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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.

We have assessed the impact of a-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of a-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction., Instituto de Investigaciones Bioquímicas de La Plata

Innovations in fluorescence microscopy of live cells involving new reagents and techniques reveal dynamic processes that were not previously observable and therefore unknown. Water soluble, bio-functionalized semiconductor quantum dots (QDs) provide advantages of much greater photostability compared to conventional fluorescent dyes, and, as a consequence, single QDs can be easily detected. QDs coupled to growth factor ligands behave similarly as the natural ligand and serve as highly fluorescent probes of the erbB family of tyrosine kinase receptors in living cells. Continuous confocal laser scanning microscopy and flow cytometry measurements of QDs combined with visible fluorescent fusions of the receptors have elucidated individual steps in the signaling cascades initiated by these receptors. This report highlights advantages and some disadvantages of QDs, such as size and blinking behavior that complicate some live cell imaging applications. The new class of noble metal nanodots constitute an attractive alternative to QDs in that they are not only highly fluorescent and photostable, but also, much smaller and non-toxic. We present a new synthesis method for the production of Au nanodots. We demonstrate that electrochemical synthesis allows the reproducible control of cluster size. The resulting clusters are more monodisperse than those formed by other methods and are stable over many months. We report their characterization using MALDI-TOF mass spectrometry and UV-VIS spectroscopy.

Semiconductor nanocrystals, or quantum dots (QDs), are exciting new fluorescent probes useful in imaging at the single molecule to the whole animal level. Some recent applications of QDs are reviewed here and the reader is directed to the original literature for further details.

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 Innovative thinking and experimentation were the hallmarks of Mack Fulwyler's approach to research. This report summarizes some of the ideas and their early realizations that he pursued in the field of imaging cytometry, work that was not published before his untimely death, although he composed the initial draft of this report. Methods Included are related experiments implemented in the programmable array microscope (PAM) devised for patterned illumination and detection, the instrument that Mack Fulwyler employed during a sabbatical leave in Gottingen in 1998. Despite being the originator of instrumentation for flow cytometry and sorting, Mack Fulwyler was intensely interested in imaging systems, recognizing their ability to resolve cellular details obscured by the whole cell signals generally acquired in flow. At one point, these interests merged with those of two other authors (I.T.Y. and T.M.J.), leading to the Image Cytometry and Sorting (ICAS) strategy and project. A major goal was uncomplicated rare cell detection and isolation using a sequential process of cellular labeling via suitable probes, whole field imaging, and selective area-restricted photoinduced reactions designed to encapsulate and/or chemically or physically tag cells in a manner permitting subsequent fractionation by bulk techniques. Results and Conclusion This publication features photoinduced polymerization, photodecaging, photoactivation, and photochromic conversion reactions carried out by Fulwyler and/or the other authors with the PAM, employing operator designated patterns and locations in various samples. Photopolymerization of polyethylene glycol-diacrylate to a gel-like structure allowing the specific selection of objects (cells) for further analysis and processing techniques was the approach explored personally by Mack Fulwyler in relation to the ICAS concept. © 2005 International Society for Analytical Cytology

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

In any society, professional or otherwise, certain individuals stand out by virtue of their ability, leadership qualities, other personal attributes, or combinations thereof. Mack Fulwyler had all of these but somehow even more. His philosophy of life, revealed in the manner with which he dealt with others, reminded one of Henry Thoreau with respect to the grandeur of nature, Thomas Edison with respect to the virtues of persistence and ingenuity, and Walt Whitman with respect to the freedom of thought and spirit. Mack Fulwyler the inventor made fundamental contributions to numerous fields of science and technology, and Mack the individual enriched our lives by being a superb friend and mentor. In this special issue of Cytometry, his colleagues acknowledge their debt and appreciation with scientific contributions closely linked to Mack’s work—in two cases, representing some of his own unpublished investigations—and with personal reminiscences about their interactions with him. We are very thankful for the opportunity to offer a unique tribute to a towering figure in the field of analytical cytometry and hope that this memorial issue will serve as an inspiration to younger students and practicing scientists. In addition, we recognize not only the memory of Mack Fulwyler but also the living presence of his wife, Carol, unflagging partner through the many successes and vicissitudes that marked Mack’s career.

Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands).PcG genes are essential genes in higher eukaryotes responsible for the maintenance of the spatially distinct repression of developmentally important regulators such as the homeotic genes. Their absence, as well as overexpression, causes transformations in the axial organization of the body. Although protein complexes have been isolated in vitro, little is known about their stability or exact mechanism of repression in vivo.We determined the translational diffusion constants of PcG proteins,dissociation constants and residence times for complexes in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with our experimental data. We were able to determine forward and reverse rate constants for complex formation. Complexes exchanged within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time,ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria.

We constructed a photoactivatable Drosophila histone 2 A variant green fluorescent fusion protein (H2AvD-paGFP) for tracking chromatin loci in living Drosophila embryos. Activation of paGFP was achieved by irradiation from a single-photon diode laser at 408 nm, but activated nuclei failed to divide. Photoconversion could also be achieved by two-photon fs pulses in the range of 780–840 nm. Viability in whole-mount embryos could only be maintained at 820 nm, at which we could activate, simultaneously track and quantitate the mobility of multiple fluorescent loci. This report constitutes the first demonstration of two-photon activation of paGFP and the use of a paGFP-fusion protein in investigations of whole organisms. Keywords: Green fluorescent protein; Histone; Chromatin dynamics; Nuclear architecture; Two-photon microscopy; Photoactivation Abbreviations: eGFP, enhanced green fluorescent protein; paGFP, photoactivatable GFP; wtGFP, wild-type GFP; H2AvD, histone 2 A variant Drosophila; FRAP, fluorescence recovery after photobleaching; DIC, differential interference contrast; MSD, mean square displacement; SNR, signal-to-noise ratio; FWHM, full-width at half-maximum

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.

The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments.

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.

Sm and Sm-like (LSm) proteins form heptameric complexes that are involved in various steps of RNA metabolism. In yeast, the Lsm1-7 complex functions in mRNA degradation and is associated with several enzymes of this pathway, while the complex LSm2-8, the composition of which largely overlaps with that of LSm1-7, has a role in pre-mRNA splicing. A human gene encoding an LSm1 homolog has been identified, but its role in mRNA degradation has yet to be elucidated. We performed subcellular localization studies and found hLSm1 predominantly in the cytoplasm. However, it is not distributed evenly; rather, it is highly enriched in small, discrete foci. The endogenous hLSm4 is similarly localized, as are the overexpressed proteins hLSm1-7, but not hLSm8. The foci also contain two key factors in mRNA degradation, namely the decapping enzyme hDcp1/2 and the exonuclease hXrn1. Moreover, coexpression of wild-type and mutant LSm proteins, as well as fluorescence resonance energy transfer (FRET) studies, indicate that the mammalian proteins hLSm1-7 form a complex similar to the one found in yeast, and that complex formation is required for enrichment of the proteins in the cytoplasmic foci. Therefore, the foci contain a partially or fully assembled machinery for the degradation of mRNA.

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 report the acquisition and deconvolution of three-dimensional spectrally resolved images in a programmable array microscope implementing a Hadamard transform fluorescence spectroscopy system with adjustable spectral resolution. A stack of 16 two-dimensional spectral images was collected at 400 nm intervals along the optical axis. The specimen consisted of a polytene chromosome spread from Drosophila melanogaster doubly labelled for the Polyhomeotic protein by indirect immunofluorescence labelling with Alexa594 and for DNA with YOYO-1. The resulting four-dimensional data set consisted of the xyz spatial dimensions (898 x 255 x 16) with a 26-point spectrum at each spatial location. The total exposure time to the sample was 34 min. The system requires the acquisition of multiple images, and thus works best with fluorophores that are resistant to photobleaching. Image deconvolution reduced the amount of out-of-focus blur by up to a factor of 8, resulting in a dramatic improvement in the visualization of the chromosome backbone and localization of the specific Polyhomeotic domains.

