Lipid Exchange Assay in Living Cells

We describe a method for using cyclodextrin to mediate exchange between lipids of the plasma membrane with exogenous lipids. This technique can be paired with experiments studying transmembrane proteins, which behave differently in lipid raft-like environments than they do in non-raft-like environments.

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane's propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.

The plasma membrane is composed of a lipid bilayer enriched with various membrane proteins, including transmembrane receptors and ion channels. Lipid domains within the membrane have been elucidated through detergent-soluble and insoluble regions identified in detergent-resistant membrane (DRM) fractionation experiments¹. The insoluble fractions were characterized by being enriched in cholesterol, tightly packed sphingomyelins and saturated phospholipids, exhibiting higher melting points, in contrast to the soluble fractions that predominantly consist of lower melting temperature and loosely packed unsaturated phospholipids. The tightly pa....

1. Preparation of Methyl-α-CD solution

1. Add 20 mL of phosphate-buffered saline (PBS) to ~10 g of MαCD powder in a glass bottle. Incubate in a warm water bath (45 °C) to dissolve, stirring occasionally until well dissolved.
   NOTE: The solution may still be cloudy due to insoluble CD species.
2. Pass solution through a 0.22 µm syringe filter. The solution will become clear.
3. Use a refractometer to determine the exact concentration of MαCD.
   1. Place 10 µL of sample on the sample area of a refractometer, illuminate it using a white incandescent bulb, and record the solution's refr....

To demonstrate the observable change in cellular lipid composition after the exchange, we performed HP-TLC on CHO IR cells following brain SM (bSM) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exchange (Figure 1). In cases where sphingomyelins like bSM are being used for exchange, an increase in the SM band intensity is apparent, along with a decrease in PC band intensity relative to the untreated control. Conversely, when exchanging phosphatidylcholines like DOPC, the PC band becomes.......

Since the conceptualization of the existence of lipid rafts in the cell membrane there have been numerous attempts to visualize them in cells and study lipid and receptor association. Experiments involving microscopy¹¹ in cells used fluorescently tagged biomarkers, usually, proteins and lipids known to associate with rafts, to visually study the localization of ordered lipid domains in the cell¹². However, the cell membrane is full of folds^13,

The authors declare no conflicts of interest.

Funding was provided by NIH grant GM 122493. CHO IR cells were a kind gift from Dr Jonathan Whittaker (Case Western Reserve University).