Phagoscopy: dissecting cell-autonomous immunity with  ex vivo electron cryo-microscopy
Our immune system provides a formidable barrier to the many microbial pathogens that we encounter every day. Yet, many pathogens have the ability to avert this barrier by invading the host cell and seeking shelter inside a phagosome whose membrane physically prevents the pathogen from being recognized and eliminated. Cell-autonomous immunity is a part of the innate immune system that fights off such pathogens. Among the antimicrobial effectors mobilized by this immune response are the Guanylate-Binding Proteins (GBPs). GBPs form dynamic supramolecular assemblies that promote lysis of phagosomes and, thus, killing of pathogens. Despite their central importance, we know very little about the molecular mechanisms of GBPs. Two fundamental questions are: (1) What is the structure and composition of GBP assemblies on membranes?, and (2) Once assembled, how do the GBPs structurally rearrange to reshape and rupture the phagosome's membrane? These questions remain unanswered because structural biology has been lacking methods for determining dynamically changing structures of proteins that are assembled in complex environments such as phagosomes. Here, I propose to take a two-pronged approach to address these questions: first, I will use cryo-EM and (single-molecule) fluorescence microscopy to elucidate the interactions and conformational changes involved in GBP oligomerization on model membranes. Second, I will visualize this pathway on native phagosomes using a recently developed ex vivo reconstitution system unique to my laboratory. By determining how GBP assemblies form on phagosome membranes, how they reshape the membrane so that it ruptures, and how these processes can be regulated and inhibited, I will derive a mechanistic model of a key effector function that cells employ to combat disease-causing pathogens. More broadly, my study will establish a novel approach for integrative imaging that will be applicable to a wide range of dynamic molecular assemblies in cells.