Search

We evaluated the effect of high hydrostatic pressure on mouse embryonic fibroblasts (MEFs) and mouse embryonic stem (ES) cells. Hydrostatic pressures of 15, 30, 60, and 90 MPa were applied for 10 min, and changes in gene expression were evaluated. Among genes related to mechanical stimuli, death-associated protein 3 was upregulated in MEF subjected to 90 MPa pressure; however, other genes known to be upregulated by mechanical stimuli did not change significantly. Genes related to cell differentiation did not show a large change in expression. On the other hand, genes related to pluripotency, such as Oct4 and Sox2, showed a twofold increase in expression upon application of 60 MPa hydrostatic pressure for 10 min. Although these changes did not persist after overnight culture, cells that were pressurized to 15 MPa showed an increase in pluripotency genes after overnight culture. When mouse ES cells were pressurized, they also showed an increase in the expression of pluripotency genes. These results show that hydrostatic pressure activates pluripotency genes in mammalian cells. This article has an associated First Person interview with the first author of the paper., Summary: Application of high hydrostatic pressure on somatic cells induce changes in gene expression including upregulation in pluripotency genes.

Fluorescent nanodiamonds (FNDs) have been attracting much attention as promising therapeutic agents and probes for bioimaging and nanosensing. For their biological applications, several hydrophilizing methods to enhance FND colloidal stability have been developed to suppress their aggregation and the nonspecific adsorption to biomolecules in complex biomedical environments. However, these methods involve several complicated synthetic and purification steps, which prohibit the use of FNDs for bioapplications by biologists. In this study, we describe a simple one-pot FND hydrophilization method that comprises coating of the surface of the nanoparticles with COOH-terminated hyperbranched polyglycerol (HPG-COOH). HPG-COOH-coated FNDs (FND-HPG-COOHs) were found to exhibit excellent dispersibility under physiological conditions despite the thinness of the 5 nm HPG-COOH layer. Biotinylated FND-HPG-COOHs specifically captured avidin molecules in the absence of nonspecific protein adsorption. Moreover, we demonstrated that FND-HPG-COOHs conjugated with antibodies can be used to selectively target integrins in fixed HeLa cells. In addition, intracellular temperature changes were measured via optically detected magnetic resonance using FND-HPG-COOHs conjugated with mitochondrial localization signal peptides. Our one-pot synthetic method will encourage the broad use of FNDs among molecular and cellular biologists and pave the way for extensive biological and biomedical applications of FNDs.

For the development of artificial cell-like machinery, liposomes encapsulating cytoskeletons have drawn much recent attention. However, there has been no report showing isothermally reversible morphological changes of liposomes containing cytoskeletons. We succeeded in reversibly changing the shape of cell-sized giant vesicles by controlling the polymerization/depolymerization state of cytoskeletal microtubules that were encapsulated in the vesicles using pressure changes. The result indicates that it is possible to manipulate artificial cell models composed of molecules such as lipids and proteins. The findings obtained in this study will be helpful in clarifying the details of cooperation between cytoskeletal dynamics and morphogenesis of biological membranes and in improving the design and construction of further advanced artificial cell-like machinery, such as drug-delivery systems. In addition, the experimental system used in this study can be applied to research to elucidate the adaptive strategy of living organisms to external stimuli and extreme conditions such as osmotic stress and high-pressure environments like the deep sea.

