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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.

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 Forster 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. © 2013 International Society for Advancement of Cytometry

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.

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.

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.

Background ErbB2 (HER-2), a member of the epidermal growth factor (EGF) receptor family, is a class I transmembrane receptor tyrosine kinase. Although erbB2 has no known physiologic ligand, it can form complexes with other members of the family and undergo transactivation of its very potent kinase activity, thereby initiating downstream signaling and cell proliferation. ErbB2 is a frequent pathologic marker in ductal invasive breast carcinomas and is targeted by using a specific humanized monoclonal antibody, trastuzumab (Herceptin). The antibody is effective in only 20% to 50% of erbB2-positive tumors, and this resistance, as yet poorly understood, constitutes a major therapeutic challenge. Methods Magnetic microspheres coated with ligands or antibodies are widely used for separation of proteins and cells and allow localized, high intensity, and precisely timed stimulation of cells. We used EGF- and trastuzumab-covered paramagnetic microspheres, quantitative confocal laser scanning microscopy, and digital image processing to investigate the (trans)activation of and local signal propagation from erbB1 and erbB2 on trastuzumab sensitive and resistant carcinoma cell lines expressing these receptors at high levels. Results On A431 cells expressing high levels of endogenous erbB1 and transfected erbB2-mYFP (A4-erbB2-mYFP F4 cell line), EGF-coupled-microspheres activated erbB1 and transactivated erbB2-mYFP. In two other cell lines with comparable erbB2 expression but lower levels of erbB1, EGF microspheres transactivated erbB2 less efficiently. Trastuzumab in solution activated erbB2 on A4-erbB2-mYFP and the trastuzumab sensitive SKBR-3 cells, but only negligibly on the resistant JIMT-1 cells that showed a 10 times higher Kd for the antibody. Nevertheless, pronounced erbB2 activation and tyrosine phosphorylation could be detected after stimulation with trastuzumab-coupled microspheres in all cell lines, although transactivation of erbB1 was negligible. Receptor phosphorylation was restricted to the immediate proximity of the microspheres, i.e., receptor clusters external to these locations remained inactive. Conclusion ErbB1 ligand and erbB2 specific antibody attached to magnetic microspheres are efficient tools in assessing erbB activation, localized signal propagation, and erbB heterodimer formation. Trastuzumab coupled to microspheres is more efficient at accessing erbB2 and activating it than trastuzumab in solution. © 2005 International Society for Analytical Cytology

The parallel (recombination) 'R-triplex' can accommodate any nucleotide sequence with the two identical DNA strands in parallel orientation. We have studied oligonucleotides able to fold back into such a recombination-like structure. We show that the fluorescent base analogs 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI) can be used as structural probes for monitoring the integrity of the triple-stranded conformation and for deriving the thermodynamic characteristics of these structures. A single adenine or guanine base in the third strand of the triplex-forming and the control oligonucleotides, as well as in the double-stranded (ds) and single-stranded (ss) reference molecules, was substituted with 2AP or 6MI. The 2AP*(T.A) and 6MI*(C.G) triplets were monitored by their fluorescence emission and the thermal denaturation curves were analyzed with a quasi-two-state model. The fluorescence of 2AP introduced into an oligonucleotide sequence unable to form a triplex served as a negative control. We observed a remarkable similarity between the thermodynamic parameters derived from melting of the secondary structures monitored through absorption of all bases at 260 nm or from fluorescence of the single base analog. The similarity suggests that fluorescence of the 2AP and 6MI base analogs may be used to monitor the structural disposition of the third strand. We consider the data in the light of alternative 'branch migration' and 'strand exchange' structures and discuss why these are less likely than the R-type triplex.

We report a system for collecting spectrally resolved fluorescent lifetime images. Frequency domain fluorescence lifetime detection was combined with two-dimensional spectral imaging in a programmable array microscope. The spectroscopic fluorescence lifetime imaging microscopy (sFLIM) system has a resolution of ∼50 (λ/Δλ) in the current arrangement and a wavelength range of ∼430–750 nm. With the sFLIM system, we recorded the lifetime spectra of rhodamine 6G, rhodamine B, and the DNA intercalation dye propidium iodide (PI) in cuvettes and an EGFP-fusion of the histone 2A variant D protein in Drosophila salivary gland explants in the presence and absence of PI. In the absence of PI, the EGFP-fusion exhibited a lifetime of 2.7 ns with little variation in wavelength. The lifetime of PI alone ranged from ∼1 ns in buffer to ∼18 ns when intercalated in the nuclei of intact cells. The combination of EGFP and PI in the Drosophila salivary gland explants exhibited strong fluorescence resonance energy transfer (FRET), a result consistent with the known nucleosomal structure of eukaryotic chromatin.

