Search

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.

Despite being catalytically defective, pseudokinases are typically essential players of cellular signalling, acting as allosteric regulators of their active counterparts. Deregulation of a growing number of pseudokinases has been linked to human diseases, making pseudokinases therapeutic targets of interest. Pseudokinases can be dynamic, adopting specific conformations critical for their allosteric function. Interfering with their allosteric role, with small molecules that would lock pseudokinases in a conformation preventing their productive partner interactions, is an attractive therapeutic strategy to explore. As a well-known allosteric activator of epidermal growth factor receptor family members, and playing a major part in cancer progression, the pseudokinase HER3 is a relevant context in which to address the potential of pseudokinases as drug targets for the development of allosteric inhibitors. In this proof-of-concept study, we developed a multiplex, medium-throughput thermal shift assay screening strategy to assess over 100 000 compounds and identify selective small molecule inhibitors that would trap HER3 in a conformation which is unfavourable for the formation of an active HER2–HER3 heterodimer. As a proof-of-concept compound, AC3573 bound with some specificity to HER3 and abrogated HER2–HER3 complex formation and downstream signalling in cells. Our study highlights the opportunity to identify new molecular mechanisms of action interfering with the biological function of pseudokinases.

Non-small cell lung cancer (NSCLC) is a complex disease often driven by activating mutations or amplification of the epidermal growth factor receptor (EGFR) gene, which expresses a transmembrane receptor tyrosine kinase. Targeted anti-EGFR treatments include small-molecule tyrosine kinase inhibitors (TKIs), among which gefitinib and erlotinib are the best studied, and their function more often imaged. TKIs block EGFR activation, inducing apoptosis in cancer cells addicted to EGFR signals. It is not understood why TKIs do not work in tumours driven by EGFR overexpression but do so in tumours bearing classical activating EGFR mutations, although the latter develop resistance in about one year. Fluorescence imaging played a crucial part in research efforts to understand pro-survival mechanisms, including the dysregulation of autophagy and endocytosis, by which cells overcome the intendedly lethal TKI-induced EGFR signalling block. At their core, pro-survival mechanisms are facilitated by TKI-induced changes in the function and conformation of EGFR and its interactors. This review brings together some of the main advances from fluorescence imaging in investigating TKI function and places them in the broader context of the TKI resistance field, highlighting some paradoxes and suggesting some areas where super-resolution and other emerging methods could make a further contribution.

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-X3-Small’ motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.FieldsStructural Biology and Biophysics / Computational Biology

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

Voltage‐gated sodium channels comprise an ion‐selective α‐subunit and one or more associated β‐subunits. The β3‐subunit (encoded by the SCN3B gene) is an important physiological regulator of the heart‐specific sodium channel, Nav1.5. We have previously shown that when expressed alone in HEK293F cells, the full‐length β3‐subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric β3‐subunits, not just at the plasma membrane, but throughout the secretory pathway. We then investigate the corresponding clustering properties of the α‐subunit and the effects upon these of the β3‐subunits. The oligomeric status of the Nav1.5 α‐subunit in vivo, with or without the β3‐subunit, has not been previously investigated. Using super‐resolution fluorescence imaging, we show that under conditions typically used in electrophysiological studies, the Nav1.5 α‐subunit assembles on the plasma membrane of HEK293F cells into spatially localized clusters rather than individual and randomly dispersed molecules. Quantitative analysis indicates that the β3‐subunit is not required for this clustering but β3 does significantly change the distribution of cluster sizes and nearest‐neighbor distances between Nav1.5 α‐subunits. However, when assayed by PLA, the β3‐subunit increases the number of PLA‐positive signals generated by anti‐(Nav1.5 α‐subunit) antibodies, mainly at the plasma membrane. Since PLA can be sensitive to the orientation of proteins within a cluster, we suggest that the β3‐subunit introduces a significant change in the relative alignment of individual Nav1.5 α‐subunits, but the clustering itself depends on other factors. We also show that these structural and higher‐order changes induced by the β3‐subunit do not alter the degree of electrophysiological gating cooperativity between Nav1.5 α‐subunits. Our data provide new insights into the role of the β3‐subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.

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.

Summary The advancement of serial cryoFIB/SEM offers an opportunity to study large volumes of near-native, fully hydrated frozen cells and tissues at voxel sizes of 10 nm and below. We explored this capability for pathologic characterization of vitrified human patient cells by developing and optimizing a serial cryoFIB/SEM volume imaging workflow. We demonstrate profound disruption of subcellular architecture in primary fibroblasts from a Leigh syndrome patient harboring a disease-causing mutation in USMG5 protein responsible for impaired mitochondrial energy production., Graphical Abstract, Highlights • Developed and optimized a serial cryoFIB/SEM volume imaging workflow • Visualized the 3D structure of an entire cell under native conditions • Revealed a disruption of cellular structures in primary LS patient fibroblasts • Demonstrated the potential for clinical phenotyping of pathogenic tissues, Serial cryoFIB/SEM offers new opportunities for structural analysis of cells and tissues under near-native conditions. Zhu et al. developed and optimized a serial cryoFIB/SEM volume imaging workflow for visualization of entire vitreous cells in 3D, and demonstrated its potential for clinical and pathological applications with primary Leigh syndrome patient cells.

