Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles

TLDR

The membrane raft hypothesis proposes that lipid bilayer heterogeneities, or domains, play key roles in cellular functions such as sorting, signaling, and trafficking, yet their characterization in live cells is difficult, so model systems like giant unilamellar vesicles have been used to optically distinguish coexisting fluid phases, mainly in simple lipid mixtures. The authors directly image giant plasma membrane vesicles to observe protein partitioning between liquid‑ordered‑like and liquid‑disordered‑like phases without detergent disruption. Giant plasma membrane vesicles form micrometer‑scale fluid phase domains that segregate at temperatures below ~25 °C, display lipid partitioning patterns analogous to model membranes, and allow observation of protein segregation—e.g., the IgE receptor FcepsilonRI preferentially localizes to liquid‑disordered‑like phases—demonstrating that GPMVs are a powerful tool for studying biological membrane heterogeneity.

Abstract

The membrane raft hypothesis postulates the existence of lipid bilayer membrane heterogeneities, or domains, supposed to be important for cellular function, including lateral sorting, signaling, and trafficking. Characterization of membrane lipid heterogeneities in live cells has been challenging in part because inhomogeneity has not usually been definable by optical microscopy. Model membrane systems, including giant unilamellar vesicles, allow optical fluorescence discrimination of coexisting lipid phase types, but thus far have focused on coexisting optically resolvable fluid phases in simple lipid mixtures. Here we demonstrate that giant plasma membrane vesicles (GPMVs) or blebs formed from the plasma membranes of cultured mammalian cells can also segregate into micrometer-scale fluid phase domains. Phase segregation temperatures are widely spread, with the vast majority of GPMVs found to form optically resolvable domains only at temperatures below approximately 25 degrees C. At 37 degrees C, these GPMV membranes are almost exclusively optically homogenous. At room temperature, we find diagnostic lipid phase fluorophore partitioning preferences in GPMVs analogous to the partitioning behavior now established in model membrane systems with liquid-ordered and liquid-disordered fluid phase coexistence. We image these GPMVs for direct visual characterization of protein partitioning between coexisting liquid-ordered-like and liquid-disordered-like membrane phases in the absence of detergent perturbation. For example, we find that the transmembrane IgE receptor FcepsilonRI preferentially segregates into liquid-disordered-like phases, and we report the partitioning of additional well known membrane associated proteins. Thus, GPMVs now provide an effective approach to characterize biological membrane heterogeneities.