A 14 nt DNA sequence 5′-AGAATGTGGCAAAG-3′ from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar–phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5′-end of the probe strand as donor and BODIPY-Texas Red on the 3′-amino group of either strand of the target duplex as acceptor. There was full protection from OsO4-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe–intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.

Background: Frequency-domain fluorescence lifetime imaging microscopy (FLIM) is finding increasing use in the analysis of biological systems. However, the calibration, determination of resolvable lifetime differences, and evaluation of artifacts have not been extensively treated. We describe a multi-point method for calibrating a frequencydomain FLIM system, characterize the minimum detectable heterogeneity and intra- and inter-image lifetime differences, discuss the statistical treatment of FLIM data, and suggest methods for minimizing artifacts. Methods: A set of solutions exhibiting single-component lifetimes suffice for accurately calibrating a reference material with a single-component lifetime, even in the absence of accurate data on the lifetimes of the individual solutions or the reference material. We used a set of rhodamine 6G solutions quenched with varying concentrations of iodide, leading to lifetimes of 0.5‐ 4.0 ns, to calibrate a 1 mM reference solution of rhodamine 6G in water. Results: We measured a value of 4.11 ns with an estimated absolute error of 60.05 ns for the rhodamine 6G reference solution. With 57.7 MHz modulation, the minimum detectable inter-image lifetime difference was 0.1‐ 0.15 ns and the minimum detectable intra-image lifetime difference was 4 ‐5 ps, allowing solutions differing in lifetime by 40 and 70 ps to be easily distinguished. The minimum detectable lifetime heterogeneity was 50 ‐ 80 ps. Evaluation of replicate measurements of single solutions demonstrated that inter-image instrument errors exceeded those predicted from intra-image statistics by more than an order of magnitude. We also measured lifetimes and heterogeneity in 4 GFP variants (WTGFP, EGFP, S65T, and EYFP) with the technique. Conclusion: The multi-point calibration method is applicable to any system consisting of single-component lifetimes. Applying the method in our FLIM microscope allowed us to demonstrate a previously unreported degree of lifetime resolution in a FLIM microscope. Cytometry 43:248 ‐260, 2001. © 2001 Wiley-Liss, Inc. Key terms: lifetime standards; rhodamine 6G; iodide quenching; FLIM; green fluorescent protein (GFP)

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry,PO Box 2841, 37018 Goettingen, GermanySubmitted 24 July 1996, accepted 8 October 1996Abstract. Using examples of hybridization both to RNA and to DNA sequences wedemonstrate that whole-mountDrosophila embryos are excellent objects with which tostudy questions about nuclear structure and function by combining FISH and confocallaser scanning microscopy. A fluorescently labelled 29-base oligonucleotide was usedto probe transcription at a locus known to be strongly induced upon heat shock. Thetranscript from this locus apparently serves to stabilize and protect nuclear proteinswhich may be needed for nuclear processes after heat or chemical stress. We found thatless than 5% of the protein is colocalized with the transcript under normal growthconditions, but more than 50% is sequestered by the transcript during heat shock. DNAprobes to genes in the bithorax complex were used to examine the relationship betweenhomologous pairing and gene expression in late stage gastrulating embryos. Analysis ofthe disposition of probes cloned in P1 vectors in embryos from mid-blastodermthroughout gastrulation allowed us to conclude that polarized nuclear organizationbreaks down after the blastoderm stage. Homologous pairing of the bithorax complexgenes proceeds during gastrulation so that at the time of germ band retraction the twoalleles are always in close proximity independent of expression of the gene or theregion along the anterior–posterior axis of the body. Finally, we demonstrate thatsmaller DNA targets can be visualized in whole mount embryos by enhancement of theFISH signal by tyramide-fluorophore deposition.Keywords: hnRNP-K, 93D locus, omega-n transcript, bithorax complex, homologouspairing, confocal laser scanning microscopy1. IntroductionMost problems in developmental biology can only beunderstood in the context of the whole animal whichprovides the stage whereon the actors, gradients ofactivators and repressors, or signal molecules and receptors,perform their choreographed and complex interplay.Dissolution of the organism into its single cells destroysthe entire program of events, disrupts the intercellularsignalling and may reset the biological clock to an entirelynew situation. Thus, whether one is defining a phenotypeor determining complex gene expression patterns, theobservation of the entire organism or at least wholeorgan systems is essential. Almost 10 years ago thereplacement of radioactive labels [1,2] by non-radioactivenucleotide derivatives, such as digoxigenin (DIG), biotin orfluorochromes [3–6], revolutionized the detection of mRNAin tissues and cells. Subsequently, Tautz and Pfeifle [7]introduced methods to simultaneously permeabilize and fixwhole Drosophila embryos so that the localization of thetranscripts could be observed in whole organisms. Thistechnique was quickly adapted to embryos from Xenopus,sea urchin and other organisms [8,9]. The result was animprovement in resolution of non-radioactive probes formRNA localization, compared with the radioactive labelmethod. This improvement enabled whole animal labelling,compared with sectioning, and the ability to simultaneouslydetect protein expression by immunochemistry. Thesetechniques have produced important data which contributeto the present understanding of basic concepts in

