Watching single molecules in action
We are physical scientists interested in developing and applying a range of new quantitative biophysical methods, based on laser fluorescence spectroscopy and scanning probe microscopy, to important problems in biology, which have not been addressed to date due to the lack of suitable tools and hence directly image biological processes down to the level of single molecules. While our experiments range from studies of individual biomolecules to living cells, no previous biological background is required for our research.
Single molecule fluorescence
In contrast to conventional experiments, which measure ensemble averaged behaviour, single-molecule measurements are able to probe individual molecules to measure variation in their properties and follow their dynamics individually. By studying molecules one at time, specific complexes in a mixture can be identified and analysed without the need for any separation. With our collaborators we are exploiting single molecule fluorescence spectroscopy to probe the conformations and function of range of biologically important molecules and processes. Thiis currently includes studying individual T-cell receptors and toll-like receptors on live cells and directly imaging how these proteins reorganise on triggering. In collaboration with Professor Dobson, we are probing the proteins aggregates involved in many neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, how these aggregates are formed and the molecular basis by which these protein aggregates damage neuronal cells.
In collaboration with Professor Korchev at Imperial College we have developed a method based on a scanning nanopipette that allows robust, high resolution, non- contact imaging of living cells, down to the level of individual protein complexes. It can also be used to probe function by performing nanoscale assays such as locally deliver controlled amounts of reagents or performing single ion channel recording. The figure shows the University of Cambridge crest written in fluorescent DNA. We are using this method to watch the details of biological process taking place on the surface of living cells and to directly observe protein aggregates damage neuronal cells .
Initiation of T cell signalling by CD45 segregation at "close contacts". Nature Immunology 17, 574 (2016).
Remarkably low affinity of CD4/major histocompatibility class 11 protein interactions. PNAS 113, 5628 (2016).
Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading. PNAS 113, E1206 (2016).
A mechanistic model of tau amyloid aggregation based on the direct observation of oligomers. Nature Communication 6, 7025 (2015).
Single molecule imaging reveals that small amyloid-beta (1-42) oligomers interact with the cellular prion protein. Chembiochem 15, 2515 (2014).
Single-molecule measurements of transient biomolecular complexes through microfluidic dilution. Analytical Chemistry 85, 6855 (2013).
Local delivery of molecules from a nanopipette for quantitative receptor mapping on live cells. Analytical chemistry 85, 9333 (2013).
Ubiquitin chain conformation regulates recognition and activity of interacting proteins. Nature 492, 266-270 (2012).
Direct Observation of the Interconversion of Normal and Toxic Forms of alpha-Synuclein. Cell 149, 1048-1059 (2012).
The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-beta(1-40) peptide. Nature structural & molecular biology 19, 79-83 (2012).
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