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

基本信息

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
项目类别：
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

项目摘要

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.

概括我们的目标是建立在任何可以利用遗传密码的模型系统中直接标记肌动蛋白的工具扩张。尽管我们对肌动蛋白和细胞骨架结构的分子成分了解很多，我们对对于这些结构的功能至关重要的肌动蛋白动力学仍然知之甚少。我们的能力建立对肌动蛋白结构的机制理解是我们细胞生物学知识的基础人类疾病。我们受到体内肌动蛋白定量测量研究工具可用性的限制动力学。由于丝内广泛，精确定位肌动蛋白上能够耐受变化的位置并不容易与超过 100 个肌动蛋白结合蛋白相互作用的界面和表面。小至 12 个氨基的基因标签酸会破坏多种细胞过程。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基-tRNA 合成酶/tRNA 对进行位点特异性整合在肌动蛋白上精心选择的位置上具有反应性侧链的非规范氨基酸 (ncAA)。使用逆需求狄尔斯-阿尔德反应（无金属点击化学的一种明显更快的变体）我们将添加荧光团与 ncAA 结合用于体内成像。我们期望能够修饰肌动蛋白内的单个氨基酸，不破坏功能，基于这样的事实，用小荧光探针共价标记的肌动蛋白是功能性的。此外，之前的工作表明，仅标记约 2% 的肌动蛋白就足以可视化大多数肌动蛋白。结构。因此，轻微的扰动和/或低整合效率不会成为证明的障碍原理实验。首先，我们将使用遗传筛选来确定 ncAA 掺入的候选位置。最初，我们将工作在强大的遗传模型生物芽殖酵母，酿酒酵母中。由于其 87% 的序列与骨骼肌动蛋白相同，酵母肌动蛋白已经被研究了数十年，提供了有关表面的大量数据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展；和，重要的是，在这笔资助的背景下，酵母的工作速度很快。一旦我们建立了原理验证，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，它提供了广泛的一系列遗传工具。遗传密码扩展已在果蝇衍生的 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 细胞使果蝇成为理想的扩展系统。成功将导致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。成功标记肌动蛋白，将导致我们对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。该方法还将提供一种新的研究密切相关的肌动蛋白亚型的方法，其独特的作用仍然知之甚少。