Co-reporter:Patrick Flagmeier;Suman De;David C. Wirthensohn;Steven F. Lee;Cécile Vincke;Serge Muyldermans;Tuomas P. J. Knowles;Sonia Ghi;Christopher M. Dobson
Angewandte Chemie 2017 Volume 129(Issue 27) pp:7858-7862
Publication Date(Web):2017/06/26
DOI:10.1002/ange.201700966
AbstractTo quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.
Co-reporter:James H. Felce, Sarah L. Latty, Rachel G. Knox, Susan R. Mattick, ... Simon J. Davis
Cell Reports 2017 Volume 20, Issue 11(Volume 20, Issue 11) pp:
Publication Date(Web):12 September 2017
DOI:10.1016/j.celrep.2017.08.072
•Systematic analysis of 60 Rhodopsin-family GPCRs reveals that most are monomers•Rhodopsin-family root ancestor GPCRs are also monomers•Across families, rates of receptor diversification correlate with stoichiometry•Skewed family structure suggests dimerization increases receptor “fitness density”The organization of Rhodopsin-family G protein-coupled receptors (GPCRs) at the cell surface is controversial. Support both for and against the existence of dimers has been obtained in studies of mostly individual receptors. Here, we use a large-scale comparative study to examine the stoichiometric signatures of 60 receptors expressed by a single human cell line. Using bioluminescence resonance energy transfer- and single-molecule microscopy-based assays, we found that a relatively small fraction of Rhodopsin-family GPCRs behaved as dimers and that these receptors otherwise appear to be monomeric. Overall, the analysis predicted that fewer than 20% of ∼700 Rhodopsin-family receptors form dimers. The clustered distribution of the dimers in our sample and a striking correlation between receptor organization and GPCR family size that we also uncover each suggest that receptor stoichiometry might have profoundly influenced GPCR expansion and diversification.Download high-res image (328KB)Download full-size image
Co-reporter:Patrick Flagmeier;Suman De;David C. Wirthensohn;Steven F. Lee;Cécile Vincke;Serge Muyldermans;Tuomas P. J. Knowles;Sonia Ghi;Christopher M. Dobson
Angewandte Chemie International Edition 2017 Volume 56(Issue 27) pp:7750-7754
Publication Date(Web):2017/06/26
DOI:10.1002/anie.201700966
AbstractTo quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aβ42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aβ peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.
Co-reporter:Marija Iljina;Liu Hong;Mathew H. Horrocks;Marthe H. Ludtmann
BMC Biology 2017 Volume 15( Issue 1) pp:57
Publication Date(Web):03 July 2017
DOI:10.1186/s12915-017-0390-6
The aggregation of the protein ɑ-synuclein (ɑS) underlies a range of increasingly common neurodegenerative disorders including Parkinson’s disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ɑS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ɑS aggregation in vitro in the presence of two ɑS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ɑS.We show that both nanobodies inhibit the formation of ɑS fibrils. Furthermore, using single-molecule fluorescence techniques, we demonstrate that nanobody binding promotes a rapid conformational conversion from more stable oligomers to less stable oligomers of ɑS, leading to a dramatic reduction in oligomer-induced cellular toxicity.The results indicate a novel mechanism by which diseases associated with protein aggregation can be inhibited, and suggest that NbSyn2 and NbSyn87 could have significant therapeutic potential.
Co-reporter:Mathew H. Horrocks, Steven F. Lee, Sonia Gandhi, Nadia K. Magdalinou, Serene W. Chen, Michael J. Devine, Laura Tosatto, Magnus Kjaergaard, Joseph S. Beckwith, Henrik Zetterberg, Marija Iljina, Nunilo Cremades, Christopher M. Dobson, Nicholas W. Wood, and David Klenerman
ACS Chemical Neuroscience 2016 Volume 7(Issue 3) pp:399
Publication Date(Web):January 22, 2016
DOI:10.1021/acschemneuro.5b00324
The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson’s disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.Keywords: biomarkers; CSF; Parkinson’s; single-molecule
Co-reporter:Mathew H. Horrocks, Laura Tosatto, Alexander J. Dear, Gonzalo A. Garcia, Marija Iljina, Nunilo Cremades, Mauro Dalla Serra, Tuomas P. J. Knowles, Christopher M. Dobson, and David Klenerman
Analytical Chemistry 2015 Volume 87(Issue 17) pp:8818
Publication Date(Web):August 10, 2015
DOI:10.1021/acs.analchem.5b01811
α-Synuclein oligomers can be toxic to cells and may be responsible for cell death in Parkinson’s disease. Their typically low abundance and highly heterogeneous nature, however, make such species challenging to study using traditional biochemical techniques. By combining fast-flow microfluidics with single-molecule fluorescence, we are able to rapidly follow the process by which oligomers of αS are formed and to characterize the species themselves. We have used the technique to show that populations of oligomers with different FRET efficiencies have varying stabilities when diluted into low ionic strength solutions. Interestingly, we have found that oligomers formed early in the aggregation pathway have electrostatic repulsions that are shielded in the high ionic strength buffer and therefore dissociate when diluted into lower ionic strength solutions. This property can be used to isolate different structural groups of αS oligomers and can help to rationalize some aspects of αS amyloid fibril formation.
