Effect of Triton X-100 on Raft-Like Lipid Mixtures: Phase Separation and Selective Solubilization

Abstract

Under certain conditions, biological membranes exhibit resistance to solubilization, even at high detergent concentration. These insoluble fragments are enriched in sphingolipids, cholesterol, and certain proteins having a preference for more organized environments. Here we investigated the effect of detergent Triton X-100 (TX-100) on raft-like lipid mixtures composed of POPC (palmitoyl oleoyl phosphatidylcholine, an unsaturated lipid), SM (sphingomyelin, a saturated lipid), and cholesterol, focusing on the detergent-induced phase separation at subsolubilizing concentration and the extent of solubilization at higher concentration. Giant unilamellar vesicles (GUVs) of POPC/SM/chol containing a fluorescent probe known to prefer the liquid-disordered phase were prepared and observed with fluorescence microscopy. A phase diagram constructed in the presence and absence of 0.1 mM TX-100 showed that the detergent induces macroscopic liquid-ordered/liquid-disordered (Lo/Ld) phase separation over a wide range of membrane composition, indicating that TX-100 has the ability to rearrange the lateral heterogeneity of the lipid mixture. The extent of solubilization of the POPC/SM/chol GUVs was quantified by measuring the vesicle size before and after the injection of a high concentration of TX-100. In parallel, the solubilization extent of large unilamellar vesicles (LUVs) was assessed by turbidity measurements. The extent of solubilization decreases significantly as the fractions of SM and cholesterol in the mixture increase. The origin of the detergent resistance is the low partitioning of TX-100 in cholesterol-rich membranes, especially in SM-containing ones, as evidenced by isothermal titration calorimetry experiments on LUVs. Our results provide a guide to future research on the effects of TX-100 on raft-like lipid mixtures.

Biophysical approaches in the study of biomembrane solubilization: quantitative assessment and the role of lateral inhomogeneity

“…6b. The results are similar to values obtained from isothermal titration calorimetry experiments (Tsamaloukas et al 2006;Caritá et al 2017). It is clear from the data that cholesterol significantly reduces the partition of TX-100 into the bilayer, and only a small amount of TX-100 is able to insert into the Lo phase.…”

“…To explore the raft-like ternary (Caritá et al 2017). To that purpose, three fixed concentrations of cholesterol were chosen (20, 30 and 40 mol%) and the proportions of POPC and SM in the mixture were varied at intervals of 10 mol%.…”

Lipid signaling to membrane proteins: From second messengers to membrane domains and adapter-free endocytosis

“…2 D illustrates the induction of MEND by Triton X-100 (TX-100, 120 µM). Like TX-100, ionic detergents such as SDS can also displace cholesterol laterally ( Caritá et al, 2017 ), and SDS induces MEND at concentrations 10-fold lower than its critical micelle concentration. These MEND responses, illustrated in Fig.…”

Controlling the Kinetics of an Enzymatic Reaction through Enzyme or Substrate Confinement into Lipid Mesophases with Tunable Structural Parameters

“…To this purpose, a set of “control” mesophases, after the first release step, was disassembled by immersion in a 24 mg/mL solution of Triton X 100. The non-ionic surfactant, at relatively high concentration [ 43 , 44 ] is able to disrupt the lipid bilayer to yield mixed micelles, with simultaneous quantitative release of AP originally contained in the liquid crystalline mesophase. The AP amount was measured in terms of enzymatic activity.…”