Fig. 7: NHSL1 negatively regulates Arp2/3 activity. | Nature Communications

Fig. 7: NHSL1 negatively regulates Arp2/3 activity.

From: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

Fig. 7

a Lifetime images of B16-F1 expressing the Arp2/3 biosensor and either empty Myc- control plasmid or Myc-tagged dominant active Rac (DA Rac). Warm colours indicate short lifetimes of the donor mTurq2, which represent high FRET efficiency and Arp2/3 activity. Representative images from 4 independent biological experiments are shown. Scale bar: 20 μm. b FRET efficiency histograms from the same representative wild-type B16-F1 (orange circles) and DA Rac (lilac upside-down triangles) cells expressing the Arp2/3 biosensor as shown in a. c Quantification of average cellular FRET efficiency that represents Arp2/3 activity from 4 independent biological repeats from control: 31 cells (orange circles); DA Rac: 26 cells (lilac upside-down triangles). Data points are the weighted average means for each cell, calculated from the FRET efficiency histogram and used instead of the normal (unweighted) mean in order to better represent the true FRET efficiency. Results are the average weighted average mean ± SEM (error bars), ****P = 0.0000000065; t = 7.379; df = 54. Unpaired, two-tailed t-test. d Lifetime images of wild-type B16-F1, NHSL1 CRISPR clone 2 and NHSL1 CRISPR clone 21 cells expressing the Arp2/3 biosensor. Warm colours indicate short lifetimes of the donor mTurq2, which represent high FRET efficiency and Arp2/3 activity. Representative images from 4 independent biological experiments are shown. Scale bar: 20 μm. e FRET efficiency histogram from the same representative wild-type B16-F1 (orange circles), NHSL1 CRISPR clone 2 (magenta squares) and NHSL1 CRISPR clone 21 (green triangles) cells expressing the Arp2/3 biosensor as shown in d. f Quantification of average cellular FRET efficiency which represents Arp2/3 activity from 4 independent biological repeats from control: 19 cells (orange circles); NHSL1 CRISPR 2: 21 cells (magenta squares); NHSL1 CRISPR 21: 11 cells (green triangles). Data points are the weighted average means for each cell, calculated from the FRET efficiency histograms. Data points represent the average weighted mean ± SEM (error bars), Kruskal–Wallis test: P = 0.0091; Kruskal–Wallis statistic: 9.407 and Dunn’s multiple comparisons test: control versus CRISPR 2 *P = 0.0136; control versus CRISPR 21 *P = 0.0260. Source data are provided as a Source Data file.

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