Red-shifted fluorescent proteins monitor enzymatic activity in live HT-1080 cells with fluorescence lifetime imaging microscopy (FLIM).

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a membrane-tethered collagenase primarily involved in the mechanical destruction of extracellular matrix proteins. MT1-MMP has also been shown to be upregulated in several types of cancers. Many coordinated functions of MT1-MMP during migration and invasion remain to be determined. In this paper, live cells from the invasive cell line HT-1080 were imaged using an intracellular Förster resonance energy transfer-based biosensor specific for MT1-MMP; a substrate specific for MT1-MMP was hybridized with the mOrange2 and mCherry fluorescent proteins to form the Förster resonance energy transfer-based sensor. The configuration of the biosensor was determined with fluorescence lifetime-resolved imaging microscopy using both a polar plot-based analysis and a rapid data acquisition modality of fluorescence lifetime-resolved imaging microscopy known as phase suppression. Both configurations of the biosensor (with or without cleavage by MT1-MMP) were clearly resolvable in the same cell. Changes in the configuration of the MT1-MMP biosensor were observed primarily along the edge of the cell following the removal of the MMP inhibitor GM6001. The intensities highlighted by phase suppression correlated well with the fractional intensities derived from the polar plot.

Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency.

A spectrograph with continuous wavelength resolution has been integrated into a frequency-domain fluorescence lifetime-resolved imaging microscope (FLIM). The spectral information assists in the separation of multiple lifetime components, and helps resolve signal cross-talking that can interfere with an accurate analysis of multiple lifetime processes. This extends the number of different dyes that can be measured simultaneously in a FLIM measurement. Spectrally resolved FLIM (spectral-FLIM) also provides a means to measure more accurately the lifetime of a dim fluorescence component (as low as 2% of the total intensity) in the presence of another fluorescence component with a much higher intensity. A more reliable separation of the donor and acceptor fluorescence signals are possible for Förster resonance energy transfer (FRET) measurements; this allows more accurate determinations of both donor and acceptor lifetimes. By combining the polar plot analysis with spectral-FLIM data, the spectral dispersion of the acceptor signal can be used to derive the donor lifetime - and thereby the FRET efficiency - without iterative fitting. The lifetime relation between the donor and acceptor, in conjunction with spectral dispersion, is also used to separate the FRET pair signals from the donor alone signal. This method can be applied further to quantify the signals from separate FRET pairs, and provide information on the dynamics of the FRET pair between different states.

Image analysis for denoising full-field frequency-domain fluorescence lifetime images.

Video-rate fluorescence lifetime-resolved imaging microscopy (FLIM) is a quantitative imaging technique for measuring dynamic processes in biological specimens. FLIM offers valuable information in addition to simple fluorescence intensity imaging; for instance, the fluorescence lifetime is sensitive to the microenvironment of the fluorophore allowing reliable differentiation between concentration differences and dynamic quenching. Homodyne FLIM is a full-field frequency-domain technique for imaging fluorescence lifetimes at every pixel of a fluorescence image simultaneously. If a single modulation frequency is used, video-rate image acquisition is possible. Homodyne FLIM uses a gain-modulated image intensified charge-coupled device (ICCD) detector, which unfortunately is a major contribution to the noise of the measurement. Here we introduce image analysis for denoising homodyne FLIM data. The denoising routine is fast, improves the extraction of the fluorescence lifetime value(s) and increases the sensitivity and fluorescence lifetime resolving power of the FLIM instrument. The spatial resolution (especially the high spatial frequencies not related to noise) of the FLIM image is preserved, because the denoising routine does not blur or smooth the image. By eliminating the random noise known to be specific to photon noise and from the intensifier amplification, the fidelity of the spatial resolution is improved. The polar plot projection, a rapid FLIM analysis method, is used to demonstrate the effectiveness of the denoising routine with exemplary data from both physical and complex biological samples. We also suggest broader impacts of the image analysis for other fluorescence microscopy techniques (e.g. super-resolution imaging).

Fluorescence lifetime imaging microscopy of Chlamydomonas reinhardtii: non-photochemical quenching mutants and the effect of photosynthetic inhibitors on the slow chlorophyll fluorescence transient.