Co-reporter:Rebecca R. Murphy, George Danezis, Mathew H. Horrocks, Sophie E. Jackson, and David Klenerman
Analytical Chemistry 2014 Volume 86(Issue 17) pp:8603
Publication Date(Web):August 8, 2014
DOI:10.1021/ac501188r
It is of significant biophysical interest to obtain accurate intramolecular distance information and population sizes from single-molecule Förster resonance energy transfer (smFRET) data obtained from biomolecules in solution. Experimental methods of increasing cost and complexity are being developed to improve the accuracy and precision of data collection. However, the analysis of smFRET data sets currently relies on simplistic, and often arbitrary methods, for the selection and denoising of fluorescent bursts. Although these methods are satisfactory for the analysis of simple, low-noise systems with intermediate FRET efficiencies, they display systematic inaccuracies when applied to more complex systems. We have developed an inference method for the analysis of smFRET data from solution studies based on rigorous model-based Bayesian techniques. We implement a Monte Carlo Markov chain (MCMC) based algorithm that simultaneously estimates population sizes and intramolecular distance information directly from a raw smFRET data set, with no intermediate event selection and denoising steps. Here, we present both our parametric model of the smFRET process and the algorithm developed for data analysis. We test the algorithm using a combination of simulated data sets and data from dual-labeled DNA molecules. We demonstrate that our model-based method systematically outperforms threshold-based techniques in accurately inferring both population sizes and intramolecular distances.
Co-reporter:Priyanka Narayan, Kira M. Holmström, Dong-Hyun Kim, Daniel J. Whitcomb, Mark R. Wilson, Peter St. George-Hyslop, Nicholas W. Wood, Christopher M. Dobson, Kwangwook Cho, Andrey Y. Abramov, and David Klenerman
Biochemistry 2014 Volume 53(Issue 15) pp:
Publication Date(Web):April 9, 2014
DOI:10.1021/bi401606f
Oligomers of the amyloid-β (Aβ) peptide have been implicated in the neurotoxicity associated with Alzheimer’s disease. We have used single-molecule techniques to examine quantitatively the cellular effects of adding well characterized Aβ oligomers to primary hippocampal cells and hence determine the initial pathway of damage. We found that even picomolar concentrations of Aβ (1–40) and Aβ (1–42) oligomers can, within minutes of addition, increase the levels of intracellular calcium in astrocytes but not in neurons, and this effect is saturated at a concentration of about 10 nM of oligomers. Both Aβ (1–40) and Aβ (1–42) oligomers have comparable effects. The rise in intracellular calcium is followed by an increase in the rate of ROS production by NADPH oxidase in both neurons and astrocytes. The increase in ROS production then triggers caspase-3 activation resulting in the inhibition of long-term potentiation. Our quantitative approach also reveals that only a small fraction of the oligomers are damaging and that an individual rare oligomer binding to an astrocyte can initiate the aforementioned cascade of responses, making it unlikely to be due to any specific interaction. Preincubating the Aβ oligomers with an extracellular chaperone, clusterin, sequesters the oligomers in long-lived complexes and inhibits all of the physiological damage, even at a ratio of 100:1, total Aβ to clusterin. To explain how Aβ oligomers are so damaging but that it takes decades to develop Alzheimer’s disease, we suggest a model for disease progression where small amounts of neuronal damage from individual unsequestered oligomers can accumulate over time leading to widespread tissue-level dysfunction.
Co-reporter:Kristina A. Ganzinger;Dr. Priyanka Narayan;Dr. Seema S. Qamar;Dr. Laura Weimann;Dr. Rohan T. Ranasinghe; Adriano Aguzzi; Christopher M. Dobson;Dr. James McColl;Dr. Peter St. George-Hyslop; David Klenerman
ChemBioChem 2014 Volume 15( Issue 17) pp:2515-2521
Publication Date(Web):
DOI:10.1002/cbic.201402377
Abstract
Oligomers of the amyloid-β peptide (Aβ) play a central role in the pathogenesis of Alzheimer’s disease and have been suggested to induce neurotoxicity by binding to a plethora of cell-surface receptors. However, the heterogeneous mixtures of oligomers of varying sizes and conformations formed by Aβ42 have obscured the nature of the oligomeric species that bind to a given receptor. Here, we have used single-molecule imaging to characterize Aβ42 oligomers (oAβ42) and to confirm the controversial interaction of oAβ42 with the cellular prion protein (PrPC) on live neuronal cells. Our results show that, at nanomolar concentrations, oAβ42 interacts with PrPC and that the species bound to PrPC are predominantly small oligomers (dimers and trimers). Single-molecule biophysical studies can thus aid in deciphering the mechanisms that underlie receptor-mediated oAβ-induced neurotoxicity, and ultimately facilitate the discovery of novel inhibitors of these pathways.
