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Superfamily I helicases are nonhexameric helicases responsible for the unwinding of nucleic acids. However, whether they unwind DNA in the form of monomers or oligomers remains a controversy. In this study, we addressed this question using direct single-molecule fluorescence visualization of Escherichia coli UvrD, a superfamily I DNA helicase. We performed a photobleaching-step analysis of dye-labeled helicases and determined that the helicase is bound to 18-basepair (bp) double-stranded DNA (dsDNA) with a 3′ single-stranded DNA (ssDNA) tail (12, 20, or 40 nt) in a dimeric or trimeric form in the absence of ATP. We also discovered through simultaneous visualization of association/dissociation of the helicase with/from DNA and the DNA unwinding dynamics of the helicase in the presence of ATP that these dimeric and trimeric forms are responsible for the unwinding of DNA. We can therefore propose a new kinetic scheme for the helicase-DNA interaction in which not only a dimeric helicase but also a trimeric helicase can unwind DNA. This is, to our knowledge, the first direct single-molecule nonhexameric helicase quantification study, and it strongly supports a model in which an oligomer is the active form of the helicase, which carries important implications for the DNA unwinding mechanism of all superfamily I helicases.

N-Methylpyrrole (Py)-N-methylimidazole-(Im) polyamides are small molecules that can recognize specific DNA sequences in the minor groove of B-form DNA with DNA recognition rules. Py favors the T, A, and C bases, excluding G and Im favors G. Lone pair of N3 in Im forms a hydrogen bond with 2 amino hydrogen of guanine. Anti-parallel pairings of Im/Py and Py/Im bind to the G•C and C•G sequence in DNA, respectively. Anti-parallel pairing of Py/Py binds to A•T and T•A degenerately. Aliphatic β-alanine (β) can be substituted for Py. Anti-parallel pairings of Py/β and β/Py bind to A•T and T•A degenerately, and anti-parallel pairings of Im/β and β/Im specify G•C and C•G, respectively. Recently, Py-Im polyamides have been conjugated with fluorophores and some of the fluorophores conjugate Py-Im polyamides could be used for specific DNA detection. In this study, we synthesized two Py-Im polyamide 1 and 2, which interact with the 145 bp DNA containing nucleosome positioning sequence 601. We conjugated cyanine dyes Cy3 or Cy5 with 1 or 2. In the absence of the target DNA, the fluorescent intensity from the fluorescence conjugate Py-Im polyamide diminished their fluorescence, compared with Cy3 or Cy5. In the presence of the target DNA or nucleosome, the fluorescent intensity from the fluorescence conjugate Py-Im polyamides increased. Furthermore, interestingly, FRET between Cy3-Py-Im polyamide and Cy5-Py-Im polyamide on nucleosome was observed. These results provide possibilities that the fluorescent conjugates of Py-Im polyamides can be used for characterization of the dynamic interactions within protein-DNA complexes.

In eukaryotic cells mRNA plays a key role in gene regulation through various processes throughout the cell. Direct observation of endogenous mRNA in living cells promises a significant comprehension of this refined process regulation. Recently a number of fluorescent probes for mRNA have been developed including our fluorescent antisense probe. However, quantitative monitoring of intracellular mRNAs has been difficult. In this study, fluorescence correlation spectroscopy (FCS) was used to quantify the hybridization reaction of fluorescent antisense probes with mRNA. When antisense probes hybridize with the target mRNA, which form large complexes, they will show slower diffusion than unbound probes in a free state. Antisense 2'-O-methyl RNA probes for GAPDH mRNA labeled with Cy3 were microinjected into the cytoplasm of COS7 cells and the fluorescence intensity of the confocal volume was analyzed by FCS. As we expected, probes hybridized with mRNA showed slower diffusion times than those of unbound probes. Two probe fractions having different diffusion times were observed, suggesting that we detected both antisense probe/mRNA-hybrid and unbound probes. The fraction ratios of bound and unbound probes were different among cells, reflecting the different concentration of expressed endogenous GAPDH mRNA ranging from 32.0 nM to 109 nM with an average of 71.7 nM. We then applied this method to the observation of mRNA decay in living cells. Time-lapse quantitative analysis of GAPDH mRNA during siRNA-mediated RNA interference allowed the monitoring of mRNA decay in live cells. These results indicate that our method will be a powerful tool for monitoring gene activity in real time.

