Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1alpha-mediated localization in living cells

The tetracysteine sequence YRECCPGCCMWR fused to the N terminus of green fluorescent protein (GFP) self-aggregates upon biarsenical labeling in living cells or in vitro. Such dye-triggered aggregates form temperature-dependent morphologies and are dispersed by photobleaching. Fusion of the biarsenical aggregating GFP to the regulatory (R) or catalytic (C) subunit of PKA traps intact holoenzyme in compact fluorescent puncta upon biarsenical labeling. Contrary to the classical model of PKA activation, elevated cAMP does not allow RIalpha and Calpha to diffuse far apart unless the pseudosubstrate inhibitor PKI or locally concentrated substrate is coexpressed. However, RIIalpha releases Calpha upon elevated cAMP alone, dependent on autophosphorylation of the RIIalpha inhibitory domain. DAKAP1alpha overexpression induced R and C outer mitochondrial colocalization and showed similar regulation. Overall, effective separation of type I PKA is substrate dependent, whereas type II PKA dissociation relies on autophosphorylation.     

Detection of mitochondrial caspase activity in real time in situ in live cells.

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP-caspase 3 substrate-YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.     

Quantitative DNA imaging in breast tumor cells by a Hadamard transform fluorescence imaging microscope.

This paper presents a novel Hadamard transform (HT) fluorescence imaging microscope by combining multiplexed imaging technique with a conventional upright fluorescence microscope for single-cell imaging and quantitative cellular analysis. The HT imaging microscope can provide 511 x 512-pixel single-cell image with high sensitivity within 21 s. In this study, the high potential value of the microscope in biomedical analysis has been demonstrated by using it to evaluate the malignancy degree of thirty cases of human breast tumors based on the measurements of cellular DNA contents, with conclusions highly accordant with pathological diagnosis. The results show that the HT microscope has the ability to analyze very small specimens and the capability of detecting very high ploidy cells, which are advantages over flow cytometry. The microscope was also successfully applied to cellular morphological analysis, and it was demonstrated that a significant linear relationship exists between tumor nuclear DNA contents and the nuclear area, and malignant and benign tumors are significantly different in both DNA contents and nuclear area. The reliability of the HT microscope in cellular DNA measurements was also investigated.     

Hierarchical structure of the energy landscape of proteins revisited by time series analysis. I. Mimicking protein dynamics in different time scales

Time series models, which are constructed from the projections of the molecular-dynamics (MD) runs on principal components (modes), are used to mimic the dynamics of two proteins: tendamistat and immunity protein of colicin E7 (ImmE7). Four independent MD runs of tendamistat and three independent runs of ImmE7 protein in vacuum are used to investigate the energy landscapes of these proteins. It is found that mean-square displacements of residues along the modes in different time scales can be mimicked by time series models, which are utilized in dividing protein dynamics into different regimes with respect to the dominating motion type. The first two regimes constitute the dominance of intraminimum motions during the first 5 ps and the random walk motion in a hierarchically higher-level energy minimum, which comprise the initial time period of the trajectories up to 20-40 ps for tendamistat and 80-120 ps for ImmE7. These are also the time ranges within which the linear nonstationary time series are completely satisfactory in explaining protein dynamics. Encountering energy barriers enclosing higher-level energy minima constrains the random walk motion of the proteins, and pseudorelaxation processes at different levels of minima are detected in tendamistat, depending on the sampling window size. Correlation (relaxation) times of 30-40 ps and 150-200 ps are detected for two energy envelopes of successive levels for tendamistat, which gives an overall idea about the hierarchical structure of the energy landscape. However, it should be stressed that correlation times of the modes are highly variable with respect to conformational subspaces and sampling window sizes, indicating the absence of an actual relaxation. The random-walk step sizes and the time length of the second regime are used to illuminate an important difference between the dynamics of the two proteins, which cannot be clarified by the investigation of relaxation times alone: ImmE7 has lower-energy barriers enclosing the higher-level energy minimum, preventing the protein to relax and letting it move in a random-walk fashion for a longer period of time.     

