Fig. 1. Interaction of β1 integrin-GFP and talin-rod-RFP by FRET. (A) β1 integrin-GFP fibroblasts were transfected with plasmids encoding mRFP conjugates of the N-terminal 433 amino acids of talin (talin 433), the C-terminal rod domain of talin (talin rod), paxillin or -actinin and plated onto fibronectin. Images show the GFP multiphoton intensity image and (where appropriate) corresponding widefield CCD camera image of the RFP expression. Control (GFP integrin alone) image demonstrates a normal GFP lifetime in the absence of acceptor. Lifetime images mapping spatial FRET across the cells are depicted using a pseudocolour scale (blue, normal GFP lifetime; red, FRET). The bar graph represents average FRET efficiency of seven cells over three independent experiments. Error bars indicate s.e.m. (B) β1 integrin-GFP fibroblasts were transfected with talin-rod-mRFP. Cells were plated onto coverslips coated with poly-L-lysine (PLL), collagen I (COL), or laminin-1 (LN) and allowed to attach and spread for 2 hours. Control (untransfected) cells or co-transfected cells were then imaged by FLIM to detect FRET. Lifetime measurements were acquired and depicted as in A. Histogram analysis of the spread of relative FRET efficiency is an average of more than eight cells from three different experiments.