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.

DNA double-strand breaks drive genomic instability. 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 have used Atomic Force Microscopy 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.

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.

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.

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.

Our mechanistic understanding of cell function depends on imaging biological processes in cells with molecular resolution. Super-resolution fluorescence microscopy plays a crucial role by reporting cellular ultrastructure with 20-30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions, which substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixatives. Cryogenic conditions are, however, underutilized because of the lack of compatible high numerical aperture (NA) objectives. Here we describe a protocol for the use of super-hemispherical solid immersion lenses (superSILs) to achieve super-resolution imaging at cryogenic temperatures with an effective NA of 2.17 and resolution of ~10 nm.

Myosin VI is involved in a variety of cellular processes ranging from endocytosis to transcription. This multi-functional potential is achieved through alternative isoform splicing and through the interaction with a diverse network of binding partners. However, the interplay between the two modes of regulation remains unexplored. To this end, we have compared two different binding partners, Dab2 and CALCOCO2/NDP52, and their interaction with two myosin VI splice isoforms. We found that both isoforms adopt an auto-inhibited state and are subsequently activated by binding partner association. However, differential regulation is achieved through a high and a low affinity binding motifs within myosin VI, with one isoform having the high affinity site blocked. This allows competition between partners and links isoform splicing with binding partner selectivity. Dab2 competition hinders the activity of nuclear myosin VI by preventing DNA binding and transcription. Moreover, re-introduction of Dab2 in the Dab2-deficient MCF-7 cells leads to a decrease in myosin VI-dependent estrogen receptor gene expression. We propose that the frequent loss of Dab2 during the onset of cancer enables a higher level of nuclear myosin VI activity, thereby driving the activity of the estrogen receptor to promote tumourgenesis.

The epidermal growth factor receptor (EGFR) is historically the prototypical receptor tyrosine kinase, being the first cloned and the first where the importance of ligand-induced dimer activation was ascertained. However, many years of structure determination has shown that EGFR is not completely understood. One challenge is that the many structure fragments stored at the PDB only provide a partial view because full-length proteins are flexible entities and dynamics play a key role in their functionality. Another challenge is the shortage of high-resolution data on functionally important higher-order complexes. Still, the interest in the structure/function relationships of EGFR remains unabated because of the crucial role played by oncogenic EGFR mutants in driving non-small cell lung cancer (NSCLC). Despite targeted therapies against EGFR setting a milestone in the treatment of this disease, ubiquitous drug resistance inevitably emerges after one year or so of treatment. The magnitude of the challenge has inspired novel strategies. Among these, the combination of multi-disciplinary experiments and molecular dynamic (MD) simulations have been pivotal in revealing the basic nature of EGFR monomers, dimers and multimers, and the structure-function relationships that underpin the mechanisms by which EGFR dysregulation contributes to the onset of NSCLC and resistance to treatment.

Super-resolution fluorescence microscopy plays a crucial role in our understanding of cell structure and function by reporting cellular ultrastructure with 20–30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions. This substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixation. Cryogenic conditions are, however, underutilised because of the lack of compatible high numerical aperture objectives. Here, using a low-cost super-hemispherical solid immersion lens (superSIL) and a basic set-up we achieve 12 nm resolution under cryogenic conditions, to our knowledge the best yet attained in cells using simple set-ups and/or commercial systems. By also allowing multicolour imaging, and by paving the way to total-internal-reflection fluorescence imaging of mammalian cells under cryogenic conditions, superSIL microscopy opens a straightforward route to achieve unmatched resolution on bacterial and mammalian cell samples., Lin Wang et al. present a new super-resolution modality using a super-hemispherical immersion lens. They achieve a 12 nm spatial resolution in cells under cryogenic conditions, which offers the technical means to study bacterial and mammalian cell samples at molecule localisation length-scales.

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

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport and cellular signalling. Recent models suggest that for these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signalling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using sub-diffraction-limited Airyscan confocal microscopy. In addition, single particle tracking reveals that these proteins move at different rates within the membrane. We show that the actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by affecting single particle dynamics of two proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for differentially regulating protein cluster size and dynamics thereby contributing to the formation and functionality of membrane nanodomains.One sentence summarySize and mobility of protein nanodomains in the plant plasma-membrane are regulated by interaction with the cell wall extracellular matrix.Significance statementThe plant plasma membrane acts as the front line for cellular perception of the environment. As such, a large number of signalling and transport proteins which perceive or transport environmental signals, developmental cues and nutrients are located within it. Recently, a number of studies have revealed that proteins located within the plasma membrane do not simply freely diffuse within its plane. Rather, proteins are localized in nanometer sized structures called nanodomains. In addition to the plasma-membrane, plant cells also have an extracellular matrix, the cell wall. Here we have shown that the cell wall has a role in regulating the dynamics and size of plasma membrane nanodomains for proteins involved in morphogenesis (PIN3) and pathogen perception (FLS2).

