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

• 批准号: 10377932
• 负责人: Christopher Akinleye Alabi
• 金额: $ 40.56万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 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/10377932
• 关键词: Address; Affinity; Alkynes; Anabolism; Antibodies; Antibody Affinity; Anticoagulation; Attenuated; Azides; Bacteria; Behavior; 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; Receptor Signaling; Reporter; Research; Resolution; Role; Sample Size; Sampling; Signal Transduction; Structure; Surface; Surface Plasmon Resonance; Swelling; System; Technology; Tissues; Validation; Yeasts; base; 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; nanometer; nanometer resolution; nanoscale; novel strategies; optical imaging; polypeptide; protein structure function; protocol development; rational design; response; sugar; tool; user-friendly

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.

项目概要/摘要 碳水化合物链，称为聚糖，是多种生物过程的基础，包括 抗凝、细胞生长和发育、细胞间通讯、免疫识别/反应以及 微生物发病机制在人类疾病（包括大多数人类疾病）中也发挥着重要作用。 现在人们认识到细胞表面聚糖的空间排列可以为生命中最关键的信息提供信息。 细胞和多细胞行为，包括生长、细胞命运转变和组织组装。 该提案的基本假设是更好地了解聚糖在健康和健康中所发挥的作用 可以通过一套全面的分子工具来对糖组进行高成像成像，从而实现疾病的诊断 近年来，出现了强大的光学成像新方法。 一种称为扩展显微镜 (ExM) 的革命性方法可实现超纳米尺度。 通过在聚电解质基质中溶胀样品来进行详细的结构成像，从而可以获得纳米级特征 不幸的是，这些方法尚未得到利用。 由于缺乏专门识别的兼容成像试剂，糖生物学的进步 生物学上重要的聚糖表位（糖表位），并且是为高级超分辨率定制的 为了解决这些挑战，本提案寻求创建一种成像方法。 称为糖复合物 ExM (GlycoExM) 的技术利用多功能化学探针 糖表位特异性亲和试剂，用于细胞聚糖的简便检测和纳米分辨率 (目标 1) 一套基于多功能的化学探针。 寡硫醚酰胺（oligoTEAs）将被开发用于代谢标记的非靶向 GlycoExM 成像 在广泛使用的共焦显微镜上观察聚糖，有效分辨率高达 15 nm（目标 2）一种方法。 从非免疫文库中分离定制亲和试剂将适用于多重选择 糖表位特异性抗体（糖体）（目标 3）糖体将针对目标 GlycoExM 进行优化。 通过蛋白质工程和oligoTEA缀合，产生可提高分辨率的试剂 将为最佳性能的试剂开发详细的细胞和组织实验方案，其中包括。 有关糖体可再生生物合成、最佳试剂浓度和经过验证的成像的信息 除了开发该试剂“操作手册”外，该项目还将使用oligoTEAs。 和糖体来探测不同法线表面真实聚糖特征的空间表达 和癌细胞系，例如，提供（1）免疫调节聚糖的超分辨图谱 糖萼和（2）推定的半乳糖凝集素晶格的第一个详细分子结构。 GlycoExM 平台有潜力在糖组范围内实现高内涵成像，同时低成本 用户友好性将确保它能够被糖科学界及其他领域广泛使用。