Direct imaging of liquid domains in membranes by cryo-electron tomography
Direct imaging of liquid domains in membranes by cryo-electron tomography
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DOI：
10.1073/pnas.2002245117
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发表时间：
2020-08-18
影响因子：
11.1
通讯作者：
Keller, Sarah L.
中科院分区：
文献类型：
--
作者：
Cornell, Caitlin E.;Mileant, Alexander;Keller, Sarah L.
脂质双层中微米尺度区域的图像为理解区域的形状、大小和分布提供了无模型证据的金标准。直接和定量评估较小（纳米级和亚微米级）液体域的相应技术受到限制。研究人员通常试图将膜蛋白的活性与其所处结构域的属性联系起来，这取决于膜结构域的识别和表征。尽管膜结构域的某些特征可以通过间接方法来探测，但这些方法通常受到数据必须在需要多个假设或参数的模型中进行分析的限制。在这里，我们通过开发和测试两种识别仿生膜中亚微米结构域的方法来解决这一挑战。这两种方法都能获得玻璃化、水合条件下三元膜的低温电子层析图。第一种方法针对无探针应用进行了优化：通过其厚度直接将域与周围的膜区分开来。该技术定量和准确地测量领域的面积分数，在良好的协议与已知的相图。第二种方法是优化的应用程序中，部署一个单一的标签成像膜的高分辨率冷冻电子断层扫描和衍射限制的光学显微镜。对于这种方法，我们测试了一组探针，发现三聚体mCherry标签表现最好，并为开发未来的高性能，两用探针指定了标准。这些发展导致了直接和定量的玻璃化，水合囊泡中的亚微米膜域的成像。
Images of micrometer-scale domains in lipid bilayers have pro-vided the gold standard of model-free evidence to understand the domains' shapes, sizes, and distributions. Corresponding tech-niques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been limited. Researchers com-monly seek to correlate activities of membrane proteins with at-tributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Al-though some features of membrane domains can be probed by indirect methods, these methods are often constrained by the lim-itation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two methods of identifying submicron domains in biomimetic membranes. Both methods le-verage cryo-electron tomograms of ternary membranes under vit-rified, hydrated conditions. The first method is optimized for probe-free applications: Domains are directly distinguished from the surrounding membrane by their thickness. This technique quantitatively and accurately measures area fractions of domains, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to direct and quantitative imaging of submicron mem-brane domains in vitrified, hydrated vesicles.