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Octopus (Optics Clustered to OutPut Unique Solutions) celebrated in June 2020 its 10th birthday. Based at Harwell, near Oxford, Octopus is an open access, peer reviewed, national imaging facility that offers successful U.K. applicants supported access to single molecule imaging, confocal microscopy, several flavours of superresolution imaging, light sheet microscopy, optical trapping and cryoscanning electron microscopy. Managed by a multidisciplinary team, Octopus has so far assisted >100 groups of U.K. and international researchers. Cross-fertilisation across fields proved to be a strong propeller of success underpinned by combining access to top-end instrumentation with a strong programme of imaging hardware and software developments. How Octopus was born, and highlights of the multidisciplinary output produced during its 10-year journey are reviewed below, with the aim of celebrating a myriad of collaborations with the U.K. scientific community, and reflecting on their scientific and societal impact.

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X

Myosin VI is involved in many cellular processes ranging from endocytosis to transcription. This multifunctional potential is achieved through alternative isoform splicing and through interactions of myosin VI with a diverse network of binding partners. However, the interplay between these two modes of regulation remains unexplored. To this end, we compared two different binding partners and their interactions with myosin VI by exploring the kinetic properties of recombinant proteins and their distribution in mammalian cells using fluorescence imaging. We found that selectivity for these binding partners is achieved through a high-affinity motif and a low-affinity motif within myosin VI. These two motifs allow competition among partners for myosin VI. Exploring how this competition affects the activity of nuclear myosin VI, we demonstrate the impact of a concentration-driven interaction with the low-affinity binding partner DAB2, finding that this interaction blocks the ability of nuclear myosin VI to bind DNA and its transcriptional activity in vitro. We conclude that loss of DAB2, a tumor suppressor, may enhance myosin VI–mediated transcription. We propose that the frequent loss of specific myosin VI partner proteins during the onset of cancer leads to a higher level of nuclear myosin VI activity.

Funder: Medical Research Council, Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways.Double-Strand Breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state.The RNA-guided Cas9 (CRISPR associated protein 9) endonuclease enzyme, is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based onin silicopredictions. This was achieved using electroporation of recombinant Cas9-guide complex which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

SummarySince the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

DNA double-strand breaks (DSBs) drive genomic instability. For efficient and accurate repair of these DNA lesions, the cell activates DNA damage repair pathways. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency.Here, we used Atomic Force Microscopy (AFM) to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.

SUMMARYDuring transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability which underpin the function these transcription factories remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI in the nanoscale organisation of RNAPII. We reveal that myosin VI acts as the molecular anchor that holds RNAPII into transcription factories. Perturbation of myosin VI leads to the disruption of RNAPII localisation, changes in chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of myosin VI in the spatial regulation of gene expression during the rapid response to changes in the cellular environment.

Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRET’s broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community., Broad uptake of smFRET has been hindered by high instrument costs and a lack of open-source hardware and acquisition software. Here, the authors present the smfBox, a cost-effective open-source platform capable of measuring precise FRET efficiencies between dyes on freely diffusing single molecules.

The dependence on model-fitting to evaluate particle trajectories makes it difficult for single particle tracking (SPT) to resolve the heterogeneous molecular motions typical of cells. We present here a global spatiotemporal sampler for SPT solutions using a Metropolis-Hastings algorithm. The sampler does not find just the most likely solution but also assesses its likelihood and presents alternative solutions. This enables the estimation of the tracking error. Furthermore the algorithm samples the parameters that govern the tracking process and therefore does not require any tweaking by the user. We demonstrate the algorithm on synthetic and single molecule data sets. Metrics for the comparison of SPT are generalised to be applied to a SPT sampler. We illustrate using the example of the diffusion coefficient how the distribution of the tracking solutions can be propagated into a distribution of derived quantities. We also discuss the major challenges that are posed by the realisation of a SPT sampler.

