Joel Swanson, Ph.D.

Central to defense against pathogenic microorganisms is the macrophage's ability to internalize fluids and particles by endocytosis, which includes the processes of phagocytosis, receptor-mediated endocytosis and macropinocytosis. This lab uses quantitative cell biological and microscopic methods to study endocytosis in macrophages. The principle goals are to delineate the mechanisms and regulation of phagocytosis and macropinocytosis, and to characterize the intravacuolar environment in the presence of pathogenic bacteria. These goals are important for understanding macrophage biology and for elucidating how microbes cause disease.

To understand how a macrophage coordinates many cell surface receptors to ingest particles by phagocytosis and fluids by macropinocytosis, we developed and applied microscopic methods for imaging signaling molecules inside living cells. These studies employ ratiometric fluorescence microscopy of macrophages expressing protein chimeras of cyan fluorescent protein or yellow fluorescent protein. Using quantitative fluorescence resonance energy transfer (FRET) microscopy to measure interactions between fluorescent proteins inside macrophages, we identified discrete stages of signaling that correspond to the distinct stages of phagosome formation. One set of GTPases was active early during phagocytosis, when actin-rich cups of membrane extend over particle surfaces. Increased concentrations of 3' phosphoinositides deactivated some of those GTPases and activated a second set, which regulate the later stages of phagosome closure. To determine the mechanism of this signal transition, we measured the magnitudes of Fc receptor-generated signals in phagosomes as a function of IgG density on particle surfaces. We discovered that threshold concentrations of 3' phosphoinositides control cellular commitment to particle ingestion and to late stages of receptor signaling. These threshold-dependent signal transitions indicate a general mechanism to organize the molecular activities in cells. Moreover, it suggests novel therapeutic strategies for directing microbicidal responses against smaller microbes, such as viruses.

We have also examined the regulation of macropinocytosis, which is a mechanism for internalization of many bacteria and viruses. We showed that macropinosomes mature inside macrophages by progressively modifying their constituent membrane proteins from those of early endosomes, to late endosomes and finally lysosomes and we determined that Salmonella entericavar. Typhimurium enters macrophages by stimulating macropinocytosis and entering via spacious phagosomes. Recently, we used quantitative fluorescence microscopy to determine that during macropinosome formation, signal amplification follows closure of cell surface ruffles into circular, cup-shaped invaginations of plasma membrane. This local, focused amplification of membrane-tethered signals in a cup-shaped domain of plasma membrane indicates that cell surface ruffles can act as diffusion barriers to phospholipids or membrane-associated proteins in the inner leaflet of the plasma membrane. Indeed, our recent measurements of diffusion by membrane-tethered, photoactivatable green fluorescent protein indicated that ruffles and macropinocytic cups limit lateral diffusion of proteins in the inner leaflet of the plasma membrane.

A long-term objective of our studies is to identify features of macrophage endocytic compartments that counteract intracellular pathogens. Using a novel method to measure lysosome damage in macrophages, we recently determined that macrophage activation induces mechanisms for repairing or resisting mechanical damage to membranes. We are currently investigating the role of this "Inducible Lysosome Renitence" in host resistance to infections.