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

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

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.

Despite surgical advances and recent progress in adjuvant therapies, the prognosis for patients with malignant brain tumors such as glioblastoma multiforme has remained poor, and the neurological deterioration suffered by most patients as a consequence of tumor progression is dramatic and severe. In addition, malignant brain tumors have95% recurrence close to the primary site of initial resection. Unfortunately, standard imaging techniques do not permit the intraoperative identification of individual or small clusters of residual tumor cells, precluding their selective removal while sparing the surrounding normal brain tissue. In this report, we show that quantum dots (QDs) coupled to epidermal growth factor (EGF) or anti-EGF receptor receptor (EGFR, Her1) specifically and sensitively label glial tumor cells in cell culture, glioma mouse models, and human brain-tumor biopsies. A clear demarcation between brain and tumor tissue at the macroscopic as well as the cellular level is provided by the fluorescence emission of the QDs.

Contains fulltext : 52013.pdf (Publisher’s version ) (Closed access) The dendritic cell (DC) specific pathogen-uptake receptor (DC-SIGN) internalizes antigens for degradation and presentation onto MHC molecules. At the cell membrane, DC-SIGN forms nanoclusters that facilitate virus capture. However, internalized viruses, such as HIV-1, escape degradation. Here, we exploit ligand-conjugated, virus-sized, highly photostable quantum dots (QDs) to monitor in living cells antigen binding, entry, and trafficking. The antigen-coated QDs specific uptake and persistence in live DCs open the possibility for tracking antigen-presenting cells in vivo.

We report on the current version of the optical sectioning programmable array microscope (PAM) implemented with a single digital micro-mirror device (DMD) spatial light modulator utilized as a mask in both the fluorescence excitation and emission paths. The PAM incorporates structured illumination and structured detection operating in synchrony. A sequence of binary patterns of excitation light in high definition format (1920×1080 elements) is projected into the focal plane of the microscope at the 18 kHz binary frame rate of the Texas Instruments 1080p DMD. The resulting fluorescent emission is captured as two distinct signals: conjugate (c, ca. “on-focus”) consisting of light impinging on and deviated from the “on” elements of the DMD, and the non-conjugate (nc, ca. “out-of-focus”) light falling on and deviated from the “off” elements. The two distinct, deflected beams are optically filtered and detected either by two individual cameras or captured as adjacent images on a single camera after traversing an image combiner. The sectioned image is gained from a subtraction of the nc image from the c image, weighted in accordance with the pattern(s) used for illumination and detection and the relative exposure times of the cameras. The widefield image is given by the sum of the c and nc images. This procedure allows a high duty cycle (typically 25-50%) of on-elements in the excitation patterns and thus functions with low light intensities, preventing saturation and minimizing photobleaching of sensitive fluorophores. The corresponding acquisition speed is also very high, limited only by the bandwidth of the camera(s) (100 fps full frame with the sCMOS camera in current use) and the optical power of the light source (lasers, large area LEDs). In contrast to the static patterns typical of SIM systems, the programmable array allows optimization of the patterns (duty cycle, feature size and distribution), thus enabling a wide range of applications, ranging from patterned photobleaching, (e.g., FRAP, FLIP) and photoactivation, spatial superresolution, automated adaptive tracking and minimization of light exposure (MLE), and photolithography.

Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with carcinogenesis. Specific mutations common in non-small cell lung cancer (NSCLC), including EGFR-L858R and EGFR-ΔL747-P753, lead to ligand-independent phosphorylation, however the molecular mechanism by which these mutations in the EGFR kinase domain confer constitutive activity remain unknown. Here, using multiple sub-diffraction-limit imaging modalities, we reveal the altered behavior of NSCLC-associated EGFR mutants within the plasma membrane_including altered receptor dimerization, dynamics, and structure_which collectively dysregulate receptor activity. Using multi-color single particle tracking (SPT) and Hidden Markov Model analysis, EGFR mutants are shown to form stable dimers in the absence of ligand and exhibit a slower mobility that is consistent with receptor signaling. These results were confirmed using two-color, single-molecule super-resolution microscopy (dSTORM) to visualize the spatial distribution of receptors. Receptor clustering was quantified by localization-based cross-correlation analysis to show ligand-induced aggregation of EGFR as well as ligand-independent aggregation of EGFR mutants. Since the receptor ectodomain is known to play a critical role in dimerization, live cell FRET measurements between the EGFR N-terminus and the plasma membrane was used to quantify changes in ectodomain structure. We found that unliganded EGFR mutants are more readily found in the extended conformation, similar to the ligand bound wild type receptor. Therefore, mutation within the kinase domain biases the structural equilibrium of the extracellular domain toward a dimer-competent state.Collectively, these data support a model where oncogenic signaling from NSCLC-associated EGFR mutants is a result of productive dimerization between non-ligand bound receptors. Furthermore, because these mutations are found in the kinase domain, this work introduces the concept that oncogenic EGFR signaling may be controlled in part by a form of “inside-out” signaling.

