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. Copyright 2018 The Author(s)Markwardt et al. ; This work was supported by NIH ( R01DK077140 , R01HL122827 , R01MH111527 , and R21OD018315 to M.A.R.), the intramural program of the National Institute of Biomedical Imaging and Bioengineering at the NIH ( 1ZIAEB000074 to H.S.), Shenzhen Innovation Funding ( JCYJ20170818164343304 and JCYJ20170816172431715 to H.L.), the National Key Technology Research and Development Program of China ( 2017YFE0104000 to H.L.), and the National Natural Science Foundation of China ( 61525106 , 61427807 , and 61701436 to H.L.). ; https://dx.doi.org/10.1016/j.celrep.2018.06.088