Abstract
The plasma membrane of eukaryotic cells is asymmetric with respect to its phospholipid composition. Analysis of the lipid composition of the outer leaflet is important for understanding cell membrane biology in health and disease. Here, a method based on cyclodextrin-mediated lipid exchange to characterize the phospholipids in the outer leaflet of red blood cells (RBCs) is reported. Methyl-α-cyclodextrin, loaded with exogenous lipids, was used to extract phospholipids from the membrane outer leaflet, while delivering lipids to the cell to maintain cell membrane integrity. Thin layer chromatography and lipidomics demonstrated that the extracted lipids were from the membrane outer leaflet. Phosphatidylcholines (PC) and sphingomyelins (SM) were the most abundant phospholipids in the RBCs outer leaflet with PC 34:1 and SM 34:1 being the most abundant species. Fluorescence quenching confirmed the delivery of exogenous lipids to the cell outer leaflet. The developed lipid exchange method was then used to remove phosphatidylserine, a phagocyte recognition marker, from the outer leaflet of senescent RBCs. Senescent RBCs with reconstituted membranes were phagocytosed in significantly lower amounts compared to control cells, demonstrating the efficiency of the lipid exchange process and its application in modifying cell–cell interactions.
Highlights
The plasma membrane of eukaryotic cells is asymmetric with respect to its phospholipid composition
While the phospholipid asymmetry of the red blood cells (RBCs) plasma membrane has been known for more than 40 years, methods to analyze the outer leaflet lipid composition still depend on enzymatic degradation of the outer leaflet lipids
We have developed a method, based on cyclodextrin-mediated lipid exchange to characterize the outer leaflet phospholipids of RBCs
Summary
The plasma membrane of eukaryotic cells is asymmetric with respect to its phospholipid composition. Sphingomyelins (SM) and phosphatidylcholines (PC) primarily reside in the membrane outer leaflet (facing the outside environment) while the inner leaflet (facing the inside of the cell) is known to mainly consist of aminophospholipids, phosphatidylserines (PS) and phosphatidylethanolamines (PE)[2,3,4,5] This phospholipid asymmetry has important implications for cell function and is known to affect a variety of processes, including apoptosis, cell division, and blood c oagulation[6,7,8,9]. Analysis of the outer leaflet lipid composition still depends on the enzymatic degradation of phospholipids This method was effectively used, in combination with freeze-etch electron microscopy, in the study of Verkleij et al to characterize the asymmetric distribution of phospholipids in the membrane of human RBCs, revealing significant levels of SM and PC in the outer leaflet and PE and PS in the inner leaflet[2]. It has been shown that the ability of phospholipases to hydrolyze membrane lipids depends on the membrane surface tension with enzymes exerting their hydrolyzing activity only at certain membrane surface tension v alues[26,27,28]
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