Extensive Bioinformatics Analyses Reveal a Phylogenetically Conserved Winged Helix (WH) Domain (Zτ) of Topoisomerase IIα, Elucidating Its Very High Affinity for Left-Handed Z-DNA and Suggesting Novel Putative Functions.

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, "bubbles", R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the "B-Z-topoII hypothesis" and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments ("Z-flipons") as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover "conformase" activity on given G(ate) B-DNA segments ("Z-flipins"), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural-functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents.

Treatment with diphenyl-pyrazole compound anle138b/c reveals that α-synuclein protects melanoma cells from autophagic cell death.

Recent epidemiological and clinical studies have reported a significantly increased risk for melanoma in people with Parkinson's disease. Because no evidence could be obtained that genetic factors are the reason for the association between these two diseases, we hypothesized that of the three major Parkinson's disease-related proteins-α-synuclein, LRRK2, and Parkin-α-synuclein might be a major link. Our data, presented here, demonstrate that α-synuclein promotes the survival of primary and metastatic melanoma cells, which is the exact opposite of the effect that α-synuclein has on dopaminergic neurons, where its accumulation causes neuronal dysfunction and death. Because this detrimental effect of α-synuclein on neurons can be rescued by the small molecule anle138b, we explored its effect on melanoma cells. We found that treatment with anle138b leads to massive melanoma cell death due to a major dysregulation of autophagy, suggesting that α-synuclein is highly beneficial to advanced melanoma because it ensures that autophagy is maintained at a homeostatic level that promotes and ensures the cell's survival.

Conformational variability of recombination R-triplex formed by the mammalian telomeric sequence.

Alignment of three nucleic acids strands, in which the third strand is identical to one of the DNA duplex strands, occurs in various cellular systems. In the case of telomeric t-loops, recognition between the DNA duplex and the homologous single strand is likely to be mediated by proteins through formation of the transient recombination-type R-triplex. Earlier, using 2-aminopurine as a fluorescent reporting base, we evaluated the thermodynamic characteristics of intramolecular R-triplex formed by a mixed nucleotide sequence. Here, we used this approach to explore a propensity of the telomeric TTAGGG repeat to form the R-triplex. The circular dichroism spectral changes detected upon formation of the R-triplex suggest that this process is accompanied by specific conformational changes in DNA, including a local destabilization of the target duplex next to a GGG run revealed by the fluorescence of the reporting 2-aminopurine base. Surprisingly, stability of the R-triplex formed by telomeric sequence depends strikingly on the counter ion, being higher for Na(+) than for Li(+). Taken together these findings indicate a significant conformational variability of telomeric DNA in the context of recombination-type R-triplex, a phenomenon of possible biological relevance.

Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer.

Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non-small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild-type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color superresolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live-cell Förster resonance energy transfer measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization.

Fluorogen activating protein-affibody probes: modular, no-wash measurement of epidermal growth factor receptors.

Fluorescence is essential for dynamic live cell imaging, and affinity reagents are required for quantification of endogenous proteins. Various fluorescent dyes can report on different aspects of biological trafficking, but must be independently conjugated to affinity reagents and characterized for specific biological readouts. Here we present the characterization of a new modular platform for small anti-EGFR affinity probes for studying rapid changes in receptor pools. A protein domain (FAP dL5**) that binds to malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the compact EGFR binding affibody ZEGFR:1907. This is a recombinant and fluorogenic labeling reagent for native EGFR molecules. In vitro fluorescence assays demonstrated that the binding of these dyes to the FAP-affibody fusions produced thousand-fold fluorescence enhancements, with high binding affinity and fast association rates. Flow cytometry assays and fluorescence microscopy demonstrated that these probes label endogenous EGFR on A431 cells without disruption of EGFR function, and low nanomolar surface Kd values were observed with the double-ZEGFR:1907 constructs. The application of light-harvesting fluorogens (dyedrons) significantly improved the detected fluorescence signal. Altering the order of addition of the ligand, probe, and dyes allowed differentiation between surface and endocytotic pools of receptors to reveal the rapid dynamics of endocytic trafficking. Therefore, FAP/affibody coupling provides a new approach to construct compact and modular affinity probes that label endogenous proteins on living cells and can be used for studying rapid changes in receptor pools involved in trafficking.

Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson's disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this "stem cell pathology" could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for development of neuroprotective strategies for PD.

Structure-function relationships of ErbB RTKs in the plasma membrane of living cells.

