Quantitative label-free imaging of membrane protein interaction kinetics on cells

细胞膜蛋白相互作用动力学的定量无标记成像

• 批准号: 8882482
• 负责人: SHAOPENG WANG
• 金额: $ 28.61万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8882482
• 关键词: Affinity; Algorithms; Beds; Binding; Biological; Biological Assay; Biological Markers; Biological Models; Biological Process; Cancer Diagnostics; Cancer Patient; Cell Count; Cell Line; Cell membrane; Cells; Data; Detection; Device or Instrument Development; Disease; Dose; Drug Receptors; Drug Targeting; Drug resistance; Drug usage; Drug-sensitive; End Point Assay; Environment; Epidermal Growth Factor Receptor; Event; Exclusion; Feedback; Frequencies; G-Protein-Coupled Receptors; Goals; Grant; Health; Human; Image; Imaging Techniques; Immobilization; Immunofluorescence Immunologic; In Situ; Industry Collaborators; Investigation; Kinetics; Label; Lead; Maps; Measurement; Measures; Membrane; Membrane Proteins; Methods; Microscopy; Molecular; Monitor; Monoclonal Antibodies; Nature; Noise; Optics; Performance; Pharmaceutical Preparations; Preclinical Drug Evaluation; Primary Neoplasm; Procedures; Protein Binding; Proteins; Real-Time Systems; Receptor Cell; Resistance; Resolution; Roche brand of trastuzumab; Sampling; Scanning; Signal Pathway; Signal Transduction; Solid; Structure; Surface; System; Tail; Techniques; Technology; Testing; Time; Validation; Work; antitumor drug; base; design; drug candidate; electric impedance; fluorescence imaging; image processing; improved; innovation; insight; millisecond; neoplastic cell; novel; panitumumab; plasmonics; pre-clinical; real world application; receptor; receptor binding; research study; response; sensor; small molecule; submicron; success; therapeutic evaluation; tool

DESCRIPTION (provided by applicant): Proteins embedded in or attached to cell membranes perform many critically important biological functions, including cell signaling, communication and the transport of vital substances into and out of cells. They are also the most important drug targets and disease biomarkers. Despite the importance, studying membrane proteins, especially quantifying their interactions with other molecules, such as drug candidates, has been a difficult challenge. Current methods rely on either labeling the proteins with fluorescent tags or extracting them from their native membrane environment, and then purifying and immobilizing them on a surface for binding kinetic studies. The former approach is an end-point-assay, which does not provide kinetics information required for quantifying protein interactions, while the latter is not only labor-intensive but also prone to alternation of the native structures and functions of the membrane proteins. This project focuses on developing a novel technique for studying and quantifying membrane protein interactions in their native cellular environment without the need of extraction, purification, or immobilization. The core of the technique is plasmonic-based electrical impedance microscopy (P-EIM) recently invented in the PIs' lab, which has several unique capabilities: 1) It is label free and can provide quantitative analysis of binding kinetics; 2) It has a high spatial resolution (sub-microns), and thus is suitable for analyzing membrane protein binding activities of single cells, and for mapping local binding kinetics of membrane proteins within a single cell; 3) It is fast (millisecond time resolution), which enables real-time tracking of cell signal transduction cascade triggered by small molecule binding to membrane proteins. Additionally, the technique allows for simultaneous plasmonic, impedance and fluorescence imaging, and combines the strengths of these methods in one system.

描述(申请人提供)：嵌入或附着在细胞膜上的蛋白质执行许多至关重要的生物学功能，包括细胞信号、通讯和重要物质的进出细胞的运输。它们也是最重要的药物靶点和疾病生物标志物。尽管膜蛋白很重要，但研究膜蛋白，特别是量化它们与其他分子的相互作用，如候选药物，一直是一个艰巨的挑战。目前的方法要么用荧光标记蛋白质，要么从天然的膜环境中提取蛋白质，然后将它们纯化并固定在表面上进行结合动力学研究。前一种方法是终点法，它不提供量化蛋白质相互作用所需的动力学信息，而后一种方法不仅劳动密集型，而且容易改变天然结构 以及膜蛋白的功能。该项目致力于开发一种新的技术来研究和定量膜蛋白在天然细胞环境中的相互作用，而不需要提取、纯化或固定化。该技术的核心是PIS实验室最近发明的基于等离子体的电阻抗显微镜(P-EIM)，它具有几个独特的功能：1)它是免标记的，可以提供对 结合动力学；2)它具有高的空间分辨率(亚微米)，因此适合于分析单个细胞的膜蛋白结合活性，并适用于绘制单个细胞内膜蛋白的局部结合动力学图；3)它具有快速(毫秒级的时间分辨率)，能够实时跟踪小分子与膜蛋白结合引发的细胞信号转导级联。此外，该技术允许同时进行等离子成像、阻抗成像和荧光成像，并在一个系统中结合了这些方法的优点。