The Lumiptosome, an engineered luminescent form of the apoptosome can report cell death by using the same Apaf-1 dependent pathway.

Autor:	Hosseini ES; Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.; Department of Nanobiotechnology, Pharmacology and Therapeutics, School of Medicine, NUI, Galway, Ireland.; Department of Nanobiotechnology, Museum National d'Histoire Naturelle, Laboratoire Structure et Instabilité des Génomes - INSERM U1154 - CNRS 7196; Laboratoire de Biophysique, Sorbonne Universités, Paris, F-75231, France., Nikkhah M; Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran., Hamidieh AA; Pediatric Cell Therapy Research Center, Tehran University of Medical Sciences, Tehran, Iran., Fearnhead HO; Department of Nanobiotechnology, Pharmacology and Therapeutics, School of Medicine, NUI, Galway, Ireland., Concordet JP; Department of Nanobiotechnology, Museum National d'Histoire Naturelle, Laboratoire Structure et Instabilité des Génomes - INSERM U1154 - CNRS 7196; Laboratoire de Biophysique, Sorbonne Universités, Paris, F-75231, France saman_h@modares.ac.ir jean-paul.concordet@mnhn.fr., Hosseinkhani S; Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-175, Iran saman_h@modares.ac.ir jean-paul.concordet@mnhn.fr.
Jazyk:	angličtina
Zdroj:	Journal of cell science [J Cell Sci] 2020 May 27; Vol. 133 (10). Date of Electronic Publication: 2020 May 27.
DOI:	10.1242/jcs.242636
Abstrakt:	Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and apoptosome formation using a split-luciferase complementary assay. Fusion of N-terminal (Nluc) and C-terminal (Cluc)-fragments of firefly luciferase to the N-terminus of human Apaf-1 was performed in HEK293 cells by using CRISPR-Cas9 technology. This resulted in a luminescent form of the apoptosome that we named 'Lumiptosome'. During Apaf-1 gene editing, a high number of knock-in events were observed without selection, suggesting that the Apaf-1 locus is important for the integration of exogenous transgenes. Since activation of caspase-9 is directly dependent on the apoptosome formation, measured reconstitution of luciferase activity should result from the cooperative association of Nluc-Apaf-1 and Cluc-Apaf-1. Time-response measurements also confirmed that formation of the apoptosome occurs prior to activation of caspase-3. Additionally, overexpression of the Bcl2 apoptosis regulator in transgenic and normal HEK293 cells confirmed that formation of the Lumiptosome depends on release of cytochrome c Thus, HEK293 cells that stably express the Lumiptosome can be utilized to screen pro- and anti-apoptotic drugs, and to examine Apaf-1-dependent cellular pathways. Competing Interests: Competing interestsThe authors declare no competing or financial interests. (© 2020. Published by The Company of Biologists Ltd.)
Databáze:	MEDLINE
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