A Z-DNA binding protein has been isolated and characterized by biochemical means from Drosophila melanogaster tissue culture cells and embryos. This protein shares the following properties with the known, cloned Drosophila topoisomerase II: (1) expression of an ATP-dependent relaxation activity on supercoiled DNA; (2) a monomer mass of 165 kDa in SDS denaturing gels; (3) a sedimentation coefficient, S20,w, of approximately 10 S for the active enzyme; (4) cross-reactivity for the respective monoclonal and polyclonal antibodies; (5) generation of covalent enzyme-DNA intermediates at preferred cutting sites in the Drosophila HSP70 intergenic spacer region; (6) inhibition of DNA relaxation activity by antitumor drugs, e.g., the etoposide VM26, and by monospecific antibodies raised against the protein; and (7) in vitro phosphorylation by a casein kinase activity. However, we have identified new properties for our topoisomerase II preparation not previously reported for the conventionally isolated enzyme: (1) The enzyme binds to Z-DNA with an affinity 2 orders of magnitude greater than that for B-DNA. (2) The binding to Z-DNA is increased 5-10-fold by GTP or GTP-gamma-S. (3) GTP and GTP-gamma-S inhibit the catalytic activity of topoisomerase II through a proposed allosteric mechanism. (4) Z-DNA inhibits the relaxation of closed circular supercoiled DNA. (5) The preparation consists of a single polypeptide chain of 165 kDa on denaturing SDS gels with no evidence of proteolytic degradation. We postulate that the Z-DNA binding activity of undegraded topoisomerase II may be important in targeting the enzyme both to structural motifs required for chromatin organization and to sites of local supercoiling. Some of these features arise during processes such as replication and gene expression and may be more frequent during embryogenesis and early development.

The aggregation state of type I Fc epsilon-receptors (Fc epsilon RI) on the surface of single living mast cells was investigated by resonance fluorescence energy transfer. Derivatization of Fc epsilon RI specific ligands, i.e., immunoglobulin E or Fab fragments of a Fc epsilon RI specific monoclonal antibody, with donor and acceptor fluorophores provided a means for measuring receptor clustering through energy transfer between the receptor probes. The efficiency of energy transfer between the ligands carrying distinct fluorophores was determined on single cells in a microscope by analyzing the photobleaching kinetics of the donor fluorophore in the presence and absence of receptor ligands labeled with acceptor fluorophores. To rationalize the energy transfer data, we developed a theoretical model describing the dependence of the energy transfer efficiency on the geometry of the fluorescently labeled macromolecular ligands and their aggregation state on the cell surface. To this end, the transfer process was numerically calculated first for one pair and then for an ensemble of Fc epsilon RI bound ligands on the cell surface. The model stipulates that the aggregation state of the Fc epsilon RI is governed by an attractive lipid-protein mediated interaction potential. The corresponding pair-distribution function characterizes the spatial distribution of the ensemble. Using this approach, the energy transfer efficiency of the ensemble was calculated for different degrees of receptor aggregation. Comparison of the theoretical modeling results with the experimental energy transfer data clearly suggests that the Fc epsilon RI are monovalent, randomly distributed plasma membrane proteins. The method provides a novel approach for determining the aggregation state of cell surface components.

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 multi-spot 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 co-expressed 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.

The combination of multiple dye-DNA interactions, a fluorescence digital imaging system with a scientific CCD camera, and multivariate image analysis allows the rapid karyotyping of fluorescent human metaphase chromosome spreads. Chromosomes are stained with the bisbenzimidazole dye Hoechst 33342 and chromomycin A3, a dye pair used frequently in bivariate flow analysis and sorting of metaphase chromosomes in suspension. The use of ratio functions involving the total and peak intensities of the two dyes provides increased resolution of the karyotype in the microscope, and it can be anticipated that the same approach could lead to improved performance with flow systems as well. High pass filtering with a Laplace operator yields characteristic banded images of the individual chromosomes, even with total fields that are less than 200 pixels on a side.

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.

