enlargement. The same effect was reached by reduction of the intra- and extracellular Ca2+ concentration. The quantification of cell enlargement after AngII stimulation in the various experiments is shown in Suppl. Figure 6D. In conclusion, our experiments with T-plastin confirm our previous observation that the AngII induced plastin redistribution and lamellipodia formation predominantly depends on the calcium binding capacity at the EF hand motifs and is independent of phosphorylation. Effects of AngII stimulation on T-plastin localization resemble that of LCP1 A.: Immunofluorescence staining of endogenous T-plastin (red) and actin (Phalloidin, green) before and 15 min after stimulation with AngII (100 nM) shows an AngII-dependent redistribution of T-plastin to the cell margins in podocytes. B.: Schematic overview of the T-plastin deletion mutant without EF-binding sites (ABD=actin binding domain). C.: Representative live cell imaging experiments of podocytes transiently transfected with fluorescent RFP-T-plastin-WT (line 1 and 3) or the deletion mutant (line 2). For the experiment in line 3 the intracellular and extracellular Ca2+-concentration was reduced using BAPTA-AM and calcium-free Ringer solution. Stimulation of the cells occurred by addition of AngII (100 nM). The image labelled “0 min” was taken before stimulation; the other pictures were taken at the indicated time points after stimulation. D.: Quantification of cell area enlargement after stimulation from live cell experiments. RFP stained cell areas at “0 “15 time point min” were determined as unstimulated values and at time point min” as stimulated values. The ratio of cell areas is shown (n ≥ 3 independent experiments, mean ± S.D., t-test, *P<0.05; **P<0.01; ***P<0.001).
enlargement. The same effect was reached by reduction of the intra- and extracellular Ca2+ concentration. The quantification of cell enlargement after AngII stimulation in the various experiments is shown in Suppl. Figure 6D. In conclusion, our experiments with T-plastin confirm our previous observation that the AngII induced plastin redistribution and lamellipodia formation predominantly depends on the calcium binding capacity at the EF hand motifs and is independent of phosphorylation. Effects of AngII stimulation on T-plastin localization resemble that of LCP1 A.: Immunofluorescence staining of endogenous T-plastin (red) and actin (Phalloidin, green) before and 15 min after stimulation with AngII (100 nM) shows an AngII-dependent redistribution of T-plastin to the cell margins in podocytes. B.: Schematic overview of the T-plastin deletion mutant without EF-binding sites (ABD=actin binding domain). C.: Representative live cell imaging experiments of podocytes transiently transfected with fluorescent RFP-T-plastin-WT (line 1 and 3) or the deletion mutant (line 2). For the experiment in line 3 the intracellular and extracellular Ca2+-concentration was reduced using BAPTA-AM and calcium-free Ringer solution. Stimulation of the cells occurred by addition of AngII (100 nM). The image labelled “0 min” was taken before stimulation; the other pictures were taken at the indicated time points after stimulation. D.: Quantification of cell area enlargement after stimulation from live cell experiments. RFP stained cell areas at time point “0 min” were determined as unstimulated values and at time point “15 min” as stimulated values. The ratio of cell areas is shown (n ≥ 3 independent experiments, mean ± S.D., t-test, *P<0.05; **P<0.01; ***P<0.001).
Supplemental_Figures.docx