Harnessing genetic code expansion to measure in vivo actin dynamics

利用遗传密码扩展来测量体内肌动蛋白动力学

• 批准号: 9813932
• 负责人: Margot E Quinlan
• 金额: $ 22.79万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9813932
• 关键词: Acetylation; Actin-Binding Protein; Actins; Alleles; Amber; Amino Acids; Amino Acyl-tRNA Synthetases; Animal Model; Animals; Biochemical; Biological; Biological Assay; Biological Models; Cell physiology; Cells; Cellular biology; Chemicals; Chemistry; Complex; Data; Development; Diels Alder reaction; Drosophila genus; Drosophila melanogaster; Explosion; Family; Filament; Fluorescent Probes; Genetic; Genetic Code; Genetic Models; Genetic Screening; Goals; Grant; Health; Image; Imagery; Knowledge; Label; Life; Measurement; Measures; Metals; Methodology; Methylation; Microfilaments; Molecular; Phosphorylation; Physiology; Plasmids; Positioning Attribute; Post-Translational Protein Processing; Process; Property; Protein Isoforms; Proteins; Publishing; Reporter; Research; Resolution; Role; Saccharomycetales; Side; Site; Speed; Structure; Surface; System; Time; Variant; Vertebral column; Work; Yeasts; actin 2; base; cell motility; cell type; cellular imaging; experimental study; fluorophore; fly; human disease; in vivo; in vivo imaging; monomer; novel strategies; response; skeletal; success; tool

Summary Our goal is to establish tools to directly label actin in any model system that can utilize genetic code expansion. Although we know a great deal about actin and the molecular components of cytoskeletal structures, we still know very little about actin dynamics that are essential to the functions of these structures. Our ability to establish mechanistic understandings of actin structures is fundamental to our knowledge of cell biology and human disease. We are limited by the availability of research tools for quantitative measurement of in vivo actin dynamics. Pinpointing a position on actin that will tolerate change is not easy due to the extensive intrafilament interfaces and the surfaces that interact with the >100 actin binding proteins. Genetic tags as small as 12 amino acids disrupt multiple cellular processes. In response to this need, we propose to take advantage of the exciting new capabilities of genetic code expansion and recently published high resolution structures of actin filaments. We will use orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs to site-specifically incorporate non-canonical amino acids (ncAAs) with reactive side chains at carefully chosen positions on actin. Using the inverse demand Diels-Alder reaction (a significantly faster variant of metal-free click chemistry) we will add fluorophores to the ncAA for in vivo imaging. We expect to be able to modify a single amino acid within actin, without disrupting function, based on the fact that actin covalently labeled with a small fluorescent probe is functional. Further, previous work shows that labeling only ~2% of actin is sufficient for visualization of most structures. Thus slight perturbations and/or low incorporation efficiency will not be a hindrance to proof-of- principle experiments. First, we will identify candidate positions for ncAA incorporation using a genetic screen. Initially, we will work in the powerful genetic model organism budding yeast, Saccaromyces cerevisiae. Because of its 87% sequence identity with skeletal actin, yeast actin has been studied for decades, providing extensive data about surface residues and powerful, yet simple, assays of actin function. Genetic code expansion is established in yeast; and, importantly, in the context of this grant, yeast work is fast. Once we have established proof-of-principle, we will shift to the fruit fly, Drosophila melanogaster. The fly is another powerful model organism that offers a broad range of genetic tools. Genetic code expansion has been demonstrated in both Drosophila-derived S2 cells and the fly. Being able to work in a relatively high throughput manner with S2 cells before moving to whole animals makes Drosophila an ideal system in which to expand. Success will result in a strategy to directly label actin in essentially every model system and tools already working in yeast and S2 cells. Success in labeling actin, will lead to major advances in our understanding of its dynamics within the cell, and provide a much needed tool to study the vast array of actin structures essential to life and health. The methodology will also provide a new approach to study closely related actin isoforms, the distinct roles of which remain poorly understood.

总结 我们的目标是建立工具，直接标记肌动蛋白在任何模型系统，可以利用遗传密码 扩张.虽然我们对肌动蛋白和细胞骨架结构的分子组成了解很多， 我们对这些结构的功能所必需的肌动蛋白动力学仍然知之甚少。的能力 建立肌动蛋白结构的机械理解是我们细胞生物学知识的基础， 人类疾病。我们受限于研究工具的可用性定量测量在体内肌动蛋白 动力学精确定位肌动蛋白的位置，将容忍变化是不容易的，由于广泛的内丝 界面和表面与>100肌动蛋白结合蛋白相互作用。基因标签小至12个氨基酸 酸破坏多种细胞过程。为了满足这一需求，我们建议利用令人兴奋的 遗传密码扩展的新能力和最近发表的肌动蛋白丝的高分辨率结构。 我们将使用正交琥珀抑制氨酰-tRNA合成酶/tRNA对， 在肌动蛋白上精心选择的位置具有反应性侧链的非典型氨基酸（ncAA）。使用 我们将添加反向需求Diels-Alder反应（无金属点击化学的显著更快的变体） 将荧光团与ncAA结合用于体内成像。我们希望能够修饰肌动蛋白中的单个氨基酸， 而不破坏功能，基于这样的事实，即用小的荧光探针共价标记的肌动蛋白， 不降低此外，以前的工作表明，标记只有约2%的肌动蛋白是足够的可视化大多数 结构.因此，轻微的扰动和/或低的掺入效率将不会成为证明的障碍。 原则实验。 首先，我们将使用遗传筛选确定ncAA掺入的候选位置。首先，我们将工作 在强大的遗传模式生物芽殖酵母，酿酒酵母。因为它87%的序列 与骨骼肌动蛋白相同，酵母肌动蛋白已经研究了几十年，提供了关于表面 残留物和强大而简单的肌动蛋白功能测定。在酵母中建立遗传密码扩增;以及， 重要的是，在这项赠款的背景下，酵母工作是快速的。一旦我们建立了原则证明，我们将 转移到果蝇，黑腹果蝇。苍蝇是另一种强大的模式生物， 一系列基因工具。遗传密码扩增已经在果蝇衍生的S2细胞和 苍蝇在转移到整个动物之前，能够以相对高通量的方式使用S2细胞 使果蝇成为一个理想的扩张系统。成功将导致一种直接标记肌动蛋白的策略， 基本上每个模型系统和工具都已经在酵母和S2细胞中工作了。成功标记肌动蛋白，将 导致我们对其在细胞内动力学的理解取得重大进展，并提供了一个急需的工具， 研究对生命和健康至关重要的大量肌动蛋白结构。该方法还将提供新的 方法来研究密切相关的肌动蛋白亚型，其独特的作用仍然知之甚少。