From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 Action
53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a specific response...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-09-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/17/10193 |
| _version_ | 1797494854521978880 |
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| author | Laura Furia Simone Pelicci Mirco Scanarini Pier Giuseppe Pelicci Mario Faretta |
| author_facet | Laura Furia Simone Pelicci Mirco Scanarini Pier Giuseppe Pelicci Mario Faretta |
| author_sort | Laura Furia |
| collection | DOAJ |
| description | 53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a specific response to numerous DNA Double-Strand Breaks (DSBs), i.e., exposure to ionizing radiation. The localization of 53BP1 protein constitutes a key to decipher the network of activities exerted in response to stress. We present here an automated-microscopy for image cytometry protocol to analyze the evolution of the DDR, and to demonstrate how 53BP1 moved from damaged sites, where the well-known interaction with the DSB marker γH2A.X takes place, to nucleoplasm, interacting with p53, and enhancing the transcriptional regulation of the guardian of the genome protein. Molecular interactions have been quantitatively described and spatiotemporally localized at the highest spatial resolution by a simultaneous analysis of the impairment of the cell-cycle progression. Thanks to the high statistical sampling of the presented protocol, we provide a detailed quantitative description of the molecular events following the DSBs formation. Single-Molecule Localization Microscopy (SMLM) Analysis finally confirmed the p53–53BP1 interaction on the tens of nanometers scale during the distinct phases of the response. |
| first_indexed | 2024-03-10T01:40:18Z |
| format | Article |
| id | doaj.art-558d9bff813e4eb4a4a22afe5002f546 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T01:40:18Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-558d9bff813e4eb4a4a22afe5002f5462023-11-23T13:24:35ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-09-0123171019310.3390/ijms231710193From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 ActionLaura Furia0Simone Pelicci1Mirco Scanarini2Pier Giuseppe Pelicci3Mario Faretta4Department of Experimental Oncology, European Institute of Oncology IRCCS, 20139 Milan, ItalyDepartment of Experimental Oncology, European Institute of Oncology IRCCS, 20139 Milan, ItalyDepartment of Experimental Oncology, European Institute of Oncology IRCCS, 20139 Milan, ItalyDepartment of Experimental Oncology, European Institute of Oncology IRCCS, 20139 Milan, ItalyDepartment of Experimental Oncology, European Institute of Oncology IRCCS, 20139 Milan, Italy53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a specific response to numerous DNA Double-Strand Breaks (DSBs), i.e., exposure to ionizing radiation. The localization of 53BP1 protein constitutes a key to decipher the network of activities exerted in response to stress. We present here an automated-microscopy for image cytometry protocol to analyze the evolution of the DDR, and to demonstrate how 53BP1 moved from damaged sites, where the well-known interaction with the DSB marker γH2A.X takes place, to nucleoplasm, interacting with p53, and enhancing the transcriptional regulation of the guardian of the genome protein. Molecular interactions have been quantitatively described and spatiotemporally localized at the highest spatial resolution by a simultaneous analysis of the impairment of the cell-cycle progression. Thanks to the high statistical sampling of the presented protocol, we provide a detailed quantitative description of the molecular events following the DSBs formation. Single-Molecule Localization Microscopy (SMLM) Analysis finally confirmed the p53–53BP1 interaction on the tens of nanometers scale during the distinct phases of the response.https://www.mdpi.com/1422-0067/23/17/10193DNA damage response53BP1p53cell-cyclefluorescence microscopyimage analysis |
| spellingShingle | Laura Furia Simone Pelicci Mirco Scanarini Pier Giuseppe Pelicci Mario Faretta From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 Action International Journal of Molecular Sciences DNA damage response 53BP1 p53 cell-cycle fluorescence microscopy image analysis |
| title | From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 Action |
| title_full | From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 Action |
| title_fullStr | From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 Action |
| title_full_unstemmed | From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 Action |
| title_short | From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 Action |
| title_sort | from double strand break recognition to cell cycle checkpoint activation high content and resolution image cytometry unmasks 53bp1 multiple roles in dna damage response and p53 action |
| topic | DNA damage response 53BP1 p53 cell-cycle fluorescence microscopy image analysis |
| url | https://www.mdpi.com/1422-0067/23/17/10193 |
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