Search

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.

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.

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 malachitegreen (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.

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.

Several cellular processes involve alignment of three nucleic acids strands, in which the third strand (DNA or RNA) is identical and in a parallel orientation to one of the DNA duplex strands. Earlier, using 2-aminopurine as a fluorescent reporter base, we demonstrated that a self-folding oligonucleotide forms a recombination-like structure consistent with the R-triplex. Here, we extended this approach, placing the reporter 2-aminopurine either in the 5'- or 3'-strand. We obtained direct evidence that the 3'-strand forms a stable duplex with the complementary central strand, while the 5'-strand participates in non-Watson-Crick interactions. Substituting 2,6-diaminopurine or 7-deazaadenine for adenine, we tested and confirmed the proposed hydrogen bonding scheme of the A*(T.A) R-type triplet. The adenine substitutions expected to provide additional H-bonds led to triplex structures with increased stability, whereas the substitutions consistent with a decrease in the number of H-bonds destabilized the triplex. The triplex formation enthalpies and free energies exhibited linear dependences on the number of H-bonds predicted from the A*(T.A) triplet scheme. The enthalpy of the 10 nt long intramolecular triplex of -100 kJ x mol(-1) demonstrates that the R-triplex is relatively unstable and thus an ideal candidate for a transient intermediate in homologous recombination, t-loop formation at the mammalian telomere ends, and short RNA invasion into a duplex. On the other hand, the impact of a single H-bond, 18 kJ x mol(-1), is high compared with the overall triplex formation enthalpy. The observed energy advantage of a 'correct' base in the third strand opposite the Watson-Crick base pair may be a powerful mechanism for securing selectivity of recognition between the single strand and the duplex.

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., Facultad de Ciencias Médicas, Instituto de Investigaciones Bioquímicas de La Plata

Figure S6 is a duplicate of Figure 5. Please see the correct Figure S6 here: Click here for additional data file.(249K, tif)

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.

This unit describes the use of quantum dots (QDs) for live-cell imaging and the use of QDs in flow cytometry for quantitative analysis of ligand binding constants and receptor density. Conventional fluorophores and visible fluorescent protein (VFP) constructs have allowed visualization of many cellular processes. However, organic and biomolecular fluorophores have limitations in their applications, due to their small Stokes’ shift and tendency to photobleach during prolonged imaging. QDs have many advantages over conventional fluorophores, including high brightness and photostability, which make them an exceptional tool for live-cell imaging. There are a large variety of commercially available QDs with different surface reactivities and characteristics. The authors have limited the laboratory protocols presented here to the use of streptavidin-coupled QDs because this gives almost universal applicability to any cell surface receptor by coupling the ligand or antibody that recognizes the receptor to biotin and visualizing the complex by use of QDs. Curr. Protoc. Cell Biol. 36:25.1.1-25.1.18. C � 2007 by John Wiley & Sons, Inc.

We report the implementation and exploitation of fluorescence polarization measurements, in the form of anisotropy fluorescence lifetime imaging microscopy (rFLIM) and energy migration Förster resonance energy transfer (emFRET) modalities, for wide-field, confocal laser-scanning microscopy and flow cytometry of cells. These methods permit the assessment of rotational motion, association and proximity of cellular proteins in vivo. They are particularly applicable to probes generated by fusions of visible fluorescence proteins, as exemplified by studies of the erbB receptor tyrosine kinases involved in growth-factor-mediated signal transduction.

We have implemented simultaneous picosecond pulsed two- and three-photon excitation of near-UV and visible absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 1064-nm emission from a pulsed Nd:YVO4 laser was used to excite the visible mitochondrial specific dye MitoTracker Orange CM-H2TMRos or a Cy3-labeled antibody by two-photon excitation, and the UV absorbing DNA dyes DAPI and the bisbenzimidazole BBI-342 by three-photon excitation, in a shared aperture SNOM using uncoated fiber tips. Both organelles in human breast adenocarcinoma cells (MCF 7) and specific protein bands on polytene chromosomes of Drosophila melanogaster doubly labeled with a UV and visible dye were readily imaged without photodamage to the specimens. The fluorescence intensities showed the expected nonlinear dependence on the excitation power over the range of 5–40mW. An analysis of the dependence of fluorescence intensity on the tip-sample displacement normal to the sample surface revealed a higher-order function for the two-photon excitation compared to the one-photon mode. In addition, the sample photobleaching patterns corresponding to one- and two-photon modes revealed a greater lateral confinement of the excitation in the two-photon case. Thus, as in optical microscopy, two-photon excitation in SNOM is confined to a smaller volume.

