Abstract
Giant plasma membrane vesicle (GPMV) isolated from a flask of RBL-2H3 cells appear uniform at physiological temperatures and contain coexisting liquid-ordered and liquid-disordered phases at low temperatures. While a single GPMV transitions between these two states at a well-defined temperature, there is significant vesicle-to-vesicle heterogeneity in a single preparation of cells, and average transition temperatures can vary significantly between preparations. In this study, we explore how GPMV transition temperatures depend on growth conditions, and find that average transition temperatures are negatively correlated with average cell density over 15°C in transition temperature and nearly three orders of magnitude in average surface density. In addition, average transition temperatures are reduced by close to 10°C when GPMVs are isolated from cells starved of serum overnight, and elevated transition temperatures are restored when serum-starved cells are incubated in serum-containing media for 12h. We also investigated variation in transition temperature of GPMVs isolated from cells synchronized at the G1/S border through a double Thymidine block and find that average transition temperatures are systematically higher in GPMVs produced from G1 or M phase cells than in GPMVs prepared from S or G1 phase cells. Reduced miscibility transition temperatures are also observed in GPMVs prepared from cells treated with TRAIL to induce apoptosis or sphingomyelinase, and in some cases a gel phase is observed at temperatures above the miscibility transition in these vesicles. We conclude that at least some variability in GPMV transition temperature arises from variation in the local density of cells and asynchrony of the cell cycle. It is hypothesized that GPMV transition temperatures are a proxy for the magnitude of lipid-mediated membrane heterogeneity in intact cell plasma membranes at growth temperatures. If so, these results suggest that cells tune their plasma membrane composition in order to control the magnitude of membrane heterogeneity in response to different growth conditions.
Highlights
Giant plasma membrane vesicles (GPMVs) isolated from cortical cytoskeleton are a powerful model system for probing some properties of the cell surface
We have shown that GPMVs isolated from RBL-2H3 cells have miscibility phase transition temperatures that depend on cell cycle position and growth conditions
We observe more subtle but systematic changes in transition temperature from GPMVs isolated from cells that are synchronized in their cell cycle, as well as lower transition temperatures in GPMVs isolated from cells undergoing apoptosis
Summary
Giant plasma membrane vesicles (GPMVs) isolated from cortical cytoskeleton are a powerful model system for probing some properties of the cell surface. Previous studies have demonstrated that cells arrested in G0 or G1 through serum starvation or contact inhibition have altered plasma membrane lipid composition [11,12] with reduced sphingomyelin content and increased diacylglycerol and ceramide levels, both conditions expected to modulate miscibility transition temperatures in purified model membranes. Another source of GPMV transition temperature heterogeneity could arise from cells being unsynchronized within the cell cycle, since there are well documented changes in lipid composition at different cell cycle positions [12,13,14,15]. The vast majority of past work characterizing plasma membrane lipids within the cell cycle has focused on the important roles of lipids as second messengers, a few studies have noted changes in the mobility of plasma membrane lipids and proteins as a function of cell cycle position [20,21] and changes in the phase behavior of total lipid extracts [15] suggesting that membrane physical properties are affected
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