Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Markwardt, Michele L., Snell, Nicole E., Guo, Min, Wu, Yicong, Christensen, Ryan, Liu, Huafeng, Shroff, Hari, Rizzo, M.A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2018
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
_version_ 1783351820519735296
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
work_keys_str_mv AT markwardtmichelel ageneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT snellnicolee ageneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT guomin ageneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT wuyicong ageneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT christensenryan ageneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT liuhuafeng ageneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT shroffhari ageneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT rizzoma ageneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT markwardtmichelel geneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT snellnicolee geneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT guomin geneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT wuyicong geneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT christensenryan geneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT liuhuafeng geneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT shroffhari geneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms
AT rizzoma geneticallyencodedbiosensorstrategyforquantifyingnonmusclemyosiniiphosphorylationdynamicsinlivingcellsandorganisms