Oxford University Department of Biochemistry
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Mark Leake
Bottom-up systems biology using multidimensional
optical proteomics

Co-workers: P. Creixell, N. Delalez, W. Linke (Muenster, Germany), M. Plank, A. Robson, G. Rosser,
E. Sadler, Q. Xue

What is the molecular basis of the cell? How do single-molecule properties in a living organism scale up to effect whole-organism functionality? Can we bridge our gap in understanding between molecular biology and cell science in a rational, predictive context? These questions pose some of the hardest and most fundamental challenges to the future of biological research, and are those we aim to address. Full understanding of processes in living organisms is only achievable if all molecular interactions are considered. Systems biology strives to cultivate a full insight into the mechanisms of living cells by investigating interactions that elicit and direct cellular events, though to date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. What we now propose is to monitor several biological systems simultaneously in a living, functioning cell using more powerful and precise single-molecule techniques, investigating systems level biology from a bottom-up molecular level, eradicating noise rife in systems biology data associated with cell population stochasticity.


Using novel microscopy techniques and state-of-the-art genetics (Nature 2006, 443, 355), we have developed means to monitor single proteins within a living, functioning cell and to observe exchange with other molecules in a complex, functioning biological system. Our objectives are now to drive these optical techniques to a much higher level to permit fast, real-time, molecular in vivo imaging of several different proteins in multiple, complex biological systems, to establish and validate mathematical models of complex systems down to the molecular level, and to push forward the genetic development of cell strains for use in these "optical proteomics" studies.

Our current experimental model consists of unicellular micro-organisms, in the form of bacteria Escherichia coli and Rhodobacter sphaeroides, ideally suited for systems biology research; they have great economic and medical importance and present drastically more tractable processes compared to multicellular organisms, but have elements which are paradigmic of all cells. We are targeting three specific systems: motility, chemotaxis and bioenergetics. They embody biological processes controlling cell movement, sensing and signal transduction, and the synthesis of the universal fuel ATP via the respiratory redox chain, and are fundamental to the functioning of a living cell.


Our primary experimental technique utilizes advanced approaches of optical microscopy, such as total-internal-reflection fluorescence (TIRF), fluorescence imaging with one nanometre accuracy (FIONA), Foerster resonance energy transfer (FRET) and fluorescence recovery after, and loss in, photobleaching (FRAP and FLIP), generally necessitating customized construction, combined with cutting-edge GFP-fusion molecular genetics technology.


Research Images

Figure 1: Key paper from Nature journal establishing single-molecule methods for optical proteomics in vivo

Figure 2: A: TIRF imaging of a functional membrane-protein complex in the rotary motor of a bacterial GFP-fusion strain. B: Photobleaching of GFP resolved into unitary steps of size ~5,400 counts associated with single GFP molecules. C: Single-molecule precise estimations for number of tagged proteins present based on this step-wise bleaching. D: FLIP and FRAP indicate rapid active turnover of the tagged protein in the complex

Figure 3: A: Monte-Carlo simulations for fluorescence intensity corresponding to the position of GFP-tagged protein before and after focussed laser bleaching of the centre of a bacterial cell. B: Comparing different trial diffusion coefficients (coloured traces) with experimental data (black squares) for recovery of intensity following FRAP

Contact: m.leake1@physics.ox.ac.uk

Graduate Student and Postdoctoral Positions: Enquiries with CV welcome

Website: http://www.physics.ox.ac.uk/users/leake

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