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

Lead Research Organisation: University of Oxford
Department Name: Oxford Physics

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
publication icon
Baker MA (2016) Domain-swap polymerization drives the self-assembly of the bacterial flagellar motor. in Nature structural & molecular biology

publication icon
Di Paolo D (2016) Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

publication icon
Nirody JA (2019) Load-dependent adaptation near zero load in the bacterial flagellar motor. in Journal of the Royal Society, Interface

publication icon
Tusk SE (2018) Subunit Exchange in Protein Complexes. in Journal of molecular biology

 
Title Electroporation of organic dye-labelled proteins in bacteria 
Description The method used to internalise proteins in live bacteria used, adapted from Crawford et al (2013), is based on electroporation where the application of an external electrical field to a low ionic strength cell suspension creates transient pores in the membrane, allowing the uptake of DNA or other macromolecules, in this case proteins. Combined with cysteine-maleimide dye labelling and single-molecule fluorescence microscopy, this is an extremely powerful tool to investigate protein intracellular behaviour and interactions for a wide variety of moieties in an even wider range of bacterial species. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact To perform an accurate study of protein dynamics, it is essential to investigate their behaviour in vivo. Most current research relies on the use of fluorescent proteins (FPs). Over the past 20 years, these have revolutionized cell biology via their use as genetically encoded protein labels. Green fluorescent protein (GFP) and its mutated allelic forms, blue, cyan and yellow FPs are expressed as fluorescent chimeric proteins in living cells, tissues and entire organisms, after transfection with the engineered vectors. Red FPs have also been isolated from other species, including coral reef organisms, and are similarly useful. The fluorescent protein technique avoids the problem of purifying, labelling and introducing proteins into cells or the task of producing specific antibodies to surface or internal antigens. These fluorescent fusions have allowed studies of the copy number, diffusion patterns and intracellular localization of proteins involved in processes such as gene expression or membrane transport. In the bacterial flagellar motor, they have been successfully used to measure stoichiometry of different components in single motors, leading to the discovery that both rotor and stator proteins exchange on a timescale of minutes with cellular pools. However, this method has limitations. (i) Being genetically encoded, all target proteins are labelled, which can be an advantage for some applications such as, for instance, estimation of cytoplasmic concentration, but is less good for tracking single molecules; (ii) FPs are much less bright (sixfold for GFP) and photo-stable (100-fold for GFP) than commercially available small organic dyes and are, therefore, far from ideal for single-molecule tracking purposes; (iii) because the FPs are relatively large, not all fusions are fully functional, limiting the interpretation of the data and the proteins available for study. For many years researchers have been trying to find smaller, more stable fluorophores that could be used inside living cells. Organic dyes remain the prime choice for in vitro experiments due to their greater photostability, small size (up to 100-fold smaller volume than FPs) and ease of intramolecular labelling (mainly through the use of cysteine residues). All these factors are particularly important for single-molecule fluorescence imaging and tracking. However, as these are not compatible with genetic fusion, a means for re-introducing externally dye-labelled proteins into cells is required for in vivo studies. Several internalization methods combining the advantages of organic labelling and in vivo detection have been introduced over the past decade, some employing relatively large polypeptide tags that are not ideal for bacterial application, while others were limited to large, single-membrane eukaryotic cells (e.g. scrape loading, syringe loading, microinjection). The internalization method we use, adapted from Crawford et al., is based on electroporation where the application of an external electrical field to a low ionic strength cell suspension creates transient pores in the membrane, allowing the uptake of DNA or other macromolecules. Combined with cysteine-maleimide dye labelling and single-molecule fluorescence microscopy, this is an extremely powerful tool to investigate protein intracellular behaviour and interactions for a wide variety of moieties in an even wider range of bacterial species. 
URL https://www.ncbi.nlm.nih.gov/pubmed/24314075
 