Dielectric spectroscopy with microwaves in the frequency range between 0.2 and 20 GHz was used to study the hydration of myosin subfragment 1 (S1). The data were analyzed by a method recently devised, which can resolve the total amount of water restrained by proteins into two components, one with a rotational relaxation frequency (fc) in the gigahertz region (weakly restrained water) and the other with lower fc (strongly restrained water). The weight ratio of total restrained water to S1 protein thus obtained (0.35), equivalent to 2100 water molecules per S1 molecule, is not much different from the values (0.3–0.4) for other proteins. The weakly restrained component accounts for about two-thirds of the total restrained water, which is in accord with the number of water molecules estimated from the solvent-accessible surface area of alkyl groups on the surface of the atomic model of S1. The number of strongly restrained water molecules coincides with the number of solvent-accessible charged or polar atoms. The dynamic behavior of the S1-restrained water during the ATP hydrolysis was also examined in a time-resolved mode. The result indicates that when S1 changes from the S1.ADP state into the S1.ADP.P1 state (ADP release followed by ATP binding and cleavage), about 9% of the weakly restrained waters are released, which are restrained again on slow P1 release. By contrast, there is no net mobilization of strongly restrained component. The observed changes in S1 hydration are quantitatively consistent with the accompanying large entropy and heat capacity changes estimated by calorimetry (Kodama, 1985), indicating that the protein surface hydrophobicity change plays a crucial role in the enthalpy-entropy compensation effects observed in the steps of S1 ATP hydrolysis.

Using a recently developed in vitro motility assay, we have demonstrated that local anesthetics directly inhibit myosin-based movement of single actin filaments in a reversible dose-dependent manner. This is the first reported account of the actions of local anesthetics on purified proteins at the molecular level. In this study, two tertiary amine local anesthetics, lidocaine and tetracaine, were used. The inhibitory action of the local anesthetics on actomyosin sliding movement was pH dependent; the anesthetics were more potent at higher pH values, and this reaction was accompanied by an increased proportion of the uncharged form of the anesthetics. QX-314, a permanently charged derivative of lidocaine, had no effect on actomyosin sliding movement. These results indicate that the uncharged form of local anesthetics is predominantly responsible for the inhibition of actomyosin sliding movement. The local anesthetics inhibited sliding movement but hardly interfered with the binding of actin filaments to myosin on the surface or with actomyosin ATPase activity at low ionic strength. To characterize the actomyosin interaction in the presence of anesthetics, we measured the binding and breaking force of the actomyosin complex. The binding of actin filaments to myosin on the surface was not affected by lidocaine at low ionic strength. The breaking force, measured using optical tweezers, was approximately 1.5 pN per micron of an actin filament, which was much smaller than in rigor and isometric force. The binding and breaking force greatly decreased with increasing ionic strength, indicating that the remaining interaction is ionic in nature. The result suggests that the binding and ATPase of actomyosin are governed predominantly by ionic interaction, which is hardly affected by anesthetics; whereas the force generation requires hydrophobic interaction, which plays a major part of the strong binding and is blocked by anesthetics, in addition to the ionic interaction.

Escherichia coli UvrD protein is a superfamily 1 DNA helicase which plays a crucial role in nucleotide excision repair and methyl-directed mismatch repair. There is a general consensus that the enzyme unwinds a duplex DNA from a 3′ end single-stranded DNA (ssDNA) tail, a gap or a nick. However, conflicting models for the unwinding mechanism have been proposed. Concerning its stoichiometry, some biochemical studies have suggested that the enzyme has optimal activity as an oligomeric form. However, a structural study has indicated that the enzyme functions as a monomer deduced from structural analysis of UvrD-DNA complexes. To address this issue, we attempted to unravel the number of UvrD molecules bound to DNA in the presence and absence of nucleotide by single-molecule fluorescence microscopy. We performed single-molecule visualization of a Cy5-labeled Cys-Ala mutant (Cy5-UvrDC640A), in which Cys52 was labeled with high specificity, bound to 18-bp duplex DNA having a 12, 20 or 40-nt ssDNA tail under several Cy5-UvrDC640A concentrations (0.5, 1.0 and 2.0 nM). We analyzed the number of Cy5 photobleaching steps to quantify the number of UvrD molecules bound to the DNA in the absence and presence of an ATP analog, ATPγS. All the distributions of the number agreed well with the predicted distributions which support the model that UvrD protein is bound to the DNA as an oligomeric form. In the presence of ATP, inefficient DNA unwinding in the absence of free Cy5-UvrDC640A in solution and higher fluorescence intensity of Cy5-UvrDC640A compared to that non-specifically attached on the surface were observed. These results indicate that an oligomer of UvrD is the active form of the helicase.