Co-reporter:Priyanka Narayan ; Kristina A. Ganzinger ; James McColl ; Laura Weimann ; Sarah Meehan ; Seema Qamar ; John A. Carver ̂; Mark R. Wilson ; Peter St. George-Hyslop ; Christopher M. Dobson
Journal of the American Chemical Society 2013 Volume 135(Issue 4) pp:1491-1498
Publication Date(Web):January 22, 2013
DOI:10.1021/ja3103567
Oligomers of the 40 and 42 residue amyloid-β peptides (Aβ40 and Aβ42) have been implicated in the neuronal damage and impaired cognitive function associated with Alzheimer’s disease. However, little is known about the specific mechanisms by which these misfolded species induce such detrimental effects on cells. In this work, we use single-molecule imaging techniques to examine the initial interactions between Aβ monomers and oligomers and the membranes of live cells. This highly sensitive method enables the visualization of individual Aβ species on the cell surface and characterization of their oligomerization state, all at biologically relevant, nanomolar concentrations. The results indicate that oligomers preferentially interact with cell membranes, relative to monomers and that the oligomers become immobilized on the cell surface. Additionally, we observe that the interaction of Aβ species with the cell membrane is inhibited by the presence of ATP-independent molecular chaperones. This study demonstrates the power of this methodology for characterizing the interactions between protein aggregates and the membranes of live neuronal cells at physiologically relevant concentrations and opens the door to quantitative studies of the cellular responses to potentially pathogenic oligomers.
Co-reporter:Richard W. Clarke ; Anna Drews ; Helena Browne
Journal of the American Chemical Society 2013 Volume 135(Issue 30) pp:11175-11180
Publication Date(Web):July 9, 2013
DOI:10.1021/ja4038406
Herpes simplex viruses display hundreds of gD glycoproteins, and yet their neutralization requires tens of thousands of antibodies per virion, leading us to ask whether a wild-type virion with just a single free gD is still infective. By quantitative analysis of fluorescently labeled virus particles and virus neutralization assays, we show that entry of a wild-type HSV virion to a cell does indeed require just one or two of the approximately 300 gD glycoproteins to be left unbound by monoclonal antibody. This indicates that HSV entry is an extraordinarily efficient process, functioning at the level of single molecular complexes.
Co-reporter:Richard W. Clarke ; Alexander Zhukov ; Owen Richards ; Nicholas Johnson ; Victor Ostanin
Journal of the American Chemical Society 2012 Volume 135(Issue 1) pp:322-329
Publication Date(Web):December 4, 2012
DOI:10.1021/ja3094586
There is an intrinsic repulsion between glass and cell surfaces that allows noninvasive scanning ion conductance microscopy (SICM) of cells and which must be overcome in order to form the gigaseals used for patch clamping investigations of ion channels. However, the interactions of surfaces in physiological solutions of electrolytes, including the presence of this repulsion, for example, do not obviously agree with the standard Derjaguin–Landau–Verwey–Overbeek (DLVO) colloid theory accurate at much lower salt concentrations. In this paper we investigate the interactions of glass nanopipettes in this high-salt regime with a variety of surfaces and propose a way to resolve DLVO theory with the results. We demonstrate the utility of this understanding to SICM by topographically mapping a live cell’s cytoskeleton. We also report an interesting effect whereby the ion current though a nanopipette can increase under certain conditions upon approaching an insulating surface, rather than decreasing as would be expected. We propose that this is due to electroosmotic flow separation, a high-salt electrokinetic effect. Overall these experiments yield key insights into the fundamental interactions that take place between surfaces in strong solutions of electrolytes.
Co-reporter:Mathew H. Horrocks, Haitao Li, Jung-uk Shim, Rohan T. Ranasinghe, Richard W. Clarke, Wilhelm T. S. Huck, Chris Abell, and David Klenerman
Analytical Chemistry 2012 Volume 84(Issue 1) pp:179
Publication Date(Web):December 6, 2011
DOI:10.1021/ac202313d
We have experimentally determined the optimal flow velocities to characterize or count single molecules by using a simple microfluidic device to perform two-color coincidence detection (TCCD) and single pair Förster resonance energy transfer (spFRET) using confocal fluorescence spectroscopy on molecules traveling at speeds of up to 10 cm s–1. We show that flowing single fluorophores at ≥0.5 cm s–1 reduces the photophysical processes competing with fluorescence, enabling the use of high excitation irradiances to partially compensate for the short residence time within the confocal volume (10–200 μs). Under these conditions, the data acquisition rate can be increased by a maximum of 38-fold using TCCD at 5 cm s–1 or 18-fold using spFRET at 2 cm s–1, when compared with diffusion. While structural characterization requires more photons to be collected per event and so necessitates the use of slower speeds (2 cm s–1 for TCCD and 1 cm s–1 for spFRET), a considerable enhancement in the event rate could still be obtained (33-fold for TCCD and 16-fold for spFRET). Using flow under optimized conditions, analytes could be rapidly quantified over a dynamic range of up to 4 orders of magnitude by direct molecule counting; a 50 fM dual-labeled model sample can be detected with 99.5% statistical confidence in around 8 s using TCCD and a flow velocity of 5 cm s–1.