Three-dimensional structure and defects in colloidal photonic crystals revealed by tomographic scanning transmission X-ray microscopy

Self-assembled colloidal crystals have attracted major attention because of their potential as low-cost three-dimensional (3D) photonic crystals. Although a high degree of perfection is crucial for the properties of these materials, little is known about their exact structure and internal defects. In this study, we use tomographic scanning transmission X-ray microscopy (STXM) to access the internal structure of self-assembled colloidal photonic crystals with high spatial resolution in three dimensions for the first time. The positions of individual particles of 236 nm in diameter are identified in three dimensions, and the local crystal structure is revealed. Through image analysis, structural defects, such as vacancies and stacking faults, are identified. Tomographic STXM is shown to be an attractive and complementary imaging tool for photonic materials and other strongly absorbing or scattering materials that cannot be characterized by either transmission or scanning electron microscopy or optical nanoscopy.     

Assembly dynamics and detailed structure of 1-propanethiol monolayers on Au(111) surfaces observed real time by in situ STM

1-Propanethiol is chosen as a model alkanethiol to probe detailed mechanisms of the self-assembled monolayer (SAM) formation at aqueous/Au(111) interfaces. The assembly processes, including initial physi- and chemisorption, pit formation, and domain growth, were recorded into movies in real-time with high resolution by in situ scanning tunneling microscopy (STM) under potential control. Two major adsorption steps were disclosed in the propanethiol SAM formation. The first step involves weak interactions accompanied by the lift of the Au(111) surface reconstruction, which depends reversibly on the electrochemical potentials. The second step is chemisorption to form a dense monolayer, accompanied by formation of pits as well as structural changes in the terrace edges. Pits emerged at the stage of the reconstruction lift and increased to a maximum surface coverage of 4.0 +/- 0.4% at the completion of the SAM formation. Well-defined triangular pits in the SAM were found on the large terraces (more than 300 nm wide), whereas few and small pinholes appeared at the terrace edge areas. Smooth edges were converted into saw-like structural features during the SAM formation, primarily along the Au(111) atomic rows. These observations suggest that shrinking and rearrangement of gold atoms are responsible for both formation of the pits and the shape changes of the terrace edges. STM images disclose a (2 square root 3 x 3)R30 degrees periodic lattice within the ordered domains. Along with electrochemical measurements, each lattice unit is assigned to contain four propanethiol molecules exhibiting different electronic contrasts, which might originate in different surface orientations of the adsorbed molecules.     

Fluorescence lifetime imaging microscope consisting of a compact picosecond dye laser and a gated charge-coupled device camera for applications to living cells.

An inverted microscope was combined with a compact dye laser with a pulse width of <190 ps and an intensified charge-coupled device (ICCD) camera with a minimum gate width of 200 ps. The resulting fluorescence lifetime imaging microscope, which has a temporal resolution of 340 ps, was used to measure the fluorescence lifetime of polymer microspherers. The results indicated a fluorescence lifetime of 0.9 ns. The present analytical instrument was also employed in an evaluation of biological cells after labeling them with SYTO 13, a fluorescent dye.     

Super-resolution imaging and real-time tracking lysosome in living cells by a fluorescent probe

Lysosomes function as important organelles within cells and their movement associates with diverse biological events, hence the real-time tracking of lysosomal movement is of great significance. However, since most lysosome fluorescent probes suffer from relatively unsatisfactory photostability, tracking lysosomal movement in real-time remains challenging. Here, we report that a naphthalimide-based fluorescent compound, namely NIMS, is a quite promising probe for lysosome imaging. The visualizing mechanism lies in the selective accumulation of NIMS in lysosomes via a protonation reaction, followed by the fluorescence enhancement due to the interactions of NIMS with proteins. Owing to its high selectivity and good photostability, NIMS was successfully applied to capture super-resolution fluorescence images of lysosomes. More importantly, real-time tracking of lysosome movement in a single living cell by NIMS was realized with a confocal laser scanning microscope. Surprisingly, even in normal culture conditions, around 2/3 of the captured lysosomes were observed to move within 5 min, indicative of the highly dynamic features of lysosomes. Thus, this probe may facilitate the understanding of the lysosome dynamics in physiological or pathological conditions.     