Fluorescence lifetime-resolved images of chlorophyll fluorescence were acquired at the maximum P-level and during the slower transient (up to 250 s, including P-S-M-T) in the green photosynthetic alga Chlamydomonas reinhardtii. At the P-level, wild type and the violaxanthin-accumulating mutant npq1 show similar fluorescence intensity and fluorescence lifetime-resolved images. The zeaxanthin-accumulating mutant npq2 displays reduced fluorescence intensity at the P-level (about 25-35% less) and corresponding lifetime-resolved frequency domain phase and modulation values compared to wild type/npq1. A two-component analysis of possible lifetime compositions shows that the reduction of the fluorescence intensity can be interpreted as an increase in the fraction of a short lifetime component. This supports the important photoprotection function of zeaxanthin in photosynthetic samples, and is consistent with the notion of a 'dimmer switch'. Similar, but quantitatively different, behaviour was observed in the intensity and fluorescence lifetime-resolved imaging measurements for cells that were treated with the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, the efficient PSI electron acceptor methyl viologen and the protonophore nigericin and. Lower fluorescence intensities and lifetimes were observed for all npq2 mutant samples at the P-level and during the slow fluorescence transient, compared to wild type and the npq1 mutant. The fluorescence lifetime-resolved measurements during the slow fluorescence changes after the P level up to 250 s for the wild type and the two mutants, in the presence and absence of the above inhibitors, were analyzed with a graphical procedure (polar plots) to determine lifetime compositions. At higher illumination intensity, wild type and npq1 cells show a rise in fluorescence intensity and corresponding rise in the species concentration of the slow lifetime component after the initial decrease following the P level. This reversal is absent in the npq2 mutant, and for all samples in the presence of the inhibitors. Lifetime heterogeneities were observed in experiments averaged over multiple cells as well as within single cells, and these were followed over time. Cells in the resting state (induced by several hours of darkness), instead of the normal swimming state, show shortened lifetimes. The above results are discussed in terms of a superposition of effects on electron transfer and protonation rates, on the so-called 'State Transitions', and on non-photochemical quenching. Our data indicate two major populations of chlorophyll a molecules, defined by two 'lifetime pools' centred on slower and faster fluorescence lifetimes.

Single-molecule studies of DNA and RNA four-way junctions.

Branched helical junctions are common in nucleic acids. In DNA, the four-way junction (Holliday junction) is an essential intermediate in homologous recombination and is a highly dynamic structure, capable of stacking conformer transitions and branch migration. Our single-molecule fluorescence studies provide unique insight into the energy landscape of Holliday junctions by visualizing these processes directly. In the hairpin ribozyme, an RNA four-way junction is an important structural element that enhances active-site formation by several orders of magnitude. Our single-molecule studies suggest a plausible mechanism for how the junction achieves this remarkable feat; the structural dynamics of the four-way junction bring about frequent contacts between the loops that are needed to form the active site. The most definitive evidence for this is the observation of three-state folding in single-hairpin ribozymes, the intermediate state of which is populated due to the intrinsic properties of the junction.

Increased stability and lifetime of the complex formed between DNA and meta-phenyl-substituted Hoechst dyes as studied by fluorescence titrations and stopped-flow kinetics.

The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH

Fluorescence energy transfer analysis of DNA structures containing several bulges and their interaction with CAP.

DNA molecules with three bulges separated by double-stranded helical sections of B-DNA were constructed to be used as substrates for DNA-protein binding assays. Fluorescence resonance energy transfer (FRET) between dye molecules attached to the 5'-ends of the DNA molecules is used to monitor the protein binding. The A5 bulge, which consists of five unpaired adenine nucleotides, alters the direction of the helical axis by approximately 80 to 90 at every bulge site. Computer molecular modeling facilitated a pre-selection of suitable helix lengths that bring the labeled ends of the three-bulge DNA molecules (60 to 70 base-pairs long) into close proximity. The FRET experiments verified that the labeled ends of the helices of these long molecules were indeed close. A series of FRET experiments was carried out with two A5 and two A7 bulge molecules. The relative positions of the bulges were varied along the central helical DNA sequence (between the bulges) in order to determine the relative angular juxtapositions of the outlying helical arms flanking the central helical region. The global structural features of the DNA molecules are manifested in the FRET data. The FRET experiments, especially those of the two-bulge series, could be interpreted remarkably well with molecular models based on the NMR structure of the A5 bulge. These models assume that the DNA molecules do not undergo large torsional conformational fluctuations at the bulge sites. The magnitude of the FRET efficiency attests to a relatively rigid structure for many of the long 5'-end-labeled molecules. The changes in the FRET efficiency of three-bulge structures containing the specific binding sequence of the catabolite activator protein (CAP) demonstrated significant deformation of the DNA upon binding of CAP. No direct interaction of CAP with the dyes was observed.

