Calibration nanotemplates as universal standards for determining protein copy number in super-resolution microscopy

项目摘要 在这项提案中，我们将开发新的方法来克服超分辨率的主要挑战之一 这使得能够在纳米级水平上定量蛋白质拷贝数。超分辨率 显微镜是一种能够揭示分子复合物的亚细胞组织的工具， 前所未有的空间分辨率。将这些复合物纳米级组织成功能单元， 对于以空间和时间控制的方式调节它们的亚细胞活性非常重要。 然而，定量多蛋白质的蛋白质拷贝数组成一直是非常具有挑战性的， 配合物蛋白质拷贝数对于调节或错误调节蛋白质功能非常重要。蛋白 可能在某种低聚组装下起作用，并且当它们的低聚组装时获得毒性功能。 如果一种化学物质的含量超过了导致疾病的临界值，因此，适当量化子系统的能力， 分子复合物中蛋白质的细胞拷贝数分布对于获得分子复合物的机制是重要的。 深入了解这些蛋白质的健康和疾病功能。这样做的主要挑战是克服 由未知的标记化学计量和复杂的荧光团化学物理学引起的伪像。 我们通过开发校准纳米模板， 超分辨率显微镜然而，缺乏标准的，易于使用的方法和试剂， 因为实验条件的可变性使得非专家很难适应这些发展。 因此，迫切需要高度标准化的方法， 定量独立于实验条件。这项提案的目标是应对这一巨大挑战 并建立一个通用的，易于使用的和通用的校准方法，可以适应的科学 社区来量化任何感兴趣的蛋白质的拷贝数分布。我们的目标是：（一）发展 使用DNA折纸的小和大蛋白质复合物的校准纳米模板以及新的 使用设计师蛋白质纳米笼的纳米模板，（ii）获取各种超分辨率的校准数据 兼容的标签策略，并确定校准功能，（iii）开发创新的概念， 标准化基于基准测试标准和方法的新用途，可以改变校准 开发了一套完整的、用户友好的、开放的、 源代码，模块化软件，包括从单分子定位到蛋白质拷贝的所有步骤 数量测定 这一建议有可能将超分辨率显微镜从一个主要的描述性工具推进到 定量和机械细胞生物学的时代。作为一个具体的例子，这里开发的方法将 使我们有可能揭示蛋白质聚集的亚细胞分布和进化， 在比迄今为止可能的时间点早得多的时间点， 可能在未来实现新的诊断和药物筛选方法。我们希望这个方法 广泛适用于大量的生物医学问题，具有广泛的影响。

Project Summary In this proposal, we will develop new methods to overcome one of the main challenges in super-resolution microscopy and enable quantification of protein copy number at the nanoscale level. Super-resolution microscopy is an enabling tool that reveals the subcellular organization of molecular complexes with unprecedented spatial resolution. The nanoscopic organization of these complexes into functional units is highly important for regulating their subcellular activity in a spatially and temporally controlled manner. However, it has been very challenging to quantify the protein copy number composition of multi-protein complexes. Protein copy number is highly important for regulating or mis-regulating protein function. Proteins may be functional below a certain oligomeric assembly and gain toxic function when their oligomeric composition crosses a critical threshold leading to diseases. Therefore, the ability to properly quantify the sub- cellular copy number distribution of proteins within molecular complexes is important for gaining mechanistic insight into healthy and diseased function of these proteins. The main challenge, in doing so, is to overcome the artefacts arising from the unknown labeling stoichiometry and complex fluorophore photophysics. We have made important leaps towards overcoming this challenge by developing calibration nanotemplates for super-resolution microscopy. However, the lack of standard, easy-to-use methods and reagents that account for variability in experimental conditions has made it difficult for non-experts to adapt these developments. Therefore, there is an immediate need for highly standardized methodologies that allow protein copy number quantification independent of experimental conditions. The goal of this proposal is to address this big challenge and establish a versatile, easy-to-use and universal calibration method that can be adapted by the scientific community to quantify the copy number distribution of any protein of interest. Our aims are: (i) to develop calibration nanotemplates for both small and large protein complexes using DNA origami as well as novel nanotemplates that use designer protein nanocages, (ii) to acquire calibration data for diverse, super-resolution compatible labeling strategies and identify calibration functions, (iii) to develop the innovative concept of standardization based on novel use of benchmarking standards and methods that can transform the calibration functions among different experimental conditions and (iv) to develop an all-integrated, user-friendly, open- source, modular software that incorporates all the steps from single molecule localization to protein copy number determination. This proposal has the potential to advance super-resolution microscopy from a mainly descriptive tool into the era of quantitative and mechanistic cell biology. As one specific example, the methods developed here will make it possible to reveal the sub-cellular distribution and evolution of protein aggregation in a highly quantitative manner in several disease states at much earlier time points than has been possible thus far, potentially enabling new diagnostic and drug screening methods in the future. We expect the method to be widely applicable to a large number of biomedical questions and have a broad impact.