A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms.
Complex 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.
['Markwardt|Michele L|ML|', 'Snell|Nicole E|NE|', 'Guo|Min|M|', 'Wu|Yicong|Y|', 'Christensen|Ryan|R|', 'Liu|Huafeng|H|', 'Shroff|Hari|H|', 'Rizzo|M A|MA|']
D000818:Animals D015374:Biosensing Techniques D017173:Caenorhabditis elegans D002465:Cell Movement D024462:Myosin Type II D010766:Phosphorylation
non-muscle myosin II myosin homotransfer FRET fluorescent protein C. elegans GFP light sheet