The elaboration of polarity is central to organismal development and to the maintenance of functional epithelia. Amongst the controls determining polarity are the PAR proteins, PAR6/aPKCι/PAR3, regulating both known and unknown effectors. Here we identify FARP2 as a "RIPR" motif dependent partner and substrate of aPKCι that is required for efficient polarisation and junction formation. Binding is conferred by a FERM/FA domain-Kinase domain interaction and detachment promoted by aPKCι dependent phosphorylation. FARP2 is shown to promote GTP loading of Cdc42 consistent with upstream regulation of the polarising PAR6-aPKCι complex. However, it is shown that aPKCι acts to promote the localised activity of FARP2 through phosphorylation. We conclude that this aPKCι−FARP2 complex formation acts as a positive feedback control to drive polarisation through aPKCι and other Cdc42 effectors

Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain., To prevent ligand-independent dimerisation the epidermal growth factor receptor (EGFR) is autoinhibited by an extracellular dimer interaction. Here, the authors use several imaging technologies and simulations to provide structural insights on the inactive species and on how intracellular mutations circumvent the autoinhibition of the basal state.

The elaboration of polarity is central to organismal development and to the maintenance of functional epithelia. Among the controls determining polarity are the PAR proteins, PAR6, aPKCι and PAR3, regulating both known and unknown effectors. Here, we identify FARP2 as a ‘RIPR’ motif-dependent partner and substrate of aPKCι that is required for efficient polarisation and junction formation. Binding is conferred by a FERM/FA domain–kinase domain interaction and detachment promoted by aPKCι-dependent phosphorylation. FARP2 is shown to promote GTP loading of Cdc42, which is consistent with it being involved in upstream regulation of the polarising PAR6–aPKCι complex. However, we show that aPKCι acts to promote the localised activity of FARP2 through phosphorylation. We conclude that this aPKCι−FARP2 complex formation acts as a positive feedback control to drive polarisation through aPKCι and other Cdc42 effectors. This article has an associated First Person interview with the first author of the paper., Summary: The exchange factor FARP2 is identified as a polarity-determining protein that is a partner and substrate of aPKCι. Phosphorylation by aPKCι is shown to control the localised action of FARP2 on Cdc42.

While targeted therapy against HER2 is an effective first-line treatment in HER2+ breast cancer, acquired resistance remains a clinical challenge. The pseudokinase HER3, heterodimerisation partner of HER2, is widely implicated in the resistance to HER2-mediated therapy. Here, we show that lapatinib, an ATP-competitive inhibitor of HER2, is able to induce proliferation cooperatively with the HER3 ligand neuregulin. This counterintuitive synergy between inhibitor and growth factor depends on their ability to promote atypical HER2-HER3 heterodimerisation. By stabilising a particular HER2 conformer, lapatinib drives HER2-HER3 kinase domain heterocomplex formation. This dimer exists in a head-to-head orientation distinct from the canonical asymmetric active dimer. The associated clustering observed for these dimers predisposes to neuregulin responses, affording a proliferative outcome. Our findings provide mechanistic insights into the liabilities involved in targeting kinases with ATP-competitive inhibitors and highlight the complex role of protein conformation in acquired resistance., eLife digest Around 20% of breast cancers are caused because cells have too many copies of a receptor protein called HER2 on their surface. HER2 is responsible for telling the cell to divide. Cells with too many of these receptors – and breast cancer cells can have up to 1000 times too many – divide uncontrollably. This causes the cancer to grow. Several successful anti-cancer drugs, such as Herceptin and Kadcyla, are used in the clinic to block the signals produced by HER2. Other drugs called kinase inhibitors prevent HER2 from building its faulty signals. However, a particular kinase inhibitor called lapatinib was not as successful in clinical trials as the medical community had hoped. Kinase inhibitors can have unexpected effects. While they can block specific signals in a cell, they can sometimes also cause new types of signals. Could this be one of the reasons behind the disappointing clinical trial results for lapatinib? By performing experiments on breast cancer cells grown in the laboratory, Claus, Patel et al. found that lapatinib can counterintuitively boost the growth of breast cancer cells. This occurs because lapatinib causes HER2 receptors to cluster together like a daisy chain along with another protein receptor of the same family, called HER3. These chains are primed to rapidly respond to a molecule called neuregulin, a growth factor that is commonly associated with breast cancer. The results presented by Claus, Patel et al. indicate that a particular subset of breast cancer patients – those whose cancer cells do not increase production of HER3 receptors – might better respond to lapatinib than others. The insights gained into what happens to HER2 when you try to block it should also influence the design of new drugs that target either HER2 or HER3.

