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Papers In Press, published online ahead of print October 1, 2008
J. Lipid Res., doi:10.1194/jlr.D800031-JLR200

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Journal of Lipid Research, Vol. 49, 2268-2275, October 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

Fluorescence-topographic NSOM directly visualizes peak-valley polarities of GM1/GM3 rafts in cell membrane fluctuations^*

Yong Chen, Jie Qin and Zheng W. Chen¹

Department of Microbiology and Immunology, Center for Primate Biomedical Research, University of Illinois College of Medicine, Chicago, Illinois

^* This work was supported by NIH RO1 grants HL64560 (to ZWC) and RR13601 (to ZWC).

Published, JLR Papers in Press, July 4, 2008.

¹ To whom correspondence should be addressed: e-mail: zchen{at}uic.edu

Simultaneous fluorescence-topographic nanoscale imaging of cell-surface molecules in the context of membrane ultra-structures has not been reported. Here, near-field scanning optical microscopy (NSOM)-based direct fluorescence-topographic imaging indicated that GM3 rafts/nanodomains (190.0 ± 49.8 nm ranging 84.5–365.0 nm) were localized predominantly on the peaks of microvillus-like protrusions in the apical membrane of GM3 + Madin-Darby canine kidney cells, whereas GM1 rafts/nanodomains (159.5 ± 63.8 nm ranging 42–360 nm) were distributed mainly on the slops of protrusions or the valleys between protrusions in the plasma membranes of GM1 + MDCK cells. The data demonstrated that gangliosides polarized not only in a well-known apical-basolateral manner but also in the more microscopic peak-valley manner, implicating unique distribution of GM1 or GM3 in cell-surface fluctuations on the apical membrane of polarized cells. The peak-valley polarities of gangliosides also implicated their different functions relevant to lipid rafts, microvilli, or cellular processes. Importantly, our study demonstrated for the first time that the NSOM-based direct fluorescence-topographic imaging is unique and powerful for elucidating nanoscale distribution of specific cell-surface molecules in membrane fluctuations.

Supplementary key words near-field scanning optical microscopy • fluorescent quantum dot • fluorescence-topographic imaging • ganglioside GM1 • ganglioside GM3 • MDCK • polarity

Abbreviations: ATCC, American Type Culture Collection; CTB, cholera toxin subunit B; DIC, differential interference contrast; EM, electron microscopy; FITC, fluorescein isothiocyanate; FWHM, full width at half maximum; MDCK, Madin-Darby canine kidney; MFI, mean fluorescence intensity; NSOM, near-field scanning optical microscopy; QD, quantum dot; ROI, region of interest

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