Growth Conditions and Cell Cycle Phase Modulate Phase Transition Temperatures in RBL-2H3 Derived Plasma Membrane Vesicles.
Growth Conditions and Cell Cycle Phase Modulate Phase Transition Temperatures in RBL-2H3 Derived Plasma Membrane Vesicles.
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DOI：
10.1371/journal.pone.0137741
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Veatch SL
中科院分区：
文献类型：
--
作者：
Gray EM;Díaz-Vázquez G;Veatch SL
从RBL-2H3细胞中分离得到的巨质膜囊泡（GPMV）在生理温度下表现均匀，在低温下包含有序液相和无序液相共存。虽然单个GPMV在这两种状态之间在明确的温度下转换，但在单个细胞制备中存在明显的囊泡到囊泡的异质性，并且不同制备的平均转换温度可能存在显着差异。在这项研究中，我们探索了GPMV转变温度如何依赖于生长条件，发现平均转变温度与平均细胞密度呈负相关，超过15°C的转变温度与平均细胞密度呈负相关，平均表面密度接近3个数量级。此外，从缺乏血清的细胞中分离出gpmv时，平均转变温度降低了近10℃，而当血清缺乏的细胞在含血清的培养基中培养12小时时，升高的转变温度又恢复了。我们还研究了通过双胸苷阻断从G1/S边界同步的细胞中分离的gpmv的转变温度变化，发现G1期或M期细胞产生的gpmv的平均转变温度系统性地高于S期或G1期细胞制备的gpmv。用TRAIL诱导细胞凋亡或鞘磷脂酶制备的GPMVs中，也观察到混相转变温度降低，在某些情况下，在这些囊泡中，在高于混相转变温度的温度下观察到凝胶相。我们的结论是，至少有一些GPMV转变温度的变化是由细胞局部密度的变化和细胞周期的不同步引起的。假设GPMV转变温度是完整细胞膜在生长温度下脂质介导的膜非均质性大小的代表。如果是这样，这些结果表明，细胞调整其质膜组成，以控制膜异质性的大小，以响应不同的生长条件。
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.