TY - JOUR
T1 - Combining TIR and FRET in Molecular Test Systems
AU - Schneckenburger, Herbert
AU - Weber, Petra
AU - Wagner, Michael
AU - Enderle, Sandra
AU - Kalthof, Bernd
AU - Schneider, Linn
AU - Herzog, Claudia
AU - Weghuber, Julian
AU - Lanzerstorfer, Peter
PY - 2019/2/1
Y1 - 2019/2/1
N2 - Pharmaceutical agents or drugs often have a pronounced impact on protein-protein interactions in cells, and in particular, cell membranes. Changes of molecular conformations as well as of intermolecular interactions may affect dipole-dipole interaction between chromophoric groups, which can be proven by measuring the Förster resonance energy transfer (FRET). If these chromophores are located within or in close proximity to the plasma membrane, they are excited preferentially by an evanescent electromagnetic wave upon total internal reflection (TIR) of an incident laser beam. For the TIR-FRET screening of larger cell collectives, we performed three separate steps: (1) setting up of a membrane associated test system for probing the interaction between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2; (2) use of the Epac-SH188 sensor for quantitative evaluation under the microscope; and (3) application of a TIR fluorescence reader to probe the interaction of GFP with Nile Red. In the first two steps, we measured FRET from cyan (CFP) to yellow fluorescent protein (YFP) by spectral analysis and fluorescence lifetime imaging (FLIM) upon illumination of whole cells (epi-illumination) as well as selective illumination of their plasma membranes by TIR. In particular, TIR excitation permitted FRET measurements with high sensitivity and low background. The Epac sensor showed a more rapid response to pharmaceutical agents, e.g., Forskolin or the A2B adenosine receptor agonist NECA, in close proximity to the plasma membrane compared to the cytosol. Finally, FRET from a membrane associated GFP to Nile Red was used to test a multi-well TIR fluorescence reader with simultaneous detection of a larger number of samples.
AB - Pharmaceutical agents or drugs often have a pronounced impact on protein-protein interactions in cells, and in particular, cell membranes. Changes of molecular conformations as well as of intermolecular interactions may affect dipole-dipole interaction between chromophoric groups, which can be proven by measuring the Förster resonance energy transfer (FRET). If these chromophores are located within or in close proximity to the plasma membrane, they are excited preferentially by an evanescent electromagnetic wave upon total internal reflection (TIR) of an incident laser beam. For the TIR-FRET screening of larger cell collectives, we performed three separate steps: (1) setting up of a membrane associated test system for probing the interaction between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2; (2) use of the Epac-SH188 sensor for quantitative evaluation under the microscope; and (3) application of a TIR fluorescence reader to probe the interaction of GFP with Nile Red. In the first two steps, we measured FRET from cyan (CFP) to yellow fluorescent protein (YFP) by spectral analysis and fluorescence lifetime imaging (FLIM) upon illumination of whole cells (epi-illumination) as well as selective illumination of their plasma membranes by TIR. In particular, TIR excitation permitted FRET measurements with high sensitivity and low background. The Epac sensor showed a more rapid response to pharmaceutical agents, e.g., Forskolin or the A2B adenosine receptor agonist NECA, in close proximity to the plasma membrane compared to the cytosol. Finally, FRET from a membrane associated GFP to Nile Red was used to test a multi-well TIR fluorescence reader with simultaneous detection of a larger number of samples.
KW - Biosensing Techniques
KW - ErbB Receptors/genetics
KW - Fluorescence Resonance Energy Transfer/methods
KW - Gene Expression
KW - Genes, Reporter
KW - Humans
KW - Luminescent Proteins/genetics
KW - Microscopy, Fluorescence
KW - Molecular Diagnostic Techniques
KW - Fluorescence spectroscopy
KW - Drug screening
KW - Living cells
KW - Total internal reflection (TIR)
KW - Förster resonance energy transfer (FRET)
KW - Microscopy
KW - Fluorescence lifetime imaging (FLIM)
KW - Fluorescence reader
UR - http://www.scopus.com/inward/record.url?scp=85061134191&partnerID=8YFLogxK
U2 - 10.3390/ijms20030648
DO - 10.3390/ijms20030648
M3 - Article
C2 - 30717378
SN - 1422-0067
VL - 20
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 3
M1 - 648
ER -