Fig 1: Model of the proposed role for Mena at the nuclear membrane.a Mena interacts with nesprin-2 at the outer nuclear membrane via the C-terminal SRs of nesprin-2. This potentiates the interactions of nesprin-2 with F-actin and lamin A/C, permitting force transmission from the actin cytoskeleton to the nuclear lamina (1). Actomyosin-based force maintains emerin tyrosine phosphorylation. Emerin tyrosine phosphorylation has been reported to prevent its LAP2–emerin–MAN1 (LEM) domain from binding to the DNA-binding protein barrier-to-autointegration factor (BAF), limiting chromatin repositioning to the nuclear lamina (2). This proposed mechanism allows genomic loci to favour chromatin decondensation and expression of specific genes, including, in metastatic Met4 cSCC cells, those involved in cancer progression, such as genes associated with cell adhesion and migration (e.g. ITGA5, PLAU), ECM organisation (e.g. FN1, MMP1) and the immune response (e.g. PTX3, C3) (3). b Mena loss reduces the connectivity between F-actin and nesprin-2 (1), diminishing force transduction to the nuclear envelope via nesprin-2. Mena loss thus results in a reduction of emerin tyrosine phosphorylation (2), which has been reported to promote BAF binding to the emerin LEM domain, and enhances recruitment of heterochromatic lamina-associated domains (LADs) to the nuclear periphery (3). Chromatin repositioning is associated with transcriptional silencing of genes with regulatory elements in specific LADs (3), such as the immunomodulatory gene PTX3 in cSCC cells. *, the nesprin-2-interacting region of Mena has not been determined. The hetero-oligomeric SUN–nesprin assembly is simplified for visualisation purposes. Question marks indicate unresolved potential mechanistic associations. Artwork was adapted from Byron, A., Griffith, B.G.C., Herrero, A. et al. Characterisation of a nucleo-adhesome. Nat Commun 13, 3053 (2022). 10.1038/s41467-022-30556-5.
Fig 2: Mena co-localises with nesprin-2 at the nuclear membrane and forms a biochemical complex.a Confocal imaging of Met4 cSCC cells. Orange arrowheads indicate Mena staining at focal adhesions; magenta arrowheads indicate Mena localisation at the nuclear membrane. Images are representative of three independent experiments. Scale bar, 20 µm; zoom (inset) scale bar, 3 µm. b Subcellular co-occurrence analysis of Mena and nesprin-2 signals determined from confocal images. MMena, co-occurrence fraction of Mena with nesprin-2 at the nuclear membrane (nucl.) or cytoplasm (cyto.). MNesprin-2, co-occurrence fraction of nesprin-2 with Mena at the nuclear membrane or cytoplasm. Black bar, median; dark grey box, 95% confidence interval; light grey silhouette, probability density. Statistical analysis, two-sided Wilcoxon signed rank test (n = 14 images from n = 3 independent experiments). c SIM imaging of Met4 cells. Position of orthogonal section (yz) shown in top-right image (xy; grey dashed line). Green arrowheads indicate co-localisation of Mena and nesprin-2 at the nuclear membrane; black arrowheads indicate co-localisation of Mena, nesprin-2 and F-actin at the nuclear membrane. Images are representative of three independent experiments. Scale bar, 3 µm; zoom (inset) scale bar, 1 µm. d STED imaging of Met4 cells. Signals in a region beneath the apical plasma membrane (dashed black boxes; bottom-left panels) were quantified (bottom-right panel). Images are representative of two independent experiments. Scale bar, 3 µm; zoom (inset) scale bar, 1 µm. WGA, wheat germ agglutinin. For (a), (c) and (d), F-actin was detected using phalloidin. Inverted lookup tables were applied. e Immunoprecipitation (IP) analysis of nesprin-2 protein complexes in Met4 cells. Anti-Mena- and anti-nesprin-2-reactive species detected by western blotting are indicated with arrowheads. High exposure of nesprin-2 blot of Met4 lysate (input) is also shown. Western blots are representative of three independent experiments. f Representation of nesprin-2 domains and the nesprin-2 constructs used for pulldown experiments. Constructs are based on the nesprin-2 giant isoform (nesprin-2G). g, h Pulldown analyses of protein complexes associated with exogenous GFP-tagged nesprin-2G(SR49–56) (g) and mini-nesprin-2G?SR3–50 (h) in Met4 cells. Western blots are representative of three independent experiments. Ctrl control.
Fig 3: Mena is required for nesprin-2 links to actin and lamin A/C and regulates nuclear morphology.a, b IP analyses of nesprin-2 protein complexes in Met4 cells, Mena-depleted Met4 cells (Met4 Mena-/-) and Met4 Mena-/- cells with Mena11a re-expressed (Met4 Mena-/- +Mena11a). Actin (a) and lamin A/C (b) were detected by western blotting; respective densitometric intensities were normalised (norm.) to nesprin-2 and expressed relative (rel.) to Met4 cells (right panels). Black bar, median; light grey box, range. Statistical analysis, Welch’s one-way ANOVA with two-stage Benjamini–Krieger–Yekutieli correction (n = 6 independent experiments) for (a), one-way ANOVA with Tukey’s correction (n = 5 independent experiments) for (b). c Spinning-disk confocal imaging of Met4 cells and Met4 Mena-/- cells in 3D collagen matrix. Orthogonal projections (xz) were extracted from 3D brightest-point projections. Nuclei were detected using DAPI; F-actin was detected using phalloidin. Inverted lookup tables were applied. Images are representative of two independent experiments. Scale bar, 20 µm. d Quantification of nuclear volume of cells in 3D matrix (see c). 3D volume renderings of exemplar nuclei detected using DAPI are displayed. Black bar, median; dark grey box, 95% confidence interval; light grey silhouette, probability density. Statistical analysis, two-sided Welch’s t-test (n = 10 cells from n = 2 independent experiments). e Spinning-disk confocal imaging of Met4 cells, Met4 Mena-/- cells and Met4 Mena-/- +GFP-Mena11a cells on 2D fibronectin matrix. Orthogonal projections were extracted from 3D brightest-point projections. Nuclei were detected using DAPI. Inverted lookup tables were applied. Images are representative of four independent experiments. Scale bar, 20 µm. f Quantification of nuclear height of cells on 2D matrix (see e). 3D volume renderings of exemplar nuclei detected using DAPI are displayed. Black bar, median; dark grey box, 95% confidence interval; light grey silhouette, probability density. Statistical analysis, Kruskal–Wallis test with Dunn’s correction (n = 92, 87 and 94 cells for Met4, Met4 Mena-/- and Met4 Mena-/- +GFP-Mena11a cells, respectively, from n = 4 independent experiments). n.s. not significant.
Supplier Page from Abcam for Anti-Nesprin 2 antibody