Are You In or Out?: Biological Rafts and Biophysical Phases.
Hammond AT, Baumgart T, Smith AK, Holowka D, Baird B, Feigenson GW
The lipid raft hypothesis (Simons'97; Brown'98) proposes that some membrane proteins are physically segregated by their preferential partitioning into regions of different membrane order (perhaps different phase) within a continuous cellular membrane. This mechanism of protein distribution could play a fundamental role in processes including membrane trafficking, signaling, and protein sorting. However, the raft hypothesis does not predict the effects that protein clustering or crosslinking may have on the function or formation of lipid rafts. Here we show that crosslinking a minor raft component can trigger large-scale changes in the membrane’s lateral organization. When cholera toxin B (CTB) binds and crosslinks its membrane receptor (the ganglioside GM 1), it can trigger cholesterol-containing model membranes to phase separate into coexisting liquid ordered (Lo) and liquid disordered (Ld) domains (Figure 1). This lipid separation causes dramatic redistribution of a transmembrane peptide. These results suggest a mechanism by which critical membrane proteins can be significantly rearranged by altering the underlying lipid order through crosslinking or clustering a small number of proteins or lipids.
The phase behavior of bilayer lipid membranes plays a major role in the field of model membrane research and in modern cell membrane biology. Model membrane research has thus far primarily focused much effort on pure lipid membranes. However, controversial hypotheses regarding the phase behavior of cell membranes call for the analysis of the phase behavior of protein-containing membranes. The raft hypothesis assumes an interplay between lipid membrane phase behavior and lateral protein distribution, which is assumed to be involved in fundamental processes such as receptor signaling, endocytosis, protein sorting, polarization, motility, and several stages in the infectious cycle of many viruses. In processes such as signal transduction (Field'97), vesicle assembly, and viral coat assembly, the lipid environment of membrane proteins changes after they bind an extracellular ligand or intracellular protein. In many cases, this change follows crosslinking or clustering of specific membrane components, and can be inhibited by cholesterol depletion using methyl-betacyclodextrin. Crosslinking is hypothesized to change the way that the crosslinked protein partitions into or out of pre-existing domains of the plasma membrane. In the present study, we demonstrate a quite different possibility: that crosslinking a membrane component can alter the phase state of the lipid bilayer membrane and consequently the partitioning behavior of membrane proteins.
Membrane phases have been studied in mixtures of membrane-forming lipids since the 1960s (Luzzati'62; Dodge'67) , and these early studies were important in developing the fluid mosaic model of cellular membranes (Singer'72) . This model proposes that the membranes of cells are fluid lipid bilayers in which proteins are free to diffuse in the plane of the membrane. Subsequent studies of biological membrane heterogeneity and the role of cholesterol refine this view and define two different fluid phases: liquid disordered (Ld, also called Lalpha) and a more ordered phase, liquid ordered (Lo) (Brown'98; Edidin'03) . The raft hypothesis proposes that some of the observed heterogeneity of biological membranes is caused by the coexistence of both fluid phases, Ld and Lo, and that Lo is the favored environment for a selected subset of proteins.
Sterols, including cholesterol, are required to form Lo membranes (Shimshick'73; Ahmed'97). Some three-component mixtures that include cholesterol can form continuous membranes that have coexisting domains of Lo and Ld on a size scale that is resolvable by light microscopy (Dietrich'01; Veatch'03). Such lipid mixtures have been used as highly simplified models of cellular membranes. We make use of a partial description of the membrane phase behavior of such a three-component mixture (Figure 2). This partial phase diagram allows us to choose lipid compositions that form GUVs with particular experimental phase behaviors (Korlach'99; Feigenson'01).
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