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Molecular toolkit for high content resolution of glycomes by expansionmicroscopy

通过膨胀显微镜实现糖组高含量分辨率的分子工具包

基本信息

批准号：
10582565
负责人：
Christopher Akinleye Alabi
金额：
$ 39.64万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10582565
关键词：
Address Affinity Alkynes Anabolism Antibodies Antibody Affinity Anticoagulation Azides Bacteria Binding Proteins Biological Biological Process Cancer cell line Carbohydrates Catalogs Cell Communication Cell surface Cells Chemicals Chemistry Code Communities Complex Coupling Custom Detection Development Directed Molecular Evolution Disease Engineering Ensure Epitopes Etiology Evolution Fluorescence Fluorescent Dyes Fluorescent Probes Galactose Binding Lectin Gel Generations Glycobiology Glycocalyx Glycoconjugates Growth Growth and Development function Health Human Image Imaging Device Imaging Techniques Imaging technology Immune Immunofluorescence Immunologic Immunoglobulin Fragments Individual Investigation Label Length Libraries Life Light Lipids Malignant Neoplasms Manuals Maps Mediation Metabolic Methods Microarray Analysis Microscope Microscopy Molecular Molecular Structure Monosaccharides Noise Normal Cell Optics Organism Pathogenesis Physiology Play Polysaccharides Process Protein Engineering Proteins Protocols documentation Reaction Reagent Reporter Research Resolution Role Sample Size Sampling Signal Transduction Structure Surface Surface Plasmon Resonance Swelling System Technology Tissues Validation Visualization Yeasts cell behavior cell growth chemical group chemical synthesis cost density established cell line experimental study fluorescence microscope human disease immunoregulation light microscopy metabolic imaging microbial microscopic imaging nanometer nanometer resolution nanoscale novel strategies optical imaging polypeptide protein function protein structure protocol development rational design receptor response structural imaging sugar superresolution imaging superresolution microscopy tool ultra high resolution

项目摘要

Project Summary/Abstract Carbohydrate chains, known as glycans, are fundamental to a wide spectrum of biological processes, including anticoagulation, cell growth and development, cell-cell communication, immune recognition/response, and microbial pathogenesis. Glycans also feature prominently in human disease, including the majority of human cancers. The spatial arrangements of glycans on the cell surface are now recognized to inform life’s most critical cellular and multicellular behaviors, including growth, cell fate transitions, and tissue assembly. Thus, the underlying hypothesis of the proposal is that a better understanding of the role that glycans play in both health and disease could be achieved with a comprehensive set of molecular tools for imaging the glycome with high spatial resolution. Recent years have witnessed the emergence of powerful new approaches for optical imaging at nanometer lengths scales. One revolutionary approach, called expansion microscopy (ExM), achieves ultra- detailed structural imaging by swelling samples in a polyelectrolyte matrix so that nanoscale features can be resolved on conventional fluorescence microscopes. Unfortunately, these approaches have yet to be harnessed for advancement of glycobiology due to a lack of compatible imaging reagents that specifically recognize biologically important glycan epitopes (glycotopes) and that are customized for advanced super-resolution microscopy methods including ExM. To address these challenges, this proposal seeks to create an imaging technology called glycoconjugate ExM (GlycoExM) that leverages multifunctional chemical probes and glycotope-specific affinity reagents for facile detection and nanometer resolution of cellular glycans and glycoconjugates. (Aim 1) A suite of multifunctional chemical probes based on multifunctional oligothioetheramides (oligoTEAs) will be developed for untargeted GlycoExM imaging of metabolically labeled glycans on widely available confocal microscopes with up to 15 nm effective resolution. (Aim 2) A method for isolating custom affinity reagents from non-immune libraries will be adapted for multiplexable selections of glycotope-specific antibodies (glycobodies). (Aim 3) Glycobodies will be optimized for targeted GlycoExM through protein engineering and oligoTEA conjugation, leading to reagents that will enhance the resolution of ExM for cells and tissues. Detailed protocols will be developed for the top performing reagents and will include information on renewable biosynthesis of glycobodies, optimal reagent concentrations, and validated imaging conditions. In addition to the development of this reagent “operator’s manual,” the project will also use oligoTEAs and glycobodies to probe the spatial expression of authentic glycan signatures on the surfaces of different normal and cancer cell lines to, for example, provide (1) super-resolved maps of immunomodulatory glycans in the glycocalyx and (2) the first detailed molecular structure of the putative galectin lattice. Overall, the proposed GlycoExM platform has the potential to enable high-content imaging on a glycome-wide scale, while the low-cost and user-friendliness will ensure that it becomes broadly accessible to the glycoscience community and beyond.

项目总结/文摘