Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy
Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy
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DOI：
10.1101/591081
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发表时间：
2019-03
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通讯作者：
Alexander H. Clowsley;William T. Kaufhold;T. Lutz;Anna Meletiou;L. D. Michele;C. Soeller
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作者：
Alexander H. Clowsley;William T. Kaufhold;T. Lutz;Anna Meletiou;L. D. Michele;C. Soeller
蛋白质等生物分子之间的相互作用是大多数细胞过程的基础。至关重要的是直接在细胞内可视化这些分子相互作用复合物，以精确显示这些相互作用发生的位置，从而提高我们对细胞调控的理解。目前可用的接近敏感的分析，这种相互作用的原位成像产生衍射限制的信号，因此排除了相互作用复合物的纳米级分布的信息。相比之下，光学超分辨率成像提供了纳米分辨率的分子分布信息，这极大地促进了我们对细胞生物学的理解。然而，目前的超分辨率荧光成像的共定位分析容易出现假阳性信号，因为蛋白质邻近性的检测直接依赖于局部光学分辨率。在这里，我们提出了邻近依赖PAINT（PD-PAINT），一种用于蛋白质对的子衍射成像的方法，其中邻近检测与光学分辨率解耦。通过锚定到目标物种的两个DNA标签的高度距离依赖性相互作用来检测邻近性。然后使用DNA-PAINT的超分辨率方法以高对比度和纳米级分辨率对标记的蛋白质对进行成像。通过粗粒度分子模拟分析了新技术的机制，并通过成像分离的心肌细胞中心脏蛋白的DNA折纸瓦片和表位进行了实验证明。我们表明，PD-PAINT可以直接集成在一个多路复用的超分辨率成像协议，并受益于基于DNA的超分辨率定位显微镜的优点，如高特异性，高分辨率和定量成像的能力。
Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in-situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present Proximity-Dependent PAINT (PD-PAINT), a method for sub-diffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA labels anchored to the target species. Labeled protein pairs are then imaged with high contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution and the ability to image quantitatively.