Coexisting Domains in the Plasma Membranes of Live Cells Characterized by Spin-Label ESR Spectroscopy

Abstract

The importance of membrane-based compartmentalization in eukaryotic cell function has become broadly appreciated, and a number of studies indicate that these eukaryotic cell membranes contain coexisting liquid-ordered (L(o)) and liquid-disordered (L(d)) lipid domains. However, the current evidence for such phase separation is indirect, and so far there has been no direct demonstration of differences in the ordering and dynamics for the lipids in these two types of regions or their relative amounts in the plasma membranes of live cells. In this study, we provide direct evidence for the presence of two different types of lipid populations in the plasma membranes of live cells from four different cell lines by electron spin resonance. Analysis of the electron spin resonance spectra recorded over a range of temperatures, from 5 to 37 degrees C, shows that the spin-labeled phospholipids incorporated experience two types of environments, L(o) and L(d), with distinct order parameters and rotational diffusion coefficients but with some differences among the four cell lines. These results suggest that coexistence of lipid domains that differ significantly in their dynamic order in the plasma membrane is a general phenomenon. The L(o) region is found to be a major component in contrast to a model in which small liquid-ordered lipid rafts exist in a 'sea' of disordered lipids. The results on ordering and dynamics for the live cells are also compared with those from model membranes exhibiting coexisting L(o) and L(d) phases.

Organization and Dynamics of Hippocampal Membranes in a Depth-Dependent Manner: An Electron Spin Resonance Study

“…51,53 Interestingly, the order parameter of 5-PC in native hippocampal membranes is comparable to the order parameter of 0.38 obtained for the same spin label in the liquid-ordered component of live RBL-2H3 cells at 25°C. 48 This is in agreement with our previous results on native hippocampal membranes using fluorescence approaches. For example, we have previously reported that the fluorescence polarization of the popular membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in native hippocampal membranes is ∼0.33 at 25°C , 22 a value that is characteristic of liquid-ordered phase in membranes.…”

“…Similar observations have been made regarding the effect of proteins on the lipid acyl chain packing and order parameter of lipid spin labels incorporated into the plasma membranes of live RBL-2H3 mast cells as well as plasma membrane vesicles derived from them. 48,51 Interestingly, this difference in order parameter between liposomes of lipid extract and native membranes is absent when 14-PC was used. This indicates that membrane proteins exert considerable influence on membrane order in the membrane interfacial Tables 1 and 2 and Experimental Section for other details.…”

Competition between line tension and curvature stabilizes modulated phase patterns on the surface of giant unilamellar vesicles: A simulation study

“…This observation implies that phase separation occurs in the POPC-containing system on the nanometer scale, thus not resolvable by ordinary light microscopy. Given the large body of data from studies of animal cell plasma membranes that supports the occurrence of L o + L d phase domains [5][6][7], and given that the size scale, shapes, and connectivities of phase-separated domains might be involved in the fundamental behaviors of animal cells, understanding the membrane in terms of a nonrandom physical mixture might be important.…”

Mind the Line Tension: New Criteria for Nanodomains in Biological Membranes

“…However, there is little experimental evidence of stable Lo domains (i.e. lipid rafts) in live cells [2,5].…”

“…Now at 20 years since the inception of the lipid raft model [2] while the underlying basis of phase separation is clear, how this is translated in a cellular context remains obscure. The abiding mystery is sustained in large part by the inherent obstacles faced in the characterization of dynamic, nanoscopic domains in vivo [4][5][6]. Much information has instead been gathered through investigation of biomimetic model systems which permit exquisite compositional control and access to a wide array of characterization techniques [3,7].…”