The temperature of a living cell changes with every cellular event. Thus, measuring intracellular temperature will contribute to the explanation of intricate biological processes and the development of novel diagnoses. Here we demonstrate the first intracellular thermometry with a newly developed fluorescent nanogel thermometer (FNT). The temperature-sensing function of FNT is undertaken by the thermo-responsive polyNIPAM unit combined with a water-sensitive fluorophore. Furthermore, two structural modifications were made taking into consideration its functions in intracellular environments. The first was gelation at a nanometer scale using a crosslinker, allowing the nanogel to remove undesirable responses originating from interactions between cellular components and the fluorophores. The second was the enrichment of ionic sulfate groups on the surface of FNT to avoid precipitation in living cells. In a spectroscopic study, the fluorescence enhancement of FNT with increasing temperature was independent of KCl concentration (100 to 200 mM), environmental pH (4 to 10), or surrounding proteins. Then FNT was microinjected into living COS7 cells, followed by imaging with an epi-fluorescence microscope. The total fluorescence intensity of FNT in single COS7 cells showed the temperature-dependent enhancement upon heating, which provides the calibration curve for intracellular thermometry using FNT. The temperature resolution was evaluated to be 0.29-0.50 °C (27-33 °C). Next, intracellular temperature variations induced by FCCP (mitochondria uncoupler) was investigated. FCCP provoked the fluorescence enhancement of FNT, indicating intracellular heating by 0.45 °C for 30 min. This result suggests that our thermometer FNT successfully detected the intrinsic and significant intracellular temperature change in response to stimulation. In conclusion, novel thermometer FNT is superior to other candidate thermometers in terms of biocompatibility (i.e., size, sensitivity, and solubility) and functional independence (i.e., negligible interactions with cellular components), enabling intracellular temperature measurement in single living cells.

The RecQ protein family, a group of highly conserved DNA helicases including Escherichia coli RecQ, Saccharomyces cerevisiae Sgs1, Shizosaccharomyces Rqh1 and five gene products in humans, plays important roles in maintaining genomic stability. A biochemical analysis showed that E. coli RecQ unwinds various short DNA substrates (19 bp) with a fork containing a gap on the leading strand more efficiently than those with a gap on the lagging strand. A genetic analysis showed that the recQ deletion suppresses the induction of SOS response and the cell filamentation in cells that carry the dnaE486 (a mutation in the DNA polymerase III α-catalytic subunit), causing high proportion of anucleated cells. These previous results suggest that RecQ functions to generate an initiating signal to recruit RecA for SOS induction at stalled replication forks, which are required for the cell cycle checkpoint. On the other hand, a recent report indicates that higher concentration of RecQ is required for unwinding long DNA than for the short DNA substrates as described above, suggesting that RecQ can unwind DNA from not only the end but also the middle of the DNA. In this study, we analyzed RecQ helicase activity using linearized plasmid DNA (2∼3 kb) with and without a fork to investigate DNA unwinding mechanism of RecQ in more detail and would like to discuss how RecQ rescues stalled replication forks.

Protein array is a powerful means to investigate protein-protein interactions. Yet current protein arrays are not versatile due to their low sensitivity (>1ng/ml) and cost-ineffectiveness.In this study, we have developed a sensitive and cost-effective protein array using a commercial fluorescence microscope. The protein array has aligned antibody-immobilized microbeads (5 μm in diameter) inside a polydimethylsiloxane (PDMS) microfluidic chip. The minimum concentration requied for fluorescence detection was determined to be several tenths of pM (about 1 pg/ml) using fluorescently-labeled glutathione-S-transferase (GST) to the protein array having α-GST antibody immobilized microbeads.Firstly, we tried detecting a recombinant protein expressed in cultured cells. We extracted cytoplasmic components of PC12 cell expressing green fluoresent protein (GFP) and labeled them with amino group reactive fluorescent dye. The labeled product was applied to the protein array having α-GFP antibody microbeads, α-β-actin antibody microbeads as positive control and α-IgE antibody microbeads as negative control. Only α-GFP and α-β-actin antibody microbeads were fluorescent, demonstrating that the protein array is capable of detecting a target protein in cytoplasmic extract containing a large number of other proteins.At present, to test its applicability to endogenous proteins, we are trying to detect expression levels of transcription factors, c-Jun and c-Fos, in Hela cell by the method mentioned above with their counterpart antibody microbeads.

