Harnessing genetic code expansion to measure in vivo actin dynamics

摘要 我们的目标是建立工具，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 扩张。尽管我们对肌动蛋白和细胞骨架结构的分子组成了解很多， 我们仍然对肌动蛋白动力学知之甚少，而肌动蛋白动力学对这些结构的功能至关重要。我们有能力 建立对肌动蛋白结构的机械理解是我们了解细胞生物学和 人类疾病。我们受到体内肌动蛋白定量测量研究工具的限制 动力学。由于肌动蛋白内有大量的微丝，准确定位其耐受变化的位置并非易事。 与&gt；100肌动蛋白结合蛋白相互作用的界面和表面。小至12个氨基酸的基因标签 酸会扰乱多种细胞过程。为了回应这种需求，我们建议利用令人兴奋的 新的遗传密码扩展能力和最近发表的肌动蛋白细丝的高分辨率结构。 我们将使用正交琥珀抑制因子氨酰-tRNA合成酶/tRNA对来定点整合 非典型氨基酸(NCAA)，在肌动蛋白上精心选择的位置带有反应性侧链。使用 反向需求Diels-Alder反应(一种比无金属点击化学快得多的变体)我们将添加 将荧光团送到NCAA进行活体成像。我们希望能够修饰肌动蛋白中的单一氨基酸， 在不破坏功能的情况下，基于用小荧光探针共价标记的肌动蛋白 功能齐全。此外，以前的工作表明，仅标记~2%的肌动蛋白就足以使大多数 结构。因此，轻微的干扰和/或低的合并效率将不会阻碍证明- 原则性实验。 首先，我们将使用基因筛查来确定NCAA纳入的候选位置。最初，我们将致力于 在强大的遗传模式生物中发芽的酵母，酿酒酵母。因为它87%的序列 酵母肌动蛋白与骨骼肌动蛋白一样，已经被研究了几十年，提供了关于表面的广泛数据。 残基和强大而简单的肌动蛋白功能分析。在酵母中建立了遗传密码扩展；并且， 重要的是，在这笔赠款的背景下，酵母工作是快速的。一旦我们确立了原则证明，我们将 换成果蝇，黑腹果蝇。苍蝇是另一种强大的模式生物，它提供了广泛的 一系列基因工具。在果蝇来源的S2细胞和 苍蝇。在转移到整个动物之前，能够以相对较高的吞吐量处理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细胞中发挥作用。成功标记肌动蛋白，威尔 导致我们在理解细胞内的动力学方面取得重大进展，并提供了亟需的工具来 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异构体的方法，其不同的作用仍然知之甚少。

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.