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.

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

ErbB1 receptors situated on cellular filopodia undergo systematic retrograde transport after binding of the epidermal growth factor (EGF) and activation of the receptor tyrosine kinase. Specific inhibitors of the erbB1 receptor tyrosine kinase as well as cytochalasin D, a disruptor of the actin cytoskeleton, abolish transport but not free diffusion of the receptor–ligand complex. Diffusion constants and transport rates were determined with single molecule sensitivity by tracking receptors labeled with EGF conjugated to fluorescent quantum dots. Retrograde transport precedes receptor endocytosis, which occurs at the base of the filopodia. Initiation of transport requires the interaction and concerted activation of at least two liganded receptors and proceeds at a constant rate mediated by association with actin. These findings suggest a mechanism by which filopodia detect the presence and concentration of effector molecules far from the cell body and mediate cellular responses via directed transport of activated receptors.

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 programmable array microscope (PAM) incorporates a spatial light modulator (SLM) placed in the primary image plane of a widefield microscope, where it is used to define patterns of illumination and/or detection. We describe the characteristics of a special type of PAM collecting two images simultaneously. The conjugate image (Ic) is formed by light originating from the object plane and returning along the optical path of the illumination light. The non-conjugate image (Inc) receives light from only those regions of the SLM that are not used for illuminating the sample. The dual-signal PAM provides much more time-efficient excitation than the confocal laser scanning microscope (CLSM) and greater utilization of the available emission light. It has superior noise characteristics in comparison to single-sided instruments. The axial responses of the system under a variety of conditions were measured and the behaviour of the novel Inc image characterized. As in systems in which only Ic images are collected (Nipkow-disc microscopes, and previously characterized PAMs), the axial response to thin fluorescent films showed a sharpening of the axial response as the unit cell of the repetitive patterns decreased in size. The dual-signal PAM can be adapted to a wide range of data analysis and collection strategies. We investigated systematically the effects of patterns and unit cell dimensions on the axial response. Sufficiently sparse patterns lead to an Ic image formed by the superposition of the many parallel beams, each of which is equivalent to the single scanning spot of a CLSM. The sectioning capabilities of the system, as given by its axial responses, were similar for a given scan pattern and for processed pseudorandom sequence (PRS) scans with the same size of the unit cell. For the PRS scans, optical sectioning was achieved by a subtraction of an Inc image or, alternatively, a scaled widefield image from the Ic image. Based on the comparative noise levels of the two methods, the non-conjugate subtraction was significantly superior. A point spread function for Ic and Inc was simulated and properties of the optical transfer functions (OTFs) were compared. Simulations of the OTF in non-conjugate imaging did not suffer from the missing cone problem, enabling a high quality deconvolution of the non-conjugate side alone. We also investigated the properties of images obtained by subjecting the Ic and Inc data to a combined maximum likelihood deconvolution.

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

The association state(s) and activities of the ErbB receptor family members in intact living cells differ widely depending upon expression levels and their distribution and interaction partners. There are contradictory views in the literature about the aggregation states and presumed structures of the receptors in the cell membrane. Fixation artifacts may account for apparent quantitative discrepancies. We obtained biophysical FRET/FLIM data on living cells that reveal structural features of ErbB1 (EGFR) and ErbB2 as well as the effects of EGF and various kinase inhibitors on these structures.We constructed transgenes in which an acyl carrier protein sequence was introduced between the signal peptide and the mature receptor protein sequence. ACP-ErbB1 behaves similarly to wild type ErbB1 with respect to EGF binding, activation and internalization. The ACP-ErbB2 lacks the capacity for binding ligands but can be transactivated as a heterodimer with ErbB1 or ErbB3. Enzymatic labeling of the specific serine in the ACP tag by fluorescent CoA substrates served as donors. The FRET acceptor was the novel membrane probe, NR12S, which is confined exclusively to the outer leaflet of the plasma membrane.Addition of NR12S to the cells led to a dramatic reduction in the fluorescence lifetime of the donor, indicating a close proximity of the N-terminus of the ErbB1 ectodomain to the plasma membrane, supporting the published autoinhibited structure. EGF addition caused a time-dependent increase in the donor lifetime (reduced FRET), in accordance with the extended dimeric ectodomain structure oberved by Xray-crystallography. The effects of kinase inhibitors on these states and on ensuing endocytosis were also studied. The influence(s) of ErB2 density and antibodies interfering with receptor dimerization were additional topics addressed in this study. TCSPC lifetime images were analyzed with Mathematica software developed for these studies.

