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Vol. 18, Issue 1, 211-228, January 2007

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Plasma Membrane Sterol Distribution Resembles the Surface Topography of Living Cells

Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark

Submitted May 22, 2006; Revised August 24, 2006; Accepted October 13, 2006
Monitoring Editor: Sean Munro

Cholesterol is an important constituent of cellular membranes. It has been suggested that cholesterol segregates into sterol-rich and -poor domains in the plasma membrane, although clear evidence for this is lacking. By fluorescence imaging of the natural sterol dehydroergosterol (DHE), the lateral sterol distribution has been visualized in living cells. The spatial labeling pattern of DHE coincided with surface structures such as ruffles, microvilli, and filopodia with correlation lengths in the range of 0.8–2.5 µm. DHE staining of branched tubules and of nanotubes connecting two cells was detected. Dynamics of DHE in folded and plane membrane regions was comparable as determined by fluorescence recovery after photobleaching. DHE colocalized with fluid membrane-preferring phospholipids in surface structures and at sites of cell attachment as well as in the cleavage furrow of dividing cells, but it was not particularly enriched in those regions. Fluorescent sterol showed homogeneous staining in membrane blebs induced by F-actin disruption. Cross-linking the ganglioside GM1—a putative raft marker—did not affect the cell surface distribution of DHE. The results suggest that spatial heterogeneities of plasma membrane staining of DHE resolvable by light microscopy reflect the cell surface topography but not phase-separated sterol domains in the bilayer plane.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-05-0445) on October 25, 2006.

Address correspondence to: Daniel Wüstner (wuestner{at}bmb.sdu.dk)

Abbreviations used: Alexa488-CTxB, Alexa488-labeled cholera toxin subunit B; BC, biliary canaliculus; -BODIPY-PC, 2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexa-decanoyl-sn-glycero-3-phosphocholine; BSA, bovine serum albumin; CE, cholesteryl ester; CHO, Chinese hamster ovary; CTxB, cholera toxin subunit B; C6-NBD-PC, 1-palmitoyl-2-[6-[(7-nitro-2–1,3-benzooxadiazol- 4-y) amino]caproyl]-sn-glycero-3-phosphatidylcholine; DHE, dehydroergosterol; DHE/MCD, dehydroergosterol/methyl--cyclodextrin complex; DiIC12, 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DiIC16, 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; GFP-PH, green fluorescent protein-tagged pleckstrin homology protein; PC, phosphatidylcholine; PCA, principal component analysis; PFA, paraformaldehyde; ROI, region of interest; SR-BI, scavenger receptor BI; Tf, transferrin.

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