Globally elevating NM2 monomer availability initiates filament assembly. (A–C) Confocal z-stacks of primary EGFP-NM2A MEF cells were acquired. Images are sum intensity projections of z-stacks and time-sum projections of 15 frames collected every 15 s in (A) and 5 s in (B). Nascent NM2 filament appearances during that period are indicated with magenta circles and tallied in the upper right corner. The green dotted line indicates the current leading edge. The orange dotted line indicates original tail location (A) and the orange box indicates frames treated with 10 μM Y-27632 (B). Scale bars = 20 μm. (C) EGFP-NM2A sum z-projections were temporally summed over 20 frames (100 s). Nascent NM2 filament appearances during that period are indicated with magenta circles and tallied in the upper right corner. Binned time periods pre- and -post (orange box) treatment are shown for JLY treatment. Scale bar = 5 μm. Intensity LUTs are indicated to the right of each panel. (D) Nascent NM2 filament appearance events pre- and post-treatment were quantified, normalized to pre-treatment appearance events per minute, for individual cells (small black dots) and the mean of three independent experiments (large color circles). Wilcoxon matched-pairs signed rank test performed comparing each cell (n = 12). P = 0.0034.