Liposomes encapsulating cytoskeletons have drawn much recent attention to develop an artificial cell-like chemical-machinery; however, as far as we know, there has been no report showing isothermally reversible morphological changes of liposomes containing cytoskeletons because the sets of various regulatory factors, that is, their interacting proteins, are required to control the state of every reaction system of cytoskeletons. Here we focused on hydrostatic pressure to control the polymerization state of microtubules (MTs) within cell-sized giant liposomes (diameters ∼10 μm). MT is the cytoskeleton formed by the polymerization of tubulin, and cytoskeletal systems consisting of MTs are very dynamic and play many important roles in living cells, such as the morphogenesis of nerve cells and formation of the spindle apparatus during mitosis. Using real-time imaging with a high-pressure microscope, we examined the effects of hydrostatic pressure on the morphology of tubulin-encapsulating giant liposomes. At ambient pressure (0.1 MPa), many liposomes formed protrusions due to tubulin polymerization within them. When high pressure (60 MPa) was applied, the protrusions shrank within several tens of seconds. This process was repeatedly inducible (around three times), and after the pressure was released, the protrusions regenerated within several minutes. These deformation rates of the liposomes are close to the velocities of migrating or shape-changing living cells rather than the shortening and elongation rates of the single MTs, which have been previously measured. These results demonstrate that the elongation and shortening of protrusions of giant liposomes is repeatedly controllable by regulating the polymerization state of MTs within them by applying and releasing hydrostatic pressure., 細胞サイズの人工膜小胞を可逆的に繰り返し変形させられることに成功 (生体運動マシナリーを使った分子ロボット構築の可能性を実証). 京都大学プレスリリース. 2016-04-19.

Two cytochromes c5 (SBcytc and SVcytc) have been derived from Shewanella living in the deep-sea, which is a high pressure environment, so it could be that these proteins are more stable at high pressure than at atmospheric pressure, 0.1 MPa. This study, however, revealed that SBcytc and SVcytc were more stable at 0.1 MPa than at higher pressure. In addition, at 0.1–150 MPa, the stability of SBcytc and SVcytc was higher than that of homologues from atmospheric-pressure Shewanella, which was due to hydrogen bond formation with the heme in the former two proteins. This study further revealed that cytochrome c551 (PMcytc) of deep-sea Pseudomonas was more stable than a homologue of atmospheric-pressure Pseudomonas aeruginosa, and that specific hydrogen bond formation with the heme also occurred in the former. Although SBcytc and SVcytc, and PMcytc are phylogenetically very distant, these deep-sea cytochromes c are commonly stabilized through hydrogen bond formation. Deep-sea cytochromes c are commonly stabilized through hydrogen bond formation.

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.

Adenosine 5'-triphosphate (ATP) is the major energy currency of all living organisms. Despite its important functions, the spatiotemporal dynamics of ATP levels inside living multicellular organisms is unclear. In this study, we modified the genetically encoded Förster resonance energy transfer (FRET)-based ATP biosensor ATeam to optimize its affinity at low temperatures. This new biosensor, AT1.03NL, detected ATP changes inside Drosophila S2 cells more sensitively than the original biosensor did, at 25 °C. By expressing AT1.03NL in Drosophila melanogaster and Caenorhabditis elegans, we succeeded in imaging the in vivo ATP dynamics of these model animals at single-cell resolution.

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 organic molecules that bind to DNA with sequence specificity and can be used as synthetic DNA-binding ligands. In this study, five hairpin eight-ring Py-Im polyamides 1-5 with different number of Im rings were synthesized, and their binding behaviour was investigated with surface plasmon resonance assay. It was found that association rate (k(a)) of the Py-Im polyamides with their target DNA decreased with the number of Im in the Py-Im polyamides. The structures of four-ring Py-Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller. As the minor groove of DNA has a helical structure, planar Py-Im polyamides need to change their conformation to fit it upon binding to the minor groove. The data explain that an increase in planarity of Py-Im polyamide induced by the incorporation of Im reduces the association rate of Py-Im polyamides. This fundamental knowledge of the binding of Py-Im polyamides to DNA will facilitate the design of hairpin Py-Im polyamides as synthetic DNA-binding modules.

