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.

摘要 后基因组时代的一个中心挑战是全面表征~20,000的细胞角色 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基因编辑流水线，使内源性标签人的大规模产生成为可能 细胞系，并为蛋白质组范围内分析蛋白质定位和相互作用网络奠定了基础。 原生蜂窝环境。在之前的资助项目中，我们建立了细胞工程平台 并展示了这种方法在创建OpenCell文库(约1300个靶向蛋白质)方面的威力 以及它的成像和蛋白质组学特征。尽管如此，在长期实时成像方面仍然存在技术挑战 蛋白质动力学和对这些电影的定量解释。在建议的重建计划中，我们计划进一步 发展了表观照明选择平面照明显微镜(ESPIM)技术，使其成为一种广泛的 适用的活细胞显微镜平台，用于低密度亚细胞动力学的高分辨率、长期成像 光漂白和光毒性。我们还将为仪器开发相应的软件包 控制和图像处理，使其能够轻松地被其他实验室采用。最后，我们将演示 这一技术平台通过细胞周期筛选异常蛋白质动力学的适用性 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。

• DOI: 10.1101/2023.12.19.572435
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Ivanov,IvanE;Hirata-Miyasaki,Eduardo;Chandler,Talon;Kovilakam,RasmiCheloor;Liu,Ziwen;Liu,Chad;Leonetti,ManuelD;Huang,Bo;Mehta,ShalinB
• 通讯作者: Mehta,ShalinB