Fig. 5

The Rap1b binding to talin is crucial for integrin activation. a The HSQC spectra of 50 μM ¹⁵N-labeled talin-F0_DM (K15A, R35A) in the absence (black) and presence of 125 μM GMP-PNP loaded Rap1b (red). b Integrin activation assay in CHO A5 cells, which stably express integrin α_IIbβ₃. Double mutations (K15A, R35A) in talin-H substantially decreases integrin activation. The data are shown as means ± S.E.M. from four independent experiments. *p < 0.05. c Static adhesion of talin^1/2dko fibroblasts, expressing either ypet alone, ypet-tagged talin WT, or ypet-tagged talin DM (K15A, R35A). Number of adherent cells was quantified by measuring absorbance of crystal violet staining. Values measured for talin WT-transduced cells were set to one in each of six independent experiments. Values are given as mean ± S.E.M. *p < 0.05 and **p < 0.01. d Spreading of talin^1/2dko fibroblasts expressing ypet (green), ypet-tagged talin WT (red), or ypet-tagged talin DM (blue) on a fibronectin-coated surface, measured 5, 15, 30 min, 1, 2 and 4 h after plating. N = 5. e Confocal images of ypet-tagged talin WT or ypet-tagged talin DM cells on FN-coated round micropatterns stained for paxillin (red) and F-actin (blue). Ypet signal is shown in green. Scale bar 10 μm. Focal adhesion area per cell area (f), focal adhesion number per cell (g), and focal adhesion size (h) of ypet-tagged talin WT and ypet-tagged talin DM cells on FN-coated micropatterns. i Ypet fluorescence intensity within paxillin-positive focal adhesion area in relation to the cellular ypet fluorescence. N = 5; 10–15 talin WT and talin DM-expressing cells in each measurement were analyzed. All values are given as mean ± S.E.M. *p < 0.05 and **p < 0.01