Studying single molecules on living cells

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• Professor David Klenerman, Department of Chemistry, University of Cambridge
• Wednesday 21 January 2009, 16:30-17:30
• Lecture Theatre 1, Department of Veterinary Medicine.

If you have a question about this talk, please contact Suzy Blows.

One major challenge in biology is to understand how the individual molecules and complexes of the cell are organised and interact to form a functional living cell. To address this problem new sensitive biophysical tools are needed that are capable of studying single molecules in complexes both in the test-tube and on or in living cells.

To determine the oligomerisation state of proteins we have used two colour single molecule coincidence detection based on the excitation of two distinct fluorophore labels on proteins with two lasers focussed to the same spot.1 This method requires no prior knowledge of the structure of any complex formed or control of fluorophore position on the molecule. We show that this method can be used to characterise the protein oligomers formed during protein misfolding, ultimately resulting in amyloid fibril formation, and can distinguish between protein monomers and dimers on the cell surface.2

Working together with Professor Yuri Korchev at Imperial College, we have developed a method for functional nanoscale mapping of the cell surface that is based on a scanned nanopipette. This allows high resolution, non-contact imaging of the soft and responsive cell surface using the ion current that flows between an electrode in the nanopipette and bath for distance feedback control.3 Recently we have made a major advance in the resolution of the topographic images, by scanning with fine quartz pipettes, so we can directly visualise protein complexes on the surface of live cells.4 The pipette can also be use to perform local nanoscale assays on the cell surface so as to perform single channel recording 5 or apply pressure to probe the mechanical properties. We have also combined high resolution topographic imaging with simultaneous recording of the fluorescence from the cell surface.6 In addition the pipette can be used for controlled voltage driven delivery and deposition of biomolecules down to the single molecule level3 and this is being used to probe the structure of the cell membrane using single molecule fluorescence tracking.

References 1. “Determination of the Fraction and Stoichiometry of Femtomolar Levels of Biomolecular Complexes in an Excess of Monomer Using Single-Molecule, Two-Color Coincidence Detection” Anal.Chem. 78, 7707-7715 (2006). 2. “Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells” PNAS 104 ,17662-17667 (2007) 3. “The scanned nanopipette: A new tool for high resolution bioimaging and controlled deposition of biomolecules”, Phys. Chem. Chem. Phys. 7 , 2859-2866 (2005) 4. “Imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy”. Angewandte Chemie-International Edition 45, 2212-2216 (2006) 5. “Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system,” Biophysical Journal 83, 3296-3303 (2002). 6. “Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: Application to single virus-like particle entry into a cell,” PNAS 99 , 16018-16023 (2002)

This talk is part of the Departmental Seminar Programme, Department of Veterinary Medicine series.

This talk is included in these lists:

• All Talks (aka the CURE list)
• Cambridge Immunology
• Cambridge Infectious Disease
• Cambridge Infectious Diseases
• Departmental Seminar Programme, Department of Veterinary Medicine
• Lecture Theatre 1, Department of Veterinary Medicine
• Vet School Seminars

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