First published online 19 November 2003
doi: 10.1242/jcs.00827
Journal of Cell Science 117, 93-103 (2004)
Published by The Company of Biologists 2004
A role for the polysialic acid – neural cell adhesion molecule in PDGF-induced chemotaxis of oligodendrocyte precursor cells
H. Zhang1,
L. Vutskits1,2,
V. Calaora1,
P. Durbec3 and
J. Z. Kiss1,*
1 Department of Morphology, University of Geneva Medical School, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland
2 Department of Anesthesiology, Pharmacology and Surgical Intensive Care, University Hospital of Geneva, 24, rue Micheli-du-Crest, CH-1211, Geneva, Switzerland
3 Laboratoire de Génétique et Physiologie du Développement, CNRS 9943, Parc Scientifique de Luminy, Case 907, F-13288 Marseille, Cedex 9, France
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Fig. 1. Characterization of purified OPCs. Cells were purified by Percoll gradient centrifugation and cultured in the presence of PDGF (5 ng/ml) and NT3 (10 ng/ml) for 5 days. Immunofluorescence staining shows that most cells exhibit A2B5 (A) and PSA (C) immunoreactivity. (B,D) Phase contrast photographs corresponding to A and C, respectively. Bar, 40 µm.
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Fig. 2. Transfilter chemotaxis (A) and chemokinesis (B) of OPCs in response to PDGF. Purified OPCs were placed in the upper wells of the Boyden chamber, and cells that had migrated to the lower side of the filter were counted. (A) OPCs showed chemotaxis to PDGF placed in the lower well. This chemotaxis was inhibited by Endo N or by anti-PSA Ab. For Endo N-treated groups, cells were treated with Endo N for 6 hours before they were prepared for migration assay. In addition, Endo N was present in both upper and lower wells during the migration assay to prevent the recovery of PSA expression. For inactivation of PSA functions, anti-PSA Ab (2 µg/ml) was also applied to both upper and lower wells of the chemotaxis chamber. Values are expressed as a percentage of control value, i.e. the migration of OPCs without PDGF (100%=400±15 cells per mm2). Data represent the mean ± s.e.m. from at least three independent experiments. *P<0.05 and **P<0.01 by two-tailed unpaired t-test, compared with PDGF alone at the same concentrations. (B) PSA removal from the cell surface has no effect on random motility of OPCs stimulated by PDGF (chemokinesis). The same concentrations of PDGF were applied to both the upper and lower wells of the microchemotaxis chamber, so as to eliminate the gradients of PDGF. Chemokinesis of OPCs increases in the presence of PDGF, but PSA removal from the cell surface does not affect this process. Data represent the mean ± s.e.m. from at least three independent experiments.
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Fig. 3. Endo N treatment has no effect on bFGF-induced chemotaxis of OPCs. The experiments were carried out as in Fig. 2. Data represent the mean ± s.e.m. from at least three independent experiments.
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Fig. 4. Effect of Endo N on the directional migration of OPCs in response to PDGF. Top view (A) and cross section (B) of Dunn chamber (drawn after Allen et al., 1998 ) with the overlying coverslip show the position of the inner well, bridge and outer well. Cells over the annular bridge between the inner and outer well of the chamber can be observed under phase-contrast optics (as shown in C). Cell migration was recorded automatically by time-lapse frame grabbing and the migration tracks were plotted in scatter diagrams (D-I) as described in Materials and Methods. The starting point for each cells is at the intersection between X and Y axes (0, 0), and data points indicate the final positions of individual cells at the end of the 6 hour recording period. Chemotaxis on poly-L-lysine (PL) (D-F,I) or laminin (G,H) was tested by placing PDGF at 100 ng/ml (D-H) or 400 ng/ml (I) in the outer well. The direction of the gradient is vertically upwards. In the presence of PDGF gradient, OPCs migrate toward PDGF and display clear directionality of migration (D,G,I). For chemokinesis (E), equal amounts of PDGF (5 ng/ml) were added in both inner and outer wells of the chamber. After PSA removal by Endo N treatment, these cells lose the chemotactic response to PDGF and migrate randomly (F,H). Arrow in C indicates the direction of the outer well of the Dunn chamber. Bar, 50 µm.
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Fig. 5. Migration tracks of control and Endo N-treated OPCs. (A) Representative time-lapse sequences of OPC migration under control conditions and in the presence of Endo N. Arrows indicate the source of PDGF. It is evident that control cells maintain the directionality of migration towards the source of PDGF, whereas Endo N-treated cells make random turns. Asterisks indicate migrating cells. (B) The migration tracks of two representative control cells and two Endo N-treated cells in the presence of PDGF gradient, taking the starting point for each cell at the intersection between X and Y axes (0, 0) and the source of PDGF at the top. Bars, 30 µm.
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Fig. 6. The migration speed (µm/hour) and FMI values under different conditions. The cell migration speed was calculated for each lapse interval and the mean speed was derived for a period of 6 hours. Data are shown as mean ± s.e.m. from at least three independent experiments (the numbers of the counted cells were indicated in Fig. 4). FMI values can be either positive or negative, depending on the direction in which cells migrate (see Materials and Methods). PL, poly-L-lysine. *P<0.01 by two-tailed unpaired t-test, significantly different from chemokinesis or chemotaxis with Endo N.
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Fig. 7. Scatter diagram showing the reversibility of the effect of Endo N treatment on OPC chemotaxis. Before the migration assay in Dunn chamber, cultures were treated with Endo N in the presence of Ara-C (5x10–6 M) and PDGF (5 ng/ml) for 24 hours, subsequently washed and further cultured in normal medium with PDGF (5 ng/ml), NT3 (10 ng/ml) and Ara-C for 16 hours. The migration assay was performed as described in Materials and Methods. The final positions of cells after 6 hours of migration were indicated with the starting point for each cell at (0, 0), with the source of PDGF (100 ng/ml) at the top. Note that cells have normal chemotactic responses to PDGF gradient in contrast to cells kept in the presence of Endo N (Fig. 4). Data from four independent experiments are shown. Speed and FMI represent the mean ± s.e.m. from these four independent experiments.
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Fig. 8. The effect of PSA removal on PDGF-induced lamellipodial formation. Control and Endo N-treated (12 hours) cells were serum starved and incubated with medium (A,B), 0.5 ng/ml PDGF (C-F) or 5 ng/ml PDGF (G,H) for 10 minutes. Cells were fixed, stained with Texas Red-phalloidin and Oregon Green DNase I to detect F-actin fibers and G-actin, respectively. Cells were examined by fluorescence confocal microscopy. Bar, 20 µm in A-D,G and H; 8 µm in E and F.
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© The Company of Biologists Ltd 2004
