Microvillar Membrane Microdomains Exist at Physiological Temperature

TLDR

Lipid rafts are hypothesized to serve as signaling platforms, yet their in‑vivo existence and function remain uncertain. The study aims to determine whether superraft analysis can be applied to other membrane microdomain systems. The authors isolated microvillar membrane rafts at 37 °C using Brij 98 and performed sequential detergent extraction at increasing temperatures to identify highly detergent‑resistant superrafts. Microvillar rafts isolated at physiological temperature have similar protein and lipid profiles to low‑temperature rafts, confirming their existence in vivo; superrafts are enriched in galectin‑4, alkaline phosphatase, and aminopeptidase N, and galectin‑4 functions as a core raft stabilizer and organizer.

Abstract

Lipid rafts (glycosphingolipid/cholesterol-enriched membrane microdomains) have been isolated as low temperature, detergent-resistant membranes from many cell types, but despite their presumed importance as lateral sorting and signaling platforms, fundamental questions persist concerning raft function and even existence in vivo. The nonionic detergent Brij 98 was used to isolate lipid rafts from microvillar membrane vesicles of intestinal brush borders at physiological temperature to compare with rafts, obtained by "conventional" extraction using Triton X-100 at low temperature. Microvillar rafts prepared by the two protocols were morphologically different but had essentially similar profiles of protein- and lipid components, showing that raft microdomains do exist at 37 degrees C and are not "low temperature artifacts. " We also employed a novel method of sequential detergent extraction at increasing temperature to define a fraction of highly detergent-resistant "superrafts." These were enriched in galectin-4, a beta-galactoside-recognizing lectin residing on the extracellular side of the membrane. Superrafts also harbored the glycosylphosphatidylinositol-linked alkaline phosphatase and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other membrane microdomain systems.