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SUMMARY:Studying single molecules on living cells - Professor David Klener
 man\, Department of Chemistry\, University of Cambridge
DTSTART:20090121T163000Z
DTEND:20090121T173000Z
UID:TALK16081@talks.cam.ac.uk
CONTACT:Suzy Blows
DESCRIPTION:One major challenge in biology is to understand how the indivi
 dual molecules and complexes of the cell are organised and interact to for
 m a functional living cell. To address this problem new sensitive biophysi
 cal tools are needed that are capable of studying single molecules in comp
 lexes both in the test-tube and on or in living cells. \n\nTo determine th
 e oligomerisation state of proteins we have used two colour single molecul
 e coincidence detection based on the excitation of two distinct fluorophor
 e labels on proteins with two lasers focussed to the same spot.1 This meth
 od requires no prior knowledge of the structure of any complex formed or c
 ontrol of fluorophore position on the molecule. We show that this method c
 an be used to characterise the protein oligomers formed during protein mis
 folding\, ultimately resulting in amyloid fibril formation\, and can disti
 nguish between protein monomers and dimers on the cell surface.2 \n\nWorki
 ng together with Professor Yuri Korchev at Imperial College\, we have deve
 loped a method for functional nanoscale mapping of the cell surface that i
 s based on a scanned nanopipette. This allows high resolution\, non-contac
 t imaging of the soft and responsive cell surface using the ion current th
 at flows between an electrode in the nanopipette and bath for distance fee
 dback control.3 Recently we have made a major advance in the resolution of
  the topographic images\, by scanning with fine quartz pipettes\, so we ca
 n 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 surf
 ace so as to perform single channel recording 5  or apply pressure to prob
 e the mechanical properties. We have also combined high resolution topogra
 phic imaging with simultaneous recording of the fluorescence from the cell
  surface.6 In addition the pipette can be used for controlled voltage driv
 en delivery and deposition of biomolecules down to the single molecule lev
 el3 and this is being used to probe the structure of the cell membrane usi
 ng single molecule fluorescence tracking.	\n	 \n\nReferences\n1.	“Determ
 ination of the Fraction and Stoichiometry of Femtomolar Levels of Biomolec
 ular Complexes in an Excess of Monomer Using Single-Molecule\, Two-Color C
 oincidence Detection” Anal.Chem. 78\, 7707-7715 (2006).\n2.	“Single-mo
 lecule level analysis of the subunit composition of the T cell receptor on
  live T cells” PNAS  104\,17662-17667 (2007)\n3.	"The scanned nanopipett
 e: A new tool for high resolution bioimaging and controlled deposition of 
 biomolecules"\, Phys. Chem. Chem. Phys. 7 \, 2859-2866 (2005)\n4.	“Imagi
 ng proteins in membranes of living cells by high-resolution scanning ion c
 onductance microscopy”. Angewandte Chemie-International Edition 45\, 221
 2-2216 (2006)\n5.	"Ion channels in small cells and subcellular structures 
 can be studied with a smart patch-clamp system\," Biophysical Journal 83\,
  3296-3303 (2002).\n6.	"Scanning surface confocal microscopy for simultane
 ous topographical and fluorescence imaging: Application to single virus-li
 ke particle entry into a cell\," PNAS 99 \, 16018-16023 (2002)\n\n
LOCATION:Lecture Theatre 1\, Department of Veterinary Medicine
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