Co-reporter:Priyanka Narayan, Sarah Meehan, John A. Carver, Mark R. Wilson, Christopher M. Dobson, and David Klenerman
Biochemistry 2012 Volume 51(Issue 46) pp:
Publication Date(Web):October 29, 2012
DOI:10.1021/bi301277k
The aberrant aggregation of the amyloid-β peptide into β-sheet rich, fibrillar structures proceeds via a heterogeneous ensemble of oligomeric intermediates that have been associated with neurotoxicity in Alzheimer’s disease (AD). Of particular interest in this context are the mechanisms by which molecular chaperones, part of the primary biological defenses against protein misfolding, influence Aβ aggregation. We have used single-molecule fluorescence techniques to compare the interactions between distinct aggregation states (monomers, oligomers, and amyloid fibrils) of the AD-associated amyloid-β(1–40) peptide, and two molecular chaperones, both of which are upregulated in the brains of patients with AD and have been found colocalized with Aβ in senile plaques. One of the chaperones, αB-crystallin, is primarily found inside cells, while the other, clusterin, is predominantly located in the extracellular environment. We find that both chaperones bind to misfolded oligomeric species and form long-lived complexes, thereby preventing both their further growth into fibrils and their dissociation. From these studies, we conclude that these chaperones have a common mechanism of action based on sequestering Aβ oligomers. This conclusion suggests that these chaperones, both of which are ATP-independent, are able to inhibit potentially pathogenic Aβ oligomer-associated processes whether they occur in the extracellular or intracellular environment.
Co-reporter:Alex Zhukov, Owen Richards, Victor Ostanin, Yuri Korchev, David Klenerman
Ultramicroscopy 2012 Volume 121() pp:1-7
Publication Date(Web):October 2012
DOI:10.1016/j.ultramic.2012.06.015
We have developed a new method of controlling the pipette for scanning ion conductance microscopy to obtain high-resolution images faster. The method keeps the pipette close to the surface during a single line scan but does not follow the exact surface topography, which is calculated by using the ion current. Using an FPGA platform we demonstrate this new method on model test samples and then on live cells. This method will be particularly useful to follow changes occurring on relatively flat regions of the cell surface at high spatial and temporal resolutions.Highlights► We introduce a new method for fast scanning ion conductance microscopy (FSCIM). ► Method based on adaptive scanning using a field programmable gate array. ► Method gives improved sampling rate compared to other SICM control methods. ► Comparison of hopping mode SICM and FSICM made on live A6 cells.
Co-reporter:David Klenerman, Yuri E Korchev, Simon J Davis
Current Opinion in Chemical Biology 2011 Volume 15(Issue 5) pp:696-703
Publication Date(Web):October 2011
DOI:10.1016/j.cbpa.2011.04.001
Determining the organisation of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signaling, is a major challenge in cell biology and requires methods with nanoscale resolution. Recent advances in fluorescence imaging both at the diffraction limit tracking single molecules and exploiting super resolution imaging have now reached a stage where they can provide fundamentally new insights. Complementary developments in scanning ion conductance microscopy also allow the cell surface to be imaged with nanoscale resolution. The challenge now is to combine the information obtained using these different methods and on different cells to obtain a coherent view of the cell surface. In the future this needs to be driven by interdisciplinary research between physical scientists and biologists.Highlights► Studying the organisation and function of the cell surface needs nanoscale methods. ► Nanoscale fluorescence imaging can now image labeled molecules on live cells. ► Fluorescence based method can be used to study protein organisation. ► Nanoscale topographic imaging of the surface of live cells is now possible. ► New insights into the cell surface organisation will come from combinations of these methods.
Co-reporter:Dr. Angel Orte;Dr. Richard W. Clarke; David Klenerman
ChemPhysChem 2011 Volume 12( Issue 3) pp:491-499
Publication Date(Web):
DOI:10.1002/cphc.201000636
Abstract
The use of Förster resonance energy transfer (FRET) as a tool to study biomolecules has been greatly enhanced by new advances in single-molecule fluorescence (SMF) techniques. This has allowed new insights into the structure and dynamics of complex biomolecular machinery. However, there are still technical drawbacks in the application of conventional SMF–FRET. Herein, we review the use of single-molecule coincidence spectroscopy to study FRET systems, an analytical variation of the conventional scheme, using one or two confocal lasers of different colours. We highlight the advantages of the coincidence spectroscopy and illustrate this with examples of its application to some biological systems of interest.
