Faculty
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Joel Swanson |
Central to defense against pathogenic microorganisms is the macrophage’s ability to internalize particles by phagocytosis. This lab uses quantitative fluorometric and microscopic methods to delineate the mechanisms and regulation of phagocytosis and to characterize the intravacuolar environment in the presence and absence of pathogenic bacteria. These goals are important not only for understanding macrophage biology, but also for elucidating basic mechanisms of microbial pathogenesis and innate resistance to infections.
To define the mechanism of Fc g receptor-mediated phagocytosis, we are developing and applying microscopic methods for imaging signaling molecules inside living macrophages. We are examining the regulation of the actin cytoskeleton during phagocytosis, focusing in particular on the contributions of Rho-family GTPases and membrane phosphoinositides. These studies employ ratiometric fluorescence microscopy of macrophages expressing cyan fluorescent protein (CFP) and chimeras of yellow fluorescent protein (YFP) plus protein domains that bind phosphoinositides specifically. We are also developing and applying quantitative methods for using fluorescence resonance energy transfer (FRET) microscopy to image protein-protein interactions inside macrophages during phagocytosis.
A long-term objective of our studies is to identify those features of macrophage endocytic compartments that counteract intracellular pathogens. Although post-phagocytic delivery of microbes into macrophage lysosomes typically leads to their degradation, some pathogenic microorganisms survive phagocytosis and evade macrophage defense mechanisms. Listeria monocytogenes is an intracellular pathogen that survives by passing from phagosomes into cytoplasm. It secretes a hemolytic protein, listeriolysin O (LLO), which mediates bacterial passage into cytoplasm. We are presently identifying the compartment permeabilized by L. monocytogenes, and determining how this compartment is altered in activated macrophages. Activation of macrophages with interferon- g plus LPS or TNF a increases their resistance to many pathogens, including L. monocytogenes. We are testing the hypothesis that increased resistance to pathogens in activated macrophages results from altered phagosome progression to lysosomes, plus localized delivery of toxic compounds into late endosome-like phagosomes. Quantitative fluorometric methods are being used to measure endocytic compartment dynamics and physiology in activated and non-activated macrophages. Fluorescence microscopic methods are being used to measure intravacuolar pH and calcium concentrations, and to localize reactive oxygen and nitrogen intermediates in individual cells. We compare listericidal and nonlistericidal macrophages, as well as macrophages from mice with induced deletions for components of the nitric oxide or reactive oxygen intermediate biosynthetic pathways. Because these studies provide direct measurements of conditions inside the vacuolar compartments of activated macrophages, their results should improve understanding of host defense mechanisms related to infection by L. monocytogenes as well as other intracellular pathogens.
Selected Publications:
Christensen, K. A., J. T. Myers and J. A. Swanson. 2002. pH-dependent regulation of lysosomal calcium in macrophages. J. Cell Sci. 115: 599-607.
Diakonova, M., G. Bokoch and J. A. Swanson. 2002. Dynamics of cytoskeletal proteins during Fcg receptor-mediated phagocytosis in macrophages. Mol. Biol. Cell 13: 402-411.
Myers, J. T. and J. A. Swanson. 2002. Calcium spikes in activated macrophages during Fcg receptor-mediated phagocytosis. J. Leukoc. Biol. 72: 677-684.
Hoppe, A. D., K. A. Christensen and J. A. Swanson. 2002. Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys. J. 83: 3652-3664.
Myers, J. T., A. W. Tsang and J. A. Swanson. 2003. Localized reactive oxygen and nitrogen intermediates inhibit escape of Listeria monocytogenes from vacuoles in activated macrophages. J. Immunol. 171: 5447-5453.
