New Technology for Tracking Proteins by Light and Electron Microscopy

项目摘要 荧光显微镜（FM）、电子显微镜（EM）和相关光的最新发展 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。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， 使细胞能够感知并响应环境。然而，这些努力受到短缺的限制 将FM-、EM-和CLEM-相容的报告化学物质连接到靶蛋白的方法。 我们将通过开发一种新技术来标记和成像多个细胞， 蛋白质一次具体来说，我们建议开发一套异源二聚体卷曲螺旋标签， 允许FM和EM的特异性蛋白质标记。我们新的多功能相互作用肽（VIP）标签将 蛋白特异性和细胞相容性。简而言之，一个螺旋（"标签"）将被遗传编码为与一个螺旋的融合物。 感兴趣的蛋白质表达后，标记的蛋白质随后将通过异二聚体形成被标记 具有高亲和力（KD <5 nM）的"探针肽"。我们发现了一组线圈，它们会自动分类成特定的对， 这将使多达四种蛋白质能够同时被标记和成像。记者可以很聪明， 用于FM的光稳定荧光团或用于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成像平台之间交替使用。我们将设计VIP 标签，以实现多蛋白质相互作用的定量分析所需的高标记效率。 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。我们将 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是一种经过充分研究的跨膜受体，也是验证我们的研究结果的理想靶点。 技术.我们对TfR2的研究将揭示TfR2在细胞内的亚细胞分布和转运的新信息。 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.开发和 通过FM验证用于成像蛋白质的一组VIP标签。目标二。使用VIP标签跟踪受体定位 和多蛋白质相互作用。 我们相信，VIP标签，一旦充分开发和验证，将是一个理想的技术， 用纳米级的精度研究蛋白质的细胞组织。此外，我们认为， 该技术对于成像与人类健康和疾病相关的细胞过程具有广泛的用途。

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.