The use of anthrolysin O and ostreolysin A to study cholesterol in cell membranes.
The use of anthrolysin O and ostreolysin A to study cholesterol in cell membranes.
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DOI：
10.1016/bs.mie.2021.01.011
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发表时间：
2021
影响因子：
--
通讯作者：
Radhakrishnan A
中科院分区：
文献类型：
--
作者：
Johnson KA;Radhakrishnan A
胆固醇是动物细胞质膜（PMs）的主要成分，占总质膜脂质的35-40摩尔%。最近对胆固醇结合细菌毒素（如antholysin O （ALOD4）结构域4）和真菌毒素（如Ostreolysin A (OlyA)）的研究揭示了PM胆固醇组织的新见解。这些研究已经定义了三个不同的PM胆固醇池-一个对膜完整性至关重要的固定池，一个可以通过OlyA检测到的鞘磷脂（SM）隔离池，以及一个可以通过ALOD4检测到的可接近池。可及性胆固醇可与蛋白质相互作用并转运到内质网（ER），并控制许多细胞信号过程，包括胆固醇稳态，Hedgehog信号传导以及细菌和病毒感染。在这里，我们详细描述了使用ALOD4和OlyA这两种可溶性和非溶性蛋白来研究动物细胞pmms中的胆固醇组织。此外，我们描述了我们开发的两个新版本的ALOD4，以增加该探针在细胞研究中的多功能性。一种是双His6和FLAG表位标记的版本，另一种是荧光版本，其中ALOD4与单体荧光蛋白Neon融合。这些新形式的ALOD4与先前描述的OlyA一起提供了一个扩展的工具集合来感知，可视化和调节pm上可接近和sm隔离的胆固醇水平，并研究这些胆固醇池在不同膜信号事件中的作用。
Cholesterol is a major component of the plasma membranes (PMs) of animal cells, comprising 35–40 mol% of total PM lipids. Recent studies using cholesterol-binding bacterial toxins such as domain 4 of Anthrolysin O (ALOD4) and fungal toxins such as Ostreolysin A (OlyA) have revealed new insights into the organization of PM cholesterol. These studies have defined three distinct pools of PM cholesterol—a fixed pool that is essential for membrane integrity, a sphingomyelin (SM)-sequestered pool that can be detected by OlyA, and a third pool that is accessible and can be detected by ALOD4. Accessible cholesterol is available to interact with proteins and transport to the endoplasmic reticulum (ER), and controls many cellular signaling processes including cholesterol homeostasis, Hedgehog signaling, and bacterial and viral infection. Here, we provide detailed descriptions for the use of ALOD4 and OlyA, both of which are soluble and non-lytic proteins, to study cholesterol organization in the PMs of animal cells. Furthermore, we describe two new versions of ALOD4 that we have developed to increase the versatility of this probe in cellular studies. One is a dual His6 and FLAG epitope-tagged version and the other is a fluorescent version where ALOD4 is fused to Neon, a monomeric fluorescent protein. These new forms of ALOD4 together with previously described OlyA provide an expanded collection of tools to sense, visualize, and modulate levels of accessible and SM-sequestered cholesterol on PMs and study the role of these cholesterol pools in diverse membrane signaling events.
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影响因子：
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作者：
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影响因子：
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作者：
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DOI：
10.1073/pnas.1309273110
发表时间：
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影响因子：
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作者：
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