We review the states of the ErbB family of receptor tyrosine kinases (RTKs), primarily the EGF receptor (EGFR, ErbB1, HER1) and the orphan receptor ErbB2 as they exist in living mammalian cells, focusing on four main aspects: (1) aggregation state and distribution in the plasma membrane; (2) conformational features of the receptors situated in the plasma membrane, compared to the crystallographic structures of the isolated extracellular domains; (3) coupling of receptor disposition on filopodia with the transduction of signaling ligand gradients; and (4) ligand-independent receptor activation by application of a magnetic field.

Magnetic nanoparticles as mediators of ligand-free activation of EGFR signaling.

BACKGROUND: Magnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells. METHODOLOGY/PRINCIPAL FINDINGS: The EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength. CONCLUSIONS/SIGNIFICANCE: We demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor ectodomain. The results suggest that activation of growth factor receptors may be triggered by ligand-independent molecular crowding resulting from overexpression and/or sequestration in membrane microdomains.

Dynamic conformational transitions of the EGF receptor in living mammalian cells determined by FRET and fluorescence lifetime imaging microscopy.

We have revealed a reorientation of ectodomain I of the epidermal growth factor receptor (EGFR; ErbB1; Her1) in living CHO cells expressing the receptor, upon binding of the native ligand EGF. The state of the unliganded, nonactivated EGFR was compared to that exhibited after ligand addition in the presence of a kinase inhibitor that prevents endocytosis but does not interfere with binding or the ensuing conformational rearrangements. To perform these experiments, we constructed a transgene EGFR with an acyl carrier protein sequence between the signal peptide and the EGFR mature protein sequence. This protein, which behaves similarly to wild-type EGFR with respect to EGF binding, activation, and internalization, can be labeled at a specific serine in the acyl carrier tag with a fluorophore incorporated into a 4'-phosphopantetheine (P-pant) conjugate transferred enzymatically from the corresponding CoA derivative. By measuring Förster resonance energy transfer between a molecule of Atto390 covalently attached to EGFR in this manner and a novel lipid probe NR12S distributed exclusively in the outer leaflet of the plasma membrane, we determined the apparent relative separation of ectodomain I from the membrane under nonactivating and activating conditions. The data indicate that the unliganded domain I of the EGFR receptor is situated much closer to the membrane before EGF addition, supporting the model of a self-inhibited configuration of the inactive receptor in quiescent cells.

Differential endocytosis and signaling dynamics of insulin receptor variants IR-A and IR-B.

Insulin signaling comprises a complex cascade of events, playing a key role in the regulation of glucose metabolism and cellular growth. Impaired response to insulin is the hallmark of diabetes, whereas upregulated insulin activity occurs in many cancers. Two splice variants of the insulin receptor (IR) exist in mammals: IR-A, lacking exon 11, and full-length IR-B. Although considerable biochemical data exist on insulin binding and downstream signaling, little is known about the dynamics of the IR itself. We created functional IR transgenes fused with visible fluorescent proteins for use in combination with biotinamido-caproyl insulin and streptavidin quantum dots. Using confocal and structured illumination microscopy, we visualized the endocytosis of both isoforms in living and fixed cells and demonstrated a higher rate of endocytosis of IR-A than IR-B. These differences correlated with higher and sustained activation of IR-A in response to insulin and with distinctive ERK1/2 activation profiles and gene transcription regulation. In addition, cells expressing IR-B showed higher AKT phosphorylation after insulin stimulation than cells expressing IR-A. Taken together, these results suggest that IR signaling is dependent on localization; internalized IRs regulate mitogenic activity, whereas metabolic balance signaling occurs at the cell membrane.

Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis.

Ligand-driven dimerizations of ErbB receptor subunits fulfill a fundamental role in their activation. We have used the number and brightness analysis technique to investigate the existence of preformed ligand-independent dimers and clusters and to characterize the initial steps in the activation of ErbB1 and ErbB2. In cells expressing 50,000-200,000 receptors, ErbB1 was monomeric in the absence of ligand stimulation, whereas in CHO cells with receptor levels >500,000 as much as 30% of ErbB1 was present as preformed dimers. EGF induced the formation of ErbB1 dimers as well as larger clusters (up to pentamers) that colocalized with clathrin-coated pits. The distribution of unstimulated ErbB2 in cells expressing 3·10(5)-10(6) receptors was fundamentally different, in that this receptor was present in preformed homoassociated aggregates containing 5-10 molecules. These constitutive ErbB2 homoclusters colocalized with caveolae, increased in size at subphysiological temperatures, but decreased in size upon EGF stimulation. We conclude that these ErbB2 clusters are promoted primarily by membrane-mediated interactions and are dispersed upon ligand stimulation.

