Abstract：
The biophysical drivers of membrane lateral heterogeneity, often termed lipid rafts, have been largely explored using synthetic liposomes or mammalian plasma membrane-derived giant vesicles. Yeast vacuoles, an organelle comparable to mammalian lysosomes, is the only in vivo system that shows stable micrometer scale phase separation in unperturbed cells. The ease of manipulating lipid metabolism in yeast makes this a powerful system for identifying lipids involved in the onset of vacuole membrane heterogeneity. Vacuole domains are induced by stationary stage growth and nutritional starvation, during which they serve as a docking and internalization site for lipid droplet energy stores. Here we describe methods for characterizing vacuole phase separation, its physiological function, and its lipidic drivers. First, we detail methodologies for robustly inducing vacuole domain formation and quantitatively characterizing during live cell imaging experiments. Second, we detail a new protocol for biochemical isolation of stationary stage vacuoles, which allows for lipidomic dissection of membrane phase separation. Third, we describe biochemical techniques for analyzing lipid droplet internalization in vacuole domains. When combined with genetic or chemical perturbations to lipid metabolism, these methods allow for systematic dissection of lipid composition in the structure and function of ordered membrane domains in living cells.
摘要：
膜横向异质性的生物物理驱动因素，通常称为脂筏，已使用合成脂质体或哺乳动物质膜衍生的巨囊泡进行了大量探索。酵母液泡,与哺乳动物溶酶体相当的细胞器，是唯一在未扰动细胞中显示稳定的微米级相分离的体内系统。在酵母中操纵脂质代谢的容易性使其成为鉴定与液泡膜异质性发作有关的脂质的强大系统。液泡结构域是由静止期生长和营养饥饿引起的，在此期间，它们充当脂滴能量储存的对接和内化位点。在这里，我们描述了表征液泡相分离的方法，它的生理功能，和它的脂类驱动因素。首先,我们详细介绍了在活细胞成像实验中稳健诱导液泡结构域形成和定量表征的方法。第二,我们详细介绍了固定阶段液泡生化分离的新方案，这允许膜相分离的脂质解剖。第三，我们描述了分析液泡域中脂质液滴内化的生化技术。当与脂质代谢的遗传或化学扰动相结合时，这些方法允许系统地解剖活细胞中有序膜结构域的结构和功能中的脂质组成。
