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Vol. 18, Issue 6, 2112-2122, June 2007

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Gangliosides GM1 and GM3 in the Living Cell Membrane Form Clusters Susceptible to Cholesterol Depletion and Chilling

Akikazu Fujita^*, Jinglei Cheng^*, Minako Hirakawa, Koichi Furukawa, Susumu Kusunoki, and Toyoshi Fujimoto^*

Departments of *Anatomy and Molecular Cell Biology and Biochemistry II, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; and Department of Neurology, Kinki University School of Medicine, Osaka 589-8511, Japan

Submitted January 26, 2007; Revised March 6, 2007; Accepted March 20, 2007
Monitoring Editor: Jean Gruenberg

Presence of microdomains has been postulated in the cell membrane, but two-dimensional distribution of lipid molecules has been difficult to determine in the submicrometer scale. In the present paper, we examined the distribution of gangliosides GM1 and GM3, putative raft molecules in the cell membrane, by immunoelectron microscopy using quick-frozen and freeze-fractured specimens. This method physically immobilized molecules in situ and thus minimized the possibility of artifactual perturbation. By point pattern analysis of immunogold labeling, GM1 was shown to make clusters of <100 nm in diameter in normal mouse fibroblasts. GM1-null fibroblasts were not labeled, but developed a similar clustered pattern when GM1 was administered. On cholesterol depletion or chilling, the clustering of both endogenous and exogenously-loaded GM1 decreased significantly, but the distribution showed marked regional heterogeneity in the cells. GM3 also showed cholesterol-dependent clustering, and although clusters of GM1 and GM3 were found to occasionally coincide, these aggregates were separated in most cases, suggesting the presence of heterogeneous microdomains. The present method enabled to capture the molecular distribution of lipids in the cell membrane, and demonstrated that GM1 and GM3 form clusters that are susceptible to cholesterol depletion and chilling.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Toyoshi Fujimoto (tfujimot{at}med.nagoya-u.ac.jp).

Abbreviations used: b-CtxB, biotinylated cholera toxin B; CI, confidence interval; CSR, complete spatial randomness; EM, electron microscopy; GAR-Fab5, colloidal gold (5-nm)-conjugated anti-rabbit IgG F(ab')2 fragment; PAG5, colloidal gold (5-nm)-conjugated protein A; MCD, methyl--cyclodextrin; NND, nearest neighbor distance; PC, phosphatidylcholine; SDS-FRL, SDS-treated freeze-fracture replicas.