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

蛋白质是活细胞的主力，它们是如何工作的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实时观察活细胞中蛋白质的活动。这需要将荧光标记添加到蛋白质分子上，而蛋白质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，它可以通过基因工程融合到任何感兴趣的蛋白质上。通过这种方法，我们学到了很多东西，它将继续成为生命科学领域的重要工具。十年来，我们的实验室一直是这一进步的一部分。我们使用荧光蛋白融合物来发现细菌鞭毛马达的组成——鞭毛马达是一种自我组装的纳米级旋转电动机，推动细菌游泳——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在机器和细胞中循环的备用零件池之间不断交换。当机器继续工作时！然而，荧光蛋白有其局限性。它们并不是特别好的荧光标签，相比之下，现在市面上可以买到的小有机染料分子更亮，在“光漂白”之前持续时间更长。这就限制了在标签褪色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，只能很容易地附着在折叠成每个蛋白质分子的分子链的两端。正因为如此，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，并且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，在人工模型系统中纯化蛋白质，使用小的有机染料而不是荧光蛋白作为标记。但是到目前为止，还不可能将这些小标记的蛋白质放入细胞内。我们最近开发了一种新方法，使我们能够将小染料标签的优点应用到活细胞中。这些蛋白质被纯化并标记为在细胞外工作，然后使用一种称为电穿孔的方法将其放入细胞中——这是一种将DNA导入细胞进行基因工程的标准方法。在发明者的帮助下，我们建议开发、利用和推广这种新方法。我们将把它应用到我们实验室当前研究中产生的一系列问题上。长期目标是将其建立为跨生物系统研究体内蛋白质行为的另一种方法。当单个信号分子在检测外部环境的感觉细胞群和对其做出反应的鞭毛马达之间穿梭时，我们已经可以在几十秒内跟踪它们。长时间观察单个分子将告诉我们这个系统如何工作的细节，我们将在至少六个相关系统上使用相同的方法，看看我们能学到什么。一如既往，对于一种新方法，我们可以期待对预期的一些证实和一些惊喜。

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.