Giri, P. | bioRxiv Science

bioRxiv2023

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context

Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy an optimized cryo-ET platform that enables cellular-structural biology in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using this platform, we reconstructed sub-nanometer resolution structures of the human cardiac muscle thin filament, a central component of the contractile machinery. Reconstructing the troponin complex, a regulatory component of the thin filament, from within cells, we identified previously unobserved conformations that highlight the structural flexibility of this regulatory complex. We next measured the impact of chemical and genetic perturbations associated with cardiovascular disease on the structure of troponin. In both cases, we found changes in troponin structure that are consistent with known disease phenotypes--highlighting the value of our approach for dissecting complex disease mechanisms in the cellular context.

biophysics↗

bioRxiv2020

Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication

Salmonellosis and listeriosis together accounted for more than one third of foodborne illnesses in the United States and almost half the hospitalizations for gastrointestinal diseases in 2018 while tuberculosis afflicted over 10 million people worldwide causing almost 2 million deaths. Regardless of the intrinsic virulence differences among Listeria monocytogenes, Salmonella enterica and Mycobacterium tuberculosis, these intracellular pathogens share the ability to survive and persist inside the macrophage and other cells and thrive in iron rich environments. Interferon-gamma (IFN-{gamma}) is a central cytokine in host defense against intracellular pathogens and has been shown to promote iron export in macrophages. We hypothesize that IFN-{gamma} decreases iron availability to intracellular pathogens consequently limiting replication in these cells. In this study, we show that IFN-{gamma} regulates the expression of iron-related proteins hepcidin, ferroportin, and ferritin to induce iron export from macrophages. Listeria monocytogenes, S. enterica, and M. tuberculosis infections significantly induce iron sequestration in human macrophages. In contrast, IFN-{gamma} significantly reduces hepcidin secretion in S. enterica and M. tuberculosis infected macrophages. Similarly, IFN-{gamma}-activated macrophages express higher ferroportin levels than untreated controls even after infection with L. monocytogenes bacilli; bacterial infection greatly down-regulates ferroportin expression. Collectively, IFN-{gamma} significantly inhibits pathogen-associated intracellular iron sequestration in macrophages and consequently retards the growth of intracellular bacterial pathogens by decreasing iron availability.

cell biology↗