Live-cell intracellular immunofluorescence

活细胞细胞内免疫荧光

基本信息

批准号：
10467023
负责人：
Xiaohu Gao
金额：
$ 21.37万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-10 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10467023
关键词：
Actins Antibodies Binding Biological Biological Markers Biological Process Biology Bypass Cations Cell Nucleus Cell Signaling Process Cell membrane Cell physiology Cells Cellular Structures Charge Chimeric Proteins Cholesterol Clinical Complex Cytoplasm Cytosol DNA Data Diffuse Dimensions Endocytosis Endosomes Evolution Exposure to Flow Cytometry Fluorescent Antibody Technique Future Goals Gold Growth Human body Image Immunofluorescence Immunologic Immunoglobulin Fragments Immunohistochemistry Immunologics Individual Intracellular Space Label Learning Lipids Liposomes Lysosomes Membrane Metabolism Molecular Molecular Biology Nature Organism Pathologic Processes Pathway interactions Peptides Polymers Process Production Proteins Protocols documentation RNA Reproduction Research Resolution Sampling Science Signal Transduction Specificity Surface System Technology Time base cell fixing cellular engineering cytotoxicity delivery vehicle drug development fluorescence imaging hydrophilicity imaging agent imaging probe innovation interest invention live cell imaging macromolecule nanobodies preservation prevent protein function real-time images small molecule temporal measurement three dimensional structure tool

项目摘要

ABSTRACT Immunofluorescence (IF) is one of the most important tools for both basic biology research and clinical sample imaging due to the broad availability of antibodies and the resolution and sensitivity of fluorescence imaging. Despite the tremendous amount of data produced by IF in the past ~60 years since its initial invention, IF suffers from a fundamental limitation: incapability of interrogating intracellular targets in live cells (cells have to be fixed first, thus only providing a snapshot of the dynamic cell signaling process). Considering the 3D structure of cells, there are far more intracellular targets with biological significance than their cell-membrane counterparts, but immunological agents such as antibodies, antibody fragments, peptides, nanobodies, and scFvs, being highly hydrophilic macromolecules, cannot spontaneously cross the cell membranes. To overcome the barriers of cell membrane and endocytosis that are extremely effective in preventing intact biomolecules to enter the cytosol, we propose to develop an intracellular protein delivery technology building on a unique concept: non- covalent cholesterol tagging. In contrast to conventional delivery technologies that are mostly based on endocytosis (inappropriate for live cell imaging due to the high background generated by imaging agents trapped in endosomes), our small-molecule tag enables proteins to permeate through the cell membrane. More importantly, this is achieved without generating pores in the membrane that cause cytotoxicity. If successful, this platform technology should open a whole new dimension for molecular biology, drug development, and cell engineering.

抽象的免疫荧光（如果）是基本生物学研究和临床样本的最重要工具之一由于抗体的广泛可用性以及荧光成像的分辨率和灵敏度而引起的成像。尽管IF在最初发明以来的过去约60年中产生了大量数据，但如果受苦从基本限制中：审问活细胞中细胞内靶标的能力（细胞必须为首先修复，因此仅提供动态单元信号过程的快照）。考虑的3D结构细胞，具有生物学意义的细胞内靶标比其细胞膜对应物具有更大的生物学意义。但是免疫学剂，例如抗体，抗体片段，肽，纳米体和SCFV，是高度亲水的大分子不能自发地越过细胞膜。克服障碍细胞膜和内吞作用，它们在防止完整的生物分子中非常有效细胞质，我们建议以独特的概念开发细胞内蛋白质递送技术建设：非 - 共价胆固醇标记。与传统交付技术相反，这些技术主要基于内吞作用（由于被捕获的成像剂产生的高背景而导致的活细胞成像不合适在内体中），我们的小分子标签使蛋白质可以通过细胞膜渗透。更多的重要的是，这是在不产生引起细胞毒性的膜上产生孔的情况下实现的。如果成功，这平台技术应为分子生物学，药物开发和细胞打开一个全新的维度工程。