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

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.

We will develop, optimize and test the use of a new method for introducing stable fluorescently labelled proteins into whole, living bacteria by electroporation. The long term aim is to establish this as an additional method for studying in vivo protein behavior across biological systems.

Specific aims:

1. Optimize delivery of functional proteins labelled with organic dyes into live bacteria by electroporation. a. Expression, purification and labelling of proteins following well-established protocols in our labs. b. Delivery of labelled proteins into cells by electroporation using a modification of the standard protocol for introducing DNA. c. Optimization of the growth and the recovery protocols.

2. Test models for signalling and signal integration in both E.coli and R. sphaeroides chemotaxis by tracking single molecules of the response regulator CheY as they bind the receptor cluster and the flagellar motor.

3. Characterize exchange dynamics of soluble components of the flagellar motor and T3SS. Protein exchange has been observed with fluorescent protein fusions, but the dwell times and trajectories of individual molecules are not known. We will measure these directly.

4. Identify the dynamics of divisome proteins in R. sphaeroides. Chromosome segregation and division proteins will be labelled and tracked, to determine their binding partners and the order of events that they orchestrate.

5. Investigate the mechanism of flagellar filament assembly. We will test between competing models of the mechanism of export of flagellar filament proteins and injectisome substrates, by direct observation of single molecules while they are exported.

6. Measure the effects of altering cytoplasmic crowding by either growth state or stress. R. sphaerodies cytoplasm contains densely packed chromoatophores under some growth conditions. We will quantify the effects of this crowding by tracking the diffusion of single labelled chemotaxis proteins.

Expectations for research of this nature:
Helping to understand in detail the molecular machinery of life, which is the goal of this proposal, is a necessary complement to the modern explosion in biological information represented by genome sequencing and other advances in molecular biology. The work will have immediate impact in the research fields of molecular motors, molecular machines, single-molecule biophysics, protein signalling, cell division and protein complexes. In addition to advancing our understanding of the fundamental principles by which molecular machines and protein signalling work, the novel techniques that we propose to develop will find applications in related fields. By bringing the many advantages of small organic dyes to bear upon research on protein tracking in live bacteria, which has hitherto been limited to fluorescent protein fusions, we will open new avenues for researchers in many fields.

Communications and engagement with beneficiaries
The beneficiaries of this research are categorized below.
1. Within industry, the greatest impact will be on companies and researchers working on biomedical problems, in the long term. We are living in the midst of a revolution in the way the life- and physical sciences interact. Developments in genetics and molecular biology in the last few decades have opened the fundamental processes of life to the quantitative scrutiny that previously was the domain of the mathematical and physical sciences. The current research proposal will advance the new paradigm of single-molecule biology, in which fundamental biological processes are investigated and understood in quantitative, mechanistic, microscopic detail. These advances will allow insights into areas such as protein signalling, cell division and protein complexes, enabling these companies to apply this knowledge in the development of new treatments for a wide variety of diseases and in a greater understanding of many signalling pathways within the human body.
2. More broadly, in the medium term, UK science, education and industry in general will benefit. People trained, techniques developed and ideas tested during the course of the project will spread into science, industry and education, enriching the scientific culture that is vital to the success of these areas of the UK economy.
3. The General Public will benefit in both the short and longer term: The scientific understanding of the mechanisms of protein machines and signalling networks is an important example to demonstrate the power of a scientific, rational approach to explain the marvels of nature. This approach is crucial if the UK is to lead the world in the modern knowledge-based economy. Communication with the general public will be through several channels: a web-site that includes a description of the research at a level suitable for an educated layman, research forums, tours of the laboratory as part of the outreach effort of the Oxford Physics department. These impacts will all be rather immediate.5. Emerging nanotechnologies and personal medicine
The long-range economic impact of this research will be in these fields, and this will benefit society as a whole. These are far enough in the future to be very difficult to predict in detail.
Exploitation and Application:
The commercial potential of the methods developed will be assessed and managed by the PI and Co-I, with the assistance of ISIS, the University of Oxford's wholly-owned technology transfer company.