Cholesterol is an essential component of the mammalian plasma membrane involved in diverse cellular processes. Our recent quantitative imaging analysis using ratiometric cholesterol sensors showed that the available cholesterol concentration in the inner leaflet of the plasma membrane (IPM) is low in unstimulated cells, and increased in a stimulus-specific manner to trigger cell signaling events. However, the transbilayer distribution of cholesterol in the plasma membrane of mammalian cells remains controversial. Here we report a systematic and rigorous evaluation of basal IPM cholesterol levels in a wide range of mammalian cells with different properties employing cholesterol sensors derived from the D4 domain of the Perfringolysin O toxin and a sterol-transfer protein, Osh4. Results consistently showed that although basal IPM cholesterol levels vary significantly among cells, they remain significantly lower than cholesterol levels in the outer leaflets. We found that IPM cholesterol levels were particularly low in all tested primary cells. These results support the universality of the low basal IPM cholesterol concentration under physiological conditions. We also report here the presence of sequestered IPM cholesterol pools, which may become available to cytosolic proteins under certain physiological conditions. We hypothesize that these pools may partly account for the low basal level of available IPM cholesterol. In conclusion, we provide new experimental data that confirm the asymmetric transbilayer distribution of the plasma membrane cholesterol, which may contribute to regulation of various cellular signaling processes at the plasma membrane.

中文翻译：

---

评估哺乳动物细胞质膜内叶中可用的胆固醇浓度。

胆固醇是参与多种细胞过程的哺乳动物质膜的重要组成部分。我们最近使用比率胆固醇传感器进行的定量成像分析表明，在未受刺激的细胞中，质膜 (IPM) 内叶的可用胆固醇浓度较低，并以刺激特异性方式增加以触发细胞信号传导事件。然而，哺乳动物细胞质膜中胆固醇的跨双层分布仍然存在争议。在这里，我们报告了使用源自 Perfringolysin O 毒素的 D4 结构域和甾醇转移蛋白 Osh4 的胆固醇传感器，对具有不同特性的各种哺乳动物细胞中的基础 IPM 胆固醇水平进行了系统和严格的评估。结果一致表明，尽管细胞间的基础 IPM 胆固醇水平差异很大，但它们仍然显着低于外叶中的胆固醇水平。我们发现 IPM 胆固醇水平在所有测试的原代细胞中都特别低。这些结果支持生理条件下低基础 IPM 胆固醇浓度的普遍性。我们还在这里报告了隔离的 IPM 胆固醇池的存在，在某些生理条件下，这些胆固醇池可能对细胞溶质蛋白可用。我们假设这些池可能部分解释了可用 IPM 胆固醇的低基础水平。总之，我们提供了新的实验数据，证实了质膜胆固醇的不对称跨双层分布，