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.

项目摘要/摘要 碳水化合物链，称为聚糖，是广泛的生物过程的基础，包括 抗凝，细胞生长和发育，细胞 - 细胞通信，免疫识别/反应以及 微生物发病机理。聚糖在人类疾病中也具有重要的特征，包括大多数人 癌症。现在认识到在细胞表面上的聚糖的空间布置，以告知人生最关键的 细胞和多细胞行为，包括生长，细胞脂肪过渡和组织组装。那， 该提案的基本假设是，更好地理解了聚糖在两​​种健康中的作用 可以通过一组全面的分子工具来实现疾病 空间分辨率。近年来见证了具有强大新方法的光学成像 在纳米长度尺度上。一种革命性的方法，称为扩展显微镜（EXM），可实现超大型 通过在聚电解质基质中溶胀样品进行详细的结构成像，以便可以是纳米级特征 在常规荧光显微镜上分析。不幸的是，这些方法尚未得到利用 由于缺乏兼容的成像试剂，糖生物学的发展 生物学上重要的聚糖表位（糖基植物），这些表位是针对高级超分辨率定制的 显微镜方法，包括EXM。为了应对这些挑战，该提议试图创建成像 利用多功能化学问题和 糖特异性亲和力试剂，用于可容纳检测和纳米分辨率的细胞聚糖和纳米分辨率 糖缀合物。 （目标1）基于多功能的多功能化学问题套件 将开发寡硫代乙醚（寡素），以用于代谢标记的非靶向糖成像 在广泛可用的共聚焦显微镜上具有多达15 nm有效分辨率的聚糖。 （目标2）一种方法 从非免疫库中隔离自定义亲和力试剂将适用于多重选择 糖特异性抗体（糖基抗体）。 （AIM 3）将针对靶向糖胶体进行优化糖基因 通过蛋白质工程和寡凝结结合，导致试剂将增强分辨率 细胞和组织的EXM。将为顶级性能试剂开发详细的协议，并将包括 有关糖基体，最佳试剂浓度和经过验证成像的可再生生物合成的信息 状况。除了开发此试剂“操作器手册”之外，该项目还将使用寡头 和糖酶以探测不同正常表面上正宗糖签名的空间表达 例如，癌细胞系提供（1）免疫调节性聚糖的超级分辨图 糖卵形和（2）假定的半乳糖素晶格的第一个详细分子结构。总体而言，提议 Glycoexm平台有可能在范围范围内实现高含量成像，而低成本 用户友好将确保GlyCoscience社区及其他地区可以广泛使用。