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A recent paper1xArai, Y. et al. Nature. 1999; 399: 446–448Crossref | PubMed | Scopus (184)See all References1 took manipulation of molecules with optical tweezers to a new level by showing that it is possible to tie knots in both actin filaments and DNA molecules (Fig. 1Fig. 1). Arai et al. attached polystyrene beads to the polymers, then used optical tweezers to manipulate these beads and hence the polymers themselves. In addition to tying knots, they recorded the tensile strength and flexibility of both the actin filaments and DNA molecules. As well as being an entertaining exercise, this approach has further practical applications. Tying a knot in a linear polymer generates a stable curvature that could be used as a basis for experimentation on, for example, the binding of associated proteins to curved versus straight polymer. Conceivably, knotting of such molecules could also be used to perturb cellular structure and observe the resulting effects.Fig. 1Tying a knot in a DNA molecule. A DNA molecule stained with POPO-3 was attached to two beads, and then movement of the beads using optical tweezers was used to generate various types of conformation, including knots, in the DNA molecule. Images 3–7 are accompanied by explanatory drawings depicting the conformation shown. The paper1xArai, Y. et al. Nature. 1999; 399: 446–448Crossref | PubMed | Scopus (184)See all References1 contains a similar set of images for actin, and other types of knot in DNA molecules. Bar, 10 μm.View Large Image | Download PowerPoint Slide

We used video-fluorescence microscopy to directly observe the sliding movement of single fluorescently labeled actin filaments along myosin fixed on a glass surface. Single actin filaments labeled with phalloidin-tetramethyl-rhodamine, which stabilizes the filament structure of actin, could be seen very clearly and continuously for at least 60 min in 02-free solution, and the sensitivity was high enough to see very short actin filaments less than 40 nm long that contained less than eight dye molecules. The actin filaments were observed to move along double-headed and, similarly, single-headed myosin filaments on which the density of the heads varied widely in the presence of ATP, showing that the cooperative interaction between the two heads of the myosin molecule is not essential to produce the sliding movement. The velocity of actin filament independent of filament length (greater than 1 micron) was almost unchanged until the density of myosin heads along the thick filament was decreased from six heads/14.3 nm to 1 head/34 nm. This result suggests that five to ten heads are sufficient to support the maximum sliding velocity of actin filaments (5 micron/s) under unloaded conditions. In order for five to ten myosin heads to achieve the observed maximum velocity, the sliding distance of actin filaments during one ATP cycle must be more than 60 nm.

Fusion proteins of a truncated mutant of myosin subfragment-1 (S1dC) and green fluorescent protein (GFP) were expressed in vitro by T7 RNA polymerase and rabbit reticulocyte lysate. Single S1dC-GFP fusion proteins were clearly seen and their individual ATP turnovers were directly monitored using low background total internal reflection fluorescence microscopy (LBTIRFM), recently developed by our laboratory. LBTIRFM using GFP as a fluorescent tag allowed us to assay functions of single protein molecules expressed in vitro. Thus, the results suggested that this method may be particularly useful to analyze functions of proteins that cannot be produced in an active form and/or in large quantities in conventional heterologous expression systems.

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.

The myosin molecule consists of two heads, each of which contains an enzymatic active site and an actin-binding site. The fundamental problem of whether the two heads function independently or cooperatively during muscle contraction has been studied by methods using an actomyosin thread, superprecipitation and chemical modification of muscle fibres. No clear conclusion has yet been reached. We have approached this question using an assay system in which sliding movements of fluorescently labelled single actin filaments along myosin filaments can be observed directly. Here, we report direct measurement of the sliding of single actin filaments along one-headed myosin filaments in which the density of heads was varied over a wide range. Our results show that cooperative interaction between the two heads of myosin is not essential for inducing the sliding movement of actin filaments.

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.