Christopher J. Tynan

Focal adhesions are sites of integrin extension

FRET experiments model integrin conformation changes in adherent cells.

Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

A ‘pocket guide’ to total internal reflection fluorescence

The phenomenon of total internal reflection fluorescence (TIRF) was placed in the context of optical microscopy by Daniel Axelrod over three decades ago. TIRF microscopy exploits the properties of an evanescent electromagnetic field to optically section sample regions in the close vicinity of the substrate where the field is induced. The first applications in cell biology targeted investigation of phenomena at the basolateral plasma membrane. The most notable application of TIRF is single‐molecule experiments, which can provide information on fluctuation distributions and rare events, yielding novel insights on the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell. This short review intends to provide a ‘one stop shop’ explanation of the electromagnetic theory behind the remarkable properties of the evanescent field, guide the reader through the principles behind building or choosing your own TIRF system and consider how the most popular applications of the method exploit the evanescent field properties.

Human Epidermal Growth Factor Receptor (EGFR) Aligned on the Plasma Membrane Adopts Key Features of Drosophila EGFR Asymmetry

ABSTRACT The ability of epidermal growth factor receptor (EGFR) to control cell fate is defined by its affinity for ligand. Current models suggest that ligand-binding heterogeneity arises from negative cooperativity in signaling receptor dimers, for which the asymmetry of the extracellular region of the Drosophila EGFR has recently provided a structural basis. However, no asymmetry is apparent in the isolated extracellular region of the human EGFR. Human EGFR also differs from the Drosophila EGFR in that negative cooperativity is found only in full-length receptors in cells. To gain structural insights into the human EGFR in situ, we developed an approach based on quantitative Förster resonance energy transfer (FRET) imaging, combined with Monte Carlo and molecular dynamics simulations, to probe receptor conformation in epithelial cells. We experimentally demonstrate a high-affinity ligand-binding human EGFR conformation consistent with the extracellular region aligned flat on the plasma membrane. We explored the relevance of this conformation to ligand-binding heterogeneity and found that the asymmetry of this structure shares key features with that of the Drosophila EGFR, suggesting that the structural basis for negative cooperativity is conserved from invertebrates to humans but that in human EGFR the extracellular region asymmetry requires interactions with the plasma membrane.

Automated multidimensional single molecule fluorescence microscopy feature detection and tracking

Characterisation of multi-protein interactions in cellular networks can be achieved by optical microscopy using multidimensional single molecule fluorescence imaging. Proteins of different species, individually labelled with a single fluorophore, can be imaged as isolated spots (features) of different colour light in different channels, and their diffusive behaviour in cells directly measured through time. Challenges in data analysis have, however, thus far hindered its application in biology. A set of methods for the automated analysis of multidimensional single molecule microscopy data from cells is presented, incorporating Bayesian segmentation-based feature detection, image registration and particle tracking. Single molecules of different colours can be simultaneously detected in noisy, high background data with an arbitrary number of channels, acquired simultaneously or time-multiplexed, and then tracked through time. The resulting traces can be further analysed, for example to detect intensity steps, count discrete intensity levels, measure fluorescence resonance energy transfer (FRET) or changes in polarisation. Examples are shown illustrating the use of the algorithms in investigations of the epidermal growth factor receptor (EGFR) signalling network, a key target for cancer therapeutics, and with simulated data.

Self-association of Calcium-binding Protein S100A4 and Metastasis

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.

EGFR oligomerization organizes kinase-active dimers into competent signalling platforms

Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling.

Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes

Background The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging. Methodology/Principal Findings A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells. Conclusions/Significance We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470–1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging.

A tale of the epidermal growth factor receptor: The quest for structural resolution on cells

The challenge of determining the architecture and geometry of oligomers of the epidermal growth factor receptor (EGFR) on the cell surface has been approached using a variety of biochemical and biophysical methods. This review is intended to provide a narrative of how key concepts in the field of EGFR research have evolved over the years, from the origins of the prevalent EGFR signalling dimer hypothesis through to the development and implementation of methods that are now challenging the conventional view. The synergy between X-ray crystallography and cellular fluorescence microscopy has become particularly important, precisely because the results from these two methods diverged and highlighted the complexity of the challenge. We illustrate how developments in super-resolution microscopy are now bridging this gap. Exciting times lie ahead where knowledge of the nature of the complexes can assist with the development of a new generation of anti-cancer drugs.

3D visualization of additive occlusion and tunable full-spectrum fluorescence in calcite

From biomineralization to synthesis, organic additives provide an effective means of controlling crystallization processes. There is growing evidence that these additives are often occluded within the crystal lattice. This promises an elegant means of creating nanocomposites and tuning physical properties. Here we use the incorporation of sulfonated fluorescent dyes to gain new understanding of additive occlusion in calcite (CaCO3), and to link morphological changes to occlusion mechanisms. We demonstrate that these additives are incorporated within specific zones, as defined by the growth conditions, and show how occlusion can govern changes in crystal shape. Fluorescence spectroscopy and lifetime imaging microscopy also show that the dyes experience unique local environments within different zones. Our strategy is then extended to simultaneously incorporate mixtures of dyes, whose fluorescence cascade creates calcite nanoparticles that fluoresce white. This offers a simple strategy for generating biocompatible and stable fluorescent nanoparticles whose output can be tuned as required.