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

校准纳米模板作为超分辨率显微镜中确定蛋白质拷贝数的通用标准

• 批准号: 9911066
• 负责人: Melike Lakadamyali
• 金额: $ 36.45万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911066
• 关键词: Address; Antibodies; Benchmarking; Biological; Blinking; Buffers; Calibration; Cell Line; Cell Nucleus; Cell physiology; Cells; Cellular biology; Communities; Complex; Computer software; Core Facility; Cytoplasm; DNA; Data; Development; Disease; Drug Screening; Ensure; Environment; Evolution; Future; Genes; Goals; Image; Imagery; Label; Measures; Membrane; Methodology; Methods; Microscope; Microscopy; Molecular; Morphologic artifacts; Multiprotein Complexes; Nature; Nuclear Pore Complex; Photobleaching; Proteins; Quality Control; Reagent; Reporter; Research Personnel; Resolution; Sampling; Standardization; Tertiary Protein Structure; Testing; Time; Validation; Work; base; diagnostic screening; dimer; experimental study; fluorophore; innovation; insight; interest; molecular assembly/self assembly; monomer; nano; nanocage; nanoscale; novel; novel diagnostics; novel strategies; open source; predictive modeling; protein aggregation; protein complex; protein distribution; protein function; single molecule; stem; stoichiometry; tool; user friendly software; user-friendly

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.

项目摘要 在此提案中，我们将开发新的方法来克服超分辨率的主要挑战之一 显微镜检查并启用纳米级水平上的蛋白质拷贝数定量。超分辨率 显微镜是一种启示工具，揭示了分子复合物的亚细胞组织 前所未有的空间分辨率。这些复合物将这些复合物分为功能单位的纳米镜组织是 对于以空间和时间控制的方式调节其亚细胞活性非常重要。 但是，量化多蛋白的蛋白质拷贝数组成是非常具有挑战性的 复合物。蛋白质拷贝数对于调节或错误调节蛋白质功能非常重要。蛋白质 当其低聚时，可能在某个寡聚组件下起作用，并获得有毒功能 组成跨越了导致疾病的关键阈值。因此，正确量化子的能力 分子络合物中蛋白质的细胞拷贝数分布对于获得机械性很重要 深入了解这些蛋白质的健康和患病功能。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光体物质产生的人工制品。 我们通过开发校准纳米复制物来克服这一挑战的重要飞跃 超分辨率显微镜。但是，缺乏说明的标准，易于使用的方法和试剂 对于实验条件的可变性，使非专家难以适应这些发展。 因此，立即需要高度标准化的方法，以允许蛋白质拷贝数 与实验条件无关的定量。该提议的目的是应对这一重大挑战 并建立一种多功能，易于使用和通用的校准方法，该方法可以由科学改编 量化任何感兴趣蛋白质的拷贝数分布。我们的目标是：（i） 使用DNA折纸和新型的小蛋白质复合物的校准纳米复合物以及新型 使用设计器蛋白质纳米元素的纳米置换物，（ii）获取多种超分辨率的校准数据 兼容标签策略并确定校准功能，（iii），以发展创新的概念 基于基准标准标准和可以改变校准的方法的新颖使用标准化 不同的实验条件和（iv）之间的功能，以开发出全面，用户友好，开放的功能 来源，模块化软件，结合了从单分子定位到蛋白质拷贝的所有步骤 数字确定。 该提案有可能将超分辨率显微镜从主要描述工具推进到 定量和机械细胞生物学的时代。作为一个具体示例，这里开发的方法将 使蛋白质聚集在高度 到目前为止，在几个疾病状态下的定量方式比到目前为止的时间早得多 未来有可能实现新的诊断和药物筛查方法。我们希望该方法是 广泛适用于许多生物医学问题，并产生广泛的影响。