Item does not contain fulltext In vivo transfection efficiency (TE) using cationic liposome/oligonucleotide (ODN) complexes is often hampered by interactions with serum components. Novel cationic lipids with different hydroxyethyl or dihydroxypropyl ammonium backbones, esterified hydrocarbon chains and hydroxy substituents have been synthesized and applied in cationic liposome formulations with and without the helper lipid DOPE (1:1, m/m). Their properties for cellular ODN delivery were determined using fluorescently labeled ODNs (F-ODNs). Cationic lipids with hydrocarbon chains esterified to non-glycerol backbones in non-vicinal configuration were completely ineffective in nuclear ODN-delivery. Instead, an increased cytoplasmic localization of F-ODNs was observed. Cationic lipids equipped with only one hydrocarbon were completely incompetent for cellular ODN delivery. In the absence of serum, all cationic lipids tested with hydrocarbon chains in vicinal configuration esterified to a glycerol backbone (the respective N-(1,2-diacyl-dihydroxypropyl)-N,N,N-trimethyl-ammoniumchlorides or N-(1,2-diacyl-dihydroxypropyl)-N(hydroxyethyl)-N,N-dimethyl-ammoniumchlori des as well as N-(1,2-diacyl-dihydroxypropyl)-N(1,2-dihydroxypropyl)-N,N-dimethyl-ammoniu mchlorides with lauroyl, myristoyl, palmitoyl, stearoyl and erucoyl chains) were able to transfect cells when combined with DOPE (20-80% nuclear fluorescence). Remarkably, only the analog esterified with two myristoyl chains was equally effective even in the absence of DOPE. By adding hydroxy groups to the N-alkyl residue, TE under serum conditions was improved yielding transfection rates of 55%, 75% and 90% for 0, 1 or 2 substituted hydroxy groups, respectively. For plasmid DNA, different requirements were identified. Again, the analog with two myristoyl chains was most effective but only in the presence of DOPE. However, the addition of hydroxy groups had no influence on the TE in the presence of serum.

The living cell mediates its internal state and the exchange of substances and information with its environment primarily via protein-protein interactions. The spatio-temporal disposition of structural, catalytic, and regulatory proteins defines the nature and functional state of the cell. Signaling mechanisms, as a prominent example, occupy a central role in this process, leading to a set of canonical questions, challenges and strategies (Table 1).

Background: The programmable array microscopes (PAMs) are a family of instruments incorporating arbitrary control of the patterns of illumination and/or detection. The PAM can be used in sectioning and nonsectioning modes, thereby constituting a useful platform for fluorescence lifetime imaging. Methods and Results: We used a PAM for acquisition of optically sectioned and widefield fluorescence lifetime images, in which contrast was increased predominantly by suppressing out-of-focus light contributions. We simulate, display, and discuss the effects of blurring and fluorophore heterogeneity on lifetime imaging in widefield and confocal configurations. Conclusion: Sectioning improves the quality of lifetime images of samples with multiple fluorophores or spatially varying F€ resonance energy transfer. q 2005 International Society for Analytical Cytology

Semiconducting nanocrystals, or quantum dots (QDs), have emerged as a new tool in physiological imaging, combining high brilliance, photostability, broad excitation but very narrow emission spectra, and surface chemistry compatible with biomolecular conjugation. In this review, we demonstrate the power of QDs in diverse applications, including long-term in vivo fluorescence imaging.

FTIR spectroscopy has been used to follow the formation of parallel stranded DNA duplexes incorporating isoG or m5isoC bases and determine their base pairing scheme. The results are discussed in comparison with data concerning anti-parallel duplexes with comparable base composition and sequence. In duplexes containing A–T and isoG–C or m5isoC–G base pairs shifts of the thymine C2 O2 and C4 O4 carbonyl stretching vibrations (to lower and higher wavenumbers, respectively, when compared to their positions in classical cis Watson–Crick (WC) base pairs) reflect the formation of trans Watson–Crick A–T base pairs. All carbonyl groups of cytosines, m5isocytosines, guanines and isoguanines are found to be involved in hydrogen bonds, indicative of the formation of isoG–C and m5isoC–G base pairs with three hydrogen bonds. Molecular modeling shows that both structures form regular right handed helices with C2′endo sugar puckers. The role of the water content on the helical conformation of the parallel duplexes has been studied by FTIR and CD. It is found that a conformational transition similar to the B → A transition observed for anti-parallel duplexes induced by a decrease of the water content of the samples can occur for these parallel duplexes. Their helical flexibility has been evidenced by FTIR studies on hydrated films by the emergence of absorption bands characteristic of A type geometry, in particular by an S-type → N-type repuckering of the deoxyribose. All sugars in the parallel duplex with alternating d(isoG–A)/d(C–T) sequence can adopt an N-type geometry in low water content conditions. The conformational transition of the parallel hairpin duplex with alternating d(isoG–A)/d(C–T) sequence was followed by circular dichroism in water/trifluoroethanol solutions and its free energy at 0 °C was estimated to be 6.6 ± 0.3 kcal mol−1.