Using total internal reflection fluorescence microscopy, we have directly observed individual interactions of single RNA polymerase molecules with a single molecule of lambda-phage DNA suspended in solution by optical traps. The interactions of RNA polymerase molecules were not homogeneous along DNA. They dissociated slowly from the positions of the promoters and sequences common to promoters at a rate of approximately 0.66 s-1, which was more than severalfold smaller than the rate at other positions. The association rate constant for the slow dissociation sites was 9.2 x 10(2) bp-1 M-1 s-1. The frequency of binding to the fast dissociation sites was dependent on the A-T composition; it was larger in the AT-rich regions than in the GC-rich regions. RNA polymerase molecules on the fast dissociation sites underwent linear diffusion (sliding) along DNA. The binding to the slow dissociation sites was greatly enhanced when DNA was released to a relaxed state, suggesting that the binding depended on the strain exerted on the DNA. The present method is potentially applicable to the examination of a wide variety of protein-nucleic acid interactions, especially those involved in the process of transcription.

We have developed a new technique for measurements of piconewton forces and nanometer displacements in the millisecond time range caused by actin-myosin interaction in vitro by manipulating single actin filaments with a glass microneedle. Here, we describe in full the details of this method. Using this method, the elementary events in energy transduction by the actomyosin motor, driven by ATP hydrolysis, were directly recorded from multiple and single molecules. We found that not only the velocity but also the force greatly depended on the orientations of myosin relative to the actin filament axis. Therefore, to avoid the effects of random orientation of myosin and association of myosin with an artificial substrate in the surface motility assay, we measured forces and displacements by myosin molecules correctly oriented in single synthetic myosin rod cofilaments. At a high myosin-to-rod ratio, large force fluctuations were observed when the actin filament interacted in the correct orientation with a cofilament. The noise analysis of the force fluctuations caused by a small number of heads showed that the myosin head generated a force of 5.9 +/- 0.8 pN at peak and 2.1 +/- 0.4 pN on average over the whole ATPase cycle. The rate constants for transitions into (k+) and out of (k-) the force generation state and the duty ratio were 12 +/- 2 s-1, and 22 +/- 4 s-1, and 0.36 +/- 0.07, respectively. The stiffness was 0.14 pN nm-1 head-1 for slow length change (100 Hz), which would be approximately 0.28 pN nm-1 head-1 for rapid length change or in rigor. At a very low myosin-to-rod ratio, distinct actomyosin attachment, force generation (the power stroke), and detachment events were directly detected. At high load, one power stroke generated a force spike with a peak value of 5-6 pN and a duration of 50 ms (k(-)-1), which were compatible with those of individual myosin heads deduced from the force fluctuations. As the load was reduced, the force of the power stroke decreased and the needle displacement increased. At near zero load, the mean size of single displacement spikes, i.e., the unitary steps caused by correctly oriented myosin, which were corrected for the stiffness of the needle-to-myosin linkage and the randomizing effect by the thermal vibration of the needle, was approximately 20 nm.

Bacterial plasmids have evolved segregation machineries to partition replicated DNA to the daughter cells at cell division. P1 phage lysogenizes as a low-copy-number plasmid in Escherichia coli. Its partition system consists of three components, a centromere-like region, parS, an adaptor protein that binds to the centromere, ParB and a partition ATPase, ParA. In general, it is known that a ParB/parS partition complex is formed when ParB oligomerize onto the centromere. This large nucleoprotein complex interacts with ParA and is thought to couple ATP hydrolysis to drive the movement and segregation of plasmids to opposite cell-halves. To understand ATP-driven DNA segregation, we reconstituted the P1 plasmid partition system in a cell-free reaction and visualized the spatiotemporal dynamics using TIRF microscopy. We coated a flow cell surface with non-specific DNA to mimic the bacterial nucleoid surface and flowed in the three-component reaction system. We found that ParA coats the artificial nucleoid creating a reference scaffold for plasmid movement. ParA assembles onto the ParB/parS complexes and anchors them onto the ParA-coated nucleoid surface. ParB stimulates ParA disassembly leading to vigorous Brownian motion of the plasmid as the complex loses bridging interactions with the nucleoid. The plasmid detaches from the nucleoid surface leaving a hole devoid of ParA, which is refilled rapidly with ParA rebinding onto the nucleoid. FRAP experiments demonstrate the dynamic exchange of proteins on the nucleoid surface and the partition complex. We present a Par partition model of ParB-stimulated ParA assembly/disassembly triggering dynamic instability leading to plasmid segregation and movement.