The subcellular three-dimensional distribution of three polycomb-group (PcG) proteins—polycomb, polyhomeotic and posterior sex combs—in fixed whole-mount Drosophila embryos was analyzed by multicolor confocal fluorescence microscopy. All three proteins are localized in complex patterns of 100 or more loci throughout most of the interphase nuclear volume. The rather narrow distribution of the protein intensities in the vast majority of loci argues against a PcG-mediated sequestration of repressed target genes by aggregation into subnuclear domains. In contrast to the case for PEV repression (Csink, A.K., and S. Henikoff. 1996. Nature. 381:529–531), there is a lack of correlation between the occurrence of PcG proteins and high concentrations of DNA, demonstrating that the silenced genes are not targeted to heterochromatic regions within the nucleus. There is a clear distinction between sites of transcription in the nucleus and sites of PcG binding, supporting the assumption that most PcG binding loci are sites of repressive complexes. Although the PcG proteins maintain tissue-specific repression for up to 14 cell generations, the proteins studied here visibly dissociate from the chromatin during mitosis, and disperse into the cytoplasm in a differential manner. Quantitation of the fluorescence intensities in the whole mount embryos demonstrate that the dissociated proteins are present in the cytoplasm. We determined that

Major histocompatibility complex (MHC) class I antigens in the plasma membranes of human T (HUT-102B2) and B (JY) lymphoma cells were probed by immunochemical reagents using fluorescence, transmission electron, and scanning force microscopies. Fluorescent labels were attached to monoclonal antibodies W6/32 or KE-2 directed against the heavy chain of HLA class I (A, B, C) and L368 or HB28 against the beta 2-microglobulin light chain. The topological distribution in the nanometer range was studied by photobleaching fluorescence resonance energy transfer (pbFRET) on single cells. A nonrandom codistribution pattern of MHC class I molecules was observed over distances of 2-10 nm. A second, nonrandom, and larger-scale topological organization of the MHC class I antigens was detected by indirect immunogold labeling and imaging by transmission electron microscopy (TEM) and scanning force microscopy (SFM). Although some differences in antigen distribution between the B- and T-cell lines were detected by pbFRET, both cell lines exhibited similar clustering patterns by TEM and SFM. Such defined molecular distributions on the surfaces of cells of the immune system may reflect an underlying specialization of membrane lipid domains and fulfill important functional roles in cell-cell contacts and signal transduction.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.

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.

The automated high-speed analysis and separation of cells on the basis of spectroscopic parameters has been applied to studies of cellular differentiation in two systems. The temporal changes following induction of differentiation by dimethylsulfoxide in the Friend virus-transformed erythroid cells were quantitated by multiparameter analysis leading to the separation of discrete subpopulations. Thus, following induction, cell size decreased as measured by light scattering, the number of H-2 histocompatibility antigen sites decreased as measured by indirect fluorescent antibody binding, the number of lectin-binding sites per cell increased as measured by fluorescein-labeled concanavalin-A and the microviscosity of the hydrocarbon region of the plasma membrane increased as determined by the fluorescence emission anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene. Cells were separated on the basis of several of these parameters and analyzed for their hemogloglobin content by benzidine staining. Examination of cells separated according to the anisotropy parameter showed that high anisotropy values were correlated with (a) small cell size, (b) positive staining with benzidine and (c) pronounced reactivity with fluorescent antibody to the erythrocyte protein spectrin. Disaggregated cells from Hydra attenuata were selectively stained with the dyes rhodanile blue, 7-(p-methoxybenzylamino)-4-nitrobenz-2-oxa-1,3-diazole and fluorescamine. Distribution analyses and preliminary separations indicated the feasibility of obtaining homogeneous classes of cell types in a viable state. The experiments with emission anisotropy represent the first analyses and separations of single cells on the basis of fluorescence polarization. Many other uses of this technique are anticipated.

The spectroscopic techniques of fluorescence resonance energy transfer and of time-resolved delayed fluorescence and phosphorescence have been introduced into a microscope equipped with a solid-state CCD camera and phase-locked excitation and emission choppers. The distribution and replication of DNA in cells has been quantitated by these methods, as well as by confocal laser scanning microscopy. Sites of replication in living cells can be uniquely identified by delayed luminescence.

PRINCIPLES OF LUMINESCENCE MICROSCOPY • • • 273 Photometric Features of a Luminescence Microscope 273 Performance Criteria 276 Practical Considerations 276 SOLID-STATE CHARGE-COUPLED DEVICE CAMERAS FOR LUMINESCENCE IMAGING .. ...... . ..... . 276 Properties of CCD Cameras 277 Applications of a CGD Camera ........ 279 Photobleaching Fluorescence Resonance Energy Transfer Digital Imaging Microscopy..... 281 Delayed Luminescence (Fluorescence and Phosphorescence) Digital Imaging Microscopy 284 SCANNING LUMINESCENCE MICROSCOPES 285 Confocal Microscopy 289 Optical Principles of Confocal Microscopy 289