Deregulated and excessive expression of epidermal growth factor receptor (EGFR or erbB1), a transmembrane receptor tyrosine kinase specific for the epidermal growth factor (EGF), is a feature and/or cause of a wide range of human cancers, and thus inhibition of its expression is potentially therapeutic. In RNA interference (RNAi), duplexes of 21-nucleotide RNAs (small interfering RNA, siRNA) corresponding to mRNA sequences of particular genes are used to efficiently inhibit the expression of the target proteins in mammalian cells. Here we show that by using RNAi the expression of endogenous erbB1 can be specifically and extensively (90%) suppressed in A431 human epidermoid carcinoma cells. As a consequence, EGF-induced tyrosine phosphorylation was inhibited and cell proliferation was reduced due to induction of apoptosis. We established an inverse correlation between the level of expressed erbB1 and EGF sensitivity on a cell-by-cell basis using flow cytometry. A431 cells expressing endogenous erbB1 were transfected with erbB1 fused C-terminally to enhanced green fluorescent protein (EGFP). Selective inhibition of the expression of the fusion protein was achieved with an siRNA specific for the EGFP mRNA, whereas the erbB1-specific siRNAs inhibited the expression of both molecules. siRNA-mediated inhibition of erbB I and other erbB tyrosine kinases may constitute a useful therapeutic approach in the treatment of human cancer. (C) 2003 Elsevier Science (USA). All rights reserved.

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.

Antigen presentation by MHC class II molecules can be enhanced by paraformaldehyde fixation of antigen-presenting cells prior to assay. This treatment might be expected to aggregate membrane proteins and thus stabilize and strengthen transient protein-protein interactions involved in intercellular cooperation. Lateral and rotational dynamics of the MHC class II antigen I-Ad on A20 cells fixed with various concentrations of paraformaldehyde were examined by fluorescence photobleaching recovery and time-resolved phosphorescence anisotropy, respectively. Probes were tetramethylrhodamine and erythrosin conjugates of MKD6 Fab fragments. Increasing concentrations of paraformaldehyde led to a progressive increase in the limiting anisotropy of I-Ad at 4 degrees C from the value of 0.042 for untreated cells, indicative of large aggregate formation, while leaving the rotational correlation time of 29 micros unchanged, a measure of the unperturbed molecule. On the other hand, the translational diffusion constants decreased from approximately 2x10(-10) cm2 s(-1), while the fractional recovery remained unchanged at about 40-50%. Taken together, these results suggest that fixation crosslinks class II molecules to each other or to other membrane proteins into structures large enough (>500,000 kDa) to diffuse translationally with perceptibly size-dependent rates. The fixation effects on both class II rotation and lateral diffusion were half-maximal at paraformaldehyde concentrations of approximately 0.2%. Possible relations between the biological effector functions of class II and the physical sizes of fixation-induced aggregates are discussed.

Many indirect methods have been developed to study the constitution and conformation of macromolecules inside the living cell. Direct analysis by Raman spectroscopy is an ideal complement to techniques using directly labelled fluorescent probes or of indirect labelling with mono- and polyclonal antibodies. The high information content of Raman spectra can characterize biological macromolecules both in solution and in crystals. The positions, intensities and linewidths of the Raman lines (corresponding to vibrational energy levels) in spectra of DNA-protein complexes yield information about the composition, secondary structure and interactions of these molecules, including the chemical microenvironment of molecular subgroups. The main drawback of the method is the low Raman scattering cross-section of biological macromolecules, which until now has prohibited studies at the level of the single cell with the exception of (salmon) sperm heads, in which the DNA is condensed to an exceptionally high degree. Ultraviolet-resonance Raman spectroscopy has been used to obtain single cell spectra (and F. Sureau and P. Y. Turpin, personal communication), but in this method absorption of laser light may impair the integrity of the sample. We have avoided this problem in developing a novel, highly sensitive confocal Raman microspectrometer for nonresonant Raman spectroscopy. Our instrument makes it possible to study single cells and chromosomes with a high spatial resolution (approximately less than 1 micron 3).

The binding activities of the 170 kDa and the 180 kDa human topoisomerases II (topo II alpha and topo II beta) to linear DNA fragments with different degrees of curvature were characterized. In gel retardation experiments it was shown that both forms of the enzyme bind preferentially to a curved 287 bp fragment, forming a detectable stable complex. The affinity for straight DNA fragments of similar length is significantly lower. Both a commercially available topo II alpha, isolated from placenta, and topo II alpha and topo II beta purified from nuclear extracts of the Namalwa lymphoma tissue culture line gave similar results. The effects of double-stranded poly[d(A-T)], poly[d(G-C)], supercoiled plasmid DNA and linear Z-DNA on the topo II-complex with curved DNA were analyzed in competition experiments. The hierarchy of affinities of the 180 kDa topo II beta for these DNAs has the order: linear left-handed DNA > supercoiled DNA > or = curved DNA >> poly[d(A-T)] > poly[d(G-C)]. The 170 kDa topo II alpha binds with similar affinity to curved DNA and linear Z-DNA > or = supercoiled DNA >> linear B-DNA. The data imply that human topoisomerase II binding is more sensitive to DNA secondary structure than to DNA sequence per se. The ability of the enzyme to preferentially recognize a wide variety of sequences in unusual secondary structures suggests a mode of targeting the enzyme in vivo to regions of high negative supercoiling.

