Triton X-100 promotes a cholesterol-dependent condensation of the plasma membrane

Abstract

The molecular components of membrane rafts are frequently defined by their biochemical partitioning into detergent-resistant membranes. In the present study, we used a combination of epifluorescence and two-photon microscopy to visualize and quantify whether this insolubility in detergent reflects a pre-existing organization of the PM (plasma membrane). We found that the treatment of cells with cold TX (Triton X-100) promotes a profound remodelling of the PM, including a rapid rearrangement of the glycosphingolipid GM1 and cholesterol into newly formed structures, only partial solubilization of fluid domains and the formation of condensed domains that cover 51% of the remaining membrane. TX does not appear to induce the coalescence of pre-existing domains; instead, the domains that remain after TX treatment seem to be newly formed with a higher degree of condensation than those observed in native membranes. However, when cholesterol was complexed physically by treatment with a second detergent, such as saponin, cholesterol did not separate into the newly formed structures, condensation of the domains was unaltered, and the relative area corresponding to ordered domains increased to occupy 62% of the remaining membrane. Our results suggest that detergent can be used to enrich ordered domains for biochemical analysis, but that TX treatment alone substantially alters the lateral organization of the PM.

An X‐ray diffraction study of model membrane raft structures

“…Current thinking favours the, so‐called, raft hypothesis, which postulates that the signalling elements are segregated and assembled in ordered lipid domains in the membrane [1–6]. This membrane heterogeneity is rationalized on the basis that, for the efficient operation of a signalling system, the protein components must be closely associated and organized in such a way that structural changes accompanying the generation of a signal are coupled to the transducing elements responsible for execution of the response [7].…”

“…The conventional protocol, irrespective of the type of membrane, has been to recover a membrane fraction that survives dissolution by Triton X‐100 treatment at 4 °C. The integrity of this method has recently been challenged [7] and alternative methods based on a milder detergent treatment that is more compatible with physiological conditions have been developed [9]. The resulting membrane raft fraction retains properties consistent with an arrangement of constituents expected of its biological progenitor.…”

Nerve Growth Factor Signaling from Membrane Microdomains to the Nucleus: Differential Regulation by Caveolins

“…Clearly, the very definition of growth factor binding-initiated translocation into, or out of lipid rafts, is dependent on the extraction protocol. While we, and others [11,12,14,17,80] used a detergent-independent method, either on post-mortem tissue or in cultured cells [13,77,78,79], used a Triton X100-dependent method, which has been shown to induce the formation of non-physiological structures [81]. This discrepancy notwithstanding, the common conclusion of these observations is that there is ligand-induced movement of the receptors between different membrane compartments.…”

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

“…As we have previously reported, TX-100 and Brij detergents disrupt the erythrocyte membrane in diverse ways (Casadei et al 2014b). TX-100 is known to preferentially solubilize phospholipids from the inner leaflet of the cell membrane (Ingelmo-Torres et al 2009;Koumanov et al 2005;Lichtenberg et al 2005) while Brij detergents are not as selective as TX-100 (Schuck et al 2003). Rodi et al (2014) have also demonstrated that TX-100 incorporates and solubilize lipid components from the erythrocyte membranes in a different way compared with CHAPS (a zwitterionic detergent).…”