The Escherichia coli RuvB hexameric ring motor proteins, together with RuvAs, promote branch migration of Holliday junction DNA. Zero mode waveguides (ZMWs) constitute of nanosized holes and enable the visualization of a single fluorescent molecule under micromolar order of the molecules, which is applicable to characterize the formation of RuvA–RuvB–Holliday junction DNA complex. In this study, we used ZMWs and counted the number of RuvBs binding to RuvA–Holliday junction DNA complex. Our data demonstrated that different nucleotide analogs increased the amount of Cy5-RuvBs binding to RuvA–Holliday junction DNA complex in the following order: no nucleotide, ADP, ATPγS and mixture of ADP and ATPγS. These results suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA–RuvB–Holliday junction DNA complex formation.

Intracellular transport of neurotrophin receptors together with neurotrophins is one of the key events of neurotrophin signaling for the growth and the survival of neurons. However, the involvement of neurotrophin signaling in the regulation of intracellular transport of neurotrophin receptors has been remained unclear. We visualized the behavior of TrkA, a receptor of nerve growth factor (NGF), by labeling with GFP in PC12 cells. We found remarkable changes of the behavior of TrkA-GFP upon the application of NGF. Before the application, only ∼37% of the fluorescent dots of TrkA showed translocations along neurites of PC12 cells. After the application, number of the dots showing the directional movement increased to ∼65%. The averaged velocities of the directional movement of TrkA-GFP dots became higher after the application of NGF. We tested the idea whether NGF binding accelerated the translocations of TrkA by simultaneously observing TrkA-GFP and fluorescently labeled NGF, Cy3.5-NGF. The velocity of TrkA-GFP dots associated with Cy3.5-NGF was remarkably higher than that of TrkA-GFP dots without Cy3.5-NGF. On the basis of these observations, we hypothesize that there is a signaling mechanism within a single vesicle that facilitates the intracellular transport of each vesicle containing the activated TrkA. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 634–649, 2011

The motor protein kinesin has two heads and walks along microtubules processively using energy derived from ATP. However, how kinesin heads are coordinated to generate processive movement remains elusive. Here we created a hybrid nanomachine (DNA-kinesin) using DNA as the skeletal structure and kinesin as the functional module. Single molecule imaging of DNA-kinesin hybrid allowed us to evaluate the effects of both connect position of the heads (N, C-terminal or Mid position) and sub-nanometer changes in the distance between the two heads on motility. Our results show that although the native structure of kinesin is not essential for processive movement, it is the most efficient. Furthermore, forward bias by the power stroke of the neck linker, a 13-amino-acid chain positioned at the C-terminus of the head, and internal strain applied to the rear of the head through the neck linker are crucial for the processive movement. Results also show that the internal strain coordinates both heads to prevent simultaneous detachment from the microtubules. Thus, the inter-head coordination through the neck linker facilitates long-distance walking.

Elucidating the dynamic organization of nuclear RNA foci is important for understanding and manipulating these functional sites of gene expression in both physiological and pathological states. However, such studies have been difficult to establish in vivo as a result of the absence of suitable RNA imaging methods. Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks. Upon in vivo electroporation, exciton-controlled sequence-specific oligonucleotide probes revealed focally concentrated endogenous 28S rRNA and U3 snoRNA at nucleoli and poly(A) RNA at nuclear speckles. Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue. Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior.

The Escherichia coli RuvA–RuvB complex promotes branch migration of Holliday junction DNA, which is the central intermediate of homologous recombination. Like many DNA motor proteins, it is suggested that RuvA–RuvB promotes branch migration by driving helical rotation of the DNA. To clarify the RuvA–RuvB-mediated branch migration mechanism in more detail, we observed DNA rotation during Holliday junction branch migration by attaching a bead to one end of cruciform DNA that was fixed to a glass surface at the opposite end. Bead rotation was observed when RuvA, RuvB, and ATP were added to the solution. We measured the rotational rates of the beads caused by RuvA–RuvB-mediated branch migration at various ATP concentrations. The data provided a K m value of 65 μM and a V max value of 1.6 revolutions per second, which corresponds to 8.3 bp per second. This real-time observation of the DNA rotation not only allows us to measure the kinetics of the RuvA–RuvB-mediated branch migration, but also opens the possibility of elucidating the branch migration mechanism in detail.

