Quantification of integrin receptor agonism by fluorescence lifetime imaging

Abstract

Both spatiotemporal analyses of adhesion signalling and the development of pharmacological inhibitors of integrin adhesion receptors currently suffer from the lack of an assay to measure integrin-effector binding and the response of these interactions to agonists. Here, we have expressed integrin-GFP and effector-mRFP pairs in living cells and quantified their association using FLIM to measure FRET. Talin-β1 and paxillin-α4 association was both ligand- and receptor activation state-dependent, and sensitive to inhibition with small molecule RGD and LDV mimetics, respectively. An adaptation of the assay revealed the agonistic activity of these small molecules and provides a new, quantitative assay for the screening of activity of small molecule integrin inhibitors.

L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro

“…Acquisition times on the order of 300 s at low excitation power were used to achieve sufficient photon statistics for fitting, avoiding either pulse pile-up or significant photobleaching. Data were analyzed as previously described (50,51). The FRET efficiency is related to the molecular separation of donor and acceptor and the fluorescence lifetime of the interacting fraction by…”

Direct Interactions with the Integrin β1 Cytoplasmic Tail Activate the Abl2/Arg Kinase

“…The talin F3 subdomain was cloned into pGEX-6P-1 from a talin cDNA. For the FRET-FLIM studies, mutants were generated in ␤1-GFP constructs previously cloned as described (17). Arg-mCherry or Arg-RFP constructs were cloned into pN1-eGFP (Clontech) or pK1 vectors where eGFP was replaced with mCherry or RFP (5 ) murine embryonic fibroblast cells were spontaneously immortalized using the 3T3 protocol by passaging at 3.75 ϫ 10 5 cells/10-cm dish every 3 days for up to 20 passages as described previously (18).…”

“…FLIM-based FRET Measurements-Experiments were performed, and FRET efficiencies were analyzed as described previously (17). Briefly, cells were transfected with a 1:1.5 or 1:2 ratio of GFP:mCherry/RFP (donor:acceptor) plasmids and allowed to express for 36 h. Cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% (w/v) Triton X-100 in PBS.…”

“…After quenching with 1 mg/ml sodium borohydride in PBS for 10 min at room temperature, the cells were washed in PBS and mounted in Mowiol containing 2.5% Dabco (Sigma-Aldrich). Time domain FLIM was performed with a multiphoton microscope system (with TE2000 microscope; Nikon) described in detail previously (17). Fluorescence lifetime imaging capability was provided by time-correlated single-photon counting electronics (SPC-700; Becker and Hickl).…”

Kindlin Binds Migfilin Tandem LIM Domains and Regulates Migfilin Focal Adhesion Localization and Recruitment Dynamics

“…Cells were fixed using 4% paraformaldehyde followed by quenching with 1 mg/ml of sodium borohy-dride and mounted onto glass slides using Fluorsave (Merck). Details of the time domain FLIM performed with a multiphoton microscope system were described previously (35). Briefly, fluorescence lifetime imaging capability was provided by timecorrelated single photon counting electronics (Becker & Hickl, SPC 700).…”