Binding partners regulate unfolding of myosin VI to activate the molecular motor.

Myosin VI is the only minus-end actin motor and it is coupled to various cellular processes ranging from endocytosis to transcription. This multi-potent nature is achieved through alternative isoform splicing and interactions with a network of binding partners. There is a complex interplay between isoforms and binding partners to regulate myosin VI. Here, we have compared the regulation of two myosin VI splice isoforms by two different binding partners. By combining biochemical and single-molecule approaches, we propose that myosin VI regulation follows a generic mechanism, independently of the spliced isoform and the binding partner involved. We describe how myosin VI adopts an autoinhibited backfolded state which is released by binding partners. This unfolding activates the motor, enhances actin binding and can subsequently trigger dimerization. We have further expanded our study by using single-molecule imaging to investigate the impact of binding partners upon myosin VI molecular organization and dynamics.

DNA damage alters nuclear mechanics through chromatin reorganization.

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.

Competition between two high- and low-affinity protein-binding sites in myosin VI controls its cellular function.

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.

The Rho family GEF FARP2 is activated by aPKCι to control tight junction formation and polarity.

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.

Supramolecular clustering of the cardiac sodium channel Nav1.5 in HEK293F cells, with and without the auxiliary ß3-subunit.

Voltage-gated sodium channels comprise an ion-selective α-subunit and one or more associated ß-subunits. The ß3-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 ß3-subunit forms trimers in the plasma membrane. We extend this result with biochemical assays and use the proximity ligation assay (PLA) to identify oligomeric ß3-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 ß3-subunits. The oligomeric status of the Nav1.5 α-subunit in vivo, with or without the ß3-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 ß3-subunit is not required for this clustering but ß3 does significantly change the distribution of cluster sizes and nearest-neighbor distances between Nav1.5 α-subunits. However, when assayed by PLA, the ß3-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 ß3-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 ß3-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 ß3-subunit and the supramolecular organization of sodium channels, in an important model cell system that is widely used to study Nav channel behavior.

A small molecule inhibitor of HER3: a proof-of-concept study.

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.

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.

Nanometric molecular separation measurements by single molecule photobleaching.

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.

Effect of phosphorylation on EGFR dimer stability probed by single-molecule dynamics and FRET/FLIM.

Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (koff). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the koff of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell's proliferation potential.

Determining the geometry of oligomers of the human epidermal growth factor family on cells with <10 nm resolution.

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.

Cell wall constrains lateral diffusion of plant plasma-membrane proteins.

A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein-protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.

Structure-function relationships and supramolecular organization of the EGFR (epidermal growth factor receptor) on the cell surface.

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.

Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers.

The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation. Here we show that drug-resistant EGFR mutations manipulate the assembly of ligand-free, kinase-active oligomers to promote and stabilize the assembly of oligomer-obligate active dimer sub-units and circumvent the need for ligand binding. We reveal the structure and assembly mechanisms of these ligand-free, kinase-active oligomers, uncovering oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Importantly, we find that the active dimer sub-units within ligand-free oligomers are the high affinity binding sites competent to bind physiological ligand concentrations and thus drive tumor growth, revealing a link with tumor proliferation. Our findings provide a framework for future drug discovery directed at tackling oncogenic EGFR mutations by disabling oligomer-assembling interactions.

Compositional Evolution of Individual CoNPs on Co/TiO2 during CO and Syngas Treatment Resolved through Soft XAS/X-PEEM.

The nanoparticle (NP) redox state is an important parameter in the performance of cobalt-based Fischer-Tropsch synthesis (FTS) catalysts. Here, the compositional evolution of individual CoNPs (6-24 nm) in terms of the oxide vs metallic state was investigated in situ during CO/syngas treatment using spatially resolved X-ray absorption spectroscopy (XAS)/X-ray photoemission electron microscopy (X-PEEM). It was observed that in the presence of CO, smaller CoNPs (i.e., ≤12 nm in size) remained in the metallic state, whereas NPs ≥ 15 nm became partially oxidized, suggesting that the latter were more readily able to dissociate CO. In contrast, in the presence of syngas, the oxide content of NPs ≥ 15 nm reduced, while it increased in quantity in the smaller NPs; this reoxidation that occurs primarily at the surface proved to be temporary, reforming the reduced state during subsequent UHV annealing. O K-edge measurements revealed that a key parameter mitigating the redox behavior of the CoNPs were proximate oxygen vacancies (Ovac). These results demonstrate the differences in the reducibility and the reactivity of Co NP size on a Co/TiO2 catalyst and the effect Ovac have on these properties, therefore yielding a better understanding of the physicochemical properties of this popular choice of FTS catalysts.

Autophagy receptor NDP52 alters DNA conformation to modulate RNA polymerase II transcription.

NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear functions for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We find that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover nuclear roles for NDP52 in gene expression and DNA structure regulation.

Human epidermal growth factor receptor (HER1) aligned on the plasma membrane adopts key features of Drosophila EGFR asymmetry.

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.

Investigating extracellular in situ EGFR structure and conformational changes using FRET microscopy.

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.

Single molecule fluorescence detection and tracking in mammalian cells: the state-of-the-art and future perspectives.

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.

Fluorescence Imaging of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer.

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.

Resolving the Effect of Oxygen Vacancies on Co Nanostructures Using Soft XAS/X-PEEM.

Improving both the extent of metallic Co nanoparticle (Co NP) formation and their stability is necessary to ensure good catalytic performance, particularly for Fischer-Tropsch synthesis (FTS). Here, we observe how the presence of surface oxygen vacancies (Ovac) on TiO2 can readily reduce individual Co3O4 NPs directly into CoO/Co0 in the freshly prepared sample by using a combination of X-ray photoemission electron microscopy (X-PEEM) coupled with soft X-ray absorption spectroscopy. The Ovac are particularly good at reducing the edge of the NPs as opposed to their center, leading to smaller particles being more reduced than larger ones. We then show how further reduction (and Ovac consumption) is achieved during heating in H2/syngas (H2 + CO) and reveal that Ovac also prevents total reoxidation of Co NPs in syngas, particularly the smallest (∼8 nm) particles, thus maintaining the presence of metallic Co, potentially improving catalyst performance.