... understanding life in molecular detail

Dr Neil Thomson

Bionanotechnology, Synthetic Biology, DNA-protein interactions, Gene Expression


My group’s research concentrates on the development and application of atomic force microscopy (AFM) to study the structure and dynamics of biological systems, particularly down to the molecular level. The central aim is to develop new AFM techniques. methods and applications to biological systems to derive new understanding of function at multiple length scales from nano to micro. We use AFM to image, measure forces and manipulate material, from biomolecules through macromolecular assemblies to cells and tissues.

Current major projects include:
  • Multiple RNA polymerase transactions on single DNA templates
  • Engineered Therapeutic Microbubbles
  • Developing and understanding dynamic atomic force microscopy
  • Nano-mechanical behaviour of complex biological assemblies

Research Areas and Collaborations

A short summary of the current research themes in my group including collaborators are listed below.

1) Developing dynamic atomic force microscopy (dAFM)

Sergio Santos and Matteo Chiesa (Masdar Institute, Abu Dhabi)

Victor Barcons and Josep Font (Universitat Politècnica de Catalunya, Barcelona, Spain)

Albert Verdaguer (CSIC, Barcelona, Spain)

Figure 1: Series of dynamic AFM images of an 800bp linear dsDNA molecule on mica taken in hydrating ambient conditions, using a) non-contact mode and d) repulsive tapping-mode and showing the bi-stable region at intermediate set-point ratios (b and c). (Reproduced from Figure 4 in Santos, Billingsley and Thomson, Nanoimaging, Methods Mol Biol, 2013, Volume 950, pp 315-341.)

Application of atomic force microscopy (AFM) to biological systems is usually carried out in a dynamic mode, where the force sensing cantilever is vibrated and the surface is detected via shifts in amplitude, frequency or phase. Dynamic AFM eliminates shearing forces that are typically damaging to soft biological materials. Understanding the dynamics of the oscillating cantilever above the surface when the tip interacts with the sample is critically important for optimizing performance and interpreting data. Through a combination of modelling and experiment we have recently developed a new imaging mode based on amplitude modulation and using humidity controlled environments can monitor and measure the hydrophilicity of single biomolecules, such as DNA. We also recently developed a technique to accurately size the AFM tip in a non-destructive in situ method. This is an important advance because the AFM tip size ultimately determines the best achievable resolution.

2) DNA-Protein Interactions

Transactions of Multiple RNA polymerases on single DNA templates

Jennifer Kirkham and William A Bonass (Oral Biology, Leeds)

Claudio Rivetti (University of Parma, Italy)

Carolyn W. Gibson (University of Pennsylvania, USA)

Figure 2: Simple model of a nested gene in vitro. Two E. coli RNA polymerases are recruited to lambdaPR promoters aligned in a convergent orientation on a linear dsDNA molecule. After addition of NTPs the polymerases transcribe towards each other causing a “collision” event. Distributions of collision events show that the polymerases do not usually end up in close proximity. (Reproduced from Figure 11 in Billingsley et al. Physical Biology 2012, 9 021001).

A nested-gene is one whose entire sequence is contained within another gene, usually oriented in the opposite sense. This leads to the possibility of convergent promoters on opposite strands of individual double-stranded DNA templates. Nested genes have wide-reaching implications for control of gene expression particularly in developing tissues and may invoke transcriptional interference. In a single molecule experiment, it is possible to directly observe what happens when two convergent RNA polymerase molecules meet on a DNA template. This AFM-based approach is helping to answer how the mechanics of polymerases interactions at the molecular level are determined and may shed light on whether two nested genes can be simultaneously expressed.

The Mechanism of Action of Topoisomerases.

Tony Maxwell (John Innes Centre, Norwich)

Sarah Harris (Physics and Astronomy, Leeds)

 

The structure and mechanism of action of E. coli DNA gyrase and other topoisomerases is being probed using AFM.  Gyrase is a member of the protein family, topoisomerases, which are responsible for controlling the topology (supercoiling and knotting) of DNA. It is a molecular motor, which uses the energy released in ATP hydrolysis to relax positive supercoils, or introduce negative supercoils into double-stranded DNA. Since gyrase is necessary to regulate and control bacterial gene expression, it is a desirable target for antibiotics. AFM imaging is determining the low-level resolution, tertiary structure of the enzyme and its relationship with DNA templates.  Details of the proteins mechanistic action can be determined through static imaging of the protein in the presence of DNA and various drug molecules and analogs of ATP.  A longer-term goal is to image topoisomerase turnover in real-time within the microscope, as has been previously achieved in my postdoctoral work for E. coli RNA polymerase. 