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.

Super-resolution fluorescence microscopy achieves 20-30 nm resolution by using liquid-immersion objectives to optimize light collection and chemical sample fixation to minimize image blurring. It is known that fluorophore brightness increases substantially under cryogenic conditions and that cryo-fixation is far superior in preserving ultrastructure. However, cryogenic conditions have not been exploited to improve resolution or sample quality because liquid immersion media freezes at the objective, losing its optical properties. Here, simply by replacing the immersion fluid with a low-cost super-hemispherical solid immersion lens (superSIL), we effortlessly achieve uperSIL microscopy delivers a straightforward route to achieve unmatched nanoscale resolution on both bacterial and mammalian cell samples, which any laboratory can effortlessly and inexpensively implement.

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

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications. DOI: http://dx.doi.org/10.7554/eLife.24903.001, eLife digest Many of the molecules that are essential for life are too small to be visible inside cells. So, scientists use large complex proteins called antibodies that bind to these molecules to detect whether they are present and show where they are in a cell. As well as being useful tools in experiments, these antibodies can be used to help identify and treat diseases. The body produces antibodies in response to an infection. The antibodies used in experiments are purified from animal blood, but this method of producing antibodies has flaws. For example, it can be difficult to make identical batches of antibody that always behave in the same way. So scientists have developed “alternative binding proteins” that can be made in the laboratory. These proteins are much less complicated and can be developed more quickly than antibodies, and can easily be adapted for a variety of uses. An alternative binding protein called an Affimer behaves in a similar way to an antibody by binding tightly to its target molecule, but is much more stable to acidity and high temperature. Tiede et al. have now tested how well the Affimer works in a wide range of different experiments that normally use antibodies to analyse the amount of a particular molecule inside a cell. The results of the tests show that the Affimer behaves in the same way as antibodies, and sometimes works more effectively. Tiede et al. show that an Affimer can help to reveal how a particular molecule works within a cell, to create detailed pictures of molecules in cells and tissues, and to identify a tumour. It can also be used alongside a new technique called ‘super-resolution microscopy’ that allows researchers to watch the activity of individual molecules. Future challenges are to test the Affimer in even more applications and to encourage its wider use by researchers, alongside other alternative binding proteins, as as replacements for some antibodies. This could ultimately lead to the development of faster and more efficient diagnostic, imaging and therapeutic tests. DOI: http://dx.doi.org/10.7554/eLife.24903.002

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.

Insights from single-molecule tracking in mammalian cells have the potential to greatly contribute to our understanding of the dynamic behavior of many protein families and networks which are key therapeutic targets of the pharmaceutical industry. This is particularly so at the plasma membrane, where the method has begun to elucidate the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell, including signal transduction, receptor recognition, cell-cell adhesion, etc. However, despite much progress, single-molecule tracking faces challenges in mammalian samples that hinder its general application in the biomedical sciences. Much work has recently focused on improving the methods for fluorescent tagging of target molecules, detection and localization of tagged molecules, which appear as diffraction-limited spots in charge-coupled device (CCD) images, and objectively establishing the correspondence between moving particles in a sequence of image frames to follow their diffusive behavior. In this review we outline the state-of-the-art in the field and discuss the advantages and limitations of the methods available in the context of specific applications, aiming at helping researchers unfamiliar with single molecules methods to plan out their experiments.

Total internal reflection fluorescence microscopy (TIRFM) has been proven to be an extremely powerful technique in animal cell research for generating high contrast images and dynamic protein conformation information. However, there has long been a perception that TIRFM is not feasible in plant cells because the cell wall would restrict the penetration of the evanescent field and lead to scattering of illumination. By comparative analysis of epifluorescence and TIRF in root cells, it is demonstrated that TIRFM can generate high contrast images, superior to other approaches, from intact plant cells. It is also shown that TIRF imaging is possible not only at the plasma membrane level, but also in organelles, for example the nucleus, due to the presence of the central vacuole. Importantly, it is demonstrated for the first time that this is TIRF excitation, and not TIRF-like excitation described as variable-angle epifluorescence microscopy (VAEM), and it is shown how to distinguish the two techniques in practical microscopy. These TIRF images show the highest signal-to-background ratio, and it is demonstrated that they can be used for single-molecule microscopy. Rare protein events, which would otherwise be masked by the average molecular behaviour, can therefore be detected, including the conformations and oligomerization states of interacting proteins and signalling networks in vivo. The demonstration of the application of TIRFM and single-molecule analysis to plant cells therefore opens up a new range of possibilities for plant cell imaging.

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.