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A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living
Michele L Markwardt1, Nicole E Snell1, Min Guo2
1Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Cell Reports
|July 26, 2018
Summary
Researchers developed a novel FRET-based method to track non-muscle myosin II (NMMII) regulatory light chain (RLC) phosphorylation in living cells and organisms. This technique visualizes myosin dynamics crucial for cell behaviors and embryonic development.
Area of Science:
- Cell Biology
- Biophysics
- Molecular Imaging
Background:
- Non-muscle myosin II (NMMII) plays a critical role in cellular functions through the dynamic phosphorylation of its regulatory light chain (RLC).
- Understanding the spatiotemporal regulation of RLC phosphorylation is essential for deciphering complex cell behaviors and developmental processes.
Purpose of the Study:
- To develop and validate a novel homotransfer fluorescence resonance energy transfer (FRET) biosensor for real-time tracking of RLC phosphorylation in living cells and organisms.
- To investigate the dynamic changes in myosin phosphorylation during cell migration and embryonic development.
Main Methods:
- Utilized fluorescent protein-tagged RLCs engineered to exhibit FRET in their dephosphorylated state.
- Employed homotransfer FRET for quantitative measurement of RLC phosphorylation dynamics.
- Integrated the FRET biosensor with advanced microscopy techniques, including polarization inverted selective-plane illumination microscopy (piSPIM).
Main Results:
- Demonstrated successful tracking of RLC phosphorylation in living fibroblasts, revealing dynamic changes at the leading edge and retracting structures.
- Observed persistent colocalization of phosphorylated myosin with activated myosin light chain kinase in migrating cells.
- Tracked myosin phosphorylation dynamics during C. elegans embryonic development, noting a shift in localization correlating with twitching onset.
- Quantitative analysis indicated that RLC phosphorylation dynamics are independent of protein expression levels.
Conclusions:
- The developed FRET-based approach provides a versatile and powerful tool for visualizing and quantifying myosin phosphorylation in living systems.
- This method enables new insights into the role of myosin dynamics in fundamental biological processes like cell migration and development.
- The findings highlight the dynamic nature of myosin phosphorylation and its independent regulation during development.
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