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Department of Chemistry

 
Portrait of dk10012

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 and we work closely with a range of biological and clinical collaborators. We are also part of the Cambridge Dementia Research centre.

Single molecule fluorescence

By studying molecules one at time, using fluorescence, 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 study a range of biologically important molecules and processes. At present our main projects are:

  • Imaging the early molecular events that lead to the triggering of T-cells, a process that underpins the adaptive immune response and is remarkably  sensitive and selective.
  • Imaging the early molecular events that lead to the triggering of Toll-like receptors, a key process in the innate immune response which plays an important role in causing inflammation in neurodegenerative diseases such as Alzheimer’s disease.
  • Imaging and characterising the protein aggregates formed in the test-tube during aggregation reactions of disease-associated proteins and those present in human samples such as cerebral spinal fluid and brain tissue. We want to determine the aggregates responsible for causing neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease in humans and the mechanisms by which they damage cells and spread through the brain.
  • Determining the structure and organisation of DNA in the nucleus of cells and  how this is regulated and changes during  cell differentiation.
  • Imaging aggregates of P53 and determining the role of these aggregates in the development and spreading of cancer.  
  • Developing and improving our imaging methods. At present our focus is developing methods to image protein aggregates at 20 nm spatial resolution and determine their composition and structure.

Scanning nanopipette

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.

 

 

 

Two new positions available

Two  PDRA positions are  available from October, 2019 . For more details and to apply see:

http://www.jobs.cam.ac.uk/job/22865/

https://www.ch.cam.ac.uk/job/22853

 

Selected Publications

Activation of Toll-like receptors nucleates assembly of the MyDDosome signaling hubeLife 7, (2018) . 

 Inhibiting the Ca2+ Influx Induced by Human CSFCell Rep 21, 3310 (2017)

Initiation of T cell signalling by CD45 segregation at "close contacts". Nature Immunology 17, 574 (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).

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).

Publications

Soluble aggregates present in cerebrospinal fluid change in size and mechanism of toxicity during Alzheimer's disease progression
S De, DR Whiten, FS Ruggeri, C Hughes, M Rodrigues, DI Sideris, CG Taylor, FA Aprile, S Muyldermans, TPJ Knowles, M Vendruscolo, C Bryant, K Blennow, I Skoog, S Kern, H Zetterberg, D Klenerman
– Acta Neuropathologica Communications
(2019)
7,
120
A cell topography-based mechanism for ligand discrimination by the T cell receptor
RA Fernandes, KA Ganzinger, JC Tzou, P Jönsson, SF Lee, M Palayret, AM Santos, AR Carr, A Ponjavic, VT Chang, C Macleod, BC Lagerholm, AE Lindsay, O Dushek, A Tilevik, SJ Davis, D Klenerman
– Proceedings of the National Academy of Sciences
(2019)
116,
14002
Different soluble aggregates of Aβ42 can give rise to cellular toxicity through different mechanisms.
S De, DC Wirthensohn, P Flagmeier, C Hughes, FA Aprile, FS Ruggeri, DR Whiten, D Emin, Z Xia, JA Varela, P Sormanni, F Kundel, TPJ Knowles, CM Dobson, C Bryant, M Vendruscolo, D Klenerman
– Nature Communications
(2019)
10,
1541
Citrullination of HP1γ 1 chromodomain affects association to chromatin
M Wiese, AJ Bannister, S Basu, W Boucher, K Wohlfahrt, MA Christophorou, ML Nielsen, D Klenerman, ED Laue, T Kouzarides
– Epigenetics & chromatin
(2019)
12,
21
Secondary nucleation and elongation occur at different sites on Alzheimer's amyloid-b aggregates
T Scheidt, U Łapińska, JR Kumita, DR Whiten, D Klenerman, MR Wilson, SIA Cohen, S Linse, M Vendruscolo, CM Dobson, TPJ Knowles, P Arosio
– Science Advances
(2019)
5,
eaau3112
Direct observation of prion protein oligomer formation reveals an aggregation mechanism with multiple conformationally distinct species
JC Sang, J-E Lee, AJ Dear, S De, G Meisl, AM Thackray, R Bujdoso, TPJ Knowles, D Klenerman
– Chem Sci
(2019)
10,
4588
Spectrally Resolved Photodynamics of Individual Emitters in Large-Area Monolayers of Hexagonal Boron Nitride
HL Stern, R Wang, Y Fan, R Mizuta, JC Stewart, L-M Needham, TD Roberts, R Wai, NS Ginsberg, D Klenerman, S Hofmann, SF Lee
– ACS Nano
(2019)
13,
4538
Increased Secondary Nucleation Underlies Accelerated Aggregation of the Four-Residue N-Terminally Truncated Aβ42 Species Aβ5–42
T Weiffert, G Meisl, P Flagmeier, S De, CJR Dunning, B Frohm, H Zetterberg, K Blennow, E Portelius, D Klenerman, CM Dobson, TPJ Knowles, S Linse
– ACS Chemical Neuroscience
(2019)
10,
2374
Filamentous aggregates are fragmented by the proteasome holoenzyme
R Cliffe, JC Sang, F Kundel, D Finley, D Klenerman, Y Ye
– Cell reports
(2019)
26,
2140
Simple and ultrafast resonance frequency and dissipation shift measurements using a fixed frequency drive
A Guha, N Sandström, VP Ostanin, W van der Wijngaart, D Klenerman, SK Ghosh
– Sensors and Actuators, B: Chemical
(2019)
281,
960
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Research Interest Groups

Telephone number

01223 336481

Email address

dk10012@cam.ac.uk