Probing hydroxyl radicals and their imaging in living cells by use of FAM-DNA-Au nanoparticles

The incorporation of gold nanoparticles (Au NPs) as quencher modules in fluorescent probes for DNA damage caused by intracellular hydroxyl radicals (HO*) is reported. Au NPs of 15 nm diameter were decorated with DNA oligomers terminating in thiol functions in their 3' positions and possessing 5' fluorophore modifications. The Au NPs, which have high extinction coefficients, functioned as excellent fluorescent quenchers in the fluorophore-Au NP composites. FRET is switched off as a factor of HO*-induced strand breakage in the single-stranded DNAs, restoring the fluorescence of the quenched fluorophores, which can be followed by spectrofluorimetry. In vitro assays with HO*-generating Fenton reagent demonstrated increases in fluorescence intensity with a linear range from 8.0 nM to 1.0 microM and a detection limit as low as 2.4 nM. Confocal microscopic imaging of macrophages and HepG2 revealed that the probe is cell-permeable and intracellular HO*-responsive. The unique combination of good selectivity and high sensitivity establishes the potential value of the probe for facilitating investigations of HO*-mediated cellular homeostasis and injury.     

A near-IR reversible fluorescent probe modulated by selenium for monitoring peroxynitrite and imaging in living cells

We have developed a near-IR reversible fluorescent probe containing an organoselenium functional group that can be used for the highly sensitive and selective monitoring of peroxynitrite oxidation and reduction events under physiological conditions. The probe effectively avoids the influence of autofluorescence in biological systems and gave positive results when tested in both aqueous solution and living cells. Real-time images of cellular peroxynitrite were successfully acquired.     

Fluorescent chemodosimeter for Cys/Hcy with a large absorption shift and imaging in living cells

A novel molecule T1 with efficient intramolecular charge transfer was designed as a fluorescent chemodosimeter for cysteine (Cys) and homocysteine (Hcy). Upon addition of Cys/Hcy, T1 exhibited greatly enhanced fluorescence intensity as well as a large absorption peak shift (70 nm), and can be used for bioimaging of Cys/Hcy in living cells and detection in human plasma by visual color change. The detection mechanism was proved by (1)H NMR, mass spectrometry analysis and Gaussian calculations.     

Detection of single atoms and buried defects in three dimensions by aberration-corrected electron microscope with 0.5-a information limit

The ability of electron microscopes to analyze all the atoms in individual nanostructures is limited by lens aberrations. However, recent advances in aberration-correcting electron optics have led to greatly enhanced instrument performance and new techniques of electron microscopy. The development of an ultrastable electron microscope with aberration-correcting optics and a monochromated high-brightness source has significantly improved instrument resolution and contrast. In the present work, we report information transfer beyond 50 pm and show images of single gold atoms with a signal-to-noise ratio as large as 10. The instrument's new capabilities were exploited to detect a buried Sigma3 {112} grain boundary and observe the dynamic arrangements of single atoms and atom pairs with sub-angstrom resolution. These results mark an important step toward meeting the challenge of determining the three-dimensional atomic-scale structure of nanomaterials.     

Three rhodamine-based "off-on" chemosensors with high selectivity and sensitivity for Fe3+ imaging in living cells

Three new rhodamine-based probes Y1-Y3 were synthesized as "off-on" chemosensors for Fe(3+) imaging in living cells. The recognizing behaviors were investigated both experimentally and computationally. The crystal structure of the complex Y3-Fe(3+) revealed that Fe(3+) preferred to coordinate with the N atom of benzothiazole moiety rather than the O atom of carboxyl group.     

Fast-response fluorescent probe with favorable water solubility for highly sensitive imaging of endogenous tyrosinase in living cells and zebrafish model

Tyrosinase(TYR)is an important polyphenolic oxidase enzyme and usually regards as a biomarker of melanoma cancer.Highly effective tracking TYR activity in vivo ...     

