Fig. 3

α2 integrins suppress Cdc42 activity upon cell–cell adhesion formation. a Representative images of control, α2 knockdown (KD) and α2KD cells pre-treated with ML141 (10 μM, 4h) plated onto laminin or Fc-E-cadherin-coated coverslips for 30 min and fixed and stained for F-actin. b Quantification of the number of filopodia per cell for cells adhered to laminin or Fc-E-cadherin. Data are from at least 30 cells per condition; representative of 3 independent experiments. c Analysis of Cdc42 activity in cell lysates from control and α2KD monolayers in 2-mM Ca²⁺ treated with either DMSO or ML141 (10 μM, 4h) by G-LISA. N=3 wells per condition; representative of 3 independent experiments. d FRET/donor ratiometric images of control and α2KD cells expressing the Cdc42 FRET biosensor taken from movies following a Ca²⁺ addition time course. White boxes highlight cell–cell junctions. e Quantification of the relative changes in Cdc42 FRET/donor ratios at junctions over time. Data are means −/+ SEM from 18 cells pooled from 3 independent experiments. f FRET/donor ratiometric images of control and α2KD cells expressing the Cdc42 GDI FRET biosensor taken from movies following a Ca²⁺ addition time course. g Quantification of the relative changes in GDI FRET/donor ratios within cells over time. h Images of RhoGDI-GFP and Y156 phosphomutants (Y>E and Y>F) expressed in WT cells, fixed and stained for E-cadherin. i Quantification of mean E-cadherin junction width from cells as in h. Scale bars, 10μm throughout. P values = ***p<0.001, **p<0.01