Genetically encoded caspase sensor and RFP-LC3 for temporal analysis of apoptosis-autophagy.
Autor: | Chandrasekharan A; Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India. Electronic address: aneeshc@rgcb.res.in., Tiwari SK; Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India., Munirpasha HA; Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India., Sivasailam A; Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India., Jayaprasad AG; Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India., Harikumar A; Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India., Santhoshkumar TR; Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O., Thiruvananthapuram, Kerala 695014, India. |
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Jazyk: | angličtina |
Zdroj: | International journal of biological macromolecules [Int J Biol Macromol] 2024 Feb; Vol. 257 (Pt 2), pp. 128807. Date of Electronic Publication: 2023 Dec 14. |
DOI: | 10.1016/j.ijbiomac.2023.128807 |
Abstrakt: | The balance between pro-death and pro-survival signaling determines the fate of cells under a variety of pathological and physiological conditions. The pro-cell death signaling, apoptosis, and survival singling, autophagy work in an integrated manner for maintaining cell integrity. Their altered balance drives pathological conditions such as cancer, inflammatory disorders, and neurodegenerative diseases. Dissecting complex crosstalk between autophagy and apoptosis requires simultaneous detection of both events at a single cell level with good temporal resolution in real-time. Here, we have used two distinct fluorescent-based probes of caspase activation and autophagy for generating such sensor cells. Cells stably expressing RFP-LC3 as an autophagy marker were further stably expressed with a FRET-based probe for caspase activation with a nuclear localization signal. The functional validation and live-cell imaging of the sensor cells using selected treatments revealed that stress that induces rapid cell death often fails to induce autophagy signaling, and slow cell death induction triggers simultaneous autophagy signaling with caspase activation. The real-time imaging revealed the time-dependent shift of cells towards caspase activation while autophagy is inhibited confirming basal autophagy confers survival against apoptosis under stress conditions. Confocal imaging also revealed that cells under 3D culture condition maintain increased autophagy over monolayer cultures. High-throughput adaptability of the system extends its application for the screening of compounds that cause caspase activation, autophagy, or both demonstrating the potential utility of the sensor probe for diverse biological applications. Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. (Copyright © 2023 Elsevier B.V. All rights reserved.) |
Databáze: | MEDLINE |
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