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

Abstract

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.

Keywords: Apaf-1; Apoptosis; Apoptosome; CRISPR-Cas9; Lumiptosome; Split luciferase.