Ann M. Wehman - US grants

Mechanisms of non-apoptotic programmed cell death and corpse clearance Project Summary/Abstract Many cells are programmed to die during development and homeostasis, but not all cells die via apoptosis. Additionally, cancer cells have proven resistant to apoptosis-promoting therapies. Therefore, it is important to understand non-apoptotic mechanisms of cell death, as well as how dying cells signal for clearance. Furthermore, corpse clearance and phagolysosomal degradation are important to avoid inflammation and auto- immune disease, but there are major gaps in our understanding of all these processes. We established that C. elegans polar bodies are non-apoptotic cells that undergo an unknown form of programmed necrosis(Fazeli et al., Cell Rep 2018)1. Using this genetic model, we propose to identify the mechanisms that lead to necrotic cell death and loss of membrane integrity in Aim 1, focusing initially on non- apoptotic caspases and scramblase regulators based on preliminary data generated by an undergraduate student. These targeted screens will identify whether polar body death shares common mechanisms with established modes of programmed necrosis or defines an undiscovered type of cell death, which may give insight into uncharacterized cell death modalities in humans during health and disease. We found that polar bodies externalize phosphatidylserine (PS) and are cleared by embryonic cells using LC3-associated phagocytosis1. However, the signaling pathways that regulate PS exposure in necrotic cells are not known. In preliminary data from an undergraduate student, we have identified at least four redundant lipid scramblases that are required for PS exposure and have reduced PS exposure by mutating them all. This approach is one of several in Aim 2 that will allow us to define the signaling pathways that regulate engulfment of the corpse as well as the recruitment of Atg8/LC3 family proteins to the phagosome. These signaling pathways may be conserved in other types of cell death, including apoptosis, providing wide-ranging insights into the mechanisms of corpse clearance. We also discovered the first role for Atg8/LC3 recruitment to the phagolysosome: promoting breakdown of the corpse membrane within the lumen1. However, how Atg8/LC3 transduces the signal and which factors carry out membrane breakdown remain a mystery. In Aim 3, we tease apart the localization and roles of Atg8/LC3 proteins and their interactors to define the mechanisms of corpse membrane breakdown within the phagolysosome. Membrane breakdown is the limiting factor for the degradation of nucleic acids and proteins within phagolysosomal cargos, which is especially important for MHC class II-expressing immune cells, where lysosomal breakdown products are used for antigen presentation. As defects in LC3-associated phagocytosis have been tied to autoimmune disease in mammals, determining how C. elegans cells promote corpse membrane breakdown can provide new insights into conserved mechanisms in humans during immune signaling, inflammation, and autoimmune disease in addition to pathogen clearance.