Fig. 9: NHSL1 increases actin retrograde flow speed and F-actin assembly rate. | Nature Communications

Fig. 9: NHSL1 increases actin retrograde flow speed and F-actin assembly rate.

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

Fig. 9

a A representative snapshot in time (02:42 min:s) of a segmented lamellipodium from a wild-type cell (WT), NHSL1 knockout cell (NHSL1 CRISPR 2) and NHSL1 knockout cells rescued with a wild-type NHSL1 construct (NHSL1 CRISPR 2 Rescue Myc-NHSL1 WT) and with a mutant NHSL1 construct (NHSL1 CRISPR 2 Rescue Myc-NHSL1 SW Mut) using a bicistronic expression plasmid also conferring resistance to blasticidin to ensure that all cells analysed expressed NHSL1 and also expressing lifeAct-EGFP. Left panels show a frame from segmented Airyscan movies; right panels show the corresponding frame from the PIV interpolated movie. Each colourmap has the same range (0–3 μm/min) and distance between vector arrows (1 μm); warmer colours represent faster flow, vector arrows are all of the unit length. Scale bar: 10 μm. Representative images shown from six independent biological experiments. b Quantification of the average speed of actin retrograde flow for each condition over all frames for each cell. Flow is significantly reduced in NHSL1 knockout cells but cannot be fully rescued by either the WT or mutant NHSL1 constructs. Results are mean ± SEM; n = 41, 32, 35 and 36 for the respective conditions (left to right on x axis), from 6 independent biological repeats. One-way ANOVA: P = 0.0001; F(3,140) = 7.312; Tukey’s multiple comparisons tests: ***P = 0.0002, ns = not significant: CRISPR 2 versus rescue Myc-NHSL1 SW Mut: nsP = 0.4285; CRISPR 2 versus rescue Myc-NHSL1 WT: nsP = 0.8275. c Quantification of the F-actin assembly rate for each condition over all frames for each cell. The F-actin assembly rate is significantly reduced in NHSL1 knockout cells but cannot be rescued by either the WT or mutant NHSL1 constructs. Results are mean ± SEM; n = 41, 32, 35 and 36 for the respective conditions (left to right on x axis), from 6 independent biological repeats. One-way ANOVA: P = 0.0059; F(3,104) = 4.400; Tukey’s multiple comparisons test: **P = 0.0044, ns = not significant: CRISPR 2 versus rescue Myc-NHSL1 SW Mut: nsP = 0.7874; CRISPR 2 versus rescue Myc-NHSL1 WT: nsP = 0.7967. Source data are provided as a Source Data file.

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