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

Authors:

Stefania Petrini, Giulia Bagnato, Michela Piccione, Valentina D’Oria, Valentina Apollonio, Marco Cappa, Claudia Castiglioni, Filippo Maria Santorelli, Teresa Rizza, Rosalba Carrozzo, Enrico Silvio Bertini, Barbara Peruzzi

Keywords:

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

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.

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