Description Flagellar filament assembly in E. coli and Salmonella 
Organisation Helmholtz Association of German Research Centres
Department Helmholtz Centre for Infection Research (HZI)
Country Germany 
Sector Public 
PI Contribution We investigate the flagellar filament assembly in E. coli through electroporation of fluorescent dye-labelled E.coli FliC and single-molecule imaging. We use our own E.coli strains, purify and label E. coli FliC ourselves and use custom built microscope suites available in our Department.
Collaborator Contribution The collaboration aims at investigating the flagellar filament assembly in Salmonella through electroporation of fluorescent dye-labelled Salmonella FliC and single-molecule imaging. The collaborators will provide suitable Salmonella strains and purified labelled or unlabelled FliC protein for us to perform electroporation and imaging on.
Impact This collaboration is multi-disciplinary among biophysics, molecular biology and biochemistry.
Start Year 2015
 
Description Investigation of interactions of CheY with the bacterial flagellar motor in E. coli 
Organisation Weizmann Institute of Science
Department Department of Biochemistry
Country Israel 
Sector Academic/University 
PI Contribution We designed and performed the experiments, plus wrote the manuscript of the outcoming paper (Di Paolo D, Afanzar O, Armitage JP, Berry RM. 2016 Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli. Phil. Trans. R. Soc. B 371: 20150492. http://dx.doi.org/10.1098/rstb.2015.0492).
Collaborator Contribution The collaborator from the Weizmann Institute contributed strains, one of the proteins used and data analysis through custom-written software. He also contributed to refining the paper.
Impact Article 1: Di Paolo D, Afanzar O, Armitage JP, Berry RM. 2016 Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli. Phil. Trans. R. Soc. B 371: 20150492. http://dx.doi.org/10.1098/rstb.2015.0492 Article 2: Afanzar O, Di Paolo D, Eisenstein M, Plochowietz A, Kapanidis AN, Berry RM and Eisenbach M. In submission. Hidden dynamics of CheY at the switch of the bacterial flagellar motor. The nature of this collaboration was multi-disciplinary among biophysics, molecular biology and biochemistry.
Start Year 2015
 
Description Investigation of interactions of CheY6 with the flagellar motor in Rhodobacter Sphaeroides 
Organisation University of Oxford
Department Nuffield Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Labelling, electroporation and imaging of proteins provided by collaborators in strains also provided by collaborators on microscope suites available to the research team. Data analysis.
Collaborator Contribution Provision of suitable Rhodobacter Sphaeroides strains and purification of one of the proteins used; provision of wet lab facilities, microscopes, chemicals and other reagents.
Impact Article in preparation: CheY6 and motor interaction in Rhodobacter Sphaeroides. This collaboration is multi-disciplinary among biophysics, molecular biology and biochemistry.
Start Year 2015
 
Description Conference for Undergraduate Women in Physics (CUWiP) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact CUWiP UK is aimed at helping undergraduate physicists who identify as women to continue in physics by providing them with the opportunity to participate in a conference focused on their development as scientists and showcasing options for their educational and professional futures. I led the tours of the laboratories and helped out with screening of candidates and general organisation.
Year(s) Of Engagement Activity 2015,2016,2017
URL https://www2.physics.ox.ac.uk/equality-and-diversity/women-in-physics-society/cuwip-uk
 
Description JAM Talks (Junior Awards for Microbiology, Birmingham University) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact The J.A.M.s are a monthly junior seminar series aimed at integrating and connecting young researchers around the world. Each month a new junior researcher is invited to present their work in a relaxed, friendly environment in an exciting and engaging way. The talk I gave was attended by about 50 postgraduate and undergraduate students.
Year(s) Of Engagement Activity 2016
URL http://www.jamtalks.com/home.html
 
Description Participation to the Bacterial Locomotion and Signal Transduction (BLAST) conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact XIII Conference on Bacterial Locomotion and Signal Transduction (BLAST) in Tucson (AZ): invited to give a talk in the "Chemotaxis" session, about 100 people in the audience. Very useful feedback and networking opportunities that led to subsequent collaborations on the topic presented.
Year(s) Of Engagement Activity 2015
URL http://blast.ucsc.edu/
 
Description Women in Physics 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact The aim of the Women in Physics is to promote the career development of women in Physics while providing a welcoming support network through increased interaction with peers, mentors and role models. Dr. Diana Di Paolo, a delegate on this grant, organized and chaired Committee meetings, coordinated events and liased with the Department in the role of Secretary first and then Vice-President.
Year(s) Of Engagement Activity 2014,2015,2016,2017
URL https://www2.physics.ox.ac.uk/equality-and-diversity/women-in-physics-society