New Technology for Tracking Proteins by Light and Electron Microscopy

通过光学和电子显微镜追踪蛋白质的新技术

• 批准号: 9753275
• 负责人: Kimberly Elizabeth Beatty
• 金额: $ 39.51万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753275
• 关键词: Address; Affinity; Binding; Binding Proteins; Caveolae; Cell Nucleus; Cell physiology; Cell surface; Cells; Cellular Structures; Chemicals; Chemistry; Chimeric Proteins; Clathrin; Clathrin-Coated Vesicles; Color; Complex; Cytosol; Development; Dimerization; Disease; Electron Microscopy; Electrons; Endocytosis; Endosomes; Engineering; Environment; Fluorescence Microscopy; Funding; Gold; Health; Hemochromatosis; Hereditary hemochromatosis; Human; Image; Imaging technology; In Vitro; Investigation; Iron; Kinetics; LIGHT protein; Label; Light; Mediating; Membrane; Membrane Proteins; Methods; Microscopy; Mission; Monitor; Multiprotein Complexes; Names; Nanostructures; Organism; Peptides; Property; Protein Chemistry; Proteins; Protocols documentation; Quantum Dots; Receptor Cell; Receptor Signaling; Reporter; Research; Research Personnel; Research Project Grants; Resolution; Sorting - Cell Movement; Specificity; Structure; TFRC gene; Technology; Transferrin; United States National Institutes of Health; Work; cellular imaging; coated pit; cross reactivity; dimer; fluorophore; high resolution imaging; imaging platform; improved; instrumentation; interest; light microscopy; macromolecule; nanoparticle; nanoscale; new technology; particle; protein aminoacid sequence; protein transport; receptor; sensor; tool; trafficking; transferrin receptor 2; uptake

Project Summary Recent developments in fluorescence microscopy (FM), electron microscopy (EM), and correlative light and EM (CLEM) offer unprecedented opportunities for illuminating cellular structures at the nanoscale. It is now feasible to visualize and quantify the spatial organization of proteins and other macromolecules that enable cells to sense and respond to their environment. However, these efforts are restricted by the shortage of methods for attaching FM-, EM-, and CLEM-compatible reporter chemistries to target proteins. We will address this limitation by developing a new technology for labeling and imaging multiple cellular proteins at once. Specifically, we propose to develop a set of heterodimeric coiled-coil tags that will allow specific protein labeling for FM and EM. Our new versatile interacting peptide (VIP) tags will be protein specific and cell compatible. Briefly, one coil (the “tag”) will be genetically-encoded as a fusion to a protein of interest. After expression, the tagged protein will be subsequently labeled via heterodimer formation with a high affinity (KD < 5 nM) “probe peptide”. We identified a set of coils that will self-sort into specific pairs, which will enable up to four proteins to be labeled and imaged simultaneously. The reporter can be bright, photostable fluorophores for FM or electron-dense nanoparticles for EM. In other words, VIP tags are modular, enabling end-users to alternate between state-of-the art FM and EM imaging platforms. We will engineer VIP tags to achieve the high labeling efficiency needed for quantitative analysis of multi-protein interactions. We will develop and validate our technology by investigating the iron-uptake machinery in cells. We will use our technology to determine the differential protein trafficking and interactions of transferrin receptors 1 and 2 (TfR1 and TfR2). TfR1 is a well-studied transmembrane receptor and an ideal target for validating our technology. Our studies of TfR2 will reveal new information on the sub-cellular distribution and trafficking of this recently discovered iron-sensing receptor. We propose to complete two Aims. Aim 1. Develop and validate a set of VIP tags for imaging proteins by FM. Aim 2. Use VIP tags for tracking receptor localization and multi-protein interactions by EM. We believe that the VIP tags, once fully developed and validated, will be an ideal technology for investigating the cellular organization of proteins with nanoscale precision. Furthermore, we believe this new technology has broad utility for imaging cellular processes related to human health and disease.

项目摘要 荧光显微镜(FM)、电子显微镜(EM)和相关光的最新进展 和EM(Clem)为在纳米尺度上照亮细胞结构提供了前所未有的机会。它是 现在可以可视化和量化蛋白质和其他大分子的空间组织 使细胞能够感知并对环境做出反应。然而，这些努力受到短缺的限制。 将FM、EM和Clem兼容的报告化学连接到靶蛋白的方法的一部分。 我们将通过开发一种新的标记和成像多个细胞的技术来解决这个限制 一下子就有蛋白质。具体地说，我们建议开发一套异二聚体螺旋线圈标签，它将 允许对FM和EM进行特定的蛋白质标记。我们新的多功能相互作用肽(VIP)标签将是 蛋白质专一性和细胞相容性。简而言之，一个线圈(“标签”)将被基因编码为与 感兴趣的蛋白质。在表达后，标记的蛋白质将随后通过异源二聚体形成而被标记 具有高亲和力(Kd&lt；5 NM)的“探针肽”。我们确定了一组线圈，它们会自动分类成特定的线圈对， 这将使多达四种蛋白质同时被标记和成像。记者可以很聪明， 用于FM的光稳定性荧光团或用于EM的电子致密纳米颗粒。换句话说，VIP标签是模块化的， 使最终用户能够在最先进的FM和EM成像平台之间切换。我们将为VIP设计工程师 标签，以实现定量分析多蛋白质相互作用所需的高标记效率。 我们将通过研究细胞中的铁吸收机制来开发和验证我们的技术。我们会 使用我们的技术来确定转铁蛋白受体1的差异蛋白运输和相互作用 和2(TfR1和TfR2)。TfR1是一个研究得很好的跨膜受体，也是验证我们的 技术我们对TfR2的研究将揭示关于亚细胞分布和贩运的新信息 这是最近发现的铁感应受体。我们建议完成两个目标。目标1.发展和 通过FM验证一组用于成像蛋白质的VIP标签。目的2.使用VIP标签跟踪受体定位 和多蛋白质相互作用的EM分析。 我们相信，VIP标签一旦完全开发和验证，将是一项理想的技术 以纳米级的精度研究蛋白质的细胞组织。此外，我们认为这一新的 在成像与人类健康和疾病相关的细胞过程方面，技术具有广泛的实用价值。