Raft Composition at Physiological Temperature and pH in the Absence of Detergents

Abstract

Biological rafts were identified and isolated at 37 degrees C and neutral pH. The strategy for isolating rafts utilized membrane tension to generate large domains. For lipid compositions that led only to microscropically unresolvable rafts in lipid bilayers, membrane tension led to the appearance of large, observable rafts. The large rafts converted back to small ones when tension was relieved. Thus, tension reversibly controls raft enlargement. For cells, application of membrane tension resulted in several types of large domains; one class of the domains was identified as rafts. Tension was generated in several ways, and all yielded raft fractions that had essentially the same composition, validating the principle of tension as a means to merge small rafts into large rafts. It was demonstrated that sphingomyelin-rich vesicles do not rise during centrifugation in sucrose gradients because they resist lysis, necessitating that, contrary to current experimental practice, membrane material be placed toward the top of a gradient for raft fractionation. Isolated raft fractions were enriched in a GPI-linked protein, alkaline phosphatase, and were poor in Na(+)-K(+) ATPase. Sphingomyelin and gangliosides were concentrated in rafts, the expected lipid raft composition. Cholesterol, however, was distributed equally between raft and nonraft fractions, contrary to the conventional view.

Plasma membranes are poised for activation of raft phase coalescence at physiological temperature

“…Although tension-based forces cannot be excluded from the current study, our unclustered PMS did not show optically resolvable domains at either 25°C or 37°C, a phenotype in keeping with the observations of Baumgart et al (18). Also, the size order of the segregation that we observe after clustering is beyond the patchy domain formation that Ayuan and Cohen (30) report, but rather a cohesive phase separation. More importantly, this GM1 phase separation was cholesteroldependent.…”

“…These findings highlight the inherent capability of the plasma membrane to phase separate while stressing that in the living cell this capacity is more strictly controlled. Increases in plasma membrane tension during cell swelling at 37°C can induce raft and nonraft components to segregate into domains comparable with the phenotypes seen with antibody cross-linking (30). Although tension-based forces cannot be excluded from the current study, our unclustered PMS did not show optically resolvable domains at either 25°C or 37°C, a phenotype in keeping with the observations of Baumgart et al (18).…”

Membrane Organization and Lipid Rafts

“…Altogether, these and other studies (Ayuyan and Cohen 2008) show that plasma membrane composition is poised for selective coalescence at physiological temperature. They highlight the inherent capability of the PM to phase separate while stressing that in the living cell this capacity is strictly controlled by the lipid and protein composition of the membrane as well as by the fact that cell membranes are not at equilibrium, being continuously perturbed by exchange events and membrane trafficking.…”

Undulations Drive Domain Registration from the Two Membrane Leaflets

“…This favors the merger of domains, i.e., minimization of boundary energy is achieved via the shortening of the boundary length (24,25). Increased domain size, in turn, is likely to augment the contribution of thermal fluctuations to domain coupling energy.…”