Homo-FRET Imaging Enables Quantification of Protein Cluster Sizes with Subcellular Resolution

Abstract

Fluorescence-anisotropy-based homo-FRET detection methods can be employed to study clustering of identical proteins in cells. Here, the potential of fluorescence anisotropy microscopy for the quantitative imaging of protein clusters with subcellular resolution is investigated. Steady-state and time-resolved anisotropy detection and both one- and two-photon excitation methods are compared. The methods are evaluated on cells expressing green fluorescent protein (GFP) constructs that contain one or two FK506-binding proteins. This makes it possible to control dimerization and oligomerization of the constructs and yields the experimental relation between anisotropy and cluster size. The results show that, independent of the experimental method, the commonly made assumption of complete depolarization after a single energy transfer step is not valid here. This is due to a nonrandom relative orientation of the fluorescent proteins. Our experiments show that this relative orientation is restricted by interactions between the GFP barrels. We describe how the experimental relation between anisotropy and cluster size can be employed in quantitative cluster size imaging experiments of other GFP fusions. Experiments on glycosylphosphatidylinisotol (GPI)-anchored proteins reveal that GPI forms clusters with an average size of more than two subunits. For epidermal growth factor receptor (EGFR), we observe that approximately 40% of the unstimulated receptors are present in the plasma membrane as preexisting dimers. Both examples reveal subcellular heterogeneities in cluster size and distribution.

Visualization of BRI1 and BAK1(SERK3) Membrane Receptor Heterooligomers during Brassinosteroid Signaling

“…Our observation that only a subset of BRI1-SERK3 heterooligomers is present after ligand depletion is comparable to findings for EGFR in mammalian cells, as demonstrated by Bader et al (2009). Notably, for bone morphogenic protein receptors, differential signaling responses (Nohe et al, 2002) and endocytic routes (Hartung et al, 2006) for preformed and ligand-induced receptor complexes were observed.…”

“…promote lateral signal propagation (Martin-Fernandez et al, 2002;Yu et al, 2002;Bader et al, 2009). Our observation that only a subset of BRI1-SERK3 heterooligomers is present after ligand depletion is comparable to findings for EGFR in mammalian cells, as demonstrated by Bader et al (2009).…”

“…The latter is a receptor involved in innate immune responses upon lipopolysaccharide perception. EGFR constitutive dimer formation in the absence of ligand was demonstrated in NIH 3T3 cells using anisotropy measurements, whereas the dimerization of TLR4 is triggered upon lipopolysaccharide binding (Saitoh et al, 2004;Bader et al, 2009). …”

“…However, receptor complex preformation is a common phenomenon in animal signaling systems. For example, the insulin receptor is expressed as a constitutive dimer due to disulfide linkages between extracellular domains (Massague et al, 1980), and also epidermal growth factor receptors (EGFRs) reside as preformed complexes in the PM of mammalian cells (Gadella and Jovin, 1995;Bader et al, 2009). Preformation of receptor oligomers has also been reported for several families of animal receptors (Springael et al, 2005;Van Craenenbroeck, 2012).…”

Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis

“…However, the different techniques, cell types, and conditions of the experiments have resulted in conflicting and contradictory results. Several studies using fixed cells and/or low temperature incubations based on hetero-or homo-FRET have indicated that <10% to as much as 50% of ErbB1 may exist as preformed dimers or oligomers (17,19,20). Inhibition of the tyrosine kinase activity of the receptor led to disaggregation of the transient dimers and clusters to monomers (18), suggesting that some measurements may have been made on nonstarved cells with preactivated receptors.…”