Fluorescent graphene oxide logic gates for discrimination of iron (3+) and iron (2+) in living cells by imaging

A combinational logic gate based on fluorescent graphene oxide has been reported to discriminate Fe(3+), Fe(2+) and their mixture in living cells by fluorescence imaging.     

Role of structural order and excess energy on ultrafast free charge generation in hybrid polythiophene/Si photovoltaics probed in real time by near-infrared broadband transient absorption

Despite the central role of light absorption and the subsequent generation of free charge carriers in organic and hybrid organic-inorganic photovoltaics, the precise process of this initial photoconversion is still debated. We employ a novel broadband (UV-Vis-NIR) transient absorption spectroscopy setup to probe charge generation and recombination in the thin films of the recently suggested hybrid material combination poly(3-hexylthiophene)/silicon (P3HT/Si) with 40 fs time resolution. Our approach allows for monitoring the time evolution of the relevant transient species under various excitation intensities and excitation wavelengths. Both in regioregular (RR) and regiorandom (RRa) P3HT, we observe an instant (<40 fs) creation of singlet excitons, which subsequently dissociate to form polarons in 140 fs. The quantum yield of polaron formation through dissociation of delocalized excitons is significantly enhanced by adding Si as an electron acceptor, revealing ultrafast electron transfer from P3HT to Si. P3HT/Si films with aggregated RR-P3HT are found to provide free charge carriers in planar as well as in bulk heterojunctions, and losses are due to nongeminate recombination. In contrast for RRa-P3HT/Si, geminate recombination of bound carriers is observed as the dominant loss mechanism. Site-selective excitation by variation of pump wavelength uncovers an energy transfer from P3HT coils to aggregates with a 1/e transfer time of 3 ps and reveals a factor of 2 more efficient polaron formation using aggregated RR-P3HT compared to disordered RRa-P3HT. Therefore, we find that polymer structural order rather than excess energy is the key criterion for free charge generation in hybrid P3HT/Si solar cells.     

Comparison of aqueous molecular dynamics with NMR relaxation and residual dipolar couplings favors internal motion in a mannose oligosaccharide.

An investigation has been performed to assess how aqueous dynamical simulations of flexible molecules can be compared against NMR data. The methodology compares state-of-the-art NMR data (residual dipolar coupling, NOESY, and (13)C relaxation) to molecular dynamics simulations in water over several nanoseconds. In contrast to many previous applications of residual dipolar coupling in structure investigations of biomolecules, the approach described here uses molecular dynamics simulations to provide a dynamic representation of the molecule. A mannose pentasaccharide, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-D-Manp, was chosen as the model compound for this study. The presence of alpha-linked mannan is common to many glycopeptides, and therefore an understanding of the structure and the dynamics of this molecule is of both chemical and biological importance. This paper sets out to address the following questions. (1) Are the single structures which have been used to interpret residual dipolar couplings a useful representation of this molecule? (2) If dynamic flexibility is included in a representation of the molecule, can relaxation and residual dipolar coupling data then be simultaneously satisfied? (3) Do aqueous molecular dynamics simulations provide a reasonable representation of the dynamics present in the molecule and its interaction with water? In summary, two aqueous molecular dynamics simulations, each of 20 ns, were computed. They were started from two distant conformations and both converged to one flexible ensemble. The measured residual dipolar couplings were in agreement with predictions made by averaging the whole ensemble and from a specific single structure selected from the ensemble. However, the inclusion of internal motion was necessary to rationalize the relaxation data. Therefore, it is proposed that although residual dipolar couplings can be interpreted as a single-structure, this may not be a correct interpretation of molecular conformation in light of other experimental data. Second, the methodology described here shows that the ensembles from aqueous molecular dynamics can be effectively tested against experimental data sets. In the simulation, significant conformational motion was observed at each of the linkages, and no evidence for intramolecular hydrogen bonds at either alpha(1-->2) or alpha(1-->3) linkages was found. This is in contrast to simulations of other linkages, such as beta(1-->4), which are often predicted to maintain intramolecular hydrogen bonds and are coincidentally predicted to have less conformational freedom in solution.