RuvB protein forms two hexameric rings that bind to the RuvA tetramer at DNA Holliday junctions. The RuvAB complex utilizes the energy of ATP hydrolysis to promote branch migration of Holliday junctions. The crystal structure of RuvB from Thermus thermophilus (Tth) HB8 showed that each RuvB monomer has three domains (N, M, and C). This study is a structure-function analysis of the three domains of RuvB. The results show that domain N is involved in RuvA-RuvB and RuvB-RuvB subunit interactions, domains N and M are required for ATP hydrolysis and ATP binding-induced hexamer formation, and domain C plays an essential role in DNA binding. The side chain of Arg-318 is essential for DNA binding and may directly interact with DNA. The data also provide evidence that coordinated ATP-dependent interactions between domains N, M, and C play an essential role during formation of the RuvAB Holliday junction ternary complex.

The behavior of single molecules of neurotrophins on growth cones was observed by the use of the fluorescent conjugate of nerve growth factor (NGF), Cy3-NGF. After the application of 0.4 nmCy3-NGF, chick dorsal root ganglion growth cones responded within 1 min of adding the stimulus by expanding their lamellipodia. Only 40 molecules of Cy3-NGF, which occupied 2s-1. The behavior of Cy3-NGF was shifted to a one-directional rearward movement toward the central region of the growth cone. The one-directional movement of Cy3-NGF displayed the same rate as the rearward flow of actin, ∼4 μm/min. This movement could be stopped by the application of the potent inhibitor of actin polymerization, latrunculin B. Molecules of Cy3-NGF were suggested to be internalized in the vicinity of the central region of the growth cone during this rearward trafficking, because Cy3-NGF remained in the growth cone after the growth cones had been exposed to an acidic surrounding medium: acidic medium causes the complete dissociation of Cy3-NGF from the receptors on the surface of growth cones. These results suggested that actin-driven trafficking of the NGF receptor complex is an essential step for the accumulation and endocytosis of NGF at the growth cone and for the retrograde transport of NGF toward the cell body.

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)–Ras–extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction.

Protein secretion, a key intercellular event for transducing cellular signals, is thought to be strictly regulated. However, secretion dynamics at the single-cell level have not yet been clarified because intercellular heterogeneity results in an averaging response from the bulk cell population. To address this issue, we developed a novel assay platform for real-time imaging of protein secretion at single-cell resolution by a sandwich immunoassay monitored by total internal reflection microscopy in sub-nanolitre-sized microwell arrays. Real-time secretion imaging on the platform at 1-min time intervals allowed successful detection of the heterogeneous onset time of nonclassical IL-1β secretion from monocytes after external stimulation. The platform also helped in elucidating the chronological relationship between loss of membrane integrity and IL-1β secretion. The study results indicate that this unique monitoring platform will serve as a new and powerful tool for analysing protein secretion dynamics with simultaneous monitoring of intracellular events by live-cell imaging.

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.

Orientation dependence of single-fluorophore intensity was exploited in order to videotape conformational changes in a protein machine in real time. The fluorophore Cy3 attached to the central subunit of F 1 -ATPase revealed that the subunit rotates in the molecule in discrete 120° steps and that each step is driven by the hydrolysis of one ATP molecule. These results, unlike those from the previous study under a frictional load, show that the 120° stepping is a genuine property of this molecular motor. The data also show that the rate of ATP binding is insensitive to the load exerted on the rotor subunit.

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.