Co-reporter:Justin A. Yeoman ; Angel Orte ; Beth Ashbridge ; David Klenerman ;Shankar Balasubramanian
Journal of the American Chemical Society 2010 Volume 132(Issue 9) pp:2852-2853
Publication Date(Web):February 11, 2010
DOI:10.1021/ja909383n
We have used single-molecule fluorescence microscopy to study the folded state of human telomerase RNA (hTR). Here we show that hTR adopts a new conformation on binding to human telomerase reverse transcriptase (hTERT) and reconstitution of an active ribonucleoprotein complex. Our data are consistent with the formation of an RNA pseudoknot in active human telomerase.
Co-reporter:Beth Ashbridge, Angel Orte, Justin A. Yeoman, Michael Kirwan, Tom Vulliamy, Inderjeet Dokal, David Klenerman and Shankar Balasubramanian
Biochemistry 2009 Volume 48(Issue 46) pp:
Publication Date(Web):October 17, 2009
DOI:10.1021/bi901373e
It has been proposed that human telomerase RNA (hTR) interacts with dyskerin, prior to assembly of the telomerase holoenzyme. The direct interaction of dyskerin and hTR has not been demonstrated and is an experimentally challenging research problem because of difficulties in expressing and purifying dyskerin in quantities that are useful for biophysical analysis. By orthogonally labeling dyskerin and hTR, we have been able to employ single-molecule two-color coincidence detection (TCCD) to observe directly the formation of a dyskerin·hTR complex. By systematic deletion of hTR subdomains, we have gained insights into the RNA sites required for interaction with dyskerin. We then investigated mutated forms of hTR and dyskerin that are associated with dyskeratosis congenita (DC), on the basis of clinical genetics studies, for their effects on the dyskerin·hTR interaction. Dyskerin mutations associated with X-linked DC resulted in significant impairment of the dyskerin·hTR interaction, whereas mutations in hTR associated with autosomal dominant (AD) DC did not affect the interaction. We propose that disruption of the dyskerin·hTR interaction may contribute to X-linked DC.
Co-reporter:Angel Orte, Richard W. Clarke and David Klenerman
Analytical Chemistry 2008 Volume 80(Issue 22) pp:8389
Publication Date(Web):October 15, 2008
DOI:10.1021/ac8009092
Single-molecule fluorescence resonance energy transfer (FRET) is commonly used to probe different conformations and conformational dynamics of single biomolecules. However, the analysis of raw burst traces is not always straightforward. The presence of a “zero peak” and the skewness of peaks at high and low FRET efficiencies in proximity ratio histograms make the accurate evaluation of the histogram a challenging task. This is further compounded by the difficulty associated with siting two fluorophores in optimal range of each other. Here we present an alternative method of analysis, based on handling coincident FRET photon bursts, that addresses these problems. In addition, we demonstrate methods to enhance coincidence levels and thus the accuracy of FRET determination: the use of dual-color excitation, including direct excitation of the acceptor fluorophore; the addition of a remote dye to the biomolecule, not involved in the FRET process; or a combination of the two. We show the advantages of dual excitation by studying several labeled double-stranded DNA samples as FRET models. This method extends the application of single-molecule FRET to more complicated biological systems where only a small fraction of complexes are fully assembled.
Co-reporter:Chao Li, Nicholas Johnson, Victor Ostanin, Andrew Shevchuk, Liming Ying, Yuri Korchev, David Klenerman
Progress in Natural Science: Materials International 2008 Volume 18(Issue 6) pp:671-677
Publication Date(Web):10 June 2008
DOI:10.1016/j.pnsc.2008.01.011
Microscopy is an essential technique for observation on living cells. There is currently great interest in applying scanning probe microscopy to image-living biological cells in their natural environment at the nanometer scale. Scanning ion conductance microscopy is a new form of scanning probe microscopy, which enables non-contact high-resolution imaging of living biological cells. Based on a scanned nanopipette in physiological buffer, the distance feedback control uses the ion current to control the distance between the pipette tip and the sample surface. However, this feedback control has difficulties over slopes on convoluted cell surfaces, which limits its resolution. In this study, we present an improved form of feedback control that removes the contribution of up to the third-order slope from the ion current signal, hence providing a more accurate signal for controlling the distance. We show that this allows faster and lower noise topographic high-resolution imaging.
Co-reporter:Samuel S. White Dr.;Shankar Balasubramanian Dr. Dr.;Liming Ying Dr.
Angewandte Chemie 2006 Volume 118(Issue 45) pp:
Publication Date(Web):19 OCT 2006
DOI:10.1002/ange.200602289
Jetzt wird's gemischt: Ein Nano-Diffusionsmischer für Einzelmolekül-Untersuchungen ermöglicht das Studium der Fluoreszenz einzelner Moleküle unter Nichtgleichgewichtsbedingungen in Lösung, ohne das Signal-Rausch-Verhältnis zu verschlechtern. Mit dieser Methode gelang die direkte Beobachtung des resonanten Fluoreszenzenergietransfers für unterschiedliche Konformationen beim Entfalten eines Quadruplex.