Although considerable progress has been made in imaging distances in cells below the diffraction limit using FRET and super-resolution microscopy, methods for determining the separation of macromolecules in the 10-50 nm range have been elusive. We have developed fluorophore localisation imaging with photobleaching (FLImP), based on the quantised bleaching of individual protein-bound dye molecules, to quantitate the molecular separations in oligomers and nanoscale clusters. We demonstrate the benefits of using our method in studying the nanometric organisation of the epidermal growth factor receptor in cells.

There is a limited range of methods available to characterize macromolecular organization in cells on length scales from 5–50 nm. We review methods currently available and show the latest results from a new single-molecule localization-based method, fluorophore localization imaging with photobleaching (FLImP), using the epidermal growth factor (EGF) receptor (EGFR) as an example system. Our measurements show that FLImP is capable of achieving spatial resolution in the order of 6 nm. Abbreviations: BM-I, bisindolylmaleimide-I; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FLImP, fluorophore localization imaging with photobleaching; PALM, photoactivated localization microscopy; PKC, protein kinase C; SNR, signal-to-noise ratio

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.

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.

Single-particle tracking (SPT) has been used and developed over the last 25 years as a method to investigate molecular dynamics, structure, interactions, and function in the cellular context. SPT is able to show how fast and how far individual molecules move, identify different dynamic populations, measure the duration and strength of intermolecular interactions, and map out structures on the nanoscale in cells. In combination with other techniques such as macromolecular crystallography and molecular dynamics simulation, it allows us to build models of complex structures, and develop and test hypotheses of how these complexes perform their biological roles in health as well as in disease states. Here, we use the example of the epidermal growth factor receptor (EGFR), which has been studied extensively by SPT, demonstrating how the method has been used to increase our understanding of the receptor’s organization and function, including its interaction with the plasma membrane, its activation, clustering, and oligomerization, and the role of other receptors and endocytosis. The examples shown demonstrate how SPT might be employed in the investigation of other biomolecules and systems.

FRET experiments model integrin conformation changes in adherent cells., Integrins undergo global conformational changes that specify their activation state. Current models portray the inactive receptor in a bent conformation that upon activation converts to a fully extended form in which the integrin subunit leg regions are separated to enable ligand binding and subsequent signaling. To test the applicability of this model in adherent cells, we used a fluorescent resonance energy transfer (FRET)–based approach, in combination with engineered integrin mutants and monoclonal antibody reporters, to image integrin α5β1 conformation. We find that restricting leg separation causes the integrin to adopt a bent conformation that is unable to respond to agonists and mediate cell spreading. By measuring FRET between labeled α5β1 and the cell membrane, we find extended receptors are enriched in focal adhesions compared with adjacent regions of the plasma membrane. These results demonstrate definitely that major quaternary rearrangements of β1-integrin subunits occur in adherent cells and that conversion from a bent to extended form takes place at focal adhesions.

Epidermal growth factor (EGF) receptor (EGFR) modulates mitosis and apoptosis through signaling by its high-affinity (HA) and low-affinity (LA) EGF-binding states. The prevailing model of EGFR activation-derived from x-ray crystallography-involves the transition from tethered ectodomain monomers to extended back-to-back dimers and cannot explain these EGFR affinities or their different functions. Here, we use single-molecule Förster resonant energy transfer analysis in combination with ensemble fluorescence lifetime imaging microscopy to investigate the three-dimensional architecture of HA and LA EGFR-EGF complexes in cells by measuring the inter-EGF distances within discrete EGF pairs and the vertical distance from EGF to the plasma membrane. Our results show that EGFR ectodomains form interfaces resulting in two inter-EGF distances ( approximately 8 nm and5.5 nm), different from the back-to-back EGFR ectodomain interface ( approximately 11 nm). Distance measurements from EGF to the plasma membrane show that HA EGFR ectodomains are oriented flat on the membrane, whereas LA ectodomains stand proud from it. Their flat orientation confers on HA EGFR ectodomains the exclusive ability to interact via asymmetric interfaces, head-to-head with respect to the EGF-binding site, whereas LA EGFRs must interact only side-by-side. Our results support a structural model in which asymmetric EGFR head-to-head interfaces may be relevant for HA EGFR oligomerization.

