Detecting multiple oligomerization states by multidimensional analysis of FRET images

Fluorescence resonance energy transfer (FRET) microscopy is commonly used to measure distances between fluorophores or to qualitatively confirm interaction of proteins. Whereas multiple FRETing oligomerization states may be characterized clearly using single-molecule techniques, cellular applications suffer from wide and immeasurable variability in concentration of each species across pixels. Several examples are presented to demonstrate the inapplicability of Gaussian mixture model fitting to normalized FRET distributions to the problem of identifying populations of pixels of similar oligomerization state ratios. Instead, a direct clustering approach is developed in which each pixel is considered as a (FRET, acceptor, donor) triplet and assigned a probability of belonging to each of several clusters. The probabilities may be used to create an informative colored map of the cell indicating the locations of the various oligomer types. Fluorescence resonance energy transfer Fluorescence resonance energy transfer (FRET) occurs when a donor fluorophore in an excited state excites a donor fluorophore in the ground state through dipole-dipole interaction, without the transfer of a photon. The donor is excited by a laser near the peak of its absorption spectrum, and FRET is detected by recording fluorescence near the peak of the acceptor emission spectrum. Both donor and acceptor fluorescence are also measured so that the efficiency and not just the strength of interaction may be computed. The distance at which resonance energy transfer is 50% efficient, called the Förster radius, is typically 20 to 90 Å. As the efficiency of energy transfer is proportional to R -6 , where R is the distance between donor and acceptor fluorophore, FRET microscopy is well-suited to the detection of oligomerization of proteins with attached fluorophores. Typical uses of this technique include measuring distances between fluorophores and qualitatively detecting covalent interaction (Lakowicz 2006). Recent studies from the Lester lab at Caltech (Drenen et al. 2008, Son et al. 2009) have used FRET microscopy to study interactions among the subunits that make up nicotinic acetylcholine receptors (nAChRs), a class of pentameric ion channels. Whereas muscle nicotinic receptors have fixed composition, neuronal receptors may be composed of various combinations of subunits. Stoichiometry as well as presence of specific subunits determines sensitivity; for instance, the ( 4)2( 2)3 receptor has EC50 ~1μM, whereas the ( 4)3( 2)2 receptor is 100 times less sensitive. Upregulation of receptors in response to chronic nicotine exposure favors the high-sensitivity stoichiometry (Lester et al., 2009, review). A method is required for determining, from FRET images, the relative abundances of the various stoichiometries. Current analytical techniques The distributions of FRET efficiencies for multiple conformational states have been characterized for single-molecule FRET studies, which require a very dilute solution of fluorophores so that each pixel contains at most one FRET pair (Best 2007). In contrast, live cells may contain tens to hundreds of FRETing oligomers, as well as unpaired donors and acceptors. Partially assembled receptors (dimers and tetramers) may also be present in unknown geometries. Whereas the overall ratio of donor to acceptor may be measured in addition to FRET efficiency (Chen et al. 2006), the presence of multiple similar stoichiometries of assembled receptors among unpaired and partially-assembled receptors has not been studied. One standard approach that hopes to distinguish among stoichiometries involves normalizing the FRET intensities according to Xia and Liu (2001):