We describe the synthesis of bordifluoropyrromethene (BODIPY), fluorescein, and related fluorescent derivatives of the beta-adrenergic ligand CGP 12177. With these probes we screened insect (Sf9) cells stably transformed with the human beta 2-adrenoceptor gene and expressing (2-3.5) x 10(5) human beta 2-adrenoceptors per cell. Among these derivatives only BODIPY-CGP gave a receptor-specific signal sufficiently strong for measuring the on- and off-rate constants and the equilibrium dissociation constant of beta-adrenoceptor-specific binding by spectrofluorometry or photon counting. Similar KD values for BODIPY-CGP binding were obtained by kinetic measurements (approx. 250 pM) and under equilibrium conditions (400 +/- 180 pM), and these were in the same range as those obtained with [3H]CGP 12177 (200 +/- 32 pM). The cell-bound fluorescence could be quenched specifically with nonfluorescent CGP 12177 to near background levels. The disposition of the beta 2-adrenoceptors in BODIPY-CGP-stained Sf9 cells was mainly restricted to the cell surface at 4 and 30 degrees C. Hence, beta-adrenoceptor-expressing cells can be stained specifically with BODIPY-CGP, and beta-adrenoceptors on a single cell can be assessed by photon counting under the fluorescence microscope. Cells can also be scanned by fluorescence-activated flow cytometry.

The physical interaction between plasma-membrane lipids and the epidermal growth factor (EGF)-receptor was investigated on single A431 human epidermoid carcinoma cells by monitoring fluorescence resonance energy transfer (FRET) between exogeneously added fluorescein-EGF (donor) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC, acceptor) using donor-photobleaching FRET-microscopy. The measured mean FRET-efficiency of 13% is indicative of such a physical interaction and exemplifies the great potential and sensitivity of time-resolved imaging fluorescence microscopy techniques for the study of lipid-receptor interactions on single cells.

Biological samples have been imaged using microscopes equipped with slow-scan CCD cameras. Examples are presented of studies based on the detection of light emission signals in the form of fluorescence and phosphorescence. They include applications in the field of cell biology: (a) replication and topology of mammalian cell nuclei; (b) cytogenetic analysis of human metaphase chromosomes; and (c) time-resolved measurements of DNA-binding dyes in cells and on isolated chromosomes, as well as of mammalian cell surface antigens, using the phosphorescence of acridine orange and fluorescence resonance energy transfer of labeled lectins, respectively.© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The rotational diffusion of epidermal growth factor (EGF) bound to its specific receptor on the surface of human carcinoma A431 cells was studied by means of time-resolved phosphorescence anisotropy measurements. The rotational mobility was measured on the total population of EGF receptors by using a saturating concentration of EGF conjugated with a phosphorescent label, erythrosin, or on the subpopulation of high-affinity EGF receptors by using a low concentration of labeled EGF. At 4 degrees C, the rotational correlation times for both the high-affinity and total (mostly low affinity) receptor populations were in the range of 60-100 microns. Elevation of the temperature to 37 degrees C resulted in a lengthening of the rotational correlation time of the total receptor population to 200-300 microns, confirming a previous study of receptor microaggregation. The high-affinity EGF receptors were completely immobilized at 37 degrees C (rotational correlation time greater than 500 microns). The data are consistent with a model involving association of the cytoskeleton with the high-affinity receptors at 37 degrees C, but not at 4 degrees C.

The temporal course of replication monitored by 2- or 5-min pulses of bromodeoxyuridine (BrdUrd) incorporation in synchronized 3T3 cells was mapped by high-resolution light microscopy employing a charge-coupled device (CCD) camera and a confocal laser scanning microscope (CLSM). The cells were labeled simultaneously with monoclonal antibodies directed against BrdUrd and nuclear lamin, and stained with the A+T-specific dye 4′,6-diamidino-2-phenylindole (DAPI). Stereoscopic reconstructions of cells showing both the lamin and BrdUrd distributions demonstrate that DNA replication occurs at discrete sites in the nucleus, the locations of which progress through a programmed sequence during S phase. Replication begins in a small number of sites in the interior of the nucleus exclusive of the nuclear membrane and proceeds rapidly in early S phase to encompass a relatively large number of small, discrete sites located throughout the nucleus, with the exception of the condensed heterochromatic regions. Replication is primarily confined to the condensed heterochromatic regions in mid-to-late S phase, and to the nuclear periphery at the end of S phase. These distinctive patterns demonstrate a programmed control of replication sites in the spatial domain in differentiated cell nuclei.