The helix-coil transition of DNA duplexes and hairpins observed by multiple fluorescence parameters.

The thermal denaturation of 8-20-bp DNA duplexes labeled with fluorescein and tetramethylrhodamine at opposing 5'-ends was investigated by monitoring the fluorescence intensity of the dyes, the fluorescence anisotropy of tetramethylrhodamine, the fluorescence resonance energy transfer between fluorescein and rhodamine, and, for the 20-bp duplex, the UV absorption. Melting experiments with the single strands of the duplexes revealed that the single strands can form hairpins stabilized by only a few base pairs. The thermal denaturation curves of the duplexes were fitted well to an extended all-or-none model assuming that only the fully base-paired duplex, the maximally base-paired hairpin, and the random coil conformations are present simultaneously. The extent-of-melting versus temperature curves derived from the different spectroscopic parameters are nearly identical, provided that the analysis of the baselines is carried out correctly; the DeltaH and DeltaS of the dissociation compare well with predictions based on nearest neighbor interaction values available in the literature. Our results imply that for all the oligonucleotides other than the 34-bp oligomer, no partially melted intermediates other than hairpins are present in the reaction mixture in amounts that can be detected by our methods. The melting of the hairpins was also studied directly using single-stranded oligonucleotides. The melting of a 34-bp duplex can be accounted for by a statistical zipper model.

Reversible stalling of transcription elongation complexes by high pressure.

We have investigated the effect of high hydrostatic pressure on the stability of RNA polymerase molecules during transcription. RNA polymerase molecules participating in stalled or active ternary transcribing complexes do not dissociate from the template DNA and nascent RNA at pressures up to 180 MPa. A lower limit for the free energy of stabilization of an elongating ternary complex relative to the quaternary structure of the free RNAP molecules is estimated to be 20 kcal/mol. The rate of elongation decreases at high pressure; transcription completely halts at sufficiently high pressure. The overall rate of elongation has an apparent activation volume (DeltaVdouble dagger) of 55-65 ml . mol-1 (at 35 degrees C). The pressure-stalled transcripts are stable and resume elongation at the prepressure rate upon decompression. The efficiency of termination decreases at the rho-independent terminator tR2 after the transcription reaction has been exposed to high pressure. This suggests that high pressure modifies the ternary complex such that termination is affected in a manner different from that of elongation. The solvent and temperature dependence of the pressure-induced inhibition show evidence for major conformational changes in the core polymerase enzyme during RNA synthesis. It is proposed that the inhibition of the elongation phase of the transcription reaction at elevated pressures is related to a reduction of the partial specific volume of the RNA polymerase molecule; under high pressure, the RNA polymerase molecule does not have the necessary structural flexibility required for the protein to translocate.

Global structure of three-way DNA junctions with and without additional unpaired bases: a fluorescence resonance energy transfer analysis.

The structure of three-way DNA junctions with and without extrahelical adenine nucleotides in one strand at the branch point of the junction (i.e., An bulges with n = 0, 1, 2, and 3) has been investigated by fluorescence resonance energy transfer. The structure of the junction without bulged nucleotides was found to have a symmetric trigonal geometry. With bulges, the arrangement of the arms becomes asymmetrical. The energy transfer results suggest a model of bulged junctions where the angle between two of the arms is significantly smaller than between the other two pairs of arms. The acute angle becomes smaller as the number of nucleotides in the bulge increases. The FRET efficiencies of the junctions are the same in the presence of Mg++ and Na+ ions.

Effect of additional unpaired bases on the stability of three-way DNA junctions studied by fluorescence techniques.

Fluorescence melting experiments were carried out to determine the relative stability of three-way DNA junctions with and without extrahelical adenine nucleotides in one strand at the branch point of the junction (i.e., An bulges where n = 0, 1, 2, and 3). The oligonucleotides were labeled with chromophores at the 5' ends of the strands. The progress of the thermal denaturation was followed by monitoring the fluorescence intensities and anisotropies of the dyes and the fluorescence resonance energy transfer between the two dyes. The results of the thermal denaturation experiments are interpreted and discussed in terms of either two-state thermodynamic models or statistical models for the thermal denaturation. The junctions all melt at the same temperature (at equal concentrations) within the error of the Tm determination, regardless of the presence, or absence, of the bulge. It is suggested that the denaturation of the helical arms begins primarily at the free ends of the helical arms and proceeds toward the branch point. The junctions, all which have 10 base pairs in each arm, possess thermal denaturation characteristics similar to duplexes with 20 arms. This leads to the proposition that for these junctions an important molecular parameter that controls the stability of the junctions is the number of base pairs between neighboring arms. The melting profiles obtained by monitoring the tetramethylrhodamine fluorescence are found to depend strongly on the nucleotide sequence in the single-stranded region.

