Biology Reference
In-Depth Information
Application of FRET and Microscopy
14.3.5 FRET Imaging of Receptor-Receptor Interaction for Intact Cells
with Subcellular Spatial Resolution........................................... 256
14.3.5.1 Zeiss LSM 780 ................................................................ 256
14.3.5.2 Spinning Disk Microscope................................................ 257
14.3.5.3 TIRF Microscope ............................................................. 259
14.3.6 Procedure Part II: Data Evaluation ............................................. 260
14.3.6.1 Evaluation Procedure ....................................................... 261
14.4 Results and Discussion ................................................................................. 263
Acknowledgments ................................................................................................... 264
References ............................................................................................................. 264
Abstract
G protein-coupled receptors (GPCRs) participate in the regulation of many cellular
processes and, therefore, represent key targets for pharmacological treatment. The
existence of GPCR homo- and heterodimers has become generally accepted, and
a growing body of evidence points to the functional importance of oligomeric com-
plexes for the receptor trafficking, receptor activation, and G protein coupling in na-
tive tissues. Quantitative molecular microscopy is becoming more and more
important to investigate such receptor-receptor interaction in their native environ-
ments. F¨rster resonance energy transfer (FRET) is thereby utilized to aim at inves-
tigating the interaction of molecules at distances beyond diffraction-limited spatial
resolution. The exact determination of the FRET signals, which are often only frac-
tions of the fluorescence signals, requires extensive experimental effort. Moreover,
the correct interpretation of FRET measurements as well as FRET data-based model-
ing represents an essential challenge in microscopy and biophysics.
In this chapter, we present and discuss variety of acquisition protocols and models
based on “linear unmixing FRET” (lux-FRET) to investigate receptor-receptor interac-
tion in livingcellswithhigh spatial and temporal resolution. Here,we showhowtoapply
lux-FRET in spectroscopic and different imaging devices, based either on spectral
detection or on filter cubes. We focus on detailed description for FRET measurements
andanalysesbasedonsophisticatedacquisitionprocedures according todifferent exper-
imental setups and also provide several examples of biological applications.
INTRODUCTION AND RATIONALE
G protein-coupled receptors (GPCRs) belong to a large and diverse family of in-
tegral membrane proteins that participate in the regulation of many cellular pro-
cesses and, therefore, represent key targets for pharmacological treatment. GPCRs
are known to be able to activate multiple downstream signaling modules at the
plasma membrane (
Gorinski et al., 2012
); however, the mechanisms regulating
multimodal GPCR-mediated signaling are still poorly understood. Until recently,
