Visualization of the molecular dynamics of lipopolysaccharide on the plasma membrane of murine macrophages by total internal reflection fluorescence microscopy.
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 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.
Department of Molecular Preventive Medicine, University of Tokyo, Tokyo, Japan.
Samia
S
Shawkat
Risuke
R
Karima
Tadashi
T
Tojo
Hisashi
H
Tadakuma
Shin-Ichiroh
S
Saitoh
Sachiko
S
Akashi-Takamura
Kensuke
K
Miyake
Takashi
T
Funatsu
Kouji
K
Matsushima
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence / Q000379:methods*; D008954:Models, Biological; D009930:Organic Chemicals / Q000494:pharmacology; D015398:Signal Transduction; D013997:Time Factors
C417664:Alexa594; D005456:Fluorescent Dyes; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D009930:Organic Chemicals