A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms
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AbstractComplex 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.
SponsorsThis 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.).
Identifier to cite or link to this itemhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85049897829&doi=10.1016%2fj.celrep.2018.06.088&partnerID=40&md5=63d7571dc7625a234cf55e9d7a599c56; http://hdl.handle.net/10713/9297
- A fluorescent protein biosensor of myosin II regulatory light chain phosphorylation reports a gradient of phosphorylated myosin II in migrating cells.
- Authors: Post PL, DeBiasio RL, Taylor DL
- Issue date: 1995 Dec
- Tyrosine Phosphorylation of the Myosin Regulatory Light Chain Controls Non-muscle Myosin II Assembly and Function in Migrating Cells.
- Authors: Aguilar-Cuenca R, Llorente-González C, Chapman JR, Talayero VC, Garrido-Casado M, Delgado-Arévalo C, Millán-Salanova M, Shabanowitz J, Hunt DF, Sellers JR, Heissler SM, Vicente-Manzanares M
- Issue date: 2020 Jul 6
- Myosin II transport, organization, and phosphorylation: evidence for cortical flow/solation-contraction coupling during cytokinesis and cell locomotion.
- Authors: DeBiasio RL, LaRocca GM, Post PL, Taylor DL
- Issue date: 1996 Aug
- Analysis of the role of Ser1/Ser2/Thr9 phosphorylation on myosin II assembly and function in live cells.
- Authors: Beach JR, Licate LS, Crish JF, Egelhoff TT
- Issue date: 2011 Dec 2
- Myosin regulatory light chains are required to maintain the stability of myosin II and cellular integrity.
- Authors: Park I, Han C, Jin S, Lee B, Choi H, Kwon JT, Kim D, Kim J, Lifirsu E, Park WJ, Park ZY, Kim DH, Cho C
- Issue date: 2011 Feb 15