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Quantification of integrin receptor agonism by fluorescence lifetime imaging.

ABSTRACT: Both spatiotemporal analyses of adhesion signalling and the development of pharmacological inhibitors of integrin receptors currently suffer from the lack of an assay to measure integrin-effector binding and the response of these interactions to antagonists. Indeed, anti-integrin compounds have failed in the clinic because of secondary side effects resulting from agonistic activity. Here, we have expressed integrin-GFP and effector-mRFP pairs in living cells and quantified their association using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). Association of talin with beta1 integrin and paxillin with alpha4 integrin was dependent on both the ligand and receptor activation state, and was sensitive to inhibition with small molecule RGD and LDV mimetics, respectively. An adaptation of the assay revealed the agonistic activity of these small molecules, thus demonstrating that these compounds may induce secondary effects in vivo via integrin activation. This study provides insight into the dependence of the activity of small molecule anti-integrin compounds upon receptor conformation, and provides a novel quantitative assay for the validation of potential integrin antagonists.

SUBMITTER: Parsons M

PROVIDER: S-EPMC3328206 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Quantification of integrin receptor agonism by fluorescence lifetime imaging.

Parsons Maddy M Messent Anthea J AJ Humphries Jonathan D JD Deakin Nicholas O NO Humphries Martin J MJ

Journal of cell science 20080201 Pt 3

Both spatiotemporal analyses of adhesion signalling and the development of pharmacological inhibitors of integrin receptors currently suffer from the lack of an assay to measure integrin-effector binding and the response of these interactions to antagonists. Indeed, anti-integrin compounds have failed in the clinic because of secondary side effects resulting from agonistic activity. Here, we have expressed integrin-GFP and effector-mRFP pairs in living cells and quantified their association usin ...[more]

PMID: 18216331

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Project description:Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a novel technique that measures in vivo autofluorescence intensity decay over time of endogenous fluorophores in the retina. The Heidelberg Engineering FLIO system was used to obtain two 30 degree scans centered on the fovea of both eyes. The FLIO system uses a 473nm blue scanning laser light source and the emitted fluorescence is detected in two wavelengths channels, short and long spectral channels (SSC, LSC). Since the mydriatic status influence the FLIO result, the impact of mydriasis on FLIO need to be clarified. In this prospective, observational study, the impact of mydriasis on measurements from fluorescence lifetime imaging ophthalmoscope (FLIO) images in normal subjects were evaluated. 12 healthy participants (24 eyes) were volunteered and all subjects were scanned twice and the mean fluorescence lifetime (?m) values were computed with dilation and without dilation on different days. Intraclass correlation coefficients (ICC) and coefficients of variation (CV) were calculated from the measured ?m in dilated, nondilated and between the dilated and non-dilated setting. Test duration was also compared and correlated with lifetimes in both settings. Repeatability was excellent for both the dilation and non-dilation settings (ICC; 0.967-0.996; 0.926-0.986, respectively). The agreement between the dilation and non-dilation settings, however, were lower (ICC; 0.688-0.970). The ?m in the non-dilation setting was significantly longer than in the dilation setting for the SSC (P<0.05). The FLIO test duration in the non-dilation setting was significantly longer than with dilation for the SSC (P <0.05). Although good repeatability in ?m measurements between imaging sessions were observed both with and without dilation, the agreement was not as good when comparing dilated with non-dilated measurements. Since FLIO without mydriasis results in longer ?m in the SSC and takes a longer time for image acquisition, maximal dilation is recommended for FLIO testing.

Dataset Information

Quantification of integrin receptor agonism by fluorescence lifetime imaging.

Publications

Quantification of integrin receptor agonism by fluorescence lifetime imaging.

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