Targeted cellular delivery of quantum dots loaded on and in biotinylated liposomes.

We describe the preparation, biophysical characterization, and receptor-mediated cellular internalization of biotinylated lipid particles (BLPs) loaded on the surface and internally with two distinct (colors) of quantum dot (QD) probes. BLPs were formulated with 1.4 and 2.7 mol % PEG-lipids containing either a fusogenic or pH-sensitive lipid to promote bilayer destabilization of endosomal membranes and favor QD cytoplasmic release. The amount of PEG was chosen to provide steric stabilization of the final construct. BLPs were loaded with a red-emitting QD(655) and surface coated with a green-emitting QD(525) conjugated to the epidermal growth factor (EGF) ligand in order to target the epidermal growth factor receptor (EGFR). The targeted and QD labeled BLPs showed strong and selective binding to EGFR-expressing tumor cell line and subsequent internalization. The dual-color QD labeling strategy and colocalization analysis allow prolonged live cell imaging of BLPs and loaded cargo independently, using a single excitation wavelength and simultaneous detection of both QDs. The chemistry of bioconjugation for the EGF ligand, the QDs, and the BLPs in a single lipid particle, involves only biotin-streptavidin interaction without requiring further purification from free EGF-QDs preformed complexes. Coupled with an encapsulated drug, the targeted and QD-labeled BLPs could provide imaging and drug delivery in a single multifunctional carrier.

Specific visualization of glioma cells in living low-grade tumor tissue.

BACKGROUND: The current therapy of malignant gliomas is based on surgical resection, radio-chemotherapy and chemotherapy. Recent retrospective case-series have highlighted the significance of the extent of resection as a prognostic factor predicting the course of the disease. Complete resection in low-grade gliomas that show no MRI-enhanced images are especially difficult. The aim in this study was to develop a robust, specific, new fluorescent probe for glioma cells that is easy to apply to live tumor biopsies and could identify tumor cells from normal brain cells at all levels of magnification. METHODOLOGY/PRINCIPAL FINDINGS: In this investigation we employed brightly fluorescent, photostable quantum dots (QDs) to specifically target epidermal growth factor receptor (EGFR) that is upregulated in many gliomas. Living glioma and normal cells or tissue biopsies were incubated with QDs coupled to EGF and/or monoclonal antibodies against EGFR for 30 minutes, washed and imaged. The data include results from cell-culture, animal model and ex vivo human tumor biopsies of both low-grade and high-grade gliomas and show high probe specificity. Tumor cells could be visualized from the macroscopic to single cell level with contrast ratios as high as 1000: 1 compared to normal brain tissue. CONCLUSIONS/SIGNIFICANCE: The ability of the targeted probes to clearly distinguish tumor cells in low-grade tumor biopsies, where no enhanced MRI image was obtained, demonstrates the great potential of the method. We propose that future application of specifically targeted fluorescent particles during surgery could allow intraoperative guidance for the removal of residual tumor cells from the resection cavity and thus increase patient survival.

Induction of terminal differentiation in melanoma cells on downregulation of beta-amyloid precursor protein.

The incidence of melanoma, the most aggressive type of skin cancer, is increasing dramatically, and an effective treatment for patients with advanced disease is as yet unavailable. Greater insight into the molecular features of primary and metastatic melanoma is required, particularly the identification of key regulatory genes that shield the tumor cells from terminal differentiation and apoptosis. The beta-amyloid precursor protein (APP) is a cell surface receptor and the transmembrane precursor of the Abeta-peptide, which has an important role in Alzheimer's disease. The study presented here provides evidence that APP is expressed at high levels in advanced-stage melanomas, and that the cells cleave APP and secrete sAPP. We show that blocking the expression of APP by RNA interference impairs the proliferation of metastatic melanoma cells and leads to their terminal and irreversible differentiation. In addition, suppressing APP expression in a metastatic melanoma cell line renders the cells susceptible to several chemotherapeutic agents. Targeting APP may thus constitute a new approach to the treatment of this disease.

Tumor-targeted quantum dots can help surgeons find tumor boundaries.

