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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

基本信息

批准号：
9983074
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
项目类别：
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/9983074
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

项目摘要

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.

项目总结