Figure 1.
Canonical signaling mechanisms do not precisely correlate with nascent NM2 filament appearances. (A) Example of NM2 filament appearance detection workflow with both cartoon and example frames. (B) Confocal imaging of Halo-NM2A (purple) and GCaMP7s (inverted grey). Left panel displays an example frame with kymograph ROI indicated by the red dotted line. Middle panels display overlay kymograph or calcium-alone kymograph. Orange arrows indicate calcium sparks and magenta arrows indicate NM2 filament appearances. Right panels display the same lamella before and after a calcium flash, demonstrating the lack of spacial precision in the cytosolic calcium. (C) Confocal imaging of Halo-NM2A (purple) and RhoA biosensor (inverted grey). The left panel displays an example frame with kymograph ROI indicated by the red dotted line. Right panels display overlay kymograph or RhoA biosensor alone kymograph. Orange arrows indicate an active RhoA signal and magenta arrows indicate NM2 filament appearance. NM2 filaments that appear before, after, or without any RhoA signal are indicated on the left kymograph and quantified in the subsequent graph. Error bars indicate standard deviation from 395 filament appearance events from 12 cells from 4 independent experiments. (D) Appearance rate of NM2 filaments relative to pretreatment for control (DMSO; green), MLCK inhibition (blue), and ROCK inhibition (purple). Small circles indicate individual cells and large circles indicate experimental means from 3 independent experiments. Wilcoxon matched-pairs signed rank test performed comparing each cell. DMSO: 12 cells, MLCKi: 12 cells, ROCKi: 9 cells. P = 0.0039 for ROCKi relative to DMSO control. (E) The upper cartoon displays a migrating fibroblast with unroofed lamella used for correlative light and electron microscopy (CLEM). The lower cartoon depicts EGFP-NM2A in filamentous form and the fluorescent signal (fire LUT) that is detected from this structure. (F) Overlay of correlated platinum replica electron micrograph (PREM) and super-resolution fluorescent micrograph of EGFP-NM2A (fire LUT) in an unroofed lamella. Orange and yellow boxes indicate the zoom inset in G–I. (G–I) Overlay, PREM alone, or EGFP-NM2A alone zoom insets from (F). Intensity LUT is indicated to the right. Yellow arrows indicate high-intensity NM2A clusters or filaments that overlay on organized, bundled actin in PREM. Yellow arrowheads indicate low intensity NM2A doublets that overlay on disorganized actin arrays without any qualitative difference from adjacent actin regions.

Canonical signaling mechanisms do not precisely correlate with nascent NM2 filament appearances. (A) Example of NM2 filament appearance detection workflow with both cartoon and example frames. (B) Confocal imaging of Halo-NM2A (purple) and GCaMP7s (inverted grey). Left panel displays an example frame with kymograph ROI indicated by the red dotted line. Middle panels display overlay kymograph or calcium-alone kymograph. Orange arrows indicate calcium sparks and magenta arrows indicate NM2 filament appearances. Right panels display the same lamella before and after a calcium flash, demonstrating the lack of spacial precision in the cytosolic calcium. (C) Confocal imaging of Halo-NM2A (purple) and RhoA biosensor (inverted grey). The left panel displays an example frame with kymograph ROI indicated by the red dotted line. Right panels display overlay kymograph or RhoA biosensor alone kymograph. Orange arrows indicate an active RhoA signal and magenta arrows indicate NM2 filament appearance. NM2 filaments that appear before, after, or without any RhoA signal are indicated on the left kymograph and quantified in the subsequent graph. Error bars indicate standard deviation from 395 filament appearance events from 12 cells from 4 independent experiments. (D) Appearance rate of NM2 filaments relative to pretreatment for control (DMSO; green), MLCK inhibition (blue), and ROCK inhibition (purple). Small circles indicate individual cells and large circles indicate experimental means from 3 independent experiments. Wilcoxon matched-pairs signed rank test performed comparing each cell. DMSO: 12 cells, MLCKi: 12 cells, ROCKi: 9 cells. P = 0.0039 for ROCKi relative to DMSO control. (E) The upper cartoon displays a migrating fibroblast with unroofed lamella used for correlative light and electron microscopy (CLEM). The lower cartoon depicts EGFP-NM2A in filamentous form and the fluorescent signal (fire LUT) that is detected from this structure. (F) Overlay of correlated platinum replica electron micrograph (PREM) and super-resolution fluorescent micrograph of EGFP-NM2A (fire LUT) in an unroofed lamella. Orange and yellow boxes indicate the zoom inset in G–I. (G–I) Overlay, PREM alone, or EGFP-NM2A alone zoom insets from (F). Intensity LUT is indicated to the right. Yellow arrows indicate high-intensity NM2A clusters or filaments that overlay on organized, bundled actin in PREM. Yellow arrowheads indicate low intensity NM2A doublets that overlay on disorganized actin arrays without any qualitative difference from adjacent actin regions.

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