Figure 1.

Ezrin silencing alters SLP-76 microcluster dynamics. Jurkat J14 cells expressing YFP-SLP-76 and transfected with siRNA control or with two different siRNA ezrin oligonucleotides (siEz-1 and siEz-2) were activated on anti-CD3-coated coverslips and subjected to live cell imaging using a spinning disc confocal microscope. (A) Total cell lysates were analysed by western blot with pooled anti-ezrin and anti-moesin Abs. Inhibition of ezrin expression was ∼80% for oligo 1 and ∼70% for oligo 2. (B–D, top panels) Projection of 3D+time images of YFP-SLP-76 microclusters recorded during 5 min at 12 images/min. (B–D, bottom panels) Kymograph representation of YFP-SLP-76 events occurring within an 8 pixel band located in the middle of each cell. Note that kymograph patterns in control cells converged towards the centre, whereas in ezrin-silenced cells remained parallel. (E) Using movies and 3D+time projections, cells were classified in three categories. Phenotype 1 corresponds to cells in which SLP-76 microclusters displayed centripetal movements (B; Supplementary Movie 1). Phenotype 3 corresponds to cells in which SLP-76 microclusters stayed rather immobile within the same subcellular areas (C; Supplementary Movie 2). Phenotype 2 corresponds to intermediate states between phenotype 1 and 3. A representative experiment is shown out of five independent experiments for (B), (C) and (E) and two for (D). Histograms in (E) show data from five independent experiments±s.d. Scale bar=5 μm.