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

Abstract

Cell membranes are not randomly organized, but rather are populated by fluctuating nanoassemblies of increased translational order termed lipid rafts. This lateral heterogeneity can be biophysically extended because cooling formaldehyde-isolated plasma membrane preparations results in separation into phases similar to the liquid-ordered (Lo) and liquid-disordered (Ld) states seen in model membrane systems [Baumgart T, et al. (2007) Proc Natl Acad Sci USA 104:3165-3170]. In this work we demonstrate that raft clustering, i.e., amplifying underlying raft-based connectivity to a larger scale, makes an analogous capacity accessible at 37 degrees C. In plasma membranes at this temperature, cholera toxin-mediated cross-linking of the raft ganglioside GM1 induced the sterol-dependent emergence of a slower diffusing micrometer-scale phase that was enriched in cholesterol and selectively reorganized the lateral distribution of membrane proteins. Although parallels can be drawn, we argue that this raft coalescence in a complex biological matrix cannot be explained by only those interactions that define Lo formation in model membranes. Under this light, our induction of raft-phase separation suggests that plasma membrane composition is poised for selective and functional raft clustering at physiologically relevant temperature.

Fig. 1.

PMS costained with Bodipy and TRITC-phalloidin. PMS (arrow A) retain attachment to remaining cell body (arrow B) (Lower) but could be imaged at a higher hemisphere where this attachment is no longer in focus (Upper). A431 cells were incubated with Bodipy and TRITC-phalloidin before PMS formation. TRITC-phalloidin only colocalized with the remaining cell body, indicating that actin cytoskeleton was separated from plasma membrane material during formation of PMS. (Scale bars, 5 μm.)

Fig. 2.

Induction of phase separation in PMS 37°C. Fluorescent raft protein analogues representing single spanning transmembrane (LAT), multispanning transmembrane (VIP17), and exo/cytoplasmic lipid-anchored (GL-GPI and Pal-Pal linkages, respectively) membrane-targeting motifs (A) did not show large-scale separation of phase at this temperature (B) or at 25°C (data not shown). (C) However, after cross-linking by fluorescent CTB (Alexa Fluor 488 or Alexa Fluor 647 conjugates), the lateral distribution of these proteins was reorganized into a micrometer-scaled emergent GM1 phase. (Scale bars, 5 μm.)

Fig. 3.

The emergent GM1 phase excludes the transferrin receptor. After clustering by CTB-Alexa Fluor 488, transferrin-Alexa Fluor 647 was added to PMS for 1 h at 37°C. Resultant LSM imaging clearly showed that the membrane-bound transferrin was not recruited to the coalesced GM1 phase, indicating that this lateral reorganization of membrane structure was selective in composition. (Scale bars, 5 μm.)

Fig. 4.

Cholesterol dependence of the GM1 phase. (A) After cross-linking by CTB at 37°C, PMS were stained for nonesterified cholesterol via incubation with filipin III. The emergent GM1 phase was colabeled, indicating an enrichment of cholesterol. (B) To assess the sterol dependence of the process, PMS were treated with methyl-β-cyclodextrin (MβCD) to extract cholesterol and then clustered by CTB-Alexa Fluor 488. Compared with the control nonextracted condition, MβCD-treated PMS failed to exhibit global/uninterrupted coalescence of a GM1 phase, rather displaying a much smaller and patchy domain distribution. This indicates that cholesterol was important in establishing the larger-scale connectivity between cross-linked GM1 (CTB clusters up to pentameric state) as seen by micromolar-scale phase separation in the control condition. (Scale bars, 5 μm.)

Fig. 5.

SFCS measurements on PMS. (a) LSM image of plasma membrane sphere with induced phase separation by CTB cross-linking and schematic of scan paths for two-focus scanning FCS. (b and c) Auto- (○, ◇) and cross-correlation curves (▾) measured in the bright (GM1 phase) and dark phase (surrounding membrane), respectively, and global fit to elliptical Gaussian model (24). (d) Diffusion coefficients measured with two-focus scanning FCS. Error bars denote the geometric mean and geometric 1σ confidence intervals of the measured diffusion coefficients. D_dark = 1.3_−0.2^+0.4 μm²/s, D_bright = 0.13_−0.06^+0.11 μm²/s.

Fig. 6.

Model for emergence of a GM1 phase. Micrometer-scale (global) coalescence of CTB-cross-linked GM1 in PMS is only possible if there is preexisting (raft-based) connectivity between the ganglioside and other select elements of the plasma membrane (Right). Without these interactions, CTB cross-linked-GM1 (CTB only clusters up to pentameric state) could not be bridged into a continuous phase (Left). Our results indicate that cholesterol is an important component of these bridging interactions and that retarded diffusion in the GM1 phase is most consistent with condensation and therefore amplification of these forces.