Visualization of the Molecular Dynamics of Lipopolysaccharide on the Plasma Membrane of Murine Macrophages by Total Internal Reflection Fluorescence Microscopy

Samia Shawkat, Risuke Karima, Tadashi Tojo, Hisashi Tadakuma, Shin-ichiroh Saitoh, Sachiko Akashi-Takamura, Kensuke Miyake, Takashi Funatsu, Kouji Matsushima
2008 Journal of Biological Chemistry  
The molecular action of Alexa 594-labeled lipopolysaccharide (LPS) from Escherichia coli was examined on living peritoneal macrophages of C57BL/6 mice by total internal reflection fluorescence microscope (TIRFM), and the molecular kinetics of LPS was analyzed. TIRFM visualization of the action of fluorescence-labeled LPS revealed an increase in the mean fluorescence intensity of LPS on the plasma membrane of wild type macrophages at 60 min after administration, indicating the oligomerization of
more » ... LPS after binding to the macrophages. Additionally, a time-dependent sharp decrease in the mean diffusion coefficient of LPS was observed. On the other hand, both mean fluorescence intensity and diffusion coefficient of LPS in cases of TLR4 ؊/؊ , MD2 ؊/؊ , MyD88 ؊/؊ , and TRIF ؊/؊ macrophages were significantly different from the corresponding values of wild type macrophage, whereas differences were also noticed among these molecule-deficient macrophages. Furthermore, statistical analysis indicated the major role of receptors (TLR4 and MD2) and intracellular signaling molecules (MyD88 and TRIF) in oligomerization and lowering of the diffusion rate of LPS on the plasma membrane of murine macrophages, respectively. Lipopolysaccharide (LPS), 2 the major constituent of the Gram-negative bacterial outer membrane, induces intense inflammatory responses. The complex of LPS and LPS-binding protein (LBP) interacts with CD14, a receptor on macrophages/monocytes and neutrophils. Membranebound CD14 (mCD14) is a glycosylphosphatidylinositol-
doi:10.1074/jbc.m801366200 pmid:18567586 fatcat:bbhuacejynho3deyehb4rflohi