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.

项目概要 在本提案中，我们将开发新方法来克服超分辨率的主要挑战之一 显微镜并能够在纳米级水平上量化蛋白质拷贝数。超分辨率 显微镜是一种揭示分子复合物亚细胞组织的工具 前所未有的空间分辨率。这些复合物以纳米级的方式组织成功能单元 对于以空间和时间控制的方式调节其亚细胞活动非常重要。 然而，量化多蛋白质的蛋白质拷贝数组成一直非常具有挑战性。 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 可能在低于某种寡聚组装时发挥功能，并且当其寡聚组装时获得毒性功能 成分超过导致疾病的关键阈值。因此，能够正确量化子 分子复合物内蛋白质的细胞拷贝数分布对于获得机制很重要 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物理学产生的伪影。 通过开发校准纳米模板，我们在克服这一挑战方面取得了重要飞跃 超分辨率显微镜。然而，缺乏标准、易于使用的方法和试剂来解释 由于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来允许蛋白质拷贝数 定量独立于实验条件。该提案的目标是应对这一巨大挑战 建立一种通用的、易于使用的、可被科学界采用的通用校准方法 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： (i) 发展 使用 DNA 折纸以及新颖的方法校准小型和大型蛋白质复合物的纳米模板 使用设计蛋白纳米笼的纳米模板，(ii) 获取多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）开发创新概念 基于基准测试标准和方法的新颖使用的标准化，可以改变校准 （iv）开发一个全集成的、用户友好的、开放的 源模块化软件，包含从单分子定位到蛋白质复制的所有步骤 数测定。 该提案有可能将超分辨率显微镜从主要描述性工具推进到 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 使得揭示蛋白质聚集的亚细胞分布和进化成为可能 在比迄今为止可能更早的时间点对几种疾病状态进行定量分析， 未来有可能实现新的诊断和药物筛选方法。我们期望该方法是 广泛适用于大量生物医学问题并产生广泛影响。