Visualization of single actin filaments by fluorescence microscopy led to the development of new in vitro assays for analysing actomyosin-based motility at the molecular level. The ability to manipulate actin filaments with a microneedle or an optical trap combined with position-sensitive detectors has enabled direct measurements of nanometre displacements and piconewton forces exerted by individual myosin molecules. To elucidate how myosin generates movement, it is necessary to understand how ATP hydrolysis is coupled to mechanical work at the level of the single molecule. But the most sensitive microscopic ATPase assay available still requires over 1,000 myosins. To enhance the sensitivity of such assays, we have refined epifluorescence and total internal reflection microscopies to visualize single fluorescent dye molecules. We report here that this approach can be used directly to image single fluorescently labelled myosin molecules and detect individual ATP turnover reactions. In contrast to previously reported single fluorescent molecule imaging methods, which used specimens immobilized on an air-dried surface, our method allows video-rate imaging of single molecules in aqueous solution, and hence can be applied to the study of many types of enzymes and biomolecules.

Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIα, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.51 ± 0.33 µm (11 ± 6 turns) of a braid was unlinked in a burst of reactions taking 8 ± 4 s, the unlinked length being essentially independent of the enzyme concentration between 0.25-37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being ~100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of ~10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break ~10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments.

Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5 pN nm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

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.

Chemical modification of the bovine heart cytochrome bc 1 complex with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) has been reported to inhibit the proton pumping activity without affecting the rate of electron transfer to ferricytochrome c. This study aims to examine the effect of EEDQ on energy-linked reversed electron transfer in the bc 1 complex reconstituted into potassium-loaded phospholipid vesicles. Generation of a valinomycin-mediated potassium-diffusion potential induced the reduction of cytochrome b in the reconstituted bc 1 complex in the presence of sodium ascorbate. The time course of the cytochrome b reduction was well correlated with that of the absorbance change of safranine, an optical probe for measuring membrane potential. Treatment of the bc 1 complex with EEDQ caused a decrease in the potential-induced reduction of cytochrome b as well as in the proton translocation activity. But a significant loss in the ubiquinol-cytochrome c reducing activity was not observed in the EEDQ-treated bc 1 complex. The time- and concentration-dependent effect of EEDQ on the reversed electron transfer was well correlated with that of the proton translocation activity of the bc 1 complex. These findings strongly support the idea that the potential-induced reversal of electron transfer is coupled to the reverse flow of protons in the cytochrome bc 1 complex.

The development of DNA microarray technology has enabled the successful probing of complex transcriptional networks on a genome-wide scale (Rhodes et al. 2002; Beltran et al. 2003; Zheng et al. 2004). In addition, various types of microarrays for monitoring molecular interactions have been developed (de Wildt et al. 2000; Zhu et al. 2001; MacBeath 2002; Wang et al. 2003). However, one critical concern with these conventional microarrays is their limitation to static analysis of molecular interactions. Because conventional microarray systems detect probe binding by quantitative end-point measurements after removal of unbound targets, they are not suited to either kinematic analysis of molecular interactions or to the analysis of interactions with high dissociation rates. A simple approach to address this concern is to monitor the molecular interactions in real-time. Another concern is the analysis of molecular interactions using very small samples. Proteins and/or nucleic acids from a selected subpopulation of cells or even from a single cell can be extracted using new technologies such as laser capture microdissection (Paweletz et al. 2001), microfabricated fluorescence-activated cell sorting (Wang et al. 2005), and other microfluidic systems (Thorsen et al. 2002; Hong et al. 2004). More importantly, recent progress in bioimaging (Sako et al. 2000; Itoh et al. 2002; Murakoshi et al. 2004; Michalet et al. 2005), allowing the study of the dynamic behavior of various proteins in living cells, has increased the need to bridge the gap between bioimaging and biochemistry at the single-cell level. Although DNA microarray technology can achieve mRNA profiling at a single-cell level by artificially amplifying target molecules by in vitro enzymatic reactions, such an approach is not robust and, more seriously, cannot be applied to proteins. A simple approach to address this is to miniaturize the molecular detection platform to the size of a single animal cell. While real-time monitoring of binding processes and extreme miniaturization of the analysis platform are independent issues, it was our intention to address both of these issues for application to single-cell biology. Here we describe the development of an array-based real-time system using fluorescence imaging technologies for analyzing molecular interactions with extremely small amounts of target molecules. This analysis platform will make it practical to analyze multiple molecular interactions in parallel using subnanoliter samples.