A method has been developed for the determination of the efficiency (E) of the fluorescence resonance energy transfer between moieties on cell surfaces by use of a computer-controlled flow cytometer capable of dual wavelength excitation. The absolute value of E may be calculated on a single-cell basis. The analysis requires the measurement of samples stained with donor and acceptor conjugated ligands alone as well as together. In model experiments HK 22 murine lymphoma cells labeled with fluorescein-conjugated concanavalin A (Con A) and/or rhodamine conjugated Con A were used to determine energy transfer histograms. Using the analytic solution to energy transfer in two dimensions, a high surface density of Con A binding sites was found that suggests that the Con A receptor sites on the cell surface are to a degree preclustered . We call this technique flow cytometric energy transfer ( FCET ).

The rotational diffusion of immunoglobulin E (IgE) bound to its specific Fc receptor on the surface of living rat basophilic leukemia cells was determined from time-resolved phosphorescence emission and anisotropy measurements. The IgE-receptor complexes are mobile throughout the range of temperatures of 5-38 degrees C. The residual anisotropy does not reach zero, indicating that the rotational diffusion is hindered. The values of rotational correlation times for each temperature are consistent with dispersed receptors rotating freely in the cell membrane and rule out any significant aggregation of occupied receptors before cross-linking by antigen or anti-IgE antibodies. The rotational correlation times decrease with increasing temperature from 65 microseconds at 5.5 degrees C to 23 microseconds at 38 degrees C. However, the degree of orientational constraint experienced by the probe is unchanged. Thus, the temperature dependence can be attributed primarily to a change in the effective viscosity of the cellular plasma membrane. The phosphorescence depolarization technique is very sensitive (our probe concentrations were 10-100 nM) and thus generally applicable to studies of surface receptors and antigens on living cells.

Isolated Chinese hamster chromosomes have been analyzed using a multiparameter computer-controlled cell sorter to obtain information about unique properties of individual chromosomes. Parameters other than DNA content were sought that would further aid in distinguishing among chromosomes. The polarized emission of the DNA-specific bis-benzimidazole dye Hoechst 33342 was measured for each class of chromosomes identified by a distinct peak, i.e., differeing in DNA content. The emission anisotropy values for all chromosome classes was constant (emission anisotropy = 0.30), and the same value was obtained for purified DNA in solution. Pulse width was found to be a good parameter for resolving chromosomes as a function of total emission in the case of the smaller chromosomes and orientation (i.e., arm length) for large chromosomes. A simple theoretical model for predicting the pulse shapes generated by arbitrarily oriented, thin, rigid rods was developed and applied to the evaluation of the experimental data.

The energy transfer efficiency E was measured between fluorescein-conjugated concanavalin A (Con A) and rhodamine-conjugated Con A bound to homogeneous tissue culture cells, the HK22 murine lymphoma cell line. Results from a flow cytometric energy transfer method (FCET) and two different steady-state fluorimeter methods were compared. The data were found to be in close agreement after careful correction of the steady-state fluorimetric measurements for contributions from dissociating ligand. The biological variability of the individual cells with respect to E was calculated using an error propagation analysis and were found to be less than the variability in the absolute amount of ligand binding per cell. FCET has a number of advantages over the fluorimetric measurements using suspensions of cells: (1) relatively labile receptor-ligand complexes can be measured; (2) the analysis can be restricted to undamaged cells by gating the data collection on the light-scattering signals; (3) heterogeneous populations of cells with respect to donor and acceptor topology can be distinguished by the correlation of E with other cellular parameters derived from additional signals or combinations thereof; and (4) the dynamics of donor-acceptor redistribution on subpopulations can be measured.

An early step in the transposition of bacteriophage Mu DNA in vitro is a DNA strand-transfer reaction that generates an intermediate DNA structure in which the Mu donor DNA and the target DNA are covalently joined. DNA replication, initiated at the DNA forks in this intermediate, generates a cointegrate product; simple insert products can also be formed from the same intermediate by degradation of a specific segment of the structure, followed by gap repair. This DNA strand-transfer reaction requires ATP, magnesium, the Mu A and Mu B proteins, and a factor supplied by an Escherichia coli cell extract. We have now shown that the host protein factor requirement can be satisfied by purified protein HU. The defined system has been used to determine the DNA substrate requirements for the reaction. The reaction requires the two Mu ends, located on the same DNA molecule, in the same relative orientation to one another as in the phage Mu genome. To participate in the strand-transfer reaction efficiently the mini-Mu plasmid, used as the transposon donor, must be supercoiled; the target DNA molecule may be supercoiled, relaxed circular, or linear.

We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands, later in bands. Most chromosomal bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.

Permanent cell lines from transplantable tumors from 7, 12-dimethylbenz(a)anthracene-induced erythroleukemia of the rat were established. These cell lines maintain their erythroid nature. Erythroid differentiation can be induced by dimethylsulfoxide. This is shown by decrease in cell size, appearance of red and benzidine positive cells, and induced synthesis of four out of six adult globin chains.

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.