Co-reporter:Andrew I. Shevchuk Dr.;Gregory I. Frolenkov Dr.;Daniel Sánchez Dr.;Peter S. James;Noah Freedman;Max J. Lab Dr.;Roy Jones Dr. Dr.;Yuri E. Korchev Dr.
Angewandte Chemie 2006 Volume 118(Issue 14) pp:
Publication Date(Web):28 FEB 2006
DOI:10.1002/ange.200503915
Berühren verboten! Die Oberfläche lebender Zellen ist weich und nachgiebig, weshalb bisher eine hochauflösende Abbildung der Zellmembran nicht möglich war. Nun gelang das kontaktlose Abbilden von Proteinkomplexen in der Plasmamembran lebender Zellen (siehe Bild) und das Verfolgen der strukturellen Reorganisation von Zellen. Dieser Durchbruch eröffnet den Zugang zu einer Vielzahl neuer Experimente in der Membran- und Zellbiologie.
Co-reporter:Andrew I. Shevchuk, Gregory I. Frolenkov, Daniel Sánchez, Peter S. James, Noah Freedman, Max J. Lab, Roy Jones, David Klenerman,Yuri E. Korchev
Angewandte Chemie International Edition 2006 45(14) pp:2212-2216
Publication Date(Web):
DOI:10.1002/anie.200503915
Co-reporter:Samuel S. White Dr.;Shankar Balasubramanian Dr. Dr.;Liming Ying Dr.
Angewandte Chemie International Edition 2006 Volume 45(Issue 45) pp:
Publication Date(Web):19 OCT 2006
DOI:10.1002/anie.200602289
Mix it up a little bit: A diffusive nanomixer for single-molecule studies has been developed that allows single-molecule fluorescence studies to be performed under nonequilibrium conditions in free solution without compromising the signal-to-noise ratio. This method has been used for direct monitoring by single-molecule fluorescence resonance energy transfer of the conformations of a quadruplex during unfolding.
Co-reporter:D. Zhou;A. Bruckbauer;C. Abell;D. Klenerman;D.-J. Kang
Advanced Materials 2005 Volume 17(Issue 10) pp:
Publication Date(Web):4 APR 2005
DOI:10.1002/adma.200401520
A facile, parallel, and controllable approach to fabricating three-dimensional microscale structures with highly fluorescent quantum dots (QDs) over centimeter-sized surface regions is presented. Highly selective layer-by-layer assembly on a surface patterned by microcontact printing produces features of up to twenty QD/polymer bilayers. Hierarchical structures such as (green-QD/red-QD)n (see Figure) and (QD1/polymer)n(QD1/QD2)m are also fabricated.
Co-reporter:Liming Ying, Andreas Bruckbauer, Dejian Zhou, Julia Gorelik, Andrew Shevchuk, Max Lab, Yuri Korchev and David Klenerman
Physical Chemistry Chemical Physics 2005 vol. 7(Issue 15) pp:2859-2866
Publication Date(Web):05 Jul 2005
DOI:10.1039/B506743J
The boundary between the physical and biological sciences has been eroded in recent years with new physical methods applied to biology and biological molecules being used for new physical purposes. We have pioneered the application of a form of scanning probe microscopy based on a scanned nanopipette, originally developed by Hansma and co-workers, for reliable non-contact imaging over the surface of a live cell. We have found that the nanopipette can also be used for controlled local voltage-driven application of reagents or biomolecules and this can be used for controlled deposition and the local delivery of probes for mapping of specific species. In this article we review this progress, focussing on the physical principles and new phenomena that we have observed, and then outline the future applications that are now possible.
Co-reporter:Kit T. Rodolfa, Andreas Bruckbauer, Dejian Zhou, Yuri E. Korchev,David Klenerman
Angewandte Chemie International Edition 2005 44(42) pp:6854-6859
Publication Date(Web):
DOI:10.1002/anie.200502338
Co-reporter:Kit T. Rodolfa, Andreas Bruckbauer, Dejian Zhou, Yuri E. Korchev,David Klenerman
Angewandte Chemie International Edition 2005 44(42) pp:6789
Publication Date(Web):
DOI:10.1002/anie.200590140
Co-reporter:Richard W. Clarke;Samuel S. White;Dejian Zhou Dr.;Liming Ying Dr. Dr.
Angewandte Chemie 2005 Volume 117(Issue 24) pp:
Publication Date(Web):10 MAY 2005
DOI:10.1002/ange.200500196
In der Falle und mit der Kamera eingefangen: Mit einer Nanopipette für die elektrodenlose Dielektrophorese ließen sich Alexa-488-markierte Proteine (Protein G und Immunglobulin G) und das freie Fluorophor reversibel einfangen, wie Weitfeld-Fluoreszenz-Imaging (siehe Bild) eindeutig belegt. Die dielektrophoretische Konzentration steigt bei diesen Fluorophor-markierten Proteinen um wenigstens den Faktor 300 an.
Co-reporter:Kit T. Rodolfa;Andreas Bruckbauer Dr.;Dejian Zhou Dr.;Yuri E. Korchev Dr. Dr.
