Figure 2.
Leading edge retractions initiate NM2 filament appearance events. (A) Sum intensity projection of z-stack and three-frame time averaging for frames collected every second. Orange-to-red gradient dotted lines indicate wave-like retraction, and magenta circles indicate subsequent NM2 filament appearance. Scale bar = 2 μm. (B) Halo-NM2A cells transiently expressing GFP-RhoBio were imaged every second. The image is a sum intensity projection of a z-stack with NM2 in purple and RhoBio in gray. Scale bar = 10 μm. The red line indicates the ROI used for the kymograph in the right panel. Yellow dotted lines connect leading edge retractions with NM2 filament appearance. (C) Quantification of the number of NM2 filament appearance events preceded by (blue) or not preceded by (green) a leading edge retraction. Error bars indicate standard deviation from 395 filament appearance events from 12 cells from 4 independent experiments. (D–F) Confocal time series of primary EGFP-NM2A MEF cells expressing FTractin-mScarlet were acquired. For D only, the FTractin-mScarlet channel is shown and for E only, EGFP-NM2A channel is shown. (D) Example frame of lamellar actin (left panel) and kymograph (right panel) of lamellar dynamics pre- and post-JL treatment through the red dotted line in the left panel. The orange dotted line indicates the time of JL addition. (E) EGFP- NM2A images 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. Three binned time periods pre- and -post (orange outline) treatment are shown for DMSO (top row) and JL (bottom row). Scale bars = 5 μm. (F) Nascent NM2 filament appearance events pre- and post-treatment, normalized to pretreatment appearance events per minute, were quantified for individual cells (small black dots) and the mean of three independent experiments (large color circles). Temporal and intensity LUTs are indicated to the right. Wilcoxon matched-pairs signed rank test performed comparing each cell (DMSO: 15 cells, JL: 16 cells). P < 0.0001.

Leading edge retractions initiate NM2 filament appearance events. (A) Sum intensity projection of z-stack and three-frame time averaging for frames collected every second. Orange-to-red gradient dotted lines indicate wave-like retraction, and magenta circles indicate subsequent NM2 filament appearance. Scale bar = 2 μm. (B) Halo-NM2A cells transiently expressing GFP-RhoBio were imaged every second. The image is a sum intensity projection of a z-stack with NM2 in purple and RhoBio in gray. Scale bar = 10 μm. The red line indicates the ROI used for the kymograph in the right panel. Yellow dotted lines connect leading edge retractions with NM2 filament appearance. (C) Quantification of the number of NM2 filament appearance events preceded by (blue) or not preceded by (green) a leading edge retraction. Error bars indicate standard deviation from 395 filament appearance events from 12 cells from 4 independent experiments. (D–F) Confocal time series of primary EGFP-NM2A MEF cells expressing FTractin-mScarlet were acquired. For D only, the FTractin-mScarlet channel is shown and for E only, EGFP-NM2A channel is shown. (D) Example frame of lamellar actin (left panel) and kymograph (right panel) of lamellar dynamics pre- and post-JL treatment through the red dotted line in the left panel. The orange dotted line indicates the time of JL addition. (E) EGFP- NM2A images 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. Three binned time periods pre- and -post (orange outline) treatment are shown for DMSO (top row) and JL (bottom row). Scale bars = 5 μm. (F) Nascent NM2 filament appearance events pre- and post-treatment, normalized to pretreatment appearance events per minute, were quantified for individual cells (small black dots) and the mean of three independent experiments (large color circles). Temporal and intensity LUTs are indicated to the right. Wilcoxon matched-pairs signed rank test performed comparing each cell (DMSO: 15 cells, JL: 16 cells). P < 0.0001.

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