A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms

Abstract

SUMMARYComplex 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.

Amphetamine Stimulates Endocytosis of the Norepinephrine and Neuronal Glutamate Transporters in Cultured Locus Coeruleus Neurons

“…We also examined the temporal response of our cells in calcium-free conditions. With a fluorescence resonance energy transfer (FRET) sensor designed to detect activated RhoA [14] we measured activation of the GTPase in response to AMPH with and without calcium (Fig. 6c).…”

Homotransfer FRET Reporters for Live Cell Imaging

“…Widefield systems can be configured for steady-state fluorescence polarization microscopy by the addition of plate polarizers into the optical pathway [27,45]. An excitation polarizer can be placed between the light source and the fluorescence filter cube.…”

“…Newly developed optical sectioning approaches can improve image signal-to-noise and provide gentler sample illumination conditions. Inverted selective plane illumination microscopy (iSPIM) [53], which illuminates the sample with a light sheet positioned perpendicularly to the collection lens, has been successfully configured to accommodate polarization microscopy [45,54]. Using this method, we were able to perform homotransfer FRET biosensor imaging over a seven-hour period in developing C. elegans embryos [45].…”

“…Inverted selective plane illumination microscopy (iSPIM) [53], which illuminates the sample with a light sheet positioned perpendicularly to the collection lens, has been successfully configured to accommodate polarization microscopy [45,54]. Using this method, we were able to perform homotransfer FRET biosensor imaging over a seven-hour period in developing C. elegans embryos [45]. Image collection speeds during this experiment approached video rate, which is at least 10 times faster than the previous optical sectioning methods that we have tested.…”

“…The regulatory light chain of myosin II naturally dimerizes until activated by phosphorylation. Fusion of this myosin subunit with a fluorescent protein permits detection of the phosphorylation event by monitoring homotransfer [45].…”