Angewandte Chemie 2005 Volume 117(Issue 42) pp:
Publication Date(Web):25 OCT 2005
DOI:10.1002/ange.200502338
Wie gemalt: Durch Verwendung doppelläufiger Pipetten und zweier unterschiedlicher Sorten Biomoleküle können hoch komplexe, im Verlauf abgestufte Miniaturbilder gemalt werden. Diese Methode der Partikelabscheidung im Submikrometerbereich umgeht Registraturprobleme, wie sie bei anderen Abscheidungstechniken normalerweise auftreten.
Co-reporter:Kit T. Rodolfa;Andreas Bruckbauer Dr.;Dejian Zhou Dr.;Yuri E. Korchev Dr. Dr.
Angewandte Chemie 2005 Volume 117(Issue 42) pp:
Publication Date(Web):25 OCT 2005
DOI:10.1002/ange.200590139
Co-reporter:Richard W. Clarke;Samuel S. White;Dejian Zhou Dr.;Liming Ying Dr. Dr.
Angewandte Chemie International Edition 2005 Volume 44(Issue 24) pp:
Publication Date(Web):10 MAY 2005
DOI:10.1002/anie.200500196
Caught in a trap and on camera: A nanopipette is used for electrodeless dielectrophoresis and clear evidence is shown, by using wide-field fluorescence imaging (see image), for the reversible trapping of Alexa-488-labeled proteins (protein G and immunoglobulin G) and also of the fluorophore alone. The dielectrophoretic concentration is enhanced by at least a factor of 300 for these fluorophore-labeled proteins.
Co-reporter:Samuel S. White;Liming Ying Dr.;Shankar Balasubramanian Dr. Dr.
Angewandte Chemie 2004 Volume 116(Issue 44) pp:
Publication Date(Web):10 NOV 2004
DOI:10.1002/ange.200460323
Reversibel hin- und herschalten lässt sich die Fluoreszenz eines Einzelmoleküls farbstoffmarkierter DNA durch das Anlegen eines elektrischen Feldes in der Spitze einer Nanopipette (siehe Bild; FRET=resonanter Fluoreszenzenergietransfer). Das elektrische Feld scheint nur die Konformation des Acceptorfarbstoffs zu beeinflussen, was dessen Quantenausbeute erheblich verändert.
Co-reporter:Samuel S. White;Liming Ying Dr.;Shankar Balasubramanian Dr. Dr.
Angewandte Chemie International Edition 2004 Volume 43(Issue 44) pp:
Publication Date(Web):10 NOV 2004
DOI:10.1002/anie.200460323
Reversible switching of the fluorescence of an individual dye-labeled DNA molecule is possible by utilizing an electric field applied in the tip of a nanopipette (see picture; FRET=fluorescence resonance energy transfer). The electric field appears to alter the conformation of the acceptor dye only, resulting in a significant change in its quantum yield.
Co-reporter:Shujie Lin, Andrew Oldfield, David Klenerman
Surface Science 2000 Volume 464(Issue 1) pp:1-7
Publication Date(Web):20 September 2000
DOI:10.1016/S0039-6028(00)00706-8
Sum-frequency generation at surfaces (SFG) was used in situ to probe the polycrystalline Cu surface during methanol synthesis at 8.0 bar and 300°C. Resonant features in the CH stretching region indicate the catalytic activity of the pure copper surface and the formation of a formate intermediate. Furthermore, it is observed that the lineshape of the formate spectrum is very different from that of formate formed under UHV conditions. This may imply a difference in the chemical nature of the surface or surface reconstruction under the realistic turnover conditions. We estimate the formate coverage to be 0.3–0.4 under turnover. This work demonstrates that SFG has the potential to bridge the pressure gap between UHV studies and studies under real catalytic conditions by recording SFG spectra on a polycrystalline (or single crystal) surface, under turnover conditions.
Co-reporter:Andreas Bruckbauer, Paul D. Dunne, Peter James, Elizabeth Howes, Dejian Zhou, Roy Jones, David Klenerman
Biophysical Journal (7 July 2010) Volume 99(Issue 1) pp:
Publication Date(Web):7 July 2010
DOI:10.1016/j.bpj.2010.03.067
We have investigated exchange of molecules between different membrane domains on a highly compartmentalized cell, the spermatozoon. Using Alexa Fluor 555-cholera toxin B-subunit we have observed clustering of preexisting GM1 gangliosides which diffused across the anterior acrosome-equatorial segment interface but did not access the postacrosome. By contrast, single lipid and protein molecules readily exchanged between all three domains, although they diffused more slowly on nearing and crossing to the postacrosome. Thus, two types of diffusion interfaces are present on sperm heads, an “open” interface and a “mass filter” interface. The latter seems to be due to a protein-cytoskeleton network.
