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

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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.