Temperature is a fundamental physical quantity that governs every biological reaction within living cells, and temperature distribution reflects cellular thermodynamics and function. In medical studies, the cellular pathogenesis of diseases (e.g., cancer) is characterized by extraordinary heat production. Therefore, intracellular temperature imaging of living cells should promote better understanding of cellular events and the establishment of novel diagnoses and therapies. However, imaging of temperature distributions in living cells has never been achieved. Here we demonstrate the first intracellular temperature imaging based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy (FLIM). The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.2 °C, respectively. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These findings demonstrate an intrinsic connection between temperature and organelle function. Thus, our intracellular temperature imaging has a significant impact on the comprehension of cell function and will provide insights into the regulatory mechanisms of intracellular signaling.

DNA segregation is an essential process that ensures that every daughter cell inherits a copy of genomic DNA. Many bacterial chromosomes and low-copy-number plasmids such as P1 in Escherichia coli have partitioning systems to separate and transport genomes towards opposite cell-halves before cell division. Only three components are required to partition plasmids: a parS sequence on the plasmid and two proteins, ParA and ParB. It was widely believed that P1 plasmids segregate similar to a mitotic mechanism in eukaryotes. Here, we propose that the partition proteins pattern the bacterial nucleoid using it as a track for plasmid motion. We reconstituted the P1 partition system in a DNA-coated flowcell to mimic an artificial nucleoid, and visualized the dynamics with TIRF microscopy. ParA and ParB coated the DNA surface uniformly. ParB binds specifically to parS site on the plasmid and stimulated the disassembly of ParA, an ATP-dependent DNA-binding protein. This generated a transient depletion zone surrounding the plasmid prior to its motion on the DNA surface. FRAP measurements showed the partition proteins exchanging rapidly on DNA without forming any filamentous structures. Our results support a reaction-diffusion based mechanism, where ParB on the plasmid chases and redistributes ParA patterns on the nucleoid and in turn drives plasmid movement.

In eukaryotic cells mRNA plays a key role in gene regulation. However, the function of mRNA is not fully understood because direct analysis of endogenous mRNAs in living cells has been difficult. We developed a method for the observation of endogenous mRNA in living cells using two fluorescently labeled linear antisense 2′O-methyl RNA oligonucleotides. When those two antisense probes, each is labeled with different fluorescent dyes, are hybridized to an adjacent sequence of the target mRNA, the distance between two fluorophores becomes close and FRET occurs.Here we applied linear antisense probes to the real time monitoring of endogenous mRNA, which will be useful in understanding the function of mRNA as well as the intracellular localization. First, two kinds of linear antisense probes were microinjected into the cytoplasm of living COS7 cells and the FRET signal from cells was recorded over time to examine the kinetics of the hybridization reaction with c-fos mRNA. The hybridization reaction of linear antisense probes proceeded quickly and time constants of linear antisense probe was estimated to be less than one minute. When using Molecular Beacon, the conventional probe for endogenous mRNAs, it took more than one hour to complete the hybridization. Next, the induction of c-fos mRNA in the cytoplasm of COS7 cells was investigated in real time using linear antisense probes. As a result, the elevation of c-fos mRNA expressed in the cytoplasm was observed within one hour after the stimulation with PMA (phorbol 12-myristate 13-acetate). In conclusion, we showed the linear antisense probes are advantageous in monitoring of mRNAs due to their prominent kinetics in hybridizing with target mRNAs in living cells.

Eukaryotic gene expression is regulated by chromatin structures and/or DNA modification such as CpG methylation. The basic unit of eukaryotic chromatin structure is a nucleosome consisting of approximately 150 bp DNA wrapped in 1.7 superhelical turns around a histone octamer. The histone octamer consists of two copies each of H2A, H2B, H3 and H4. Posttranslational histone modifications such as acetylation, methylation, phosphorylation and ubiquitylation regulate chromatin structure, resulting in activation or repression of gene expression. On the other hand, CpG methylation represses gene expression and is essential for silencing of parasitic DNA, genomic imprinting and embryogenesis. During DNA replication, methylated CpGs are converted into hemi-methylated CpGs and newly replicated CpGs should be methylated to inherit methylation pattern. DNA methyltransferase 1 (Dnmt1) is the enzyme to methylate hemi-methylated CpG regions. Uhrf1 is methylated CpG binding protein and interacts with Dnmt1, followed by recruitment of Dnmt1 to hemi-methylated CpG regions. SRA domain of Uhrf1 is responsible for hemi-methylated CpG binding activity. We characterize the process of hemi-methylated CpG recognition by SRA domain using Single-Molecule technique, and in this symposium, we will show our present data.