Nuclear N-WASP Induces Actin Polymerization in the Nucleus with Cortactin as an Essential Factor

Abstract

Nuclear actin polymerization was reported to control different nuclear processes, but its regulation is poorly understood. Here, we show that N-WASP can trigger the formation of nuclear N-WASP/F-actin nodules. While a cancer hotspot mutant of N-WASP lacking the VCA domain (V418fs) had a dominant negative function on nuclear F-actin, an even shorter truncation mutant found in melanoma (R128*) strongly promoted nuclear actin polymerization. Nuclear localization of N-WASP was not regulated by the cell cycle and increasing nuclear F-actin formation by N-WASP had no obvious influence on replication. However, nuclear N-WASP/F-actin nodules colocalized partially with RNA Pol II clusters. N-WASP-dependent actin polymerization promoted the maturation of RNA Pol II clusters, with the short truncation mutant R128* unexpectedly showing the strongest effect. Nuclear N-WASP nodules including V418fs colocalized with WIP and cortactin. Importantly, cortactin binding was essential but not sufficient for F-actin formation, while WIP binding was required for actin polymerization by R128*. These data reveal a cortactin-dependent role for N-WASP in the regulation of nuclear F-actin and indicate contrasting nuclear effects for N-WASP mutants found in cancer.

Keywords: N-WASP; cortactin; nuclear F-actin.

Figure 1

Endogenous nuclear N-WASP colocalizes with F-actin and the establishment of an image-based platform to analyze the role of N-WASP in nuclear actin polymerization. (A) Confocal fluorescent staining of untransfected U2OS cells for endogenous N-WASP and F-actin (Scale bar: 5 µm). (B) Schematic presentation of murine N-WASP constructs used. N-WASP domains are indicated (WH1/EVH1: WASP homology/Ena-VASP homology domain; BR: Basic region; GBD: GTPase binding domain). All N-WASP constructs had an N-terminal HA tag to facilitate detection. (C) Western blot of lysates of U2OS cells transfected with indicated N-WASP constructs or empty vector for HA. GAPDH was used as loading control (n: 1).

Figure 2

Nuclear N-WASP promotes nuclear F-actin in WT U2OS cells. (A) Confocal fluorescent staining of U2OS cells, untransfected (UTF) or transfected with the indicated N-WASP constructs. Cells were stained for DNA (DAPI), F-actin (phalloidin), and transfected N-WASP (HA). Arrowheads indicate nuclear nodules of F-actin or N-WASP (Scale bar: 5 µm). (B) Pearson colocalization analysis of nuclear N-WASP nodules with F-actin was performed on ten representative nuclei per group exhibiting clear N-WASP nodules. Each dot represents an individual cell (one-way ANOVA with Tukey’s post hoc test; ****: p < 0.0001). (C) Normalized nuclear F-actin levels in U2OS cells transfected with the indicated constructs, based on confocal imaging. Each dot represents an individual transfected cell (total number of cells pooled from two independent experiments analyzed for each construct: 88, 73, 60, 51, 86, 58, and 72; one-way ANOVA with Tukey’s post hoc test; *: p < 0.05; **: p < 0.01; ****: p < 0.0001).

Figure 3

N-WASP promotes nuclear F-actin visualized by actin chromobody. (A) Orthogonal views from different planes (x/y, x/z, and y/z) of confocal microscope Z-stacks of U2OS cells transfected with the indicated N-WASP constructs and stained for DNA (DAPI), lamin A/C, and transfected N-WASP (HA). Arrows indicate nuclear N-WASP nodules (scale bar: 5 µm). (B) Super-resolution microscopy imaging of L229P-transfected U2OS cells stained for HA (N-WASP) and lamin A/C (Scale bar: 5 μm). (C) Representative confocal images of U2OS cells stably expressing GFP–NLS–nuclear actin–chromobody (U2OS–GFP–nAC), untransfected (UTF) or transfected with HA-tagged L229P N-WASP, and stained for DAPI (DNA), GFP + anti GFP (actin), HA (N-WASP), and phalloidin (F-actin). Arrowheads indicate colocalization of HA-tagged N-WASP with nuclear actin structures identified by both actin chromobody and phalloidin staining (scale bar: 5 μm).

Figure 4

N-WASP promotes nuclear F-actin in an Arp2/3-dependent manner. (A) Confocal fluorescent staining of U2OS cells transfected with the indicated N-WASP constructs treated with DMSO or the Arp2/3 inhibitor CK-666 (100 µM, 1 h). Cells were stained for DNA (DAPI), F-actin (phalloidin), and transfected N-WASP (HA). Arrowheads indicate nuclear N-WASP nodules (scale bar: 5 µm). (B) Colocalization analysis of nuclear N-WASP nodules with F-actin was performed on ten representative nuclei per group, exhibiting clear N-WASP nodules. Each dot represents an individual cell. Pearson’s correlation coefficient was used to quantify colocalization (one-way ANOVA with Tukey’s post hoc test; ****: p < 0.0001).

Figure 5

Nuclear N-WASP fraction is independent of cell cycle. (A) Correlation of nuclear N-WASP fraction with DNA amount indicated by DAPI intensity. The coefficient of determination, R2, indicates the strength of the linear correlation. (B) Cell cycle analysis was performed using an EdU incorporation assay to assess nuclear N-WASP fractions across cell cycle phases. Cells were stained for DAPI (DNA content), EdU (S phase marker), and HA (N-WASP WT). The upper panel shows representative images (scale bar: 20 µm). The scatter plot illustrates gating applied to distinguish G1, S, and G2/M phases presented in the adjacent bar graph. The dot plot on the lower right displays nuclear N-WASP fractions for each cell cycle phase in WT N-WASP-transfected cells (cells analyzed: 12,937, 2041; three independent experiments; one-way ANOVA with Tukey’s post hoc test).

