Single-molecule fluorescence microscopy of intracellular protein dynamics in live bacteria without fluorescent proteins

无荧光蛋白的活细菌细胞内蛋白质动力学的单分子荧光显微镜

• 批准号: BB/N006070/1
• 负责人: Richard Berry
• 金额: $ 56.87万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN006070%2F1
• 关键词: Single; molecule; fluorescence; microscopy; intracellular

Proteins are the workhorses of living cells, and how they work is a topic of enormous importance. Currently, one of the best ways to find out is to watch proteins going about their business in living cells, in real time, using fluorescent microscopy. This requires fluorescent labels to be added to protein molecules, which are not themselves visible against the background of the rest of the cell. The current state-of-the-art is to use fluorescent proteins, which can be fused to any protein of interest by genetic engineering. A huge amount has been learned by this method, and it will continue to be a vital tool across the life sciences. Our lab has been part of this progress for a decade. We have used fluorescent protein fusions to discover the composition of the bacterial flagellar motor - a self-assembled nano-scale rotary electrical motor that propels swimming bacteria - and to discover that most of the protein molecules that make up this and other large biological machines are constantly exchanging between the machine and a pool of circulating spare parts in the cell. While the machine continues to work!Fluorescent proteins however have their limitations. They are not particularly good fluorescent labels, compared to small organic dye molecules that are now commercially available which are brighter and last longer before "photobleaching". This limits how much can be learned about the behavior of each labelled protein molecule, before the label bleaches and the protein molecule becomes invisible again. Also, fluorescent proteins are big and can only easily be attached at either end of the molecular chain that folds up to make each protein molecule. Because of this, they usually compromise the function of the chosen protein, and often completely abolish it. By contrast, organic dyes are much smaller and can be added anywhere on the protein surface by genetically engineering an appropriate tag for them to stick to. For these reasons, most investigations of proteins done OUTSIDE of living cells, with purified proteins in artificial model systems, use small organic dyes and not fluorescent proteins as labels. But until now it has not been possible to put these small-labelled proteins INSIDE cells.A new method has recently been developed in our building that allows us to bring the advantages of small dye labels to work inside live cells. The proteins are purified and labelled as for work outside cells, and then put into cells using a method called electroporation - which is a standard way of getting DNA into cells for genetic engineering. With the help of its inventors, we propose to develop, exploit and popularize this new method. We will bring it to bear on a range of questions arising from the current research in our labs. The long term aim is to establish this as an additional method for studying in vivo protein behavior across biological systems. We can already track single signaling molecules for tens of seconds as they shuttle between the sensory cluster that detects the external environment and the flagellar motor that responds to it. Watching individual molecules for long times will tell us in detail how this system works, and we will use the same method on at least half a dozen related systems to see what we can learn. As always with a new method, we can expect some confirmations of what was expected and some surprises.

蛋白质是活细胞的主要组成部分，它们如何工作是一个非常重要的话题。目前，最好的方法之一是使用荧光显微镜观察蛋白质在活细胞中的工作，在真实的时间。这需要将荧光标记添加到蛋白质分子上，这些分子本身在细胞其余部分的背景下是不可见的。目前最先进的技术是使用荧光蛋白，其可以通过基因工程融合到任何感兴趣的蛋白质。通过这种方法已经学到了很多东西，它将继续成为生命科学的重要工具。十年来，我们的实验室一直是这一进步的一部分。我们已经使用荧光蛋白融合来发现细菌鞭毛马达的组成-一种自组装的纳米级旋转电动马达，推动游泳的细菌-并发现构成这种和其他大型生物机器的大多数蛋白质分子不断在机器和细胞中循环的备用部件之间交换。当机器继续工作的时候！然而，荧光蛋白有其局限性。它们不是特别好的荧光标记，与现在市售的小有机染料分子相比，它们在“光漂白”之前更亮且持续时间更长。这限制了在标签漂白和蛋白质分子再次变得不可见之前，对每个标记的蛋白质分子的行为可以了解多少。此外，荧光蛋白很大，只能很容易地连接在分子链的两端，折叠起来，使每个蛋白质分子。因此，它们通常会损害所选蛋白质的功能，甚至完全破坏它。相比之下，有机染料要小得多，可以通过基因工程设计一个合适的标签来附着在蛋白质表面的任何地方。由于这些原因，大多数在活细胞外进行的蛋白质研究，在人工模型系统中使用纯化的蛋白质，使用小的有机染料而不是荧光蛋白作为标记。但是直到现在还不可能把这些小标记的蛋白质放在细胞内。最近在我们的大楼里开发了一种新方法，使我们能够把小染料标记的优点带到活细胞内。蛋白质经过纯化和标记，以便在细胞外工作，然后使用一种称为电穿孔的方法将其放入细胞中--这是将DNA送入细胞进行基因工程的标准方法。在其发明者的帮助下，我们建议开发、利用和推广这种新方法。我们将用它来解决我们实验室目前研究中出现的一系列问题。长期目标是将其建立为研究生物系统中体内蛋白质行为的额外方法。当单个信号分子穿梭于感知外界环境的感觉簇和对外界环境做出反应的鞭毛运动之间时，我们已经可以跟踪它们几十秒，长时间观察单个分子将详细地告诉我们这个系统是如何工作的，我们将在至少六个相关系统上使用同样的方法，看看我们能学到什么。和往常一样，对于一种新方法，我们可以期待一些对预期的确认和一些惊喜。

项目成果

Imaging of Single Dye-Labeled Chemotaxis Proteins in Live Bacteria Using Electroporation.

使用电穿孔对活细菌中单染料标记的趋化蛋白进行成像。

• DOI: 10.1007/978-1-4939-7577-8_19
• 发表时间: 2018
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Di Paolo D

Mutations targeting the plug‐domain of the Shewanella oneidensis proton‐driven stator allow swimming at increased viscosity and under anaerobic conditions

• DOI: 10.1111/mmi.13499
• 发表时间: 2016-12
• 期刊: Molecular Microbiology
• 影响因子: 3.6
• 作者: Susanne Brenzinger;L. Dewenter;Nicolas Delalez;Oliver Leicht;Volker Berndt;A. Paulick;R. Berry;M. Thanbichler;J. Armitage;Berenike Maier;K. Thormann

Measurement of Macromolecular Crowding in Rhodobacter sphaeroides under Different Growth Conditions.

• DOI: 10.1128/mbio.03672-21
• 发表时间: 2022-02-22
• 期刊: mBio
• 影响因子: 6.4
• 作者: Khoo JH;Miller H;Armitage JP
• 通讯作者: Armitage JP

The power of three spatial dimensions.

三个空间维度的力量。

• DOI: 10.1038/s41579-019-0260-z
• 发表时间: 2019
• 期刊: Nature reviews. Microbiology
• 作者: Khoo JH

Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli.

• DOI: 10.1098/rstb.2015.0492
• 发表时间: 2016-11-05
• 期刊: Philosophical transactions of the Royal Society of London. Series B, Biological sciences
• 作者: Di Paolo D;Afanzar O;Armitage JP;Berry RM
• 通讯作者: Berry RM