Imaging-Based Molecular Interaction Between Src and Lamin A/C Mechanosensitive Proteins in the Nucleus of Laminopathic Cells

Abstract

Laminopathies represent a wide range of genetic disorders caused by mutations in gene-encoding proteins of the nuclear lamina. Altered nuclear mechanics have been associated with laminopathies, given the key role of nuclear lamins as mechanosensitive proteins involved in the mechanotransduction process. To shed light on the nuclear partners cooperating with altered lamins, we focused on Src tyrosine kinase, known to phosphorylate proteins of the nuclear lamina. Here, we demonstrated a tight relationship between lamin A/C and Src in skin fibroblasts from two laminopathic patients, assessed by advanced imaging-based microscopy techniques. With confocal laser scanning and Stimulated Emission Depletion (STED) microscopy, a statistically significant higher co-distribution between the two proteins was observed in patients' fibroblasts. Furthermore, the time-domain fluorescence lifetime imaging microscopy, combined with Förster resonance energy transfer detection, demonstrated a decreased lifetime value of Src (as donor fluorophore) in the presence of lamin A/C (as acceptor dye) in double-stained fibroblast nuclei in both healthy cells and patients' cells, thereby indicating a molecular interaction that resulted significantly higher in laminopathic cells. All these results demonstrate a molecular interaction between Src and lamin A/C in healthy fibroblasts and their aberrant interaction in laminopathic nuclei, thus creating the possibilities of new diagnostic and therapeutic approaches for patients.

Keywords: FLIM/FRET analysis; STED-microscopy; Src tyrosine kinase; fluorochrome lifetime; lamin A/C; laminopathies; mechanosensitive proteins; nuclear envelope proteins.

Figure 1

Confocal microscopy imaging of Src (in green) and lamin A/C (in red) in control and laminopathic fibroblasts. (A) Src immunofluorescence showed a punctate and diffuse distribution in the nuclear and cytoplasmic compartments, with a higher concentration in the nuclei, both in healthy cells and patients’ cells. Orange pixels showed the overlay of Src and Lamin A/C fluorescence in doubled-stained cells. Colocalization masks of double-stained cells (white pixels) showed the Src-lamin A/C co-distribution both at the nuclear envelope (arrows and arrowheads) and in the nucleoplasm. (B) Intensity line profiles of Src (green) and lamin A/C (red) across the focal central plane, as indicated by the white dotted line of representative nuclei in the overlay images. Scale bars: 10 μm for all images except insets (5 μm). (C) Src mean fluorescence intensity decreased in patients’ nuclei, significantly in Pt 1, compared to controls (* p < 0.05).

Figure 2

STED nanoscopy of Src and lamin A/C in nuclei from healthy and laminopathic fibroblasts. (A) In the healthy control nuclei, Src labeling (green) was thickened at the nuclear periphery, with a diffuse and dotted distribution in the nuclear matrix, whereas some anomalous aggregates were observed (arrows) in patients’ nuclei. Alterations in the structural organization of the lamin A/C (red) meshworks have been seen in several nuclei of the fibroblasts of patient 1 and patient 2 (arrowheads). Colocalization masks (yellow) showed the co-distribution of Src and lamin A/C at the nuclear rim in all samples and a higher concentration in the nucleoplasm of patients’ cells (high magnification of insets). Bars: 5 µm and 2 µm. (B) Mean values of the overlap coefficient quantified in STED images of double-stained fibroblast nuclei. (** p < 0.01; * p < 0.05).

Figure 3

FLIM and FLIM-FRET microscopy of Src and lamin A/C in healthy and laminopathic fibroblast nuclei. (A) Fluorescence lifetime imaging of Src-AF488 donor in the absence (τ_D, left panel) or in the presence (τ_DA, right panel) of a lamin A/C-AF594 acceptor. Different τ values were visualized via color code lifetime scale bar (from 2.0 to 3.5 ns of range). (B) Src-AF488 mean lifetime values in the absence of the acceptor (τ_D) showed a significant increase in laminopathic nuclei compared to controls (**** p < 0.0001 in Pt 1; ** p < 0.01 in Pt 2). (C) The statistical analysis of the Src-AF488 mean τ_D values in the two specific ROIs revealed significant lifetime changes between controls and patients’ nuclei, both in the lamina and in the nuclear matrix regions (**** p < 0.0001; * p < 0.05). (D) Src-AF488 donor lifetime in the presence of the acceptor molecule (τ_DA, amplitude weighted lifetime) was significantly decreased in all samples, with a greater extent in patients’ fibroblasts (**** p < 0.0001). (E–G) Quantified FRET efficiency values (mean ± sem) of the Src-AF488 and lamin A/C-AF 594 pair obtained in all selected ROIs (E), at the nuclear rim (F) and in the nucleoplasm (G) in controls (gray dots), in Pt 1 (red dots) and Pt 2 (green dots) nuclei (**** p < 0.0001; ** p < 0.01; * p < 0.05).