Indirect visualization of endogenous nuclear actin by correlative light and electron microscopy (CLEM) using an actin-directed chromobody

Abstract

Actin fulfills important cytoplasmic but also nuclear functions in eukaryotic cells. In the nucleus, actin modulates gene expression and chromatin remodeling. Monomeric (G-actin) and polymerized actin (F-actin) have been analyzed by fluorescence microscopy in the nucleus; however, the resolution at the ultrastructural level has not been investigated in great detail. We provide a first documentation of nuclear actin in mouse fibroblasts by electron microscopy (EM). For this, we employed correlative light and electron microscopy on the same section using actin-directed nanobodies recognizing endogenous monomeric and polymeric actin proteins (so-called nuclear Actin-chromobody-GFP; nAC-GFP). Indeed, using this strategy, we could identify actin proteins present in the nucleus. Here, immunogold-labeled actin proteins were spread throughout the entire nucleoplasm. Of note, nuclear actin was complementarily localized to DAPI-positive areas, the latter marking preferentially transcriptionally inactive heterochromatin. Since actin aggregates in rod structures upon cell stress including neurodegeneration, we analyzed nuclear actin at the ultrastructural level after DMSO or UV-mediated cell damage. In those cells the ratio between cytoplasmic and nuclear gold-labeled actin proteins was altered compared to untreated control cells. In summary, this EM analysis (i) confirmed the presence of endogenous nuclear actin at ultrastructural resolution, (ii) revealed the actin abundance in less chromatin-dense regions potentially reflecting more transcriptionally active euchromatin rather than transcriptionally inactive heterochromatin and (iii) showed an altered abundance of actin-associated gold particles upon cell stress.

Keywords: Actin; EM; Immunogold; Nanobody; Neurodegeneration; Nucleus.

Fig. 1

Correlative light and electron microscopy approach. a NIH3T3 cells were spread on sapphire discs placed on coverslips and transfected with the nAC-GFP construct. b Examples of GFP signal of cells expressing nAC-GFP in the nucleus. Arrows point at filamentous structures. c Cells were cryo-fixed by high-pressure freezing, freeze substituted and LR-gold embedded, followed by cutting 100 nm sections that were picked up onto copper grids. d Staining was performed with anti-GFP-directed antibodies recognizing the GFP-tag of the chromobody nAC-GFP. Two different secondary antibodies, conjugated with either 6 nm gold particles or fluorophores were applied. e Fluorescent cells (red circle) were marked on the grids to allow re-examination of the same cell by EM. f A mosaic of single high-resolution EM pictures was prepared from a section. g Gold particles were false-color-labeled in yellow. Scale-bar b = 2 μm

Fig. 2

Localization and structure of endogenous nuclear actin in fibroblasts (NIH3T3 cells). a Overview of the distribution of all yellow false-colored gold particles labeling actin in a nuclear section of a cell expressing the nAC-GFP construct. The nAC-GFP construct recognizing actin gave signals predominantly in the nucleoplasm (surrounded by the dashed line) and only few signals were present in the cytoplasm (labeled in magenta). The composite image was derived from merging 72 single high-magnification pictures. Actin signals were spread all over the nucleoplasm with certain territories being spared (see b, c). b The same nucleus shown in (a) was labeled with fluorescent antibodies (green) recognizing the nAC-GFP and chromatin was stained with DAPI (blue). Actin signals were localized outside regions of prominent DAPI staining. c The picture in (b) was additionally co-localized with the yellow-colored gold particles. There was a high degree of co-localization of fluorescent and immunogold-labeled actin signals. d, e Higher magnifications of boxes depicted in (a) and (d). Gold particles were found as individual particles, in duplets and clusters of several particles. Many actin signals appeared to assemble in duplets [arrows in (e), but see (f) for a word of caution]. The dashed line in (d) labels the cytoplasmic-to-nuclear border. f Gold-labeled secondary antibodies accumulated on empty grids also in duplets (arrows in f) in the absence of incubation with first antibodies directed against GFP. This suggests nonspecific aggregation of secondary antibodies. g–j An untransfected cell not expressing the nAC-GFP construct was subjected to the same staining procedure as the cell in (a–e). No gold particles were visible suggesting the absence of nonspecific binding of the first and secondary antibodies (g, j). The cell was positive for DAPI (h) but did not show any GFP fluorescence (i). Scale-bars a–c, g–i = 2 μm; d, j = 200 nm; e, f = 50 nm

Fig. 3

Analysis of nuclear actin localization in DMSO-treated NIH3T3 cells. a Overview of the distribution of yellow or magenta false-colored gold particles labeling actin in the nucleus and cytoplasm, respectively, of a DMSO-treated cell expressing the nAC-GFP construct. The nAC-GFP construct recognizing actin gave comparable signals in the nucleoplasm and cytoplasm. The dashed line marks the nuclear border. b The same nucleus shown in (a) was labeled with fluorescent antibodies (green) recognizing the nAC-GFP and chromatin was stained with DAPI (blue). c The picture in (b) was also co-localized with the yellow-colored gold particles. There was a high degree of co-localization of fluorescent and immunogold-labeled actin signals. d Higher magnifications of box depicted in (a). Gold particles were found as individual particles but also as clusters of several particles in both the cytoplasm and nucleus. The dashed line labels the cytoplasmic-to-nuclear border. Scale-bars a–c = 2 μm; d = 200 nm

Fig. 4

Localization and structure of endogenous nuclear actin in UV-irradiated NIH3T3 cells. a, b An untreated (a) and a UV-irradiated (b) cells were stained for DAPI (blue) and γ-H2AX (orange), labeling DNA double-strand breaks. UV irradiation enhanced DNA damage compared to untreated cells. c Co-localization of GFP fluorescence, DAPI and false-colored gold particles (yellow). There was a strong co-localization of fluorescent and immunogold-labeled actin signals. d A composite picture depicting the distribution of nuclear yellow or cytoplasmic magenta false-colored gold particles of an UV-irradiated cell expressing the nAC-GFP construct. More false-colored gold particles were observed in the cytoplasm compared to the nucleus. The dashed line marks the nuclear border. e Higher magnifications of box depicted in d. Many gold particles accumulated in clusters in both the cytoplasm and nucleus. The dashed line labels the cytoplasmic-to-nuclear border. Scale-bars a, b = 10 μm; c, d = 2 μm; e = 200 nm

Fig. 5

Quantification of co-localization and abundance of actin-associated gold particles. a Co-localization of fluorescent signals derived from GFP and DAPI in the nucleus of untreated and DMSO-treated cells. Only 1–2% of both signals co-localized and the majority of signals were complementary. b, c Quantification gold particles/μm² (b) or total number of gold particles/section (c) for cytoplasm and nucleus for the different conditions indicated. In untreated cells, more gold particles were present in the nucleus. DMSO treatment enhanced abundance of gold particles in cytoplasm and nucleus. After UV irradiation, more gold particles were found in the cytoplasm. Each symbol in (a–c) reflects one section of one cell analyzed. ns, no significant difference, asterisks denote the degree of significant difference