Figure 3

Inside-out integrin signalling in oligodendrocytes. Panel A shows an RT-PCR experiment on cDNA obtained from cultured oligodendrocytes, grown as described in the Methods. Lane 1 – size markers (nt); lane 2 – ADAM9 (control); lane 3 – R-Ras. Panel B shows a western blot of lysates from 3 different sets of cultured oligodendrocytes (lanes 1–3) using anti-R-Ras antibodies. All 3 lanes show a band at the predicted size (23 kD) of R-Ras (arrow). Size markers (kDa) shown on the left. These 2 experiments therefore confirm that oligodendrocytes express R-Ras. Panel C shows the effect of expressing constitutively active R-Ras (R-Ras38V) in oligodendrocytes, while Panel D shows the effect of expressing the dominant-negative R-Ras43N; all 4 micrographs in each panel show representative examples of transfected cells visualised by antibodies against EGFP (green) and CNPase (red). Note the different morphologies observed, with elaborate processes and large sheets seen in some cells expressing R-Ras38V and stunted processes in some expressing R-Ras43N. The effects of R-Ras38V and R-Ras43N at different laminin-2 concentrations are quantified in Panels F and G, with control cells (expressing wild-type R-Ras) in these experiments shown in panel E. These show the increased complexity of R-Ras38V cells at all laminin-2 concentrations when compared with wild-type cells, in contrast to the decreased complexity of the R-Ras43N cells. Single and double asterix indicate significance levels of p < 0.05 and p < 0.01 respectively, calculated using Student's paired t-test. Scale bars in panels C and D are 20 μm.