Giant Plasma Membrane Vesicles: An Experimental Tool for Probing the Effects of Drugs and Other Conditions on Membrane Domain Stability

Abstract

Giant plasma membrane vesicles (GPMVs) are isolated directly from living cells and provide an alternative to vesicles constructed of synthetic or purified lipids as an experimental model system for use in a wide range of assays. GPMVs capture much of the compositional protein and lipid complexity of intact cell plasma membranes, are filled with cytoplasm, and are free from contamination with membranes from internal organelles. GPMVs often exhibit a miscibility transition below the growth temperature of their parent cells. GPMVs labeled with a fluorescent protein or lipid analog appear uniform on the micron-scale when imaged above the miscibility transition temperature, and separate into coexisting liquid domains with differing membrane compositions and physical properties below this temperature. The presence of this miscibility transition in isolated GPMVs suggests that a similar phase-like heterogeneity occurs in intact plasma membranes under growth conditions, albeit on smaller length scales. In this context, GPMVs provide a simple and controlled experimental system to explore how drugs and other environmental conditions alter the composition and stability of phase-like domains in intact cell membranes. This chapter describes methods to generate and isolate GPMVs from adherent mammalian cells and to interrogate their miscibility transition temperatures using fluorescence microscopy.

Understanding T cell signaling using membrane reconstitution

“…Given the limitations of purely synthetic systems listed above, supported plasma membrane sheets (PMSs) or giant plasma membrane vesicles may be used as alternative reconstitution platforms to obtain insights complementary to those from purely synthetic systems. Produced by mechanical sheering of cells, PMSs presumably retain the lipid and protein composition of the plasma membrane .…”

The C99 domain of the amyloid precursor protein is a disordered membrane phase-preferring protein

“…However, these approaches are known to have multiple pitfalls (19,41,42,91,92), resulting in lack of conclusive evidence for the raft association of C99. Because methods are still not available to reliably investigate the association of proteins with rafts in living cells we here visualized and quantified the affinity of C99 for raft versus nonraft domains in cell-derived GPMVs (51,52,54,62). While GPMVs do not fully recapitulate all of the features of living cell membranes, they have proven to be a valuable tool to investigate mechanisms that control the affinity of proteins for raft versus non-raft domains, and to our knowledge represent the best currently available model to address this question (49,60,61,(93)(94)(95)(96).…”

“…To further investigate this issue, we here utilize giant plasma membrane vesicles (GPMVs) as a model. Derived from the plasma membrane of live cells, these μm-sized vesicles have served as a useful model system to investigate mechanisms that regulate membrane phase behavior and control the localization of lipids and membraneassociated proteins with raft versus non-raft domains (48)(49)(50)(51)(52)(53)(54). Similar to biomimetic model membranes such as GUVs, GPMVs can undergo fluid-fluid phase separation into two coexisting domains: a raft-like ordered (Lo-like) phase and non-raft disordered (Ld-like) phase.…”

Characterization of Single Protein Dynamics in Cell Plasma Membrane Derived Polymer Cushioned Lipid Bilayers

“…In many cases, it is easier or even more informative to study intrinsic membrane properties (dynamics and organization) in an in vitro system without living cells. [19][20][21][22][23][24][25][26][27][28] Several cell-free membrane platforms have been demonstrated with different degrees of complexity that can mimic the cell membranes to various levels. For example, membrane proteins were reconstituted into model membranes where their dynamics and functions were examined.…”

“…To mimic the complex composition of native cell membranes, giant plasma membrane vesicle (GPMV), a cell-derived vesicle of a diameter of tens of micrometers, is a widely used as a cell-free system for studying various membrane properties, including membrane dynamics, phase separation, and protein behaviors. [24][25]35 The GPMV largely preserves the membrane composition and molecular orientation, making it a unique platform to study membrane proteins in native environments. Unfortunately, the spherical shape of the GPMV is less compatible with optical microscope observation and other surface-based analytical methods.…”