Visualizing lipid structure and raft domains in living cells with two-photon microscopy

TLDR

Cellular membranes are organized into lipid rafts—clusters of cholesterol and sphingolipids that form liquid‑ordered domains distinct from the surrounding liquid‑disordered membrane. The study aims to directly visualize membrane lipid structure in living cells using two‑photon microscopy. Two‑photon microscopy combined with image deconvolution was used to image sub‑micron lipid domains and compare them to detergent‑resistant membranes, revealing that cholesterol controls raft coverage and fluidity. In macrophages, liquid‑ordered domains occupy 10–15 % of the surface, localize to protrusions and contacts, and their coverage and fluidity are driven by cholesterol, confirming phase separation and supporting the lipid‑raft hypothesis.

Abstract

The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell-cell contacts and cover 10-15% of the cell surface at 37 degrees C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 microm2), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis.