Abstract：
Organelles feature characteristic lipid compositions that lead to differences in membrane properties. In cells, membrane ordering and fluidity are commonly measured using the solvatochromic dye Laurdan, whose fluorescence is sensitive to lipid packing. As a general lipophilic dye, Laurdan stains all hydrophobic environments in cells; therefore, it is challenging to characterize membrane properties in specific organelles or assess their responses to pharmacological treatments in intact cells. Here, we describe the synthesis and application of Laurdan-derived probes that read out the membrane packing of individual cellular organelles. The set of organelle-targeted Laurdans (OTL) localizes to the ER, mitochondria, lysosomes, and Golgi compartments with high specificity while retaining the spectral resolution needed to detect biological changes in membrane ordering. We show that ratiometric imaging with OTLs can resolve membrane heterogeneity within organelles as well as changes in lipid packing resulting from inhibition of trafficking or bioenergetic processes. We apply these probes to characterize organelle-specific responses to saturated lipid stress. While the ER and lysosomal membrane fluidity is sensitive to exogenous saturated fatty acids, that of mitochondrial membranes is protected. We then use differences in ER membrane fluidity to sort populations of cells based on their fatty acid diet, highlighting the ability of organelle-localized solvatochromic probes to distinguish between cells based on their metabolic state. These results expand the repertoire of targeted membrane probes and demonstrate their application in interrogating lipid dysregulation.
摘要：
细胞器具有导致膜性质差异的特征性脂质组成。在细胞中，膜有序性和流动性通常使用溶剂化变色染料Laurdan，其荧光对脂质包装敏感。作为一种通用的亲脂性染料，Laurdan对细胞中的所有疏水环境进行染色；因此，表征特定细胞器中的膜特性或评估其对完整细胞中药物治疗的反应是具有挑战性的。这里,我们描述了Laurdan衍生的探针的合成和应用，这些探针读出了单个细胞器的膜包装。以细胞器为目标的Laurdans（OTL）集定位到ER，线粒体,溶酶体,和高尔基区室具有高特异性，同时保留了检测膜有序生物学变化所需的光谱分辨率。我们表明，使用OTL的比率成像可以解决细胞器内的膜异质性以及由于抑制运输或生物能过程而导致的脂质包装变化。我们应用这些探针来表征对饱和脂质应激的细胞器特异性反应。虽然ER和溶酶体膜流动性对外源性饱和脂肪酸敏感，线粒体膜受到保护。然后，我们使用ER膜流动性的差异来根据脂肪酸饮食对细胞群进行分类，强调细胞器定位溶剂化探针根据细胞的代谢状态区分细胞的能力。这些结果扩展了靶向膜探针的库，并证明了它们在询问脂质失调中的应用。
