Homo-FRET imaging as a tool to quantify protein and lipid clustering.

Homo-FRET, Förster resonance energy transfer between identical fluorophores, can be conveniently measured by observing its effect on the fluorescence anisotropy. This review aims to summarize the possibilities of fluorescence anisotropy imaging techniques to investigate clustering of identical proteins and lipids. Homo-FRET imaging has the ability to determine distances between fluorophores. In addition it can be employed to quantify cluster sizes as well as cluster size distributions. The interpretation of homo-FRET signals is complicated by the fact that both the mutual orientations of the fluorophores and the number of fluorophores per cluster affect the fluorescence anisotropy in a similar way. The properties of the fluorescence probes are very important. Taking these properties into account is critical for the correct interpretation of homo-FRET signals in protein- and lipid-clustering studies. This is be exemplified by studies on the clustering of the lipid raft markers GPI and K-ras, as well as for EGF receptor clustering in the plasma membrane.

Ligand-induced EGF receptor oligomerization is kinase-dependent and enhances internalization.

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ∼40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.

Homo-FRET imaging enables quantification of protein cluster sizes with subcellular resolution.

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.

EGF induces rapid reorganization of plasma membrane microdomains.

The plasma membrane of mammalian cells is composed of a great variety of different lipids which are laterally organized into lipid domains. The segregation of lipids into domains has been studied in great detail in vesicles but domain formation of lipids in the plasma membrane of live cells is still unclear. We have previously used fluorescence lifetime imaging microscopy to study the colocalization of the receptor for EGF with the ganglioside GM1 and the GPI-anchored green fluorescent protein. Here we have used this technology to study the effect of EGF on the organization of GM1 in the plasma membrane. Our data show that stimulation of the cell with EGF induces rapidly a strong increase in colocalization of GM1 molecules, suggesting the formation of large lipid domains. These results support the notion that activation of EGFR signaling may result in the formation of signaling platforms.

EGF induces coalescence of different lipid rafts.

The suggestion that microdomains may function as signaling platforms arose from the presence of growth factor receptors, such as the EGFR, in biochemically isolated lipid raft fractions. To investigate the role of EGFR activation in the organization of lipid rafts we have performed FLIM analyses using putative lipid raft markers such as ganglioside GM1 and glycosylphosphatidylinositol (GPI)-anchored GFP (GPI-GFP). The EGFR was labeled using single domain antibodies from Llama glama that specifically bind the EGFR without stimulating its kinase activity. Our FLIM analyses demonstrate a cholesterol-independent colocalization of GM1 with EGFR, which was not observed for the transferrin receptor. By contrast, a cholesterol-dependent colocalization was observed for GM1 with GPI-GFP. In the resting state no colocalization was observed between EGFR and GPI-GFP, but stimulation of the cell with EGF resulted in the colocalization at the nanoscale level of EGFR and GPI-GFP. Moreover, EGF induced the enrichment of GPI-GFP in a detergent-free lipid raft fraction. Our results suggest that EGF induces the coalescence of the two types of GM1-containing microdomains that might lead to the formation of signaling platforms.

Imaging of protein cluster sizes by means of confocal time-gated fluorescence anisotropy microscopy.

A time-resolved fluorescence anisotropy imaging method for studying nanoscale clustering of proteins or lipids was developed and evaluated. It is based on FRET between the identical fluorophores (homo-FRET), which results in a rapid depolarization of the fluorescence. The method employs the time-resolved fluorescence anisotropy decays recorded in a confocal microscope equipped with pulsed excitation and time-gated detection. From the decay the limiting anisotropy r(inf) was derived, which is a direct measure for the number of fluorophores per cluster. The method was evaluated by imaging GPI-GFP, a lipid raft marker. Small clusters were observed in the plasma membrane while the cytoplasm and the Golgi contained predominantly monomers.