Despite surgical advances and recent progress in adjuvant therapies, the prognosis for patients with malignant brain tumors such as glioblastoma multiforme has remained poor, and the neurological deterioration suffered by most patients as a consequence of tumor progression is dramatic and severe. In addition, malignant brain tumors have >>95% recurrence close to the primary site of initial resection. Unfortunately, standard imaging techniques do not permit the intraoperative identification of individual or small clusters of residual tumor cells, precluding their selective removal while sparing the surrounding normal brain tissue. In this report, we show that quantum dots (QDs) coupled to epidermal growth factor (EGF) or anti-EGF receptor receptor (EGFR, Her1) specifically and sensitively label glial tumor cells in cell culture, glioma mouse models, and human brain-tumor biopsies. A clear demarcation between brain and tumor tissue at the macroscopic as well as the cellular level is provided by the fluorescence emission of the QDs.

Fluorescence recovery after photobleaching and photoconversion in multiple arbitrary regions of interest using a programmable array microscope.

Photomanipulation (photobleaching, photoactivation, or photoconversion) is an essential tool in fluorescence microscopy. Fluorescence recovery after photobleaching (FRAP) is commonly used for the determination of lateral diffusion constants of membrane proteins, and can be conveniently implemented in confocal laser scanning microscopy (CLSM). Such determinations provide important information on molecular dynamics in live cells. However, the CLSM platform is inherently limited for FRAP because of its inflexible raster (spot) scanning format. We have implemented FRAP and photoactivation protocols using structured illumination and detection in a programmable array microscope (PAM). The patterns are arbitrary in number and shape, dynamic and adjustable to and by the sample characteristics. We have used multispot PAM-FRAP to measure the lateral diffusion of the erbB3 (HER3) receptor tyrosine kinase labeled by fusion with mCitrine on untreated cells and after treatment with reagents that perturb the cytoskeleton or plasma membrane or activate coexpressed erbB1 (HER1, the EGF receptor EGFR). We also show the versatility of the PAM for photoactivation in arbitrary regions of interest, in cells expressing erbB3 fused with the photoconvertible fluorescent protein dronpa.

Quantitative single particle tracking of NGF-receptor complexes: transport is bidirectional but biased by longer retrograde run lengths.

The retrograde transport of nerve growth factor (NGF) in neurite-like processes of living differentiated PC12 cells was studied using streptavidin-quantum dots (QDs) coupled to monobiotin-NGF. These reagents were active in differentiation, binding, internalization, and transport. Ten-35% of the QD-NGF-receptor complexes were mobile. Quantitative single particle tracking revealed a bidirectional step-like motion, requiring intact microtubules, with a net retrograde velocity of 0.054+/-0.020 microm/s. Individual runs had a mean velocity of approximately 0.15 microm/s at room temperature, and the run times were exponentially distributed. The photostability and brightness of QDs permit extended real-time analysis of individual QDbNGF- receptor complexes trafficking within neurites.

Ligand-conjugated quantum dots monitor antigen uptake and processing by dendritic cells.

The dendritic cell (DC) specific pathogen-uptake receptor (DC-SIGN) internalizes antigens for degradation and presentation onto MHC molecules. At the cell membrane, DC-SIGN forms nanoclusters that facilitate virus capture. However, internalized viruses, such as HIV-1, escape degradation. Here, we exploit ligand-conjugated, virus-sized, highly photostable quantum dots (QDs) to monitor in living cells antigen binding, entry, and trafficking. The antigen-coated QDs specific uptake and persistence in live DCs open the possibility for tracking antigen-presenting cells in vivo.

Biotin-ligand complexes with streptavidin quantum dots for in vivo cell labeling of membrane receptors.

The unique fluorescence properties of quantum dots (QDs), particularly their large extinction coefficients and photostability, make them ideal probes for tracking proteins in live cells using real-time visualization. We have shown that QDs conjugated to epidermal growth factor act as functional ligands for their receptor, erbB1. Here, we describe protocols for (1) conjugation of streptavidin-QDs to biotinylated ligand, (2) formation of ligand-QD-receptor complexes, and (3) quantification of binding and internalization of receptor complex using both high-resolution fluorescence microscopy and flow cytometry.

Drosophila under the lens: imaging from chromosomes to whole embryos.

Microscopy has been a very powerful tool for Drosophila research since its inception, proving to be essential for the evaluation of mutant phenotypes, the understanding of cellular and tissue physiology, and the illumination of complex biological questions. In this article we review the breadth of this field, making note of some of the seminal papers. We expand on the use of microscopy to study questions related to gene locus and nuclear architecture, presenting new data using fluorescence in-situ hybridization techniques that demonstrate the flexibility of Drosophila chromosomes. Finally, we review the burgeoning use of fluorescence in-vivo imaging methods to yield quantitative information about cellular processes.