Fig. 1: γ-actin controls the expression and junctional accumulation of β-actin through NM2A. | Nature Communications

Fig. 1: γ-actin controls the expression and junctional accumulation of β-actin through NM2A.

From: A feedback circuitry involving γ-actin, β-actin and nonmuscle myosin-2 A controls tight junction and apical cortex mechanics

Fig. 1

a, d Immunofluorescence (IF) analysis and relative fluorescence intensity (RFI) quantifications (on the right of IF panels) of β-actin (top panels, increased, WT: n = 84, γ-actin-KO: n = 85) and phalloidin-labelled F-actin (bottom panels, not changed, WT and γ-actin-KO: n = 50 for) (a) NM2A (top panels, increased, WT: n = 82, γ-actin-KO: n = 78) and NM2B (bottom panels, not changed, WT: n = 65, γ-actin-KO: n = 70) (d) (red) at junctions in mixed cultures of WT (blue dots) and γ-actin-KO (red dots) distinguished via γ-actin (green), from 2 to 3 independent experiments. b, c, e, f Immunoblot (IB) analysis (b, e) and relative densitometric quantifications (c, f) of protein level of β-actin (increased) and pan-actin (not changed) (b, c) NM2A (increased) and NM2B (not changed) (e, f) in lysates of WT (blue dots) or γ-actin-KO (red dots) MDCK cells (3 distinct clonal lines) from 3 independent experiments. β-tubulin was used as a loading control. Dots shows replicates and bars represent mean ± SD. Indicated p-values are obtained from a two-sided one-way Anova test. g IF microscopy analysis and RFI quantifications of β-actin (red) at junctions in WT (top panels, siControl (blue dots): n = 85, siNM2A (purple dots): n = 83) or γ-actin-KO (bottom panels, siControl (red dots): n = 96, siNM2A (yellow dots): n = 93) cells upon NM2A depletion, distinguished via NM2A (green), from 3 independent experiments. Depletion of NM2A decreases β-actin localization. h IF microscopy analysis and RFI quantifications of β-actin (red) at junctions in WT (top panels, siControl (blue dots): n = 124, siNM2B (orange dots): n = 119) or γ-actin-KO (bottom panels, siControl (red dots): n = 63, siNM2B (green dots): n = 59) cells upon NM2B depletion, distinguished via NM2B (green), from 2-3 independent experiments. Depletion of NM2B does not alter β-actin localization. a, d, g, h Double arrows indicate increased β-actin or NM2A labelling. Arrows indicate normal β-actin, phalloidin, NM2A or NM2B labelling (as in WT cells). Arrowheads indicate loss of γ-actin or β-actin labelling in KO or KD cells, respectively. Scale bar = 20 µm. PLEKHA6 (cyan) is used as a junctional marker. Dots shows replicates and bars represent mean ± SD. Indicated p-values are obtained from a two-sided unpaired Mann-Whitney test. Source data for this figure are provided as a Source Data file.

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