Figure 6

Enhanced RNA Pol II clustering in response to serum stimulation correlates with nuclear actin polymerization of N-WASP. (A) Representative images of widefield microscopy of U2OS cells transfected with indicated N-WASP constructs and stained for DNA (DAPI), N-WASP (HA), and RNA Pol II. A white box with a dashed line indicates an RNA Pol II nodule, which is zoomed in on the right. RNA Pol II cluster colocalization with N-WASP nodules shows high cell-to-cell variation. Colocalization occurred in the presence and absence of serum stimulation (scale bar: 5 μm). Colocalization was quantified for these nuclei by Pearson’s correlation coefficient. (B) Colocalization of nuclear N-WASP with RNA Pol II for all constructs was determined by Pearson’s correlation coefficient under normal growth (cells analyzed: 141, 47, 61, 52, 62) and serum stimulation conditions (cell analyzed: 120, 46, 56, 77, 40). Data are pooled from three independent experiments and each dot represents a single nucleus (one-way ANOVA with Tukey’s post hoc test; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). (C) Quantitative analysis showing normalized number, size, integrated intensity, and mean intensity of RNA Pol II clusters per nucleus in U2OS cells with clear N-WASP nodules under normal growth (cells analyzed: 141, 47, 61, 52, 62) and serum stimulation conditions (cells analyzed: 120, 46, 56, 77, 40). Normalization was performed by dividing transfected (TF) cells by untransfected (UTF) cells.

Figure 7

Colocalization of Nuclear N-WASP with WIP and cortactin. (A) Confocal images of U2OS cells transfected with indicated N-WASP constructs, stained for DNA (DAPI), N-WASP (HA), and either WIP or cortactin. Arrowheads indicate nuclear N-WASP nodules (scale bar: 5 µm). (B) Colocalization analysis of nuclear N-WASP nodules with nuclear WIP and cortactin was performed on ten representative nuclei per group exhibiting clear N-WASP nodules, determined by Pearson’s correlation coefficient. Each dot represents an individual cell (one-way ANOVA with Tukey’s post hoc test; *: p < 0.05).

Figure 8

WIP is not essential for N-WASP-dependent nuclear actin polymerization. (A) Western blot for indicated proteins showing efficient loss of WIP protein in WIP KO U2OS cells (n = 3; two-tailed Student’s t-test; ns: p > 0.05; **: p < 0.01). (B) R128* expression analysis in WIP KO relative to parallel transfected WT cells analyzed by quantification of HA intensity of transfected cells (n: 4/6; two-tailed Student’s t-test; *: p < 0.05). (C) Confocal fluorescent microscopy of WIP KO U2OS cells, untransfected (UTF) or transfected with the indicated N-WASP constructs and stained for DNA (DAPI), F-actin (phalloidin), and transfected N-WASP (HA). Arrowheads indicate nuclear N-WASP or F-actin nodules (scale bar: 5 µm). (D) Colocalization analysis of nuclear N-WASP nodules with nuclear F-actin in WIP KO cells was performed on ten representative nuclei per group exhibiting clear N-WASP nodules, determined by Pearson’s correlation coefficient. Each dot represents an individual cell (one-way ANOVA with Tukey’s post hoc test; ****: p < 0.0001). (E) Nuclear N-WASP fraction based on fluorescence staining in WIP KO U2OS cells transfected with the indicated constructs, analyzed via wide-field microscopy (n = 12, 5, 5, 8, 6, 8; total cells analyzed: 1060, 849, 1053, 478, 360, 467. One-way ANOVA with Tukey’s post hoc test *: p < 0.05; **: p < 0.01; ****: p < 0.0001).

Figure 9

Cortactin is crucial for N-WASP-dependent nuclear actin polymerization. (A) Western blot for indicated proteins showing efficient loss of cortactin protein in cortactin KO U2OS cells (n: 3/5; two-tailed Student’s t-test, *: p < 0.05; ***: p < 0.001). (B) R128* expression analysis in cortactin KO relative to parallel transfected WT cells analyzed by quantification of HA intensity of transfected cells (n: 4/6; two-tailed Student’s t-test; *: p < 0.05). (C) Confocal fluorescent microscopy of cortactin KO U2OS cells, untransfected (UTF) or transfected with the indicated N-WASP constructs and stained for DNA (DAPI), F-actin (phalloidin), and transfected N-WASP (HA). Arrowheads indicate nuclear N-WASP nodules (scale bar: 5 µm). (D) Colocalization analysis of nuclear N-WASP nodules with nuclear F-actin in cortactin KO cells was performed on ten representative nuclei per group exhibiting clear N-WASP nodules, determined by Pearson’s correlation coefficient. Each dot represents an individual cell (one-way ANOVA with Tukey’s post hoc test; not significant (ns): p > 0.05). (E) Nuclear N-WASP fraction based on fluorescence staining in cortactin KO U2OS cells transfected with the indicated constructs, analyzed via wide-field microscopy. Each dot represents data from an independent experiment, with over 40 cells analyzed per experiment (n = 9, 4, 4, 6, 6, 7; total cells analyzed: 970, 757, 801, 886, 248, 321; one-way ANOVA with Tukey’s post hoc test; not significant (ns): p > 0.05; *: p < 0.05; ****: p < 0.0001).