From Cell Populations to Molecular Complexes: Multiplexed Multimodal Microscopy to Explore p53-53BP1 Molecular Interaction

• Published in: International journal of molecular sciences Vol. 25; no. 9; p. 4672
• Main Authors: Pelicci, Simone, Furia, Laura, Pelicci, Pier Giuseppe, Faretta, Mario
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.05.2024
• Subjects: 53BP1; Automation; Biomedical research; Cell cycle; Cell Line, Tumor; DNA damage; DNA damage response; Fluorescence microscopy; Humans; Laboratories; Localization; Microscopy; Microscopy, Fluorescence - methods; Mitochondria - metabolism; p53; Photographic industry; Protein Binding; RNA polymerase; Single Molecule Imaging - methods; super-resolution microscopy; Tumor proteins; Tumor Suppressor p53-Binding Protein 1 - metabolism; Tumor Suppressor Protein p53 - metabolism; United Kingdom; Massachusetts; Japan; DNA damage response; cell cycle; 53BP1; fluorescence microscopy; image analysis; DNA PAINT microscopy; super-resolution microscopy; p53
• DOI: 10.3390/ijms25094672
• ISSN: 1422-0067; 1661-6596

Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules' interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of 53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a 53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative 53BP1 functions to support p53 activity.