Genetically encoded fluorescent biosensors have revolutionized the study of signal transduction by enabling the real-time tracking of signaling activities in live cells. Investigating the interaction between signaling networks has become increasingly important to understanding complex cellular phenomena, necessitating an update of the biosensor toolkit to allow monitoring and perturbing multiple activities simultaneously in the same cell. We therefore developed a new class of fluorescent biosensors based on homo-FRET, deemed FLuorescence Anisotropy REporters (FLAREs), which combine the multiplexing ability of single-color sensors with a quantitative, ratiometric readout. Using an array of color variants, we were able to demonstrate multiplexed imaging of three activity reporters simultaneously in the same cell. We further demonstrate the compatibility of FLAREs for use with optogenetic tools as well as intravital two-photon imaging.
SUMMARYComplex cell behaviors require dynamic control over non-muscle myosin II (NMMII) regulatory light chain (RLC) phosphorylation. Here, we report that RLC phosphorylation can be tracked in living cells and organisms using a homotransfer fluorescence resonance energy transfer (FRET) approach. Fluorescent protein-tagged RLCs exhibit FRET in the dephosphorylated conformation, permitting identification and quantification of RLC phosphorylation in living cells. This approach is versatile and can accommodate several different fluorescent protein colors, thus enabling multiplexed imaging with complementary biosensors. In fibroblasts, dynamic myosin phosphorylation was observed at the leading edge of migrating cells and retracting structures where it persistently colocalized with activated myosin light chain kinase. Changes in myosin phosphorylation during C. elegans embryonic development were tracked using polarization inverted selective-plane illumination microscopy (piSPIM), revealing a shift in phosphorylated myosin localization to a longitudinal orientation following the onset of twitching. Quantitative analyses further suggested that RLC phosphorylation dynamics occur independently from changes in protein expression.
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.
The release of insulin from the pancreas is tightly controlled by glucokinase (GCK) activity that couples β-cell metabolism to changes in blood sugar. Despite having only a single glucose-binding site, GCK displays positive glucose cooperativity. Ex vivo structural studies have identified several potential protein conformations with varying levels of enzymatic activity, yet it is unclear how living cells regulate GCK cooperativity. To better understand the cellular regulation of GCK activation, we developed a homotransfer Förster resonance energy transfer (FRET) GCK biosensor and used polarization microscopy to eliminate fluorescence crosstalk from FRET quantification and improve the signal-to-noise ratio. This approach enhanced sensor contrast compared to that seen with the heterotransfer FRET GCK reporter and allowed observation of individual GCK states using an automated method to analyze FRET data at the pixel level. Mutations known to activate and inhibit GCK activity produced distinct anisotropy distributions, suggesting that at least two conformational states exist in living cells. A high glucose level activated the biosensor in a manner consistent with GCK's enzymology. Interestingly, glucose-free conditions did not affect GCK biosensor FRET, indicating that there is a single low-activity state, which is counter to proposed structural models of GCK cooperativity. Under low-glucose conditions, application of chemical NO donors efficiently shifted GCK to the more active conformation. Notably, GCK activation by mutation, a high glucose level, a pharmacological GCK activator, or S-nitrosylation all shared the same FRET distribution. These data suggest a simplified model for GCK activation in living cells, where post-translational modification of GCK by S-nitrosylation facilitates a single conformational transition that enhances GCK enzymatic activity.
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