Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy
Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy
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DOI：
10.1017/s1431927619000606
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发表时间：
2019-08-01
影响因子：
2.8
通讯作者：
Briggs, John A. G.
中科院分区：
文献类型：
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作者：
Metskas, Lauren Ann;Briggs, John A. G.
相关光电子显微镜（CLEM）已成为一种流行的技术相结合的蛋白质特异性标记的荧光与电子显微镜，在室温和低温。由于已知的延长的三重态持续时间、光谱位移和通过低温CLEM物镜的减少的光子捕获的问题，低温下的荧光应用通常限于标记的蛋白质寡聚体的定位。在这里，我们考虑荧光团的特性和行为，可以使更多的扩展应用。我们描述了二烷基carbocanine DiD，和它的自动淬火共振能量转移（RET），可以用来区分在低温下的脂质双层的融合状态。通过调整已建立的融合试验，以在冷冻CLEM条件下工作，我们确定了流感病毒样颗粒和荧光标记的脂质囊泡之间的融合区域的冷冻EM网格。这一结果表明，cryo-CLEM可用于定位功能，除了标记的蛋白质，荧光自动淬灭RET可以成功地纳入cryo-CLEM的方法。在膜融合应用的情况下，该方法提供了独立于来自cryo-EM的形态描述的功能状态的正交确认和桥接室温动力学测定和cryo-EM图像的方式。
Correlated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with electron microscopy, both at room and cryogenic temperatures. Fluorescence applications at cryo-temperatures have typically been limited to localization of tagged protein oligomers due to known issues of extended triplet state duration, spectral shifts, and reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics and behaviors that could enable more extended applications. We describe how dialkylcarbocanine DiD, and its autoquenching by resonant energy transfer (RET), can be used to distinguish the fusion state of a lipid bilayer at cryo-temperatures. By adapting an established fusion assay to work under cryo-CLEM conditions, we identified areas of fusion between influenza virus-like particles and fluorescently labeled lipid vesicles on a cryo-EM grid. This result demonstrates that cryo-CLEM can be used to localize functions in addition to tagged proteins, and that fluorescence autoquenching by RET can be incorporated successfully into cryo-CLEM approaches. In the case of membrane fusion applications, this method provides both an orthogonal confirmation of functional state independent of the morphological description from cryo-EM and a way to bridge room-temperature kinetic assays and the cryo-EM images.