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.

Dimerisation, oligomerisation, and clustering of receptor molecules are important for control of the signalling process. There has been a lack of suitable methods for the study and quantification of these processes in cells. Here we describe a protocol for a method that we have named "fluorophore localisation imaging with photobleaching" (FLImP), which uses single molecule localisation and single-step photobleaching to determine the separation of two fluorophores with a resolution of 7 nm or better. We describe the procedures required for the collection of FLImP data, and point out some of the pitfalls that must be avoided for the collection of high resolution data. We also present recent data obtained using FLImP, showing that the intracellular domain of the Epidermal Growth Factor Receptor is not required in the basal state for the receptor to form ordered inactive oligomers in the plasma membrane.

The lateral resolution of photocurrent techniques such as light-addressable potentiometric sensors (LAPS) or scanning photo-induced impedance microscopy (SPIM) is limited by the properties of the semiconductor material used. We investigated metal-insulator-semiconductor (MIS) structures based on amorphous silicon (a-Si) prepared as a thin layer on transparent glass substrates. It was shown that a sub-micrometer resolution can be achieved for this material, which is much better than the results for single crystalline Si. Some limitations caused by light scattering in the structure were observed.

We have used fluorescence resonance energy transfer (FRET) to follow the process of capsid disassembly for adenovirus (Ad) serotype 5 (Ad5) in living CHO-CAR cells. Ad5 were weakly labeled on their capsid proteins with FRET donor and acceptor fluorophores. A progressive decrease in FRET efficiency recorded during Ad5 uptake revealed that the time course of Ad5 capsid disassembly has two sequential protein dissociation rates with half-times of 3 and 60min. Fluorescence anisotropy measurements of the segmental motions of fluorophores on Ad5 indicate that the first rate is linked to the detachment from the capsid of the protruding, flexible fiber proteins. The second rate was shown to report on the combined dissociation of protein IX, penton base, and hexons, which form the rigid icosahedral capsid shell. Fluorescence lifetime imaging microscopy measurements using a pH-sensitive probe provided information on the pH of the microenvironment of Ad5 particles during intracellular trafficking, and confirmed that the fast fiber dissociation step occurred at the onset of endocytosis. The slower dissociation phase was shown to coincide with the escape of Ad5 from endocytic compartments into the cytosol, and its arrival at the nuclear membrane. These results demonstrate a rapid, quantitative live-cell assay for the investigation of virus-cell interactions and capsid disassembly.

Fluorescence resonance energy transfer (FRET) was used to reveal aspects of the mechanism of signal transduction by epidermal growth factor receptors (EGFR). The superpositions of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha) and an antibody fragment (29.1) to the carbohydrate extremity of the receptor's ectodomain as measured by FRET, show that 14% of EGFRs in A431 cells are oligomerized before growth factor binding. After binding growth factor and signaling, these oligomers dissociate before releasing growth factor. Time courses of the FRET-derived distances between constitutively oligomerized EGFRs during signal transduction show a transient structural change in the extracellular domain, which occurs simultaneously with the production of intracellular Ca2+ signals. The FRET measurements also show a slow increase in oligomerization of EGFR monomers after growth factor binding. The structural change found in the extracellular domain of oligomeric EGFRs is similar to that shown by others for EPO, Neu, Fas, and tumor necrosis factor receptors, and may therefore be a common property of the transduction of the receptor-mediated signals.

We describe an instrument designed to monitor molecular motions in multiphasic, weakly fluorescent microscopic systems. It combines synchrotron radiation, a low irradiance polarized microfluorimeter, and an automated, multiframing, single-photon-counting data acquisition system, and is capable of continually accumulating subnanosecond resolved anisotropy decays with a real-time resolution of about 60 s. The instrument has initially been built to monitor ligand–receptor interactions in living cells, but can equally be applied to the continual measurement of any dynamic process involving fluorescent molecules, that occurs over a time scale from a few minutes to several hours. As a particularly demanding demonstration of its capabilities, we have used it to monitor the environmental constraints imposed on the peptide hormone epidermal growth factor during its endocytosis and recycling to the cell surface in live cells.

