, a minimum of 300 cells was scored from three independent experiments. Bar, 10 µm. See also supplementary material video 1.

View larger version (81K): [in a new window]   Fig. 2. Cathodal polarization of Golgi requires actin polymerization and signalling of Src and PI 3-kinases. (A,C,D) EF-induced Golgi polarization was abrogated by incubation of the cells with Brefeldin A (BFA), Cytochalasin D (CyD), Wortmannin (WM, PI 3-kinase inhibitor) and PP2 (Src inhibitor). Toxin B, LS203 (cdc42 inhibitor) and Y27632 (ROCK inhibitor) decreased Golgi polarization significantly (P<0.0001), but not completely (P<0.0001) (A). (B) The percentage of cells with their morphology polarized towards the cathode was also scored. For drug treatment, CHO cells were pretreated with PP2 (20 µM), BFA (1 µM, 5 µM), CyD (0.2 µM), Y27632 (50 µM) or LS203 (100 ng/ml) for 2 hours, then subjected to an EF of 300 mV/mm for 3 hours, fixed and triple-stained with phalloidin (green), GM130 (red) and DAPI (blue). Golgi polarization was scored as in Fig. 1G. More than 200 cells from three independent experiments were scored for each condition. Bar, 10 µm.

View larger version (22K): [in a new window]   Fig. 4. The net translocation of CHO cells in an EF. Cells were followed for a 3-hour period in an EF (B-H) or without an EF (A). Each cell's position at t=0 is represented by the origin (0,0), with the final position of each cell at 3 hours plotted as a single point on the graph. The radius of each circle represents 100 µm (the average cell length is 25 µm). n, the total number of cells at a given condition. Values of directedness (the average cosine of the distribution) ±s.e.m. are indicated in the upper right corner of each plot. (C-H) Treated with different inhibitors then in an EF with continuous presence of the same drug. EF, 300 mV/mm.

View larger version (28K): [in a new window]   Fig. 5. Cathodal Golgi polarization and electrotaxis of CHO cells in an EF. (A) Time course of directional cell migration, morphologic polarization and Golgi redistribution. (B) Correlation of Golgi polarization with morphologic polarization and directedness of cell migration. (C) Correlation of Golgi polarization with migration rates in an EF. In B and C, CHO cells were pretreated as indicated with BFA (1 µM, 5 µM), CyD (0.2 µM), PP2 (20 µM), Y27632 (50 µM), Toxin B (10 ng/ml) or LS203 (100 ng/ml) for 2 hours and subjected to an EF for 3 hours with presence of the drug. Cell migration, morphologic polarization and Golgi polarization were analysed as in Materials and methods. Each data point in A represents mean±s.e.m. from at least 200 cells from three independent experiments.

View larger version (99K): [in a new window]   Fig. 3. Phospho-Akt and phospho-Src are distributed to the cathodal facing side of CHO cells in a DC EF. CHO cells were fixed after 1 hour in an EF of 300 mV/mm (B,D) or without an EF (A,C) and labelled with anti-phospho-Src (pSrc, red, A,B) or anti-Phospho-Akt antibody (pAkt, red, C,D). Nuclei were labelled with DAPI (blue).

View larger version (74K): [in a new window]   Fig. 6. Golgi polarization lags behind membrane protrusion, but can significantly enhance directional cell migration when Golgi polarizes in the EF direction. (A) Both the cell morphology and the Golgi of the cell at the left polarized downward before the onset of EF. (B) Active membrane protrusion happened at the cathodal side 30 minutes after onset of the EF (arrowheads). (C) Only the protrusion next to the Golgi persisted (arrow head). (D) The cell then moved and polarized as well as having the Golgi polarized towards the cathode. After this, net migration towards the cathode increased markedly. The directedness value and displacement speed along the x axis were significantly higher than that before the Golgi polarized in the EF direction (compare J with H and I). Note that the cell on the right did not move and the Golgi did not repolarize. (K,L) Golgi polarization towards the cathode significantly increases the trajectory speed, displacement speed and x-axis displacement speed, and directedness (*P<0.05). n=8 from three independent experiments. See also supplementary material video 2.

View larger version (67K): [in a new window]   Fig. 7. An electric field of 300 mV/mm predominates over other directional cues in directing Golgi polarization in the scratch-wound model. Monolayer culture of CHO cells were wounded and allowed to heal for 4 hours (A) without an EF; (B) with an EF applied in the normal healing direction, or (C) opposite to the normal healing direction (the EF was applied 1 hour after wounding for 3 hours). (C) An EF applied against the default healing direction polarized the Golgi away from the wound, completely ignoring the wound direction. Some degree of F-actin polarization in EF direction can be seen (C, arrowheads). The three cells on the right in C have been focally enhanced to show clearer cell contours. BFA completely inhibited both scratch wounding and EF-induced Golgi polarization (D-G). The cells were triple-labelled with F-actin (green), GM130 (red) and DAPI (blue). Cells along the wound edge were scored as polarized if the Golgi was orientated to the right (for A,B,D,E) or to the left (for C,F) as in Fig. 1G. The percentage of polarized cells±s.e.m. was calculated from the means of three independent experiments from 255595 cells (G). Bar, 10 µm.

View larger version (82K): [in a new window]   Fig. 8. An electric field of 300 mV/mm predominates over other directional cues in directing cell migration in the scratch wound model. Migration trajectories of individual cells are indicated by red lines and direction and endpoint of a 3-hour experiment indicated by arrows. (A) No EF control, wound edge moved into the wound. (B) an EF applied with the field vector in normal healing direction enhanced cell migration into the wound (P<0.001). (C) An EF applied with the field vector opposite to the normal healing direction directed the cells at the wound edge to migrate away from the wound. BFA significantly decreased but did not completely abolish directional cell migration induced by wounding and/or an applied EF (D-G). Directness was calculated from 60 cells collected from three independent experiments (G). See also supplementary material video 3.