The programmable array microscope (PAM) is a powerful tool combining the capabilities of nearly all previously described optical sectioning techniques in a single microscope. Not only can the user create optical sections of threedimensional objects, but the PAM's unique adaptive optical strategy allows a user to select the best sectioning method for a particular sample or experimental need. The key to the PAM is a spatial light modulator (SLM). This device, when placed in the image plane of a microscope, can be used to create optical sectioning, generate spatial encoding masks, and/or define regions of interest.

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.

Type I and type II DNA-topoisomerases are essential enzymes that mediate replication, transcription, re combination, and mitosis in multicellular eukaryotes but the extent of their interchange for specific reactions in vivo is controversial. Expression patterns for topoisomerase I and topoisomerase II during the embryogenesis of Drosophila melanogaster were compared with patterns of DNA replication and expression of the histone genes. In late oogenesis the maternally supplied top2 mRNA was evenly distributed throughout the egg with elevated levels at the posterior tip, a pattern that is maintained in syncytial blastoderm embryos. During gastrulation, top2 mRNA became differentially localized only to regions of DNA replication, including new expression in the gonads preceding mitosis/meiosis. Significantly higher levels of top2 mRNA were found in mitotic compared to endoreplicating tissues. The total histone mRNA was exclusively associated with DNA replication but, in cont rast to top2 mRNA, mitotic and endoreplicating cells contained similar expression levels with no expression in the gonads. Striking differences exist between the distribution of the top2 mRNA and topoisomerase If protein. The protein localizes to all evolving nuclei where it persists throughout embryogenesis. A high level of top1 mRNA transcript was present without differential tissue distribution throughout embryogenesis.

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)

We have implemented continuous-wave two-photon excitation of near-UV absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 647-nm emission of an Ar-Kr mixed gas laser was used to excite the UV-absorbing DNA dyes DAPI, the bisbenzimidazole Hoechst 33342, and ethidium bromide in a shared aperture SNOM with uncoated fiber tips. Polytene chromosomes of Drosophila melanogaster and the nuclei of 3T3 Balb/c cells labeled with these dyes were readily imaged. The fluorescence intensity showed the expected nonlinear (second order) dependence on the excitation power in the range of 8–180mW. We measured the fluorescence intensity as a function of the tip-sample displacement in the direction normal to the sample surface in the single- and two-photon excitation modes (SPE, TPE). The fluorescence intensity decayed faster in TPE than in SPE.

The Drosophila protein Hrb57A has sequence homology to mammalian heterogenous nuclear ribonucleoprotein (hnRNP) K proteins. Its in vivo distribution has been studied at high resolution by confocal laser scanning microscopy (CLSM) in embryos injected with fluorescently labeled monoclonal antibody. Injection of antibody into living embryos had no apparent deleterious effects on further development. Furthermore, the antibody-protein complex could be observed for more than 7 cell cycles in vivo, revealing a dynamic redistribution from the nucleus to cytoplasm at each mitosis from blastoderm until hatching. The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos. The Hrb57A protein was recruited to the 93D locus upon heat shock and thus serves as an in vivo probe for the activity of the gene in diploid cells of the embryo. Observations during heat shock revealed considerable mobility within interphase nuclei of this transcription site. Furthermore, the reinitiation as well as the down regulation of transcriptional loci in vivo during the recovery from heat shock could be followed by the rapid redistribution of the hnRNP K during stress recovery. These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.

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

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

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

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

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

Clustering of the mast cell function-associated antigen (MAFA) on the surface of rat mucosal type mast cells line 2H3 (RBL-2H3) leads to suppression of the secretory response induced by the type I Fc epsilon receptor (Fc epsilon RI). In order to establish a possible association between MAFA and Fc epsilon RI we measured fluorescence resonance energy transfer (FRET) between the MAFA-specific monoclonal antibody (mAb) G63 and Fc epsilon RI-bound ligands as well as between Fc epsilon RI-bound ligands themselves using the donor photobleaching FRET (pbFRET) technique. Average FRET efficiencies between 6 and 9% were determined after low-temperature incubation with fluorescent dye conjugated mAb G63 bound to MAFA (donor) and IgE bound to Fc epsilon RI (acceptor) on RBL-2H3 cells. Subsequent cross-linking of IgE by a polyvalent antigen caused no change in FRET efficiencies. These results suggest that the MAFA is located in the vicinity of the Fc epsilon RI on resting cells, and that clustering of the Fc epsilon RI leads to no significant change in the proximity of the two molecular species. In view of the sequence motif identified in the cytosolic tail of the MAFA and the observed changes in its phosphorylation upon antigen stimulation (Guthmann et al., Proc. Natl. Acad. Sci. USA 1995, 92: 9397-9401), the present study suggests that the secretory response inhibition by MAFA interferes with the signal transduction cascade initiated via the Fc epsilon RI. An additional finding was that clustering of the Fc epsilon RI by antigen showed a clear increase in the efficiency of FRET between Fc epsilon RI-bound IgE molecules conjugated with fluorescent donor and acceptor.