Dimerization and higher-order oligomerization are believed to play an important role in the activation of the EGFR (epidermal growth factor receptor). Understanding of the process has been limited by the lack of availability of suitable methods for the measurement in cells of distances in the range 10–100 nm, too short for imaging methods and too long for spectroscopic methods such as FRET. In the present article, we review the current state of our knowledge of EGFR oligomerization, and describe results from a new single-molecule localization method that has allowed the quantitative characterization of the distribution of EGFR–EGFR distances in cells. Recent data suggest the involvement of cortical actin in regulating the formation of EGFR complexes.

Deregulation of the epidermal growth factor receptor (EGFR) signalling has been correlated with the development of a variety of human carcinomas. Receptor dimerization and phosphorylation are amongst the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change which unfolds an ectodomain loop required for dimerization. It may also induce important allosteric changes in the cytoplasmic domain. Despite these findings, ensemble-averaging methods could not resolve the details of receptor activation in situ. Here, we used two-color single-molecule imaging to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKI) and phosphatase-based manipulation of EGFR phosphorylation on live cells. Ligand bound receptors were tracked on the plasma membrane with a sophisticated Bayesian segmentation tracking algorithm and the distribution of dimer lifetimes was fitted to a single-exponential to extract dimer off-rates (koff). Our data show that, pre-treatment with type I TKI, gefitinib (active conformation binder) stabilizes the EGFR homodimer. Over-expression of EGFR specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. When a nonactivating anti-EGFR antibody, Snap-425 single-chain variable fragment that competes for EGF binding was used as a ligand, no significant difference in the koff of the dimer could be detected. Conformational changes of the cytoplasmic part of the receptor upon gefitinib pre-treatment were also confirmed by changes in Fluorescence Resonance Energy Transfer (FRET) efficiency detected by ensemble FRET/FLIM (Fluorescence Lifetime Intensity Microscopy). These results provide direct evidence that receptor phosphorylation is linked to dimer stability. The phosphorylated and non-phosphorylated receptors exhibit different kinetics and cytoplasmic conformations which may account for the heterogeneity in ligand-binding affinity observed through a concave-up Scatchard plot.

We describe an instrument based on the novel combination of synchrotron radiation, a high sensitivity time‐resolved microfluorimeter, and a multiframe single photon counting data acquisition system. This instrument has been designed specifically to measure kinetic events in live cells using fluorescence resonance energy transfer, and is capable of rapidly collecting multiple consecutive decay profiles from a small number of fluorophores. The low irradiance on the samples (

Optical aberrations degrade image quality in fluorescence microscopy, including for single-molecule based techniques. These depend on post-processing to localize individual molecules in an image series. Using simulated data, we show the impact of optical aberrations on localization success, accuracy and precision. The peak intensity and the proportion of successful localizations strongly reduces when the aberration strength is greater than 1.0 rad RMS, while the precision of each of those localisations is halved. The number of false-positive localisations exceeded 10% of the number of true-positive localisations at an aberration strength of only ~0.6 rad RMS when using the ThunderSTORM package, but at greater than 1.0 rad RMS with the Radial Symmetry package. In the presence of coma, the localization error reaches 100 nm at ~0.6 rad RMS of aberration strength. The impact of noise and of astigmatism for axial resolution are also considered. Understanding the effect of aberrations is crucial when deciding whether the addition of adaptive optics to a single-molecule microscope could significantly increase the information obtainable from an image series.

Understanding how signal inputs and outputs are organised in membrane protein signalling networks is an important question in biology. The current goal is to derive methods that would allow the 'watching' of these network proteins in action and at atomic resolution to see details of their structure. This requires the addition of a 'time' dimension to structural biology so that the spatio-temporal parameters of all atoms in each protein can be described in detail. This is a huge challenge that in cell-free systems has begun to be partially addressed through dynamic experiments combined with molecular simulations. However, in cells, the functions of particular structural motifs are not just constrained by Brownian motions, energy landscapes and thermodynamics, but also by the local availability of partners in subcellular compartments and the boundary constraints imposed by cell environments, for example in the plasma membrane, with its 2D dimensionality, local curvature and electric fields. To understand protein function in cells, observations have to be made in the only physiologically-relevant 'laboratory', the cell. This adds levels of complexity to an already vast challenge.

