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

Abstract

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.

EGF induces coalescence of different lipid rafts

“…We and others have previously shown that GPI-GFP exists in small cholesterol-dependent nanoclusters (Bader et al, 2007;Sharma et al, 2004). These anisotropy experiments suggest the existence of small clusters of three to four GPI-GFP molecules per microdomain.…”

EGFR endocytosis requires its kinase activity and N-terminal transmembrane dimerization motif

“…A detailed description of our confocal anisotropy imaging setup and homo-FRET method was published previously (Bader et al, 2007;Bader et al, 2009;Hofman et al, 2010). Briefly, a 473 nm solid-state diode laser (Becker and Hickl, BDL-473-SMC) with a pulse repetition rate of 80 MHz polarized by a linear polarizer (Meadowlark, Frederick, CO) was coupled into a confocal microscope (Nikon C1, Japan).…”

Design and application of a confocal microscope for spectrally resolved anisotropy imaging

“…FRET causes depolarization of the sensitized emission because of the transfer of excitation energy to a second fluorophore the orientation of which is not constrained by the photoselection rules of the directly excited fluorophores. A decrease in the anisotropy can be measured when identical fluorophores are in close proximity (homo-FRET) allowing protein oligomerization to be detected [7,26]. Recently, Rizzo and Piston [25] demonstrated that mapping the FRET-dependent depolarization in hetero-FRET experiments could be also useful to measure protein-protein interactions.…”

“…Furthermore, many fluorophores exhibit altered emission spectra as a function of the molecular environment thus allowing quantitative sensing applications [5]. Also fluorescence anisotropy and fluorescence lifetime have been used for sensing applications [6,7] and, less frequently, for unmixing [8,9]. The development of a sensor for the combined detection of fluorescence emission and excitation spectra, anisotropy and lifetime would enable multiplexed sensing capabilities that could be exploited for biological assays.…”