Lipid Rafts Exist as Stable Cholesterol-independent Microdomains in the Brush Border Membrane of Enterocytes

Gert H Hansen,Lissi Immerdal,Evy Thorsen,Lise-Lotte Niels-Christiansen,Birthe T Nystrøm,Erland J.F Demant,E Michael Danielsen

doi:10.1074/jbc.m102667200

Gert H Hansen, Lissi Immerdal + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m102667200

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Abstract

Glycosphingolipid/cholesterol-rich membranes ("rafts")can be isolated from many types of cells, but their existence as stable microdomains in the cell membrane has been elusive. Addressing this problem, we studied the distribution of galectin-4, a raft marker, and lactase, a protein excluded from rafts, on microvillar vesicles from the enterocyte brush border membrane. Magnetic beads coated with either anti-galectin-4 or anti-lactase antibodies were used for immunoisolation of vesicles followed by double immunogold labeling of the two proteins. A morphometric analysis revealed subpopulations of raft-rich and raft-poor vesicles by the following criteria: 1) the lactase/galectin-4 labeling ratio/vesicle captured by the anti-lactase beads was significantly higher (p < or = 0.01) than that of vesicles captured by anti-galectin-4 beads, 2) subpopulations of vesicles labeled by only one of the two antibodies were preferentially captured by beads coated with the respective antibody (p < or = 0.01), 3) the average diameter of "galectin-4 positive only" vesicles was smaller than that of vesicles labeled for lactase. Surprisingly, pretreatment with methyl-beta-cyclodextrin, which removed >70% of microvillar cholesterol, did not affect the microdomain localization of galectin-4. We conclude that stable, cholesterol-independent raft microdomains exist in the enterocyte brush border.

Highlights

Ever since the “membrane cluster” hypothesis was proposed well over a decade ago [1], the biological relevance of glycosphingolipid/cholesterol-rich “raft” microdomains has been debated
Addressing this problem, we studied the distribution of galectin-4, a raft marker, and lactase, a protein excluded from rafts, on microvillar vesicles from the enterocyte brush border membrane
A morphometric analysis revealed subpopulations of raft-rich and raft-poor vesicles by the following criteria: 1) the lactase/galectin-4 labeling ratio/vesicle captured by the anti-lactase beads was significantly higher (p < 0.01) than that of vesicles captured by anti-galectin-4 beads, 2) subpopulations of vesicles labeled by only one of the two antibodies were preferentially captured by beads coated with the respective antibody (p < 0.01), 3) the average diameter of “galectin-4 positive only” vesicles was smaller than that of vesicles labeled for lactase

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—DynabeadsTM M-500 Subcellular were purchased from Dynal (Oslo, Norway). Electron Microscopy—Microvillar membrane vesicles from native and cholesterol-depleted mucosal explants captured onto antibodycoated magnetic beads were washed three times in 0.1 M sodium phosphate buffer (PB), pH 7.2 and fixed in 2% paraformaldehyde/0.1% glutaraldehyde in PB for 1 h at 4 °C. After a rinse in TBS containing 0.25% BSA (3 ϫ 10 min), the vesicles/magnetic beads were incubated in sheep anti-rabbit IgG conjugated to 7 nm gold particles [35] for 1 h. After a rinse in PB (3 ϫ 5 min) and TBS (2 ϫ 5 min) the vesicles/magnetic beads were treated with 3% BSA in TBS (30 min) at 4 °C before incubation in rabbit anti-pig lactase for 1 h. Chloroform/methanol extraction of total lipid in native and methyl-␤-cyclodextrin-treated microvillar vesicle membranes was carried out as described previously [26, 40]. Cholesterol was detected with a CuSO4/H3PO4 reagent and glycolipids were detected with a ␣-naphtol spray reagent

RESULTS AND DISCUSSION

TABLE I Morphometric analysis of immunoisolated microvillar vesicles

Native vesicles captured by