The defining feature of a programmable array microscope (PAM) is the presence of a spatial light modulator in the image plane. A spatial light modulator used singly or as a matched pair for both illumination and detection can be used to generate an optical section. Under most conditions, the basic optical properties of an optically sectioning PAM are similar to those of rotating Nipkow discs. The method of pattern generation, however, is fundamentally different and allows arbitrary illumination patterns to be generated under programmable control, and sectioning strategies to be changed rapidly in response to specific experimental conditions. We report the features of a PAM incorporating a digital micromirror device, including the axial sectioning response to fluorescent thin films and the imaging of biological specimens. Three axial sectioning strategies were compared: line scans, dot lattice scans and pseudo-random sequence scans. The three strategies varied widely in light throughput, sectioning strength and robustness when used on real biological samples. The axial response to thin fluorescent films demonstrated a consistent decrease in the full width at half maximum (FWHM), accompanied by an increase in offset, as the unit cells defining the patterns grew smaller. Experimental axial response curves represent the sum of the response from a given point of illumination and cross-talk from neighbouring points. Cross-talk is minimized in the plane of best focus and when measured together with the single point response produces a decrease in FWHM. In patterns having constant throughput, there appears to be tradeoff between the FWHM and the size of the offset. The PAM was compared to a confocal laser scanning microscope using biological samples. The PAM demonstrated higher signal levels and dynamic range despite a shorter acquisition time. It also revealed more structures in x-z sections and less intensity drop-off with scanning depth.

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.

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.

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).

Transvection is the phenomenon by which the expression of a gene can be controlled by its homologous counterpart in trans, presumably due to pairing of alleles in diploid interphase cells. Transvection or trans-sensing phenomena have been reported for several loci in Drosophila, the most thoroughly studied of which is the Bithorax-Complex (BX-C). It is not known how early trans-sensing occurs nor the extent or duration of the underlying physical interactions. We have investigated the physical proximity of homologous genes of the BX-C during Drosophila melanogaster embryogenesis by applying fluorescent in situ hybridization techniques together with high-resolution confocal light microscopy and digital image processing. The association of homologous alleles of the BX-C starts in nuclear division cycle 13, reaches a plateau of 70% in postgastrulating embryos, and is not perturbed by the transcriptional state of the genes throughout embryogenesis. Pairing frequencies never reach 100%, indicating that the homologous associations are in equilibrium with a dissociated state. We determined the effects of translocations and a zeste protein null mutation, both of which strongly diminish transvection phenotypes, on the extent of diploid homologue pairing. Although translocating one allele of the BX-C from the right arm of chromosome 3 to the left arm of chromosome 3 or to the X chromosome abolished trans-regulation of the Ultrabithorax gene, pairing of homologous alleles surprisingly was reduced only to 20-30%. A zeste protein null mutation neither delayed the onset of pairing nor led to unpairing of the homologous alleles. These data are discussed in the light of different models for trans-regulation. We examined the onset of pairing of the chromosome 4 as well as of loci near the centromere of chromosome 3 and near the telomere of 3R in order to test models for the mechanism of homologue pairing.

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.

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.

The regulation of DNA topology by topoisomerase II from Drosophila melanogaster has been studied extensively by biochemical methods but little is known about its roles in vivo. We have performed experiments on the inhibition of topoisomerase II in living Drosophila blastoderm embryos. We show that the enzymatic activity can be specifically disrupted by microinjection of antitopoisomerase II antibodies as well as the epipodophyllotoxin VM26, a known inhibitor of topoisomerase II in vitro. By labeling the chromatin of live embryos with tetramethylrhodamine-coupled histones, the effects of inhibition on nuclear morphology and behaviour was followed in vivo using confocal laser scanning microscopy. Both the antibodies and the drug prevented or hindered the segregation of chromatin daughter sets at the anaphase stage of mitosis. In addition, high concentrations of inhibitor interfered with the condensation of chromatin and its proper arrangement into the metaphase plate. The observed effects yielded non-functional nuclei, which were drawn into the inner yolk mass of the embryo. Concurrently, undamaged nuclei surrounding the affected region underwent compensatory division, leading to the restoration of the nuclear population, and thereby demonstrating the regulative capacity of Drosophila blastoderm embryos.