Ion-induced folding of the hammerhead ribozyme: a fluorescence resonance energy transfer study.

The ion-induced folding transitions of the hammerhead ribozyme have been analysed by fluorescence resonance energy transfer. The hammerhead ribozyme may be regarded as a special example of a three-way RNA junction, the global structure of which has been studied by comparing the distances (as energy transfer efficiencies) between the ends of pairs of labelled arms for the three possible end-to-end vectors as a function of magnesium ion concentration. The data support two sequential ion-dependent transitions, which can be interpreted in the light of the crystal structures of the hammerhead ribozyme. The first transition corresponds to the formation of a coaxial stacking between helices II and III; the data can be fully explained by a model in which the transition is induced by a single magnesium ion which binds with an apparent association constant of 8000-10 000 M-1. The second structural transition corresponds to the formation of the catalytic domain of the ribozyme, induced by a single magnesium ion with an apparent association constant of approximately 1100 M-1. The hammerhead ribozyme provides a well-defined example of ion-dependent folding in RNA.

Nucleic acid structure and recognition.

We review the global structures adopted by branched nucleic acids, including three- and four-way helical junctions in DNA and RNA. We find that some general folding principles emerge. First, all the structures exhibit a tendency to undergo pairwise coaxial helical stacking when permitted by the local stereochemistry of strand exchange. Second, metal ions generally play an important role in facilitating folding of branched nucleic acids. These principles can be applied to functionally important branched nucleic acids, such as the Holliday DNA junction of genetic recombination, and the hammerhead ribozyme in RNA.

Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes.

Fluorescence steady-state and lifetime experiments have been carried out on duplex and single-stranded DNA molecules labeled at the 5' ends with 5-carboxytetramethylrhodamine (TMRh). The temperature and ionic strength of the solutions were varied over large ranges. The results reveal at least three well-defined states of the TMRh-DNA molecules for the single-stranded as well as for the double-stranded DNA molecules. Two states are fluorescent, with lifetimes in the range of 0.5-1 ns and 2.5-3 ns. A third state of TMRh-DNA does not fluoresce (a dark species of TMRh-DNA). The distribution of the TMRh-DNA molecules among these three states is strongly temperature and ionic strength dependent. Estimates are made of some reaction parameters of the multistate model. The results are discussed in terms of the photophysics of TMRh, and consequences of the multiple conformers of TMRh-DNA for studies involving fluorescence studies with TMRh-labeled DNA are considered.

Heterogeneity of E. coli RNA polymerase revealed by high pressure.

The activity and subunit association of Escherichia coli RNA polymerase has been investigated by high pressure techniques (up to 2000 atm). The extent of subunit dissociation in the presence and absence of DNA was monitored by carrying out electrophoresis directly at elevated pressure. The degree of inactivation brought about by high pressure was determined by measuring the enzyme activity following decompression. The loss of activity if the enzyme molecules are not actively involved in transcription is correlated with the extent of association of the polymerase subunits. At any particular pressure only a fraction of polymerase molecules becomes inactivated; the remaining fraction retains its original activity characteristics. If the enzyme molecules are actively involved in transcription when the high pressure is applied, the RNA synthesis can be completely halted, but the elongation activity is fully recovered on decompression. The experimental results are consistent with the existence of a broad distribution of protein conformers that are differentially sensitive to the level of pressure. RNA polymerase molecules that display similar catalytic properties have large differences in their free energy of subunit association in the absence of substrates.

Inactivation of simian immunodeficiency virus by hydrostatic pressure.

The inactivation of the simian immunodeficiency viruses SIVmac251 and SIVagm by pressures of 150 and 250 MPa was determined. The extent of inactivation depended on the time that the virus was subjected to compression as well as the level of the pressure and at 150 Mpa reached 5 log10 dilution units after approximately 10 hr. The inactivations, which were uniformly carried out at room temperature, were independent of the concentration of the virus. Possible applications of pressure inactivation for molecular biological and clinical use are discussed.