Publisher Summary This chapter describes the quantitative implementation of fluorescence resonance energy transfer (FRET) in the light microscope. In this process, energy is transferred from an excited donor D fluorophore to an acceptor A in close proximity. The efficiency varies inversely with the sixth power of the separation, thereby confining the range of finite energy transfer to distances generally

Left-handed helical conformations can be adopted by the poly[d(A-C)•d(G-U)] family of DNAs bearing at least one methyl or halogen (bromine, iodine) substitution at the pyrimidine C5 heterocyclic position. A representative polynucleotide is poly[d(A-br5C)•d(G-T)]. Elevation of salt concentration and temperature shifts the equilibrium in favor of the left-handed state. The spectral changes (u.v. absorption, c.d., NMR, Raman) accompanying the highly co-operative and reversible R(right)-L(left) transitions are similar to those observed with poly[d(G-C)]. Some, but not all, antibodies raised against the left-handed members of the poly[d(G-C)] family also recognize the new class of polymers, thus establishing the existence of sequence-specific determinants in the binding of certain anti-Z DNA immunoglobulins. The latter have been used to establish the presence of left-handed regions in natural plasmid and viral DNAs and in the giant salivary gland polytene chromosomes of Chironomus thummi thummi. The Z-DNA specific immunofluorescence in fixed polytene chromosomes has been quantitated by laser scanning and photon counting and is particularly intense in certain regions, such as the telomeres.

Enzymatically synthesized poly[d(G-br5C)] was used to prepare specific polyclonal and monoclonal anti-Z DNA IgGs. The binding specificities of these antibodies were characterized using left-handed polynucleotides with the sequences d(G-x5C)n and d(A-x5C)n.d(G-T)n (mean = aza, methyl, bromo, or iodo). Polyclonal anti-poly[d(G-br5C)] IgG binds the convex surface of the Z helix as evidenced by the strong requirement for a methyl or halogen group at the C5 position of cytosine. Little or no anti-poly[d(G-br5C)] IgG binding occurs to left-handed DNAs carrying a phosphorothioate substitution in the dGpdC bond or an N-5 aza substitution in the cytosine ring. Anti-poly[d(G-br5C)] IgG can stabilize transient Z DNA structures in both polymer families, thereby displacing the equilibrium in solution between the right-and left-handed DNA conformations. Anti-poly[d(G-br5C)] IgG binding sites are found in all tested covalently closed circular natural DNAs (Form I) at their extracted negative superhelical densities, but not in any of the corresponding relaxed Form II or linear Form III DNAs. Binding of anti-poly[d(G-br5-C)] IgG leads to a reduction in the electrophoretic mobility of Form I DNA (e.g. SV40, phi X174, or pBR322) and to the formation of dimers comprised of the bivalent antibody and two supercoiled Form I DNA molecules. The dimers are converted to monomers by DTT treatment. The formation of IgG-DNA complexes is dependent on external conditions (ionic strength, temperature), the properties of the DNA (torsional stress, sequence), and the immunoglobulin (specificity, valency, and concentration). Higher order oligomeric species, indicative of two or more left-handed segments per DNA molecule are formed in reactions of anti-poly[d(G-br5C)] IgG with M13 RF I DNA but not with SV40, pBR322, or phi X174 DNAs. However, oligomers of the latter are generated with other anti-Z DNA IgGs having a broader spectrum of anti-Z DNA reactivity. Conditions which destabilize natural Z sequences in deproteinized supercoiled genomes are: monovalent salt concentrations at or above the 'physiological' range, high temperature, and topological relaxation with DNA gyrase (in the absence of ATP) or with type I topoisomerases. DNA gyrase (plus ATP) catalyses an increase in DNA negative superhelical density which leads to greater anti-Z DNA IgG binding, indicating the formation of additional left-handed regions. Polytene chromosomes of insect larvae bind anti-poly[d(G-br5C)] IgG specifically and stably at Z DNA sites. The distribution of this IgG binding differs in certain regions from that displayed by anti-Z DNA IgG probes with other sequence specificities.(ABSTRACT TRUNCATED AT 400 WORDS)

Publisher Summary This chapter discusses the in vivo labeling of macromolecules and other cellular constituents and their measurement by fluorescence microscopy. An inherent pitfall in this approach is that either the labeling and/or observation technique may perturb the cell such that the information one derives is misleading or outright erroneous. The problem certainly applies to the determination of DNA content and cell cycle progression in living cells. The chapter describes the techniques for in vivo measurements; an implicit requirement is that all experiments must be carefully controlled in the sense of demonstrating that the conditions for labeling and measurement do not impair cell viability. In some cases, this condition cannot be met and there is no satisfactory alternative but to use techniques based on fixation and labeling protocols that help to take a snapshot in time of the DNA metabolism in the living cell. The chapter also presents the use of fluorescence digital imaging microscopy (FDIM) for measuring DNA, its metabolism, and its structure.