Mapping endogenous protein dynamics in living cells

绘制活细胞内源蛋白质动态图

• 批准号: 10735776
• 负责人: Bo Huang
• 金额: $ 34.72万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735776
• 关键词: 3-Dimensional; Adoption; CRISPR/Cas technology; Cell Cycle; Cell Line; Cell physiology; Cells; Cellular biology; Collaborations; Collection; Computer software; Confocal Microscopy; Cytoplasmic Granules; Data; Data Set; Development; Exhibits; Fluorescence; Fostering; Funding; Generations; Genes; Genomics; Goals; Heterogeneity; Human Cell Line; Human Genome; Image; Immunoprecipitation; Integration Host Factors; Java; Learning; Libraries; Light; Lighting; Mammalian Cell; Maps; Mass Spectrum Analysis; Measurement; Microscope; Microscopy; Optics; Output; Pathologic; Performance; Phase; Photobleaching; Phototoxicity; Physics; Process; Production; Protein Analysis; Protein Dynamics; Proteins; Proteome; Proteomics; Pythons; Regulation; Resolution; Role; Shapes; Signal Transduction; System; Techniques; Time; Virus Diseases; cell behavior; cellular engineering; cellular imaging; computational platform; confocal imaging; denoising; design; experimental study; image processing; imaging platform; improved; instrument; interest; live cell microscopy; movie; nanobodies; protein complex; protein distribution; real-time images; screening; spatiotemporal; technology platform; tool; transfer learning; usability

ABSTRACT A central challenge of the post-genomic era is to comprehensively characterize the cellular role of the ~20,000 proteins encoded in the human genome. For this purpose, we have developed the FP11 tags and a high- efficiency CRISPR/Cas9 gene editing pipeline, enabling large-scale generation of endogenously tagged human cell lines, and paying the way to proteome-wide analysis of protein localization and interaction networks in a native cellular context. In the previously funded preject, we have established the cellular engineering platform and demonstrated the power of such an approach in creating the OpenCell library (~1300 targeted proteins) and its imaging and proteomics characterization. Still, technical challenges remain in long-term live imaging of protein dynamics and quantitative interpretation of these movies. In the proposed renewal, we plan to further develop the epi-illumination selective plane illumination microscopy (eSPIM) technique, turning it into a widely applicable live cell microscopy platform for high-resolution, long-term imaging of subcellular dynamics with low photobleaching and phototoxicity. We will also develop the corresponding software packages for instrument control and image processing to enable its easy adoption by other labs. Finally, we will demonstrate the applicability of this technological platform by screening for unusual protein dynamics through cell cycle using the OpenCell library.

抽象的 后基因组时代的一个核心挑战是全面描述约 20,000 个细胞的细胞作用 人类基因组中编码的蛋白质。为此，我们开发了 FP11 标签和高 高效的 CRISPR/Cas9 基因编辑管道，能够大规模生成内源标记的人类 细胞系，并为蛋白质定位和相互作用网络的蛋白质组范围分析提供了途径 原生细胞环境。在之前资助的项目中，我们建立了细胞工程平台 并展示了这种方法在创建 OpenCell 库（约 1300 个目标蛋白）方面的强大功能 及其成像和蛋白质组学表征。尽管如此，长期实时成像仍然存在技术挑战 这些电影的蛋白质动力学和定量解释。在拟议的更新中，我们计划进一步 开发落射照明选择性平面照明显微镜 (eSPIM) 技术，使其成为一种广泛应用的技术 适用的活细胞显微镜平台，用于亚细胞动力学的高分辨率、长期成像，且低 光漂白和光毒性。我们还将开发相应的仪器软件包 控制和图像处理，使其易于被其他实验室采用。最后，我们将演示 该技术平台的适用性是通过细胞周期筛选不寻常的蛋白质动力学 OpenCell 库。

项目成果

Mantis: high-throughput 4D imaging and analysis of the molecular and physical architecture of cells.

Mantis：细胞分子和物理结构的高通量 4D 成像和分析。

• 发表时间: 2023-12-19
• 作者: Ivanov, Ivan E;Hirata;Chandler, Talon;Kovilakam, Rasmi Cheloor;Liu, Ziwen;Liu, Chad;Leonetti, Manuel D;Huang, Bo;Mehta, Shalin B
• 通讯作者: Mehta, Shalin B