3) Protein-Protein Interactions

Sheena Radford (Astbury Centre, Leeds)

David Brockwell (Biochemistry, Leeds)

Dek Woolfson (Bristol University, UK)

Ronald Melki (CNRS, Gif-sur-Yvette, France)

Geoff Howlett (University of Melbourne, Australia)

Figure 3: Montage of an amyloid fibril self-assembled in vitro from beta2-microglobulin at low pH.

The morphological and mechanical properties of amyloid and related bio-fibrils.

The structure and formation of amyloid fibrils of the protein b2-microglobulin (b2m) has been characterized by AFM in conjunction with complementary biophysical and biochemical techniques. AFM is a particularly suitable tool for studying structures formed in heterogeneous aggregating protein solutions, which are unsuitable for ensemble measurements. Acidification of b2m induces amyloid formation in vitro and variations in environmental conditions produce different fibril morphologies.  Aggregation pathways have been elucidated through imaging of the self-assembling species quenched at different time points. In the longer term, assembly can be followed in real-time in the AFM under a variety of biologically relevant conditions, as steps toward mimicking amyloidogenesis in vivo.

We have investigated the mechanical properties of amyloid-like fibrils and related fibrils using AFM to probe individual fibrils in a bending or extensional configuration. These bio-fibrils are typically about 10 nm in diameter and can be several tens or hundreds of microns long, which makes them suitable candidates for nanoscale structural elements in new materials, such as composites or networks. The aim is to test the homogeneity of mechanical response along and between fibrils with the goal of relating mechanical properties to structure. This research has been funded by the BBSRC and assesses the suitability of these biological structures as novel materials.

4) Bacterial Research

Cyanobacterial surface structure and gliding motility

David G. Adams (Biological Sciences, Leeds)

Simon Connell (Physics and Astronomy, Leeds)

Figure 4: a) 3D render of an AFM image of filamentous cyanobacteria folded back on itself (Image size: 30x30mm; Z-range: 90nm). b) Surface topographical image of protein filaments hypothesized to be the molecular motors associated with surface gliding motility. (Image size: 500x500nm; Z-range: 90nm)

Knowledge of bacterial cell surface ultrastructure is based largely on the observation of dead cells and we therefore have no idea of the true nature of the surface in live cells. The development of atomic force microscopy (AFM) has provided the opportunity to study, for the first time, the surface of living bacteria. We were the first to succeed in immobilising filamentous, motile cyanobacteria and scanning the living cells under liquid. This project is employing AFM scanning to a range of live cyanobacteria, immobilised under liquid, to visualise the topography of the cell surface and the functioning of hypothesised molecular motors that drive gliding motility. Additionallly, changes in surface properties during physiological and developmental changes in the bacteria are under study. This project has been funded by the Leverhulme Trust and the EPRSC Doctoral Training Centre at the physical-life science interface.

Detailed research programme                  Close ▲
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Reader in Biological Physics and Bionanotechnology (Leeds) 2
BSc (Bristol) PhD (Bristol)
NATO postdoctoral fellowship (1995); EPSRC advanced research fellow (2000-2005)

Postdoc (UCSB, USA) 1995-1997
Postdoc (EPFL, Switzerland) 1999
EPSRC Advanced Research Fellow (Leeds) 2000-2005
University Research Fellow (Leeds) 2005-2009

EC Stoner 8.50d
School of Physics
0113 343 7289
n.h.thomson@leeds.ac.uk
www.mnp.leeds.ac.uk/nthomson

Selected Publications

  1. Santos S., Guang L., Souier T., Gadelrab K., Chiesa M. and Thomson N.H. “A method to provide rapid in situ determination of tip radius in dynamic atomic force microscopy.” Rev. Sci. Instrum. (2012) 83, 043707.

  2. Billingsley D.J., Bonass W.A, Crampton N., Kirkham J. and Thomson N.H. “Single molecule studies of DNA transcription using atomic force microscopy.” Physical Biology 9 (2012) 021001

  3. Santos S., Stefancich M., Hernandez H., Chiesa M. and Thomson N.H. “The hydrophilicity of a single DNA molecule.” J. Phys. Chem. C (2012) 116 (4) 2807-2818.

  4. Grant C.A., Brockwell D.J., Radford S.E., Thomson N.H. “Tuning the elastic modulus of hydrated collagen fibrils” Biophysical Journal (2009) 97 (11) 2985-2992.