MαCD-based plasma membrane outer leaflet lipid exchange in mammalian cells to study insulin receptor activity.

Signaling receptors on the plasma membrane, such as insulin receptor, can have their activity modulated to some extent by their surrounding lipids. Studying the contribution of membrane lipid properties such as presence of ordered lipid domains or bilayer thickness on the activity of receptors has been a challenging objective in living cells. Using methyl-alpha cyclodextrin-mediated lipid exchange, we are able to alter the lipids of the outer leaflet plasma membrane of mammalian cells to investigate the effect of the properties of the exchanged lipid upon receptor function in live cells.

Lipid-driven interleaflet coupling of plasma membrane order regulates FcεRI signaling in mast cells.

Interleaflet coupling-the influence of one leaflet on the properties of the opposing leaflet-is a fundamental plasma membrane organizational principle. This coupling is proposed to participate in maintaining steady-state biophysical properties of the plasma membrane, which in turn regulates some transmembrane signaling processes. A prominent example is antigen (Ag) stimulation of signaling by clustering transmembrane receptors for immunoglobulin E (IgE), FcεRI.

Loss of plasma membrane lipid asymmetry can induce ordered domain (raft) formation.

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.

Using cyclodextrin-induced lipid substitution to study membrane lipid and ordered membrane domain (raft) function in cells.

Methods for efficient cyclodextrin-induced lipid exchange have been developed in our lab. These make it possible to almost completely replace the lipids in the outer leaflet of artificial membranes or the plasma membranes of living cells with exogenous lipids. Lipid replacement/substitution allows detailed studies of how lipid composition and asymmetry influence the structure and function of membrane domains and membrane proteins.

Phospholipid exchange shows insulin receptor activity is supported by both the propensity to form wide bilayers and ordered raft domains.

Insulin receptor (IR) is a membrane tyrosine kinase that mediates the response of cells to insulin. IR activity has been shown to be modulated by changes in plasma membrane lipid composition, but the properties and structural determinants of lipids mediating IR activity are poorly understood. Here, using efficient methyl-alpha-cyclodextrin mediated lipid exchange, we studied the effect of altering plasma membrane outer leaflet phospholipid composition upon the activity of IR in mammalian cells.

Replacing plasma membrane outer leaflet lipids with exogenous lipid without damaging membrane integrity.

We recently introduced a MαCD-based method to efficiently replace virtually the entire population of plasma membrane outer leaflet phospholipids and sphingolipids of cultured mammalian cells with exogenous lipids (Li et al, (2016) Proc. Natl. Acad.

Sterol structure dependence of insulin receptor and insulin-like growth factor 1 receptor activation.

The plasma membrane is a dynamic environment with a complex composition of lipids, proteins, and cholesterol. Areas enriched in cholesterol and sphingolipids are believed to form lipid rafts, domains of highly ordered lipids. The unique physical properties of these domains have been proposed to influence many cellular processes.