Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles

Abstract

The membrane raft hypothesis postulates the existence of lipid bilayer membrane heterogeneities, or domains, supposed to be important for cellular function, including lateral sorting, signaling, and trafficking. Characterization of membrane lipid heterogeneities in live cells has been challenging in part because inhomogeneity has not usually been definable by optical microscopy. Model membrane systems, including giant unilamellar vesicles, allow optical fluorescence discrimination of coexisting lipid phase types, but thus far have focused on coexisting optically resolvable fluid phases in simple lipid mixtures. Here we demonstrate that giant plasma membrane vesicles (GPMVs) or blebs formed from the plasma membranes of cultured mammalian cells can also segregate into micrometer-scale fluid phase domains. Phase segregation temperatures are widely spread, with the vast majority of GPMVs found to form optically resolvable domains only at temperatures below Ϸ25°C. At 37°C, these GPMV membranes are almost exclusively optically homogenous. At room temperature, we find diagnostic lipid phase fluorophore partitioning preferences in GPMVs analogous to the partitioning behavior now established in model membrane systems with liquid-ordered and liquid-disordered fluid phase coexistence. We image these GPMVs for direct visual characterization of protein partitioning between coexisting liquidordered-like and liquid-disordered-like membrane phases in the absence of detergent perturbation. For example, we find that the transmembrane IgE receptor FcRI preferentially segregates into liquid-disordered-like phases, and we report the partitioning of additional well known membrane associated proteins. Thus, GPMVs now provide an effective approach to characterize biological membrane heterogeneities.liquid-disordered ͉ liquid-ordered ͉ membrane domains ͉ membrane heterogeneity ͉ rafts

What do diffusion measurements tell us about membrane compartmentalisation? Emergence of the role of interprotein interactions

“…Model membrane experiments have shown that liquidordered microdomains in fluid membranes can exist and that proteins can preferentially segregate into the fluid or the ordered phase domain [25,26]. However, biological membranes are much more complex than synthetic membranes, and the presence of lipid rafts in intact cells still remains to be proved.…”

“…A consensus definition of membrane rafts emerged from the Keystone Symposium on Lipid Rafts and Cell Function (March [23][24][25][26][27][28]2006): "Membrane rafts are small (10-200nm), heterogeneous, highly dynamic, sterol-and sphingolipid-enriched domains that compartmentalise cellular processes. Small rafts can sometimes be stabilised to form larger platforms through protein-protein and protein-lipid interactions" [41].…”

“…Many papers suggested that lipid rafts were involved in various cellular processes such as signal transduction, apoptosis or protein sorting [20,21]. They have also been shown to play a role in HIV or influenza viruses infection processes [22][23][24].Model membrane experiments have shown that liquidordered microdomains in fluid membranes can exist and that proteins can preferentially segregate into the fluid or the ordered phase domain [25,26]. However, biological membranes are much more complex than synthetic membranes, and the presence of lipid rafts in intact cells still remains to be proved.…”

Lipid rafts, cholesterol, and the brain

“…The best studied examples include Ras, influenza hemagglutinin, and components of the high affinity IgE receptor signaling pathway (Wilson et al 2002;Parton and Hancock 2004;Wilson et al 2004;Hess et al 2005) (Takeda et al 2003). In addition, large-scale lipid phase separation can be observed in plasma membrane blebs (Baumgart et al 2007). Although these approaches have provided valuable information about lipid rafts and related membrane microdomains, their use is limited to fixed or perturbed cells.…”

“…For example, some proteins expected to associate with L o domains on the basis of their association with DRMs in cells show little partitioning when studied in liposomes (Shogomori et al 2005). With the recent development of methods to examine lipid domain segregation in plasma membrane blebs, it should be possible to begin to examine this model in more detail (Baumgart et al 2007;Sengupta et al 2007). …”