Abstract
In some cases, lipids in one leaflet of an asymmetric artificial lipid vesicle suppress the formation of ordered lipid domains (rafts) in the opposing leaflet. Whether this occurs in natural membranes is unknown. Here, we investigated this issue using plasma membrane vesicles (PMVs) from rat leukemia RBL-2H3 cells. Membrane domain formation and order was assessed by fluorescence resonance energy transfer and fluorescence anisotropy. We found that ordered domains in PMVs prepared from cells by N-ethyl maleimide (NEM) treatment formed up to ∼37°C, whereas ordered domains in symmetric vesicles formed from the extracted PMV lipids were stable up to 55°C, indicating the stability of ordered domains was substantially decreased in intact PMVs. This behavior paralleled lesser ordered domain stability in artificial asymmetric lipid vesicles relative to the corresponding symmetric vesicles, suggesting intact PMVs exhibit some degree of lipid asymmetry. This was supported by phosphatidylserine mislocalization on PMV outer leaflets as judged by annexin binding, which indicated NEM-induced PMVs are much more asymmetric than PMVs formed by dithiothreitol/paraformaldehyde treatment. Destroying asymmetry by reconstitution of PMVs using detergent dilution also showed stabilization of domain formation, even though membrane proteins remained associated with reconstituted vesicles. Similar domain stabilization was observed in artificial asymmetric lipid vesicles after destroying asymmetry via detergent reconstitution. Proteinase K digestion of proteins had little effect on domain stability in NEM PMVs. We conclude that loss of PMV lipid asymmetry can induce ordered domain formation. The dynamic control of lipid asymmetry in cells may regulate domain formation in plasma membranes.
Highlights
It is widely accepted that the plasma membrane (PM), and likely even some internal cellular membranes, have the capacity to form coexisting liquid ordered (Lo state) lipid domains and liquid disordered (Ld state) lipid domains [1, 2]
Because we found the plasma membrane vesicles (PMVs) induced by N-ethyl maleimide (NEM) results in contamination with small cell debris or cytosolic components that resulted in high backgrounds when measuring DPH fluorescence, in Figs. 1 and 2 the background intensities were subtracted for the Fluorescence resonance energy transfer (FRET) measurements
At which ordered domains “melt”/become miscible with liquid-disordered state (Ld) lipids, there is a decrease in the average distance between DPH molecules that were localized in the ordered domains and FRET acceptors that were in the disordered domains
Summary
It is widely accepted that the plasma membrane (PM), and likely even some internal cellular membranes, have the capacity to form coexisting liquid ordered (Lo state) lipid domains (rafts) and liquid disordered (Ld state) lipid domains [1, 2]. Dynamic changes in lipid asymmetry have the potential to alter domain formation [9,10,11]. This is because the interleaflet coupling of lipid physical properties can alter ordered domain formation when one leaflet rich in ordered domain-forming lipids contacts a leaflet unable to spontaneously form ordered domains. Lipid asymmetry in which only one leaflet has the ability to form ordered domains can result in either induction or suppression of ordered domain formation, depending upon lipid composition [9, 10, 12,13,14]
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