Homotransfer FRET Reporters for Live Cell Imaging

Abstract

Förster resonance energy transfer (FRET) between fluorophores of the same species was recognized in the early to mid-1900s, well before modern heterotransfer applications. Recently, homotransfer FRET principles have re-emerged in biosensors that incorporate genetically encoded fluorescent proteins. Homotransfer offers distinct advantages over the standard heterotransfer FRET method, some of which are related to the use of fluorescence polarization microscopy to quantify FRET between two fluorophores of identical color. These include enhanced signal-to-noise, greater compatibility with other optical sensors and modulators, and new design strategies based upon the clustering or dimerization of singly-labeled sensors. Here, we discuss the theoretical basis for measuring homotransfer using polarization microscopy, procedures for data collection and processing, and we review the existing genetically-encoded homotransfer biosensors.

Imaging of fluorescence anisotropy during photoswitching provides a simple readout for protein self-association

“…Although homo-FRET studies typically report changes in anisotropy, reporting efficiencies would be beneficial since these do not rely on specific instrument parameters and could be more easily reproduced across laboratories. To this end, several previous publications have discussed quantitative relationships between steady-state anisotropy measurements and energy transfer efficiency [26][27][28][29] . In some of his seminal studies, Weber described the relationship between polarization, fluorophore concentration, and the average fluorophore distance 26 .…”

“…Unfortunately, these equations produce disparate values over the anisotropy range possible for an isotropic population of molecules (−0.2-0.4). More importantly, those equations [26][27][28][29] are derived with the assumption that the average value of κ 2 , the dipole orientation factor, is equal to 2/3. While this is a reasonable assumption for small fluorophores with rotational correlation times much faster than their fluorescence lifetime, this is not valid for much larger fluorescent proteins which rotate very little during their fluorescence lifetimes 30,31 .…”

“…Although equations converting anisotropy values into FRET efficiencies are available in the literature [27][28][29] , we were uncertain that assumptions made for those equations would be valid for our data using photoswitchable fluorescent proteins. Therefore, the unique capability of a psAFRET experiment to provide r et1 and r et0 values for each sample prompted us to derive a separate equation to convert these two values into drFRET, a value akin to but distinct from a hetero-FRET efficiency.…”

The extracellular matrix and Ca(2+)signaling mechanisms

“…Elimination of the cells showing features of Ca 2+ signaling dysregulation and careful selection of healthy cells should be considered as a reliable way for predicting the efficacy of such muscle treatment in terms of a longterm cell survival. Further refinement and new insights in the underlying mechanisms could be obtained in the near future from the experiments based on the usage of genetically encoded calcium indicators and from the analysis of the changes in calcium homeostasis by fluorescent ratiometric biosensors in live cell imaging (Horikawa 2015, Snell et al 2018.…”

FRET Microscopy in Yeast

“…The principles of FRET allow its measurement by several different microscopy or spectroscopy techniques [3,4,8,[77][78][79][80][81][82][83][84]. They can be divided into methods following changes in donor or acceptor fluorescence intensity, donor or acceptor fluorescence polarization/anisotropy, and the lifetime of the donor fluorescence (Figure 4).…”