We report progress on the construction of an optical sectioning programmable array microscope (PAM) implemented with a digital micro-mirror device (DMD) spatial light modulator (SLM) utilized for both fluorescence illumination and detection. The introduction of binary intensity modulation at the focal plane of a microscope objective in a computer controlled pixilated mode allows the recovery of an optically sectioned image. Illumination patterns can be changed very quickly, in contrast to static Nipkow disk or aperture correlation implementations, thereby creating an optical system that can be optimized to the optical specimen in a convenient manner, e.g. for patterned photobleaching, photobleaching reduction, or spatial superresolution. We present a third generation (Gen-3) dual path PAM module incorporating the 25 kHz binary frame rate TI 1080p DMD and a newly developed optical system that offers diffraction limited imaging with compensation of tilt angle distortion.

The spatial distribution of no-on transient A (NONA), a protein associated with specific puffs on polytene chromosomes, was followed in nuclei of living Drosophila embryos by microinjection of fluorescently labeled monoclonal antibody to NONA. The injected antibodies remained active until the larval stage, revealing the distribution of the NONA protein throughout embryogenesis. Most injected animals completed embryonic development and hatched as normal larvae. NONA was restricted to the cytoplasm until the end of cycle 11. We document an active uptake of the NONA-antibody complex into early interphase nuclei from nuclear cycle 14 onwards, following each mitosis. Significant differences in the distribution of the protein between fixed and living embryos were apparent, particularly at high resolution. The NONA protein was localized in the nuclei of living embryos at discrete sites, most of which lay at the periphery and some of which were tightly clustered. The constellation of sites changed with time; in some nuclei these changes were fast whereas in other nuclei the pattern was quite stable. These data suggest that specific protein complexes associated with active interphase chromatin, and possibly chromatin in general, are mobile in the living organism.

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.

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

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.

Quantum dots (QDs) are colloidal inorganic semiconductor nanocrystals composed typically of a CdSe, CdS or CdTe core and a ZnS shell. There are many advantages in the use of QDs as fluorophores: they can be excited over a broad spectral range and they have narrow emission bands that can be tuned from ultraviolet to infrared by adjusting size and composition. Their bright emission fluorescence and resistance to photobleaching make QDs ideal for single-particle detection and permit imaging over prolonged time periods. Because of these advantages, QDs are finding increasing use in in vivo and in vitro studies [1].

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.

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50-200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.

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

We report on a new generation, commercial prototype of a programmable array optical sectioning fluorescence microscope (PAM) for rapid, light efficient 3D imaging of living specimens. The stand-alone module, including light source(s) and detector(s), features an innovative optical design and a ferroelectric liquid-crystal-on-silicon (LCoS) spatial light modulator (SLM) instead of the DMD used in the original PAM design. The LCoS PAM (developed in collaboration with Cairn Research, Ltd.) can be attached to a port of a(ny) unmodified fluorescence microscope. The prototype system currently operated at the Max Planck Institute incorporates a 6-position high-intensity LED illuminator, modulated laser and lamp light sources, and an Andor iXon emCCD camera. The module is mounted on an Olympus IX71 inverted microscope with 60-150X objectives with a Prior Scientific x,y, and z high resolution scanning stages. Further enhancements recently include: (i) point- and line-wise spectral resolution and (ii) lifetime imaging (FLIM) in the frequency domain. Multiphoton operation and other nonlinear techniques should be feasible. The capabilities of the PAM are illustrated by several examples demonstrating single molecule as well as lifetime imaging in live cells, and the unique capability to perform photoconversion with arbitrary patterns and high spatial resolution. Using quantum dot coupled ligands we show real-time binding and subsequent trafficking of individual ligand-growth factor receptor complexes on and in live cells with a temporal resolution and sensitivity exceeding those of conventional CLSM systems. The combined use of a blue laser and parallel LED or visible laser sources permits photoactivation and rapid kinetic analysis of cellular processes probed by photoswitchable visible fluorescent proteins such as DRONPA.