Current models suggest that ligand-binding heterogeneity in HER1 [human EGFR (epidermal growth factor receptor] arises from negative co-operativity in signalling HER1 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 current crystal structure of the isolated extracellular region of HER1. This receptor also differs from the Drosophila EGFR in that negative co-operativity is found only in full-length receptors in cells. Structural insights into HER1 in epithelial cells, derived from FLIM (fluorescence lifetime imaging microscopy) and two-dimensional FRET (Förster resonance energy transfer) combined with Monte Carlo and molecular dynamics simulations, have demonstrated a high-affinity ligand-binding HER1 conformation consistent with the extracellular region aligned flat on the plasma membrane. This conformation shares key features with that of the Drosophila EGFR, suggesting that the structural basis for negative co-operativity is conserved from invertebrates to humans, but that, in HER1, the extracellular region asymmetry requires interactions with the plasma membrane.

The crystallographic structures of functional fragments of ErbBs have provided excellent insights into the geometry of growth factor binding and receptor dimerization. By placing together receptor fragments to build structural models of entire receptors, we expect to understand how these enzymes are allosterically regulated; however, several predictions from these models are inconsistent with experimental evidence from cells. The opening of this gap underlines the need to investigate intact ErbBs by combining cellular and structural studies into a full picture.

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.

This report describes a comparative X-ray diffraction study of the supramolecular structure of frog sartorius and semitendinosus muscles. For sarcomere lengths of 2.7 microns and below the X-ray diffraction diagrams of each muscle type are very similar; the only differences being that the diffraction diagram for semitendinosus muscles exhibit the presence of a broad diffraction band or a cluster of diffraction orders at a spacing of ca. 230.0 nm and, also, they lack a periodicity of ca. 102.0 nm. For sarcomere lengths greater than 2.7 microns disruption of the sarcomere from sartorius muscle occurs as seen by the loss of sampling in the diffraction diagram. The semitendinosus muscle can be stretched to much longer lengths (in excess of 3.0 microns) before a loss of sampling is detected. The data also shows that in the case of the semitendinosus muscle for long sarcomere lengths transverse bands of mass are able to move without retaining a defined distance to either the Z or the M lines. This is not observed in the case of the sartorius muscle. Thus, at resolutions between ca. 3.6 microns and 7.50 nm significant ultrastructural differences between these two muscles are apparent. The data suggest that the ability of these mass bands to move may be responsible for the differences in the development of passive tension exhibited by these two muscles.

We combine single molecule fluorescence orientation imaging with single-pair fluorescence resonance energy transfer microscopy, using a total internal reflection microscope. We show how angles and FRET efficiencies can be determined for membrane proteins at the single molecule level and provide data from the epidermal growth factor receptor system in cells.

Epidermal growth factor receptor (EGFR; ErbB1, HER1 in humans) is a receptor tyrosine kinase triggering signals across the plasma membranes of cells to determine cell fate. We have used molecular dynamics simulations to investigate structural models of ErbB1 ectodomains. We show that, with minor rearrangements, the ErbB1 back-to-back dimer can align almost flat on the cell membrane. This is in contrast to the traditional picture of ErbB1 dimers standing proud of the membrane, but in line with recent FRET and EM experiments. Interaction with the membrane leads to conformational changes in the dimer, which further stabilize the back-to-back interface. On the membrane, two dimers can associate forming a tetramer. This is enabled by a head-to-head interface, involving the ligand binding side of the ectodomain, and which significantly enhances ligand binding. A weak head-to-head interface has been seen in crystal structures, but is found to stabilise appreciably in our simulation. We also find that the domains IV, connecting the receptor to the membrane, weakly interact with each other. These simulations illustrate some of the flexibility of the ErbB1 ectodomains, and may help to explain recent experimental results.