The native form of Drosophila melanogaster DNA topoisomerase II was purified from Schneider's S3 tissue culture cells and studied with two supercoiled minicircle preparations, mini and mini-CG, 354 bp and 370 bp in length, respectively. Mini-CG contains a d(CG)7 insert which assumes a left-handed Z-DNA conformation in negative supercoiled topoisomers with a negative linking number difference - delta Lk greater than or equal to 2. The interactions of topoisomerase II with topoisomer families of mini and mini-CG were studied by band-shift gel electrophoresis in which the individual topoisomers and their discrete or aggregated protein complexes were resolved. A monoclonal anti-Z-DNA IgG antibody (23B6) bound and aggregated only mini-CG, thereby confirming the presence of Z-DNA. Topoisomerase II bound and relaxed mini-CG more readily than mini. In both cases, there was a preference for more highly negatively supercoiled topoisomers. The topoisomerase II inhibitor VM-26 induced the formation of stable covalent DNA-protein intermediates. In addition, the non-hydrolyzable GTP analogue GTP gamma S inhibited the binding and relaxation activities. Experiments to detect topoisomerase cleavage sites failed to elicit specific loci on either minicircle preparation. We conclude that Drosophila topoisomerase II is able to bind and process small minicircles with lengths as short as 360 bp and negative superhelix densities, - sigma, which can exceed 0.1. Furthermore, the enzyme has a preferential affinity for topoisomers containing Z-DNA segments and relaxes these molecules, presumably by cleavage external to the inserts. Thus, a potentially functional relationship between topoisomerase II, an enzyme regulating the topological state of DNA-chromatin in vivo, and left-handed Z-DNA, a conformation stabilized by negative supercoiling, has been established.

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.

The cell surface receptor for epidermal growth factor (EGFR) is one of the most studied integral membrane proteins. The receptor is widely distributed in cells and tissues of mammalian and avian tissues and plays an important role in growth control. Binding of the epidermal growth factor (EGF) to EGFR initiates a complex biological response, which includes self-phosphorylation of the receptor due to an intrinsic tyrosine kinase activity, phosphorylation of other membrane proteins, increased intake of metabolites, and increased proliferation. Complete amino acid sequence of EGFR revealed a high degree of homology with viral oncogenes and allowed tentative identification of an external hormone binding domain, a transmembrane domain, and a cytoplasmic domain that includes tyrosine kinase activity. EGF binding induces rapid aggregation of EGFR, a process which was also observed on other receptor systems. These and other observations led to a hypothesis that microaggregation of EGFR is a necessary prerequisite for the biological response of EGF. A direct approach to study the processes of oligomerization of cell membrane proteins is to measure their mobility under various conditions. The lateral mobility of the EGFR was studied on mouse 3T3 fibroblasts and on A431 cells. However, an examination of the equations for the lateral and rotational diffusion in membranes shows that only rotational diffusion is strongly dependent on the size of the diffusing entity. A method of measuring protein rotational diffusion by time-resolved phosphorescence has proved to be very useful in the analysis of both in vivo and in vitro systems. The authors apply this method to study the mobility of EGFR on living A431 cells and membrane preparations.© (1991) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