Co-reporter:Maksym Tsytlonok, Shehu M. Ibrahim, Pamela J.E. Rowling, Wenshu Xu, ... Laura S. Itzhaki
Structure (6 January 2015) Volume 23(Issue 1) pp:190-198
Publication Date(Web):6 January 2015
DOI:10.1016/j.str.2014.10.023
•Single-molecule FRET reveals previously hidden conformations of AnkyrinR•We show that ankyrin repeats of AnkyrinR switch between high- and low-FRET states•Switching is controlled by an unstructured “safety pin” from an adjacent subdomain•The results suggest that the order-disorder transition regulates AnkyrinR activityHere, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.
Co-reporter:Albert Chiou, Peter Hägglöf, Angel Orte, Allen Yuyin Chen, Paul D. Dunne, Didier Belorgey, Susanna Karlsson-Li, David A. Lomas, David Klenerman
Biophysical Journal (21 October 2009) Volume 97(Issue 8) pp:
Publication Date(Web):21 October 2009
DOI:10.1016/j.bpj.2009.07.057
Neuroserpin is a member of the serine proteinase inhibitor superfamily. It can undergo a conformational transition to form polymers that are associated with the dementia familial encephalopathy with neuroserpin inclusion bodies and the wild-type protein can inhibit the toxicity of amyloid-β peptides in Alzheimer's disease. We have used a single molecule fluorescence method, two color coincidence detection, to determine the rate-limiting steps of the early stages of the polymerization of fluorophore-labeled neuroserpin and have assessed how this process is altered in the presence of Aβ1–40. Our data show that neuroserpin polymerization proceeds first by the unimolecular formation of an active monomer, followed by competing processes of both polymerization and formation of a latent monomer from the activated species. These data are not in keeping with the recently proposed domain swap model of polymer formation in which the latent species and activated monomer are likely to be formed by competing pathways directly from the unactivated monomeric serpin. Moreover, the Aβ1–40 peptide forms a weak complex with neuroserpin (dissociation constant of 10 ± 5 nM) that increases the amount of active monomer thereby increasing the rate of polymerization. The Aβ1–40 is displaced from the complex so that it acts as a catalyst and is not incorporated into neuroserpin polymers.
Co-reporter:Paul D. Dunne, Ricardo A. Fernandes, James McColl, Ji Won Yoon, John R. James, Simon J. Davis, David Klenerman
Biophysical Journal (19 August 2009) Volume 97(Issue 4) pp:
Publication Date(Web):19 August 2009
DOI:10.1016/j.bpj.2009.05.046
We present a general method called dynamic single-molecule colocalization for quantitating the associations of single cell surface molecules labeled with distinct autofluorescent proteins. The chief advantages of the new quantitative approach are that, in addition to stable interactions, it is capable of measuring nonconstitutive associations, such as those induced by the cytoskeleton, and it is applicable to situations where the number of molecules is small.
Co-reporter:Ji Won Yoon, Andreas Bruckbauer, William J. Fitzgerald, David Klenerman
Biophysical Journal (15 June 2008) Volume 94(Issue 12) pp:
Publication Date(Web):15 June 2008
DOI:10.1529/biophysj.107.116285
Single molecule tracking is widely used to monitor the change in position of lipids and proteins in living cells. In many experiments in which molecules are tagged with a single or small number of fluorophores, the signal/noise ratio may be limiting, the number of molecules is not known, and fluorophore blinking and photobleaching can occur. All these factors make accurate tracking over long trajectories difficult and hence there is still a pressing need to develop better algorithms to extract the maximum information from a sequence of fluorescence images. We describe here a Bayesian-based inference approach, based on a trans-dimensional sequential Monte Carlo method that utilizes both the spatial and temporal information present in the image sequences. We show, using model data, where the real trajectory of the molecule is known, that our method allows accurate tracking of molecules over long trajectories even with low signal/noise ratio and in the presence of fluorescence blinking and photobleaching. The method is then applied to real experimental data.
Co-reporter:Richard W. Clarke, Nilah Monnier, Haitao Li, Dejian Zhou, Helena Browne, David Klenerman
Biophysical Journal (15 August 2007) Volume 93(Issue 4) pp:
Publication Date(Web):15 August 2007
DOI:10.1529/biophysj.107.106351
We present a single virion method to determine absolute distributions of copy number in the protein composition of viruses and apply it to herpes simplex virus type 1. Using two-color coincidence fluorescence spectroscopy, we determine the virion-to-virion variability in copy numbers of fluorescently labeled tegument and envelope proteins relative to a capsid protein by analyzing fluorescence intensity ratios for ensembles of individual dual-labeled virions and fitting the resulting histogram of ratios. Using EYFP-tagged capsid protein VP26 as a reference for fluorescence intensity, we are able to calculate the mean and also, for the first time to our knowledge, the variation in numbers of gD, VP16, and VP22 tegument. The measurement of the number of glycoprotein D molecules was in good agreement with independent measurements of average numbers of these glycoproteins in bulk virus preparations, validating the method. The accuracy, straightforward data processing, and high throughput of this technique make it widely applicable to the analysis of the molecular composition of large complexes in general, and it is particularly suited to providing insights into virus structure, assembly, and infectivity.
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