Multiwire detectors have certain characteristics that can be used to advantage, particularly in small-angle diffraction or scattering experiments on biological systems. The size of the active region of the detector and the total number of pixels can be conveniently matched to the dimensions of the synchrotron beam and to the optics of the beam line camera. Furthermore, the large dynamic range inherent in single-photon counting systems can be exploited to ensure the collection of high-quality data even when the scattering factor varies over several orders of magnitude. Linear and two-dimensional detectors are in regular use at the Daresbury Synchrotron Radiation Source, all using the delay-line readout method and a standardised data acquisition system which includes facilities for time-resolved measurements. The development of a new system (now at the prototype stage) to operate at the very high count rates available from synchrotron sources is outlined in this paper.

Quantification of the oligomerisation state and conformational changes of the epidermal growth factor receptor in its constitutive and active states are critical to fully understand their signal transduction pathway. We show the ability of single-molecule fluorescence microscopy to determine this information and its implementation by combining multidimensional emission optics with total internal reflection fluorescence microscopy. Importantly, the signalling process is analysed in live cells under physiological conditions.

Increased levels of the homodimeric calcium-binding protein, S100A4, have been shown to cause a metastatic phenotype in at least three independent model systems of breast cancer and its presence in carcinoma cells has been shown to be associated with a reduction in the survival of patients suffering from a range of different cancers. S100A4 has been shown to interact in vitro with another member of the S100 family of proteins, S100A1. The purpose of the present study was to find out whether S100A1 could affect S100A4 function. Fluorescence resonance energy transfer was used to show the interaction of S100A4 and S100A1 in living cells and the binding affinities between S100A4 and S100A1 were determined using a biosensor. S100A1 reduced the S100A4 inhibition of nonmuscle myosin A self-association and phosphorylation in vitro. S100A1 reduced S100A4 induced motility and growth in soft agar and metastasis in vivo. The results show for the first time that interactions between different S100 proteins can affect cancer-related activity, and that the presence of S100A1 protein in carcinoma cells might modulate the effect of S100A4 on their metastatic abilities.

Elevated levels of the calcium-binding regulatory protein, S100A4, have been shown to be causative of a metastatic phenotype in models of cancer metastasis and to be associated with reduced patient survival in breast cancer patients. Recombinant S100A4 protein interacts in vitro in a calcium-dependent manner with the heavy chain of non-muscle myosin isoform A at a protein kinase C phosphorylation site. At present, the mechanism of metastasis induction by S100A4 in vivo is almost completely unknown. The binding of S100A4 to a C-terminal recombinant fragment of non-muscle myosin heavy chain in living HeLa cells has now been shown using confocal microscopy, fluorescence lifetime imaging microscopy and time-correlated single-photon counting. The association between S100A4 and non-muscle myosin heavy chain was studied by determining fluorescence resonance energy transfer-derived changes in the fluorescence lifetime of enhanced cyan fluorescent protein fused to S100A4 in the presence of a recombinant fragment of the C-terminal region of non-muscle myosin heavy chain (rNMMHCIIA) fused to enhanced yellow fluorescent protein. There was no interaction between the non-muscle myosin heavy chain fragment and a calcium-binding-deficient mutant of S100A4 protein which has been shown to be defective in the induction of metastasis in model systems in vivo. The results demonstrate, for the first time, not only direct interaction between S100A4 and a target rNMMHCIIA in live mammalian cells, but also that the interaction between S100A4 and the non-muscle myosin heavy chain in vivo could contribute to the mechanism of metastasis induction by a high level of S100A4 protein.

The fourth generation light source (4GLS) is a new synchrotron radiation facility proposed for the United Kingdom. It is based on an energy recovery linac, and will consist of a suite of instruments providing radiation from the soft x-ray to the far infrared. In addition to undulator sources, three free electron lasers (FELs) are proposed. Two cavity-based lasers will provide infrared and vacuum-ultraviolet (VUV) radiation, and one FEL will use the self-amplification of spontaneous emission (SASE) phenomenon to produce extreme-ultraviolet (XUV) radiation. The combination of sources will provide unprecedented wavelength coverage, power, and timing structure. The 4GLS facility is expected to have great potential for many areas of research in the biomedical sciences. We discuss some potential biomedical applications of 4GLS FELs in, for example, the areas of macromolecular conformational dynamics, imaging, and radiation damage, and show where the unique properties of this combination of sources will benefit these areas of research.