Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)
Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)
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DOI：
10.3791/53154
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发表时间：
2015-12-01
影响因子：
1.2
通讯作者：
Nan, Xioalin
中科院分区：
文献类型：
--
作者：
Nickerson, Andrew;Huang, Tao;Nan, Xioalin
蛋白质-蛋白质相互作用（PPI）是生物学的关键分子事件。然而，在细胞中以足够的分辨率和灵敏度可视化 PPI 仍然是一个挑战，因为传统光学显微镜的分辨率仅限于 250 nm 左右。通过将双分子荧光互补 (BiFC) 与光激活定位显微镜 (PALM) 相结合，可以在细胞中以单分子灵敏度和纳米空间分辨率可视化 PPI。 BiFC 是一种常用的通过荧光对比度可视化 PPI 的技术，其中涉及将荧光蛋白分裂成两个非荧光片段。 PALM 是一种最新的超分辨率显微镜技术，用于在纳米和单分子尺度上对生物样品进行成像，它使用光可转换荧光探针，例如光活化荧光蛋白 (PA-FP)。 BiFC-PALM 通过拆分 PAmCherry1（一种与 PALM 兼容的 PA-FP）进行演示，具有单体性质、良好的单分子亮度、高对比度以及化学计量测量的实用性。当在氨基酸 159 和 160 之间拆分时，PAmCherry1 可制成 BiFC 探针，可在 37°C 下高效重构，对 PPI 具有高特异性和低非特异性重构。以 Ras-Raf 相互作用为例，展示 BiFC-PALM 如何帮助探测纳米尺度和单分子分辨率的相互作用。还可以使用单分子追踪 (smt-) PALM 来追踪它们在活细胞中的扩散。在此协议中，讨论了设计 BiFC-PALM 融合蛋白时要考虑的因素，解释了样品制备、图像采集和数据分析步骤，并展示了一些示例性结果。 BiFC-PALM 具有高空间分辨率、特异性和灵敏度，是研究完整生物样品中 PPI 的有用工具。
Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to similar to 250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 degrees C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.