Model of Mero-S100A4. Model of the apo and Ca²⁺-bound Mero-S100A4. Helices 3 and 4 of one monomer are shown in red, and the merocyanine dye attached to Cys81 is shown in yellow. Merocyanine attached to only one monomer is shown for simplicity. The bound calcium ions are depicted as pink spheres.

Mero-S100A4 does not regulate the monomer–polymer equilibrium of myosin-IIA filaments. (A) Promotion of myosin-IIA filament disassembly by wild-type and Mero-S100A4. At a ratio of one wild-type S100A4 dimer per myosin-IIA rod, most of the myosin-IIA rods are present in the supernatant. A ratio of five Mero-S100A4 dimers per myosin-IIA rod has no effect on the myosin-IIA filaments. (B) Inhibition of myosin-IIA filament assembly by wild-type and Mero-S100A4. At a ratio of one S100A4 dimer per myosin-IIA rod, wild-type S100A4 inhibits the assembly of myosin-IIA monomers into filaments. Mero-S100A4 does not inhibit the assembly of the myosin-IIA rods at a ratio of five Mero-S100A4 dimers per myosin-IIA rod.

Identification of small molecule inhibitors using the Mero-S100A4. (A) Hit compounds identified in a screen of a library of FDA-approved drugs. Compounds that inhibited the Ca²⁺-induced fluorescence increase of the Mero-S100A4 to a similar extent as EGTA were considered to be hits. (B) IC₅₀ values for inhibition of the Ca²⁺-induced fluorescence increase of the bio-sensor. The data represents the best fit to a three parameter logistic equation from two independent experiments.

S100A4 activity in LPA-stimulated fibroblasts. (A) Cells were injected with a 1:2 ratio of Fluo-S100A4:Mero-S100A4, stimulated with LPA for the indicated times and fixed. A representative cell for each time point is shown with purple to red signifying low to high activation. At 1 min poststimulation, S100A4 activation is restricted to cell extensions with no activated S100A4 detected in the cell body. This pattern of activation was very similar in the 6 cells examined at 1 min and the 8 cells examined at 5 min. (B) Cells were injected with a 1:2 ratio of Fluo-S100A4:Mero-S100A4 in a buffer containing 1 mM EGTA, stimulated with LPA for the indicated times and fixed. A representative cell for each time point is shown with purple to red signifying low to high activation. No activation of S100A4 is detected in cells 1 min poststimulation (10 cells were examined). Scale bar = 20 μm.

Mero-S100A4 reports activation by Ca²⁺. (A) Structure of the I-SO merocyanine dye. (B) Fluorescence excitation and emission spectra of 5 μM Mero-S100A4 dimer. Dashed line: Mero-S100A4 in the presence of EGTA. Solid line: Mero-S100A4 in the presence of Ca²⁺. Dotted line: Mero-S100A4 in the presence of Ca²⁺ and myosin-IIA. Mero-S100A4 exhibits a 3-fold increase in fluorescence upon Ca²⁺ addition. The addition of Ca²⁺ and a 10-fold molar excess of myosin-IIA results in a slight red shift, but no additional increase in fluorescence intensity. (C) Mero-S100A4 was added to a fibroblast lysate in the presence of EGTA or calcium. The data represent the average peak intensity at 634 nm for three independent experiments and the standard deviation.

Wild-type and Mero-S100A4 bind Ca²⁺ with the same affinities. Competition assay with the chelator 5,5′Br₂-BAPTA was used to examine the affinity of Mero-S100A4 for Ca²⁺. The decrease in absorbance was monitored at 263 nm for a mixture containing 25 μM 5,5′Br₂-BAPTA and either 25 μM wild-type S100A4 (A) or Mero-S100A4 (B). The data represent 6–8 titrations from 4 independent experiments. The insets show the saturation curve representation for the best fit in Caligator. (C) Ca²⁺ titration experiments were carried out using a Ca²⁺/EGTA buffering system and 5 μM Mero-S100A4 dimer. The data represent four independent experiments.

Far-UV circular dichroism spectra of wild-type, C3R/C86S and Mero-S100A4. The spectra show wild-type (solid line), C3R/C86S (dashed line) and Mero-S100A4 (dotted line) at a protein concentration of 12.5 μM dimer.

Hit compounds disrupt the interaction of S100A4 and myosin-IIA. (A) A sedimentation assay was used to examine the ability of hit compounds to attenuate the destabilization of myosin-IIA filaments by S100A4. At a molar ratio of one wild-type S100A4 dimer per myosin-IIA rod, ~90% of the myosin-IIA rods are recovered in the supernatant. Values represent the mean and the standard error of the mean from at least four independent experiments. 1, trifluoperazine; 2, prochlorperazine; 3, loperamide; 4, bepridil, and 5, fluphenazine. (B) Fluorescence anisotropy measurements of S100A4 binding to FITC-MIIA^1908–1923. Values represent the mean and the standard deviation from three independent experiments. A K_d of 1.7 ± 0.2 μM was determined from the fit to a single site saturation binding curve. (C) Titration experiment with trifluoperazine showing a loss of anisotropy as TFP competes off the bound FITC-MIIA^1908–1923. The values represent the mean and the standard deviation from three independent experiments. The data were fit to a three parameter logistic equation to obtain the EC₅₀. (D) Bar graph representation of anisotropy competition assays with lead compounds. Values represent the mean and the standard deviation for the endpoint of the titration (600 μM compound) from three independent experiments. The numbers above the bar indicate the K_i in units of μM.

S100A4 is activated during cell retraction. The panels show different time points (hours:minutes) during random cell movement over fibronectin. Upper panels: DIC. Lower panels: ratio of Mero over FITC signal. An area of high activation is prominent during retraction. The area of elevated activation persists after the start of a new protrusion in the same area (arrowhead). Elevated Mero-S100A4 ratios were observed in cellular retractions in 5 cells examined. Scale bar = 10 μm. The ratio image was scaled by setting the lower and upper threshold levels to exclude the lower 7% and upper 3%, respectively, and a linear pseudocolor lookup table was applied where the upper threshold limit value was divided by the lower threshold limit value to show the dynamic range of ratio signal.