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

Figure 1.. A FRET Assay for RLC Phosphorylation. (A) Phosphorylation of NMMII RLCs results in dissociation of RLC homodimers, actin binding, and diminished FRET. (B) REF52 fibroblasts expressing mCer3-RLC wereplated on uncoated or fibronectin (FN)-treated glass coverslips and imaged using widefield microscopy. (C) The fluorescence anisotropy of mCer3-RLC (n = 10 biological replicates) was measured in cells plated on uncoated or FN-treated glass coverslips (t test, ****p < 0.0001, n = 15 cells, bars indicate SD). (D) RLC phosphorylation sites (Ser19 and Thr18) (Ikebe et al., 1986) were mutated to alanines (AA) to prevent phosphorylation or aspartates (DD) to mimic RLC phosphorylation and expressed in REF52 cells. Fluorescence anisotropies were measured in REF52 cells expressing mutant mCer3-RLCs (t test as in C, ****p < 0.0001, n = 28 biological replicates for AA and 27 for DD, bars indicate SD). (E) REF52 cells expressing mCer3-RLC were platedon FN and fixed. (F) A ratio image was generated and pseudocoloredto show high anisotropy (i.e., phosphorylated) regions in blue. Some regions contain both high and low anisotropy pixels, making identification of phosphorylated myosin structures in the…

Figure 2.. MLCK Activity and RLC Phosphorylation. (A and B) REF52 fibroblasts expressing WT mCherry-RLC (A) or mCherry-RLC(AA) (B) were left untreated or incubated with 20 μM ML-7 to inhibit MLCK (5 min; n > 18 biological replicates; bars indicate SD; ****p < 0.0001 t test). (C) A REF52 cell coexpressing an MLCK biosensor and mCherry-RLC was plated on an uncoated glass coverslip and treated with 90 mM KCl to activate MLCK. MLCK biosensor (CFP, YFP) and mCherry-RLC polarization (P, S) images before stimulation are shown. Increases in MLCK CFP/ YFP FRET-ratios and mCherry-RLC anisotropies are represented by the indicated color scheme. Scale bar, 10 μm. Activation of both sensors was observed in the cytoplasm (white arrows). Activation of MLCK in the nucleus (oval) was also observed. (D and E) Normalized ratio changes for MLCK (CFP/YFP) and myosin RLC (P/S) are plotted for selected regions of interest for the cytoplasm (D), and the nucleus (E). KCL was added ∼5 min from the initiation of the experiment. (F and G) Data were normalized to the mean of the first 10 ratios. Changes in nuclear and cytoplasmic mCherry-RLC (F) and MLCK (G) were quantified across 10 cells. (H and I) Control experiments…

Figure 3.. MLCK Activity and RLC Phosphorylation in Cell Migration (A) MLCK activity, represented by a ratio image, and myosin phosphorylation,indicated by normalized subtraction, are shown for a randomly migrating REF52 over a 30-min period. (B) Kymographs were generated for pixels positioned along the orange line toshow changes over time. Arrows identify colocalized MLCK activation and RLC phosphorylation. Scale bars, 10 μm. (C) RLC anisotropy values within 3 mm of the detectable cellular edge were elevated compared to mutant RLC(AA) (n > 10 biological replicates; **p < 0.01 by t test; bars indicate mean and SD). (D) The MLCK sensor was similarly activated at the edges of WT and mutantRLC expressing cells in the same region (p > 0.05, t test; bars indicate SD). FRET ratios were normalized to values in the middle of the cytoplasm.

Figure 4.. RLC Phosphorylation during Retraction of a Cell Protrusion (A–C) Localized changes in RLC phosphorylation were stimulated using a microfluidic pipette to deliver 10 ng/mL PDGF to one portion of a REF52 cell expressing mCer3-RLC. (A) Rhodamine (in red) was used to mark the area of stimulation for several minutes before addition of PDGF. The P image of mCer3-RLC presented(cyan). (B) Fluorescence anisotropies are represented bythe indicated color scale. Pixel intensities from the yellow bar (top right) were used to create a kymograph. Scale bar, 10 μm. (C) Application of rhodamine (red) and PDGF (cyan) are indicated by the bars. The white arrows mark a PDGF-dependent shift in phosphorylated RLC that precedes an increase in the rate of retraction (pink arrow). Rapid myosin dephosphorylation during this phase is indicated by the blue arrow. (D) RLC anisotropy values (black) and were calculated within 3 μm of the retracting edge for cells expressing WT RLC or AA RLC. Displacement of the edge from the initial position is indicated in blue (n = 10 biological replicates, bars indicate SEM, a reference baseline at y = 0 [gray] was drawn for clarity). PDGF was added at time 0. WT RLC…

Figure 5.. RLC Phosphorylation during Embryonic Development in C. elegans (A–J) Changes in phosphorylated GFP-MLC-4 distribution were followed using piSPIM. Embryos were imaged starting at the first signs of GFP-MLC-4 expression. Z stacks were taken every 5 min until hatching. Comma (A and B), pre-twitching (C and D) and post-twitching (E and F) 2-fold, late elongation (G and H), and post-cuticle induction (I and J) stages are shown. Maximum intensity projections derived from image volumes for deconvolved S polarization images are shown alongside normalized subtraction images that indicate the location of GFP-MLC-4 phosphorylation (r > 0.27) (A, C, E, G, and I). Parallel striations in (C) and (E) result from under-sampling in the z dimension. Boxed regions of interest were enlarged (B, D, F, H, and J). In the comma stage, (B) phosphorylated myosin is organized around cell exterior (arrows and dashed lines) without apparent preference. Beginning in the twitching stage, myosin phosphorylation primarily becomes organized longitudinally to the embryo axis (compare pre-twitch, D, and post-twitch, F); arrows highlight the transverse organization in the pre-twitch state that is lost once twitching…

Figure 6.. Quantification of GFP-MLC-4 FRET Dynamics during C. elegans Embryonic Development (A) To calculate the anisotropies of the two GFPMLC-4 conformational states, we compiled data from a piSPIM imaging session of a GFP-MLC-4 expressing embryo. The embryo was imaged from comma to hatching, with one 60-frame z stack acquired every 5 min over an ∼7 hr period. Pixel anisotropy values from ∼5,000 images were pooled together, and the distribution of values was fit to a two-component Gaussian distribution (gray). Individual components for the FRET (blue) and phosphorylated states (red) are shown. (B) Calculated mean anisotropy values for eachstate are shown from 5 biological replicates. Bars indicate mean ± SD. (C) Changes in GFP-MLC-4 levels throughout the development was quantified by calculating the total fluorescence for each time point (n = 3 biological replicates, bars = SEM, developmental times were normalized to hatching [min]). (D and E) Pixel anisotropy values for each time point were then fit to a two-state Gaussian model. Peak amplitudes for each state were then used to calculate the relative percentage of phosphorylated pixels (D, n = 3 biological replicates, bars = SEM), and…