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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. Fluorescen...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117825/ https://www.ncbi.nlm.nih.gov/pubmed/30044973 http://dx.doi.org/10.1016/j.celrep.2018.06.088 |
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author | Markwardt, Michele L. Snell, Nicole E. Guo, Min Wu, Yicong Christensen, Ryan Liu, Huafeng Shroff, Hari Rizzo, M.A. |
author_facet | Markwardt, Michele L. Snell, Nicole E. Guo, Min Wu, Yicong Christensen, Ryan Liu, Huafeng Shroff, Hari Rizzo, M.A. |
author_sort | Markwardt, Michele L. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6117825 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
record_format | MEDLINE/PubMed |
spelling | pubmed-61178252018-08-31 A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms Markwardt, Michele L. Snell, Nicole E. Guo, Min Wu, Yicong Christensen, Ryan Liu, Huafeng Shroff, Hari Rizzo, M.A. Cell Rep Article 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. 2018-07-24 /pmc/articles/PMC6117825/ /pubmed/30044973 http://dx.doi.org/10.1016/j.celrep.2018.06.088 Text en This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
spellingShingle | Article Markwardt, Michele L. Snell, Nicole E. Guo, Min Wu, Yicong Christensen, Ryan Liu, Huafeng Shroff, Hari Rizzo, M.A. A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms |
title | A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms |
title_full | A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms |
title_fullStr | A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms |
title_full_unstemmed | A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms |
title_short | A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms |
title_sort | genetically encoded biosensor strategy for quantifying non-muscle myosin ii phosphorylation dynamics in living cells and organisms |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117825/ https://www.ncbi.nlm.nih.gov/pubmed/30044973 http://dx.doi.org/10.1016/j.celrep.2018.06.088 |
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