Synthetic Sufficiency Models of Cell Death for Immunology 2260195

Abstract

Abstract Introduction Regulated cell death is essential for tissue homeostasis and host defense. Many death pathways culminate in the loss of plasma membrane integrity which is orchestrated by distinct executioner proteins. We aim to define the cell-intrinsic programs that determine a single cell’s decision between survival and death downstream of executioners for apoptosis, pyroptosis, and necroptosis. We are also interested in regulation of the terminal NINJ1-mediated membrane rupture. Methods We engineered a suite of synthetic sufficiency models that allow inducible and temporal control of constitutively active death executioners. These executioners include truncated BID (tBID) to trigger mitochondrial apoptosis, the N terminal fragment of gasdermin D (NT-GSDMD) for pyroptosis, a phosphomimetic mutant of MLKL for necroptosis, and overexpressed wild-type NINJ1 to model terminal lytic rupture. Using chemogenetic induction or transient transfection, we precisely initiate each program and quantify cell fate outcomes by live-cell imaging, flow cytometry, and bulk enzymatic assays. Results Phenotypic analysis reveals distinct and expected membrane behaviors across models. Annexin V staining and DRAQ7 uptake delineate apoptotic cells that expose phosphatidylserine (Annexin V+/DRAQ7⁻) before secondary necrosis, while pyroptotic, necroptotic, and NINJ1-driven cells display early DRAQ7 positivity consistent with rapid membrane perforation or rupture. LDH release assays confirm minimal lysis during apoptosis but high lysis in lytic death models. ATP quantification shows apoptotic cells maintain intermediate energetic levels, whereas pyroptotic, necroptotic, and NINJ1-driven cells rapidly lose energetic viability. Conclusion Together, these synthetic death tools provide a modular framework to dissect the distinct kinetics, molecular features, and immunogenic potential of each cell death program. Funding Source K.R.R supported by the Catherine Othieno Sempa Post-Baccalaureate Fellowship, C.L.E. supported by the Ragon Early independence Fellowship, Karin Grunebaum Cancer Research Foundation Faculty Research Fellowship, and NIH grant R35GM159938 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)