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.

Platelet α-granule cargo release is fundamental to both hemostasis and thrombosis. Granule matrix hydration is a key regulated step in this process, yet its mechanism is poorly understood. In endothelial cells, there is evidence for 2 modes of cargo release: a jack-in-the-box mechanism of hydration-dependent protein phase transitions and an actin-driven granule constriction/extrusion mechanism. The third alternative considered is a prefusion, channel-mediated granule swelling, analogous to the membrane “ballooning” seen in procoagulant platelets. Using thrombin-stimulated platelets from a set of secretion-deficient, soluble N-ethylmaleimide factor attachment protein receptor (SNARE) mutant mice and various ultrastructural approaches, we tested predictions of these mechanisms to distinguish which best explains the α-granule release process. We found that the granule decondensation/hydration required for cargo expulsion was (1) blocked in fusion-protein-deficient platelets; (2) characterized by a fusion-dependent transition in granule size in contrast to a preswollen intermediate; (3) determined spatially with α-granules located close to the plasma membrane (PM) decondensing more readily; (4) propagated from the site of granule fusion; and (5) traced, in 3-dimensional space, to individual granule fusion events at the PM or less commonly at the canalicular system. In sum, the properties of α-granule decondensation/matrix hydration strongly indicate that α-granule cargo expulsion is likely by a jack-in-the-box mechanism rather than by gradual channel-regulated water influx or by a granule-constriction mechanism. These experiments, in providing a structural and mechanistic basis for cargo expulsion, should be informative in understanding the α-granule release reaction in the context of hemostasis and thrombosis.

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