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 epidermal growth factor receptor (EGF-R) of human A431 cells bears an antigenic determinant that is closely related to the human blood group A carbohydrate structure. Labeling studies with blood group A reactive anti-EGF-R monoclonal antibodies and various lectins revealed that A431 cultures are heterogeneous with respect to blood group A expression. We have isolated clonal variants of these cells that either express (A431A+ cells) or completely lack (A431A- cells) the blood group A specific N-acetyl-D-galactosamine (GalNAc) residue. We show that this difference is due to the absence of a UDP-GalNAc:Gal transferase activity in A431A- cells. Subsequently, we have compared EGF-R functioning in these cell lines. Scatchard analysis of EGF-binding shows that in A431A- cells 6.3% of the EGF-R belongs to a high affinity subclass (Kd = 0.4 nM) while in A431A+ this subclass represents only 3.2% of the total receptor pool. The elevated level of high affinity receptors in A431A- cells is accompanied by a parallel increase in receptor protein- tyrosine kinase activity. In membrane preparations of A431A- cells, receptor autophosphorylation as well as phosphorylation of a tyrosine-containing peptide substrate is 2-3-fold higher as compared with A431A+ cells. In intact A431A-cells, the difference in receptor activity is measured as a 2-3-fold elevated level of receptor phosphorylation and a 2-3-fold higher abundance of phosphotyrosine in total cellular protein in A431A- cells. In addition, [35S]methionine pulse-chase experiments showed a ligand-independent increase in turnover of EGF-R in A431A- cells: the receptor's half life in these cells is 10 h as compared with 17 h in A431A+ cells. Our results suggest a possible involvement of GalNAc residue(s) in determining EGF-R affinity, protein-tyrosine kinase activity and turnover in A431 cells. Furthermore, our results indicate that high affinity EGF-R are the biologically active species with respect to protein-tyrosine kinase activity.

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.

Developments in microscope, sensor, and image-processing technologies have led to integrated systems for the quantification of low-light-level emission signals from biological samples. Specificity is provided in the form of monoclonal antibodies and other ligands or enzyme substrates conjugated with efficient fluorophores. Fluorescent probes are also available for cellular macromolecular constituents and for free ions of biological interest such as H+ and Ca2+. The entire spectrum of photophysical phenomena can be exploited. Representative data are presented from studies of DNA conformation and architecture in polytene chromosomes and from studies of receptor-mediated endocytosis, calcium distribution, and the organization of the contractile apparatus in muscle cells.

SUMMARY We have measured the kinetics of specific globin mRNA and Friend virus (FV) RNA synthesis by hybridization to immobilized cDNA after induction of differentiation of two erythroleukemia cell lines (F4N, BS) by butyrate and Me,SO. The induction with butyrate in these cell lines occurs very rapidly (16-24 h). Cell cycle analysis was made of the populations throughout induction by flow cytofluorometry. The kinetics of commitment of cell populations to terminal differentiation by butyrate was determined by removal of inducer at various times and scoring of benzidine staining cells (hemoglobin producing). In addition, the cell cycle dependence of commitment was determined by flow sorting out of Gl and S+G2 cells various times after addition of inducer and scoring benzidine-stained colonies after growth in methylcellulose. Cells exposed to inducer were also sorted by cell cycle phase using an elutriator rotor. The amount of globin mRNA synthesis in the different cell populations was then determined. I. It was found that an g-12 h period in butyrate was required before (a) globin specific mRNA was synthesized; and (b) commitment to differentiation occurred. The time course of globin mRNA synthesis was positively correlated with Gl arrest, as has been also found by others. 2. The increase of FV RNA synthesis was not found during Gl arrest. It occurred early and before commitment. 3. Commitment of cells to irreversible differentiation upon butyrate induction occurs only during the Gl phase of the cell cycle. 4. Globin mRNA synthesis occurs first only in Gl cells. 5. Globin mRNA is synthesized later in all phases of the cell cycle. These data suggest that

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.

The sensitivity to agglutination by several plant lectins has been studied during the induced erythroid differentiation of Friend erythroleukemic cells in culture. In addition, the number of lectin receptors on the cell has been measured. It is shown that early during the differentiation, there is an increase in agglutinability while the receptor density remains constant. In the later phase of the differentiation process, the cells lose their sensitivity to agglutination while the receptor number and density increases. These changes were not observed on nonerythroid mastocytoma culture cells. Two nondifferentiating variants of the FL cells were shown to have altered sensitivities to agglutination by ConA.