CAREER: Multicolor imaging of single mRNA translational heterogeneity in living cells

职业：活细胞中单个 mRNA 翻译异质性的多色成像

• 批准号: 1845761
• 负责人: Timothy Stasevich
• 金额: $ 92.62万
• 依托单位: Colorado State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1845761&HistoricalAwards=false
• 关键词: CAREER; Multicolor; imaging; single; mRNA

The goal of this project is to understand how translation of mRNA to protein is orchestrated at the single molecule level in living cells. Through the creation of open-source tools for fluorescence microscopy, long-standing models of gene regulation will be tested by direct visualization, to better understand and ultimately control protein synthesis in both natural and engineered living systems. This project will provide students and junior scientists with an interdisciplinary and international research experience that will train them in advanced single-molecule microscopy, biosensor engineering, and biophysical modeling of gene regulation. This project will also incorporate inexpensive, open-source video microscopes into a multi-pronged STEM outreach and education effort designed to engage a diverse population of K-12 students and teachers with exciting citizen science. Although protein synthesis is essential to life, the real-time dynamics of mRNA translation to protein have been difficult to capture in vivo. This problem has left many open questions about how protein synthesis is regulated in living systems, particularly at the level of mRNA translation initiation and elongation. This project will answer such questions by combining single-molecule microscopy with the creation of multicolor biosensors that literally light up the heterogeneity of protein synthesis in living cells, one protein at a time. With this powerful combination of technologies, conflicting models of codon usage and open reading frame selection at the level of single mRNA translation will be directly visualized and tested for the first time. This work will lay the foundation for real-time analysis of protein synthesis in vivo, with broad application to both basic research and the biotechnology industry.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

该项目的目标是了解mRNA到蛋白质的翻译是如何在活细胞中的单分子水平上协调的。通过创建用于荧光显微镜的开源工具，将通过直接可视化来测试长期存在的基因调控模型，以更好地理解并最终控制自然和工程生命系统中的蛋白质合成。该项目将为学生和初级科学家提供跨学科和国际研究经验，培养他们先进的单分子显微镜，生物传感器工程和基因调控的生物物理建模。该项目还将把廉价的开源视频显微镜纳入多管齐下的STEM推广和教育工作中，旨在让K-12学生和教师的多样化人群参与令人兴奋的公民科学。 尽管蛋白质合成对生命至关重要，但mRNA翻译为蛋白质的实时动态很难在体内捕获。这个问题留下了许多关于蛋白质合成如何在生命系统中调节的开放问题，特别是在mRNA翻译起始和延伸的水平上。该项目将通过将单分子显微镜与生物传感器的创建相结合来回答这些问题，这些生物传感器可以照亮活细胞中蛋白质合成的异质性，一次一个蛋白质。通过这种强大的技术组合，在单个mRNA翻译水平上密码子使用和开放阅读框架选择的冲突模型将首次直接可视化和测试。这项工作将为实时分析体内蛋白质合成奠定基础，并广泛应用于基础研究和生物技术产业。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

A Multi-color Bicistronic Biosensor to Compare the Translation Dynamics of Different Open Reading Frames at Single-molecule Resolution in Live Cells

一种多色双顺反子生物传感器，用于比较活细胞中单分子分辨率下不同开放阅读框的翻译动力学

• DOI: 10.21769/bioprotoc.4096
• 发表时间: 2021
• 期刊: BIO-PROTOCOL
• 影响因子: 0.8
• 作者: Koch, Amanda;Morisaki, Tatsuya;Stasevich, Timothy
• 通讯作者: Stasevich, Timothy

Generation and diversification of recombinant monoclonal antibodies.

• DOI: 10.7554/elife.72093
• 发表时间: 2021-12-31
• 期刊: eLife
• 影响因子: 7.7
• 作者: DeLuca KF;Mick JE;Ide AH;Lima WC;Sherman L;Schaller KL;Anderson SM;Zhao N;Stasevich TJ;Varma D;Nilsson J;DeLuca JG
• 通讯作者: DeLuca JG