Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, United States.
ACS Chem Biol. 2024 Aug 16;19(8):1773-1785. doi: 10.1021/acschembio.4c00249. Epub 2024 Jul 28.
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.
细胞器具有特征性的脂质组成，导致膜性质的差异。在细胞中，膜有序性和流动性通常使用溶剂化变色染料 Laurdan 进行测量，其荧光对脂质堆积敏感。作为一种通用的亲脂性染料，Laurdan 染色细胞中所有的疏水环境；因此，在完整细胞中对特定细胞器中的膜性质进行特征描述或评估它们对药物处理的反应具有挑战性。在这里，我们描述了衍生自 Laurdan 的探针的合成和应用，这些探针可读取单个细胞细胞器的膜堆积情况。该组细胞器靶向的 Laurdan（OTL）定位于内质网、线粒体、溶酶体和高尔基体，具有高度特异性，同时保留了检测膜有序性生物变化所需的光谱分辨率。我们表明，OTL 的比率成像可以分辨细胞器内的膜异质性以及由于运输或生物能量过程抑制而导致的脂质堆积变化。我们将这些探针应用于表征细胞器对饱和脂质应激的特异性反应。虽然内质网和溶酶体膜的流动性对外源饱和脂肪酸敏感，但线粒体膜的流动性受到保护。然后，我们利用内质网膜流动性的差异，根据其脂肪酸饮食对细胞群体进行分类，突出了细胞器定位的溶剂化变色探针根据其代谢状态区分细胞的能力。这些结果扩展了靶向膜探针的范围，并证明了它们在研究脂质失调方面的应用。
