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中的主要挑战之一 这一技术可以在显微镜下观察蛋白质的拷贝数，并能够在纳米级别上量化蛋白质拷贝数。超分辨率 显微镜是揭示分子络合物亚细胞组织的一种使能工具 前所未有的空间分辨率。将这些络合物纳米组织成功能单元是 对于以空间和时间受控的方式调节它们的亚细胞活动非常重要。 然而，多蛋白质拷贝数组成的量化一直是一个极具挑战性的问题。 复合体。蛋白质拷贝数对于调节或误调节蛋白质功能是非常重要的。蛋白质 可能在某一低聚物组装下具有功能性，当其低聚物时获得有毒功能 构成跨越了导致疾病的关键门槛。因此，正确量化亚项的能力- 蛋白质在分子复合体中的细胞拷贝数分布是获得机制的重要因素 洞察这些蛋白质的健康和疾病功能。在这样做的过程中，主要挑战是克服 由未知的标记化学计量学和复杂的荧光团光物理引起的伪影。 我们已经在克服这一挑战方面取得了重要的飞跃，开发了用于 超分辨率显微镜。然而，缺乏标准的、易于使用的方法和试剂来解释 因为实验条件的多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来允许蛋白质拷贝数 不依赖于实验条件的量化。这项提案的目标是应对这一巨大挑战 建立一种通用、易用、通用性强、科学适用的定标方法 以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 用DNA折纸和新型的方法校准小分子和大分子蛋白质复合体的纳米模板 使用设计的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 兼容的标签策略和识别校准功能，(Iii)发展创新概念 基于标杆标准和方法的新使用的标准化，可以改变校准 在不同的实验条件下实现不同的功能；(4)开发一个全面集成的、用户友好的、开放的. 源代码，模块化软件，整合了从单分子定位到蛋白质复制的所有步骤 数字决定。 这一提议有可能将超分辨率显微镜从主要的描述性工具推进到 量化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 使揭示蛋白质聚集的亚细胞分布和进化成为可能 在比目前可能的时间点更早的几个疾病状态中以定量的方式， 有可能在未来实现新的诊断和药物筛选方法。我们预计这种方法是 广泛适用于大量的生物医学问题并产生广泛的影响。

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.