Introduction: Efficient apoptotic cell clearance after injury, chemotherapy, and other triggers of programmed cell death is required to avoid inflammation and further tissue damage. However the behaviors of engulfment signaling molecules and their contributions towards non-inflammatory resolution are poorly understood. In Drosophila glial cells the receptor Draper is proposed to drive engulfment of apoptotic neurons. An orthologous receptor, MEGF10, has been proposed to work in mammalian glia. Both Draper and the T cell receptor complex (TCR) are proposed to recruit downstream molecules to the plasma membrane but functional similarities and differences between T cell and engulfment signaling have not been described. We have reconstituted engulfment in order to uncover the mechanism of this important, conserved signaling system.
Experimental systems: By expressing Draper in otherwise inept phagocytes we have dramatically increased the engulfment of apoptotic cells or physiological mimics that expose natural ligands. We have also generated an inducible ligand-receptor system that provides a platform to monitor rapid responses of Draper signaling that are otherwise hard to capture. We utilize a supported lipid bilayer system and total internal fluorescence (TIRF) imaging to monitor the localization of Draper and downstream signaling molecules at the plasma membrane.
Results: Remarkable recent work in T cells has led to the development of chimeric antigen receptors (CARs) that link specific ligands to heterologous intracellular signaling pathways in order to kill tumor cells. We have taken a similar approach to engulfment signaling by engineering chimeric engulfment receptors. An inducible heterologous receptor-ligand pair is used in our chimeric engulfment receptor system. Like Draper, the engineered chimeric receptor recruits signaling machinery to the plasma membrane and drives engulfment. Our results indicate ligand flexibility in the system and that the mechanism of activation required for endogenous Draper can be mimicked by engineered chimeric receptors.
Using our TIRF assay we have discovered that Draper forms dynamic clusters similar to TCR and that clustered receptors recruit cytosolic signaling proteins to the plasma membrane. Importantly, simultaneous imaging of engulfment receptors and filamentous-actin (F-actin) has revealed that engulfment receptors drive F-actin remodeling that is distinct from behavior seen during TCR signaling. Our TIRF work has revealed both similarities and significant differences between the cellular changes set into motion by Draper signaling as compared to T cell triggering.
Conclusions: We have built reconstitution platforms to study engulfment signaling and are in a unique position to understand the essential factors and rate-limiting steps of clearance. We have discovered that engineered chimeric receptors drive efficient engulfment and that engulfment signaling is sparked by formation of dynamic clusters of signaling molecules. We are hopeful that the lessons we learn from our reconstitution systems can be applied to activate non-inflammatory clearance of apoptotic cells after injury or extensive cell death.
Citation Format: Adam Williamson, Ron Vale. Engineering approaches to uncover the mechanism of apoptotic cell clearance by a conserved signaling system. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A165.