Detecting Microdomains in Intact Cell Membranes

Abstract

Current models for cellular plasma membranes focus on spatial heterogeneity and how this heterogeneity relates to cell function. In particular, putative lipid raft membrane domains have been postulated to exist based in large part on the results that a significant fraction of the membrane is detergent insoluble and that molecules facilitating key membrane processes like signal transduction are often found in the detergent-resistant membrane fraction. Yet, the in vivo existence of lipid rafts remains extremely controversial because, despite being sought for more than a decade, evidence for their presence in intact cell membranes is inconclusive. In this review, a variety of experimental techniques that have been or might be used to look for lipid microdomains in intact cell membranes are described. Experimental results are highlighted and the strengths and limitations of different techniques for microdomain identification and characterization are assessed.

The human serotonin1A receptor exhibits G-protein-dependent cell surface dynamics

“…Monitoring diffusion of cell surface molecules represents a powerful approach to detect their organization on the plasma membrane [25,26]. In the light of the proposed significance of a spatiotemporally restricted environment in modulating receptor and G-protein interaction (see above), we have utilized the previously characterized serotonin 1A receptor tagged to the enhanced yellow fluorescence protein (EYFP) [27] to analyze its cell surface dynamics (diffusion properties) using the technique of fluorescence recovery after photobleaching (FRAP) [28,29].…”

Lipid rafts, cholesterol, and the brain

“…Such techniques can be used to monitor raft-related events with high spatial and temporal resolution without perturbing the integrity of the membrane (Lommerse et al 2004;Jacobson et al 2007). One such approach is fluorescence (or Förster) resonance energy transfer (FRET), used to test the hypothesis that raft proteins or lipids are enriched in domains with sub-micron dimensions (reviewed in (Kenworthy 2002;Lommerse et al 2004;Lagerholm et al 2005;Rao and Mayor 2005;Silvius and Nabi 2006)). In this technique, proteins or lipids of interest are labeled with fluorophores known as a donor and acceptor.…”

“…Alternatively, proteins may diffuse as part of a raft complex (Pralle et al 2000). These effects have been studied using techniques sensitive to lateral diffusion such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single molecule tracking (reviewed in (Lommerse et al 2004;Kenworthy 2005;Lagerholm et al 2005)). …”

Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation

“…While detergent-resistant membrane extraction has historically been used as a benchmark for membrane raft association [15,25,27], we note that more recent work would suggest that detergent-resistance can be a subjective approach because of its dependence on type and concentration of detergent, temperature and cell type [16,27]. In spite of this caveat, it remains a straightforward and effective means of isolating individual membrane raft proteins and complexes [16] subject to later verification.…”

“…All samples were labeled with anti-NCAM and co-stained with either fluorescently labeled cholera toxin subunit B (CTB), anti-caveolin-3, or anti-syndecan-4 ( Figure 1). CTB binds to ganglioside M1 (GM 1 ), a glycosphingolipid considered to be a specific marker of membrane rafts [27,62]. Caveolin-3, or M-caveolin, is a muscle-specific isoform of caveolin that localizes to caveolae, a subset of membrane rafts, in the plasma membrane (reviewed in [63]).…”