The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure

Abstract

stability of membranes, and membrane shape ( 4-10 ). Therefore, biologically realistic asymmetric vesicles could be widely useful in model membrane studies. However, artifi cial membranes produced in the laboratory usually have a symmetric distribution of lipids in the inner and outer leafl ets of the bilayer. Asymmetric artifi cial membranes have been diffi cult to produce, although some important progress has been made ( 11-16 ). We recently developed a robust method in which methyl-␤ -cyclodextrin (M ␤ CD)-induced lipid exchange is used to prepare model membrane vesicles with lipid asymmetry, and we have applied the method to produce asymmetric small, large, and giant unilamellar vesicles (17)(18)(19).In the present study, we extended the range of this method by using it to construct asymmetric vesicles containing SM in the outer leafl et and various species of PCs in the inner leafl et. PC acyl chain structure was systematically varied, and how this variation affects the ability to form asymmetric membranes was assayed. We found that whether the resulting vesicles were asymmetric [SM outside/ PC inside (SMo/PCi)] depended upon PC acyl chain structure. Despite this limitation, asymmetric vesicles with a wide range of acyl chain structures could be produced. This is an important advance that should allow new applications of asymmetric vesicles. These studies also allowed analysis of the effect of lipid type upon membrane structure and behavior. We investigated the origin of the acyl chain dependence of asymmetry by measuring the transverse diffusion rate of the various PCs used. There was a correlation between asymmetry and transverse diffusion. Asymmetric vesicles could be prepared only when PCs with slow transverse diffusion were used. It was found that the presence of two polyunsaturated acyl chains or overly short acyl chains prevented the maintenance of asymmetry. This Abstract Lipid asymmetry, the difference in inner and outer leafl et lipid composition, is an important feature of biomembranes. By utilizing our recently developed M ␤ CDcatalyzed exchange method, the effect of lipid acyl chain structure upon the ability to form asymmetric membranes was investigated. Using this approach, SM was effi ciently introduced into the outer leafl et of vesicles containing various phosphatidylcholines (PC), but whether the resulting vesicles were asymmetric (SM outside/PC inside) depended upon PC acyl chain structure. Vesicles exhibited asymmetry using PC with two monounsaturated chains of >14 carbons; PC with one saturated and one unsaturated chain; and PC with phytanoyl chains. Vesicles were most weakly asymmetric using PC with two 14 carbon monounsaturated chains or with two polyunsaturated chains. To defi ne the origin of this behavior, transverse diffusion (fl ip-fl op) of lipids in vesicles containing various PCs was compared. A correlation between asymmetry and transverse diffusion was observed, with slower transverse diffusion in vesicles containing PCs that supported lipid asymmetry. Thus, asymmetric vesic...

The dependence of lipid asymmetry upon polar headgroup structure

“…Samples were scanned stepwise with 4°C steps, between 16°C and by M ␤ CD-induced exchange have great potential for various applications because the procedure is not unduly complicated, they can be prepared with a variety of lipid compositions, and they can be used to create asymmetric vesicles with various sizes as well as reconstituted vesicles with membrane-inserted peptides (43)(44)(45) and proteins. We recently extended this method by generating asymmetric vesicles containing SMs as outer leafl et lipids and a large variety of PCs with different acyl chain structures as inner leafl et lipids ( 46 ). It was found that M ␤ CD-induced lipid exchange was always effi cient.…”

“…The percent of SM in exchange vesicles was calculated from HP-TLC as described previously ( 46 ). The samples for HP-TLC were from the exchange SUV-containing fractions from the Sepharose CL-2B column.…”

“…Exchange (asymmetric) small unilamellar vesicles (SUVs) were prepared as described previously ( 46 ). First, donor multilamellar vesicles (MLVs), the population from which SM was to be exchanged with the outer leafl et of the acceptor SUVs, were prepared .…”

“…The percent of SM by TLC was evaluated after calculating the intensity of SM bands. The lipids in the vesicles were quantifi ed by measuring TLC spot intensity versus a standard curve for each lipid generated on the same TLC plate as described previously ( 46 ).…”

Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids

“…We previously developed use of CDs to carry out efficient outer leaflet lipid exchange in model membrane vesicles, defining conditions such that the entire outer lipid monolayer (leaflet) of a membrane lipid bilayer can be replaced with exogenous lipids in situ, using MβCD or hydroxypropyl (HP)αCD without disturbing the lipid composition of the inner leaflet (12)(13)(14)(15)(16)(17)(18). This allowed preparation of artificial vesicles that mimic natural membranes by having both natural lipid composition and asymmetry.…”