Comparing Phase Transition Temperatures of Giant Plasmid Membrane Vesicles with Different Preparation Methods

Abstract

Giant plasma membrane vesicles (GPMVs) are traditionally made by incubating cells with a buffer containing a reducing agent and a low concentration of formaldehyde. When GPMVs are prepared in this way from RBL-2H3 cells, the vesicles appear uniform around physiological temperatures, but phase separate into coexisting liquid-ordered and liquid-disordered phases at lower temperatures. When the reducing agent dithiothreitol (DTT) is used, the GPMVs typically phase separate in the range 15°C - 20°C, where transition temperature is defined as the temperature where half of the vesicles produced by a cell population contain coexisting liquid phases. Significantly lower transition temperatures (typically ∼0C) are found when DTT is replaced with either glutathione or N-ethylmaleimide (NEM), as has been observed previously for the case of NEM (Levental et al. 2010). GPMVs were also prepared using a method that does not utilize either a reducing agent or formaldehyde, but instead uses a hypertonic chloride salt solution that results in the secretion of vesicles similar in size to those of the reducing agent methods (Del Piccolo et al. 2012). GPMVs made in this way have very low transition temperatures, typically <0C. In addition to lower transition temperatures, GPMVs prepared using NEM, glutathione, or through osmotic stress all contain a slightly increased surface fraction of liquid-ordered phase at low temperatures. These results are consistent with the previous conclusion that DTT induces biochemical changes in inner leaflet proteins that result in elevated transition temperatures and a reduced protein partitioning with liquid-ordered phase lipids (Levental et al. 2010).