Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress
Targeted Solvatochromic Fluorescent Probes for Imaging Lipid Order in Organelles under Oxidative and Mechanical Stress
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DOI：
10.1021/jacs.0c10972
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发表时间：
2021-01-08
影响因子：
15
通讯作者：
Klymchenko, Andrey S.
中科院分区：
文献类型：
--
作者：
Danylchuk, Dmytro, I;Jouard, Pierre-Henri;Klymchenko, Andrey S.
生物膜构成了活细胞所有区室的基础，因此，监测其脂质组织对于了解细胞状态和活性至关重要。然而，特别是在活细胞的不同细胞器中的脂质组织的感测和成像仍然具有挑战性。在这里，我们设计了一个阵列的溶剂化显色探针的基础上尼罗红轴承配体的内质网，线粒体，溶酶体，高尔基体，质膜，脂滴的特异性靶向。这些极性敏感的探针检测的脂质顺序的变化，通过改变它们的发射最大值，证明了在模型膜的荧光光谱。在与参考细胞器标记物的共定位显微镜实验中，它们表现出良好的细胞器选择性。使用双色荧光显微镜，新探针能够成像活细胞中细胞器的局部极性。为了排除探针设计对膜性质的敏感性的偏倚效应，我们在显微镜下校准了模型膜中的所有探针，这使得能够第一次定量描述每个感兴趣的细胞器中的脂质顺序。胆固醇提取/富集证实了探针感测脂质顺序的能力，揭示了胆固醇贫乏的细胞器特别受其富集的影响。探针还显示，氧化和机械应力产生的局部极性和脂质顺序的变化是每个细胞器的特征，线粒体和溶酶体是特别敏感的压力。新的探针构成了一个强大的工具箱，用于监测细胞对单个细胞器膜水平的物理和化学刺激的反应，这仍然是细胞研究中一个未充分探索的方向。
Biomembranes constitute a basis for all compartments of live cells, and therefore, the monitoring of their lipid organization is essential for understanding cell status and activity. However, the sensing and imaging of lipid organization specifically in different organelles of live cells remain challenging. Here, we designed an array of solvatochromic probes based on Nile Red bearing ligands for specific targeting of the endoplasmic reticulum, mitochondria, lysosomes, Golgi apparatus, plasma membranes, and lipid droplets. These polarity-sensitive probes detected variations in the lipid order by changing their emission maximum, as evidenced by fluorescence spectroscopy in model membranes. In colocalization microscopy experiments with reference organelle markers, they exhibited good organelle selectivity. Using two-color fluorescence microscopy, the new probes enabled imaging of the local polarity of organelles in live cells. To exclude the biased effect of the probe design on the sensitivity to the membrane properties, we calibrated all probes in model membranes under the microscope, which enabled the first quantitative description of the lipid order in each organelle of interest. Cholesterol extraction/enrichment confirmed the capacity of the probes to sense the lipid order, revealing that organelles poor in cholesterol are particularly affected by its enrichment. The probes also revealed that oxidative and mechanical stresses produced changes in the local polarity and lipid order that were characteristic for each organelle, with mitochondria and lysosomes being particularly stress sensitive. The new probes constitute a powerful toolbox for monitoring the response of the cells to physical and chemical stimuli at the level of membranes of individual organelles, which remains an underexplored direction in cellular research.