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J. Biol. Chem., Vol. 283, Issue 34, 22962-22971, August 22, 2008

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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^¶, and Kouji Matsushima¹

From the Department of Molecular Preventive Medicine, Graduate School of Medicine, and the Environmental Science Center, University of Tokyo, Tokyo 113-0033, Japan, the ^||Division of Infectious Genetics, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan, and the ^¶Department of Physics, School of Science and Engineering, Waseda University, Tokyo 169-8555, Japan

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.

Received for publication, February 20, 2008 , and in revised form, June 3, 2008.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Movies S1–S9.

¹ To whom correspondence should be addressed: Dept. of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyu-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-3431; Fax: 81-3-5684-2297; E-mail: koujim{at}m.u-tokyo.ac.jp.

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