Live Cell Plasma Membranes Do Not Exhibit a Miscibility Phase Transition over a Wide Range of Temperatures

Abstract

Lipid:cholesterol mixtures derived from cell membranes, as well as their synthetic reconstitutions, exhibit well defined miscibility phase transitions and critical phenomena near physiological temperatures. This suggests that lipid:cholesterol-mediated phase separation plays a role in the organization of live cell membranes. However, macroscopic lipid phase separation is not generally observed in cell membranes and the degree to which properties of isolated lipid mixtures are preserved in the cell membrane remain unknown. A fundamental property of phase transitions is that the variation of tagged particle diffusion with temperature exhibits an abrupt change as the system passes through the transition, even when the two phases are distributed in a nanometer-scale emulsion. We support this using a variety of Monte-Carlo and atomistic simulations on model lipid membrane systems. However, temperature dependent fluorescence correlation spectroscopy of labeled lipids and membrane-anchored proteins in live cell membranes show a consistently smooth increase of the diffusion coefficient as a function of temperature. We find no evidence of a discrete miscibility phase transition throughout a wide range of temperatures: 14 -37 °C. This contrasts the behavior of giant plasma membrane vesicles (GPMVs) blebbed from the same cells, which do exhibit phase transitions and macroscopic phase separation. Fluorescence lifetime analysis of a DiI probe in both cases reveals a significant environmental difference between the live cell and the GPMV. Taken together, these data suggest the live cell membrane may avoid the miscibility phase transition inherent to its lipid constituents by actively regulating physical paramters, such as tension, in the membrane.

Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins

“…2C and F). This suggests that the plasma membrane domains containing these proteins do not undergo a miscibility phase transition across the temperature range we studied, in agreement with a recent FCS study in live cells [15]. …”

The Continuing Mystery of Lipid Rafts

“…Similarly, numerous reports suggest that raft and non-raft membrane peptides exhibit qualitatively similar confinement behaviors 130, 137, 138 . Another study showed that diffusion varied smoothly with temperature for a range of different probe molecules, concluding that there was no evidence for an in situ phase transition in live cells 139 . Yet another found that probe localization and diffusion was not affected by the presence of macroscopic raft-like domains generated by binding raft proteins to a patterned surface 140 .…”

Emerging role of chemoprotective agents in the dynamic shaping of plasma membrane organization

“…To specifically further assess how exogenous treatments affect the plasma membrane, we generated giant plasma membrane vesicles (GPMVs) which retain most of the full diversity of native membrane components, but lack cytoskeletal attachment[161]. This type of reductionist approach allows us to probe specific questions regarding the interaction between diet-derived bioactives and lipid membranes without the complication of compensatory mechanisms imparted by the live cell such as membrane tension[162] and cytoskeletal remodeling[108]. GPMVs were isolated from YAMC-HRasG12V cells pre-labeled with Di4 and incubated with varying doses of (+)-catechin and procyanidin B2 (Figure 2C&D).…”