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First published online January 23, 2008
doi: 10.1242/10.1242/jcs.018440

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Quantification of integrin receptor agonism by fluorescence lifetime imaging

Maddy Parsons^1,*,, Anthea J. Messent^2,*,, Jonathan D. Humphries², Nicholas O. Deakin² and Martin J. Humphries^2,

¹ Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guys Campus, London, SE1 1UL, UK
² Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

View larger version (39K): [in this window] [in a new window]   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.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Ligand regulation of binding of β1 integrin and talin rod. (A) β1 integrin-GFP fibroblasts were transfected with talin-rod-mRFP. Cells were plated onto coverslips coated with poly-L-lysine (PLL), and allowed to attach and spread for 2 hours. Cells were then incubated with 4.2 µm beads coated with FN or anti-β1 antibodies (12G10, mAb13 or K20) for 30 minutes and subsequently imaged using FLIM as in Fig. 1. Bar graph represents average FRET efficiency of 16 cells per ligand. Efficiency was calculated using a mask of constant area around each bead region for local analysis of FRET. Error bars indicate s.e.m. (B) Cells were prepared as in A, and following incubation with beads, samples were fixed, permeabilised and stained with phalloidin-Alexa Fluor 568 to detect F-actin. Cells were then imaged by confocal microscopy.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Ligand binding to the 4 integrin extracellular domain regulates its association with paxillin. (A) B16F1 mouse melanoma cells were transfected with 4-integrin-GFP and paxillin-mRFP. Cells were then plated onto FN- or PLL-coated coverslips and imaged by multiphoton FLIM as before. (B) The same cells were plated onto H120-coated coverslips followed by treatment with DMF vehicle control or stated small molecule inhibitors for 30 minutes and imaged by multiphoton FLIM. Histogram analysis of relative spread of FRET efficiencies is a mean of 18 cells per treatment compared with 4-integrin-GFP alone control. (C) Human A375-SM melanoma cells were transfected with 4-integrin-GFP and paxillin-mRFP and plated onto a monolayer of TNF-treated activated HUVEC cells. Cells were allowed to adhere for 60 minutes, and were then treated with DMF vehicle control or stated small molecule inhibitors for 30 minutes. Cells were then imaged using multiphoton FLIM as before.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Small molecule inhibitors induce integrin signalling. (A) A375-SM cells were transfected with 4-integrin-GFP and paxillin-mRFP and plated onto coverslips coated with PLL. Cells were then incubated with K20 or mAb13 antibody-coated beads for 30 minutes, followed by treatment with DMF or small molecule inhibitor as indicated. Cells were then imaged using multiphoton FLIM. Cumulative FRET efficiency data from a masked pre-set region around the bead in ten cells per treatment is shown in the histogram. (B) Histogram demonstrating FRET efficiency of 4-paxillin association at K20 beads over a dose-response range of compounds S976162 or S9916197. Results are means ± s.e.m. (C) β1-integrin-GFP fibroblasts were transfected with talin-rod-mRFP. Cells were then plated onto coverslips coated with PLL and allowed to attach and spread for 1 hour. Cells were then incubated with beads coated with K20 for 30 minutes, and treated with either the vehicle control DMF or V0519 compound, and subsequently imaged using FLIM as before. Cumulative FRET efficiency data from a masked pre-set region around the bead in 12 cells per treatment is shown in the histogram.