Binding modes of helicase to various DNA substrates were visualized by single-molecule fluorescence imaging on silicate coverslips coated with poly(ethylene glycol) (PEG) which minimizes the backgr...

Publisher Summary This chapter discusses the single molecule imaging and nanomanipulation of biomolecules. The methods described in the chapter are applied to examining single nucleotidase reactions of other enzymes. It is possible to suspend a single DNA in solution by manipulation with dual optical traps, as well as to see single fluorescently labeled RNA polymerases. Directly observing the reading process of the DNA genetic information by a single RNA polymerase molecule is not just a dream now, but realistic. The techniques for single molecule imaging and manipulation is very powerful for studies not only of motility of motor proteins, but also of molecular genetics, signal transduction and processing in cell, dynamic molecular process of proteins.

Real time imaging of mRNA in living cell is powerful tool in investigating the role of mRNA. Here we developed the method for visualizing specific mRNAs in living cell. The proposed method contains hybridization of antisense nucleic acids with mRNA transcripted inside cell. As antisense nucleic acid, 2'O-methyl RNA and Peptide Nucleic Acid (PNA) were chosen because these artificial nucleic acids are nuclease resistant and have high affinity to mRNA. Two antisense probes labelled with different fluorescent dyes were designed and prepared. When two antisense probes were hybridized to target human c-fos mRNA, two fluorescent dyes became very close and fluorescence resonance energy transfer (FRET) occurred, resulting in changes in fluorescence spectra. Such FRET signals were detected when 2'O-methyl RNA and Peptide Nucleic Acid were used as antisense probes. The biggest signal was detected when 2'O-methyl RNA as donor probe and PNA as acceptor probe were used. The timecourse study indicated two antisense probes can hybridize fast enough to subject to real time imaging.

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.

To understand the underlying mechanism of force generation by myosin motor, it is crucial to know which part of the molecule is essential for the process. Recent structure determination of myosin motor domain at atomic resolution has revealed that the domain comprises two smaller domains, the "ATPase domain" consisting of only an N-terminal segment of the heavy chain and the "neck domain" consisting of a long α-helix of the heavy chain and two light chains. This atomic structure begs the question of whether both domains are required for force generation. To answer it, we genetically truncated the head to generate a recombinant fragment composed of the "ATPase domain" alone. The truncated head drove sliding movement of actin filaments and generated force in a novel in vitro assay system, which allows us to hold a specific site of the head on a glass surface. These results indicate that the compact ATPase domain functions as a force-generating machinery of the myosin motor.

We have developed a high resolution force measurement system in vitro by manipulating a single actin filament attached to a microneedle. The system could resolve forces less than a piconewton, and has time resolution in the submillisecond range. We have used this system to detect force fluctuations produced by individual molecular interactions. We observed large force fluctuations during isometric force generations. Noise analysis of the force fluctuations showed that the force was produced by stochastic and independent attachment-detachment cycles between actin and myosin heads, and one force-generating attachment-detachment cycle corresponded to each ATPase cycle. But, the force fluctuations almost completely disappeared during sliding at the velocities of 20 to 70% of the maximum one at zero load. The analysis indicated that myosin heads produced an almost constant force for most (probably > 70%) of the ATPase cycle time, i. e., the duty ratio > 0.7. Since the myosin step size was given as (velocity) × (the duty ratio) × (the ATPase cycle time, 30 ms), it was calculated to be 40 to 110 nm, corresponding to velocities of 20 to 70 % of the maximum one (9μm/s), respectively. These values are much greater than the displacement by a single attachment-detachment cycle of actomyosin (10–20 nm), indicating that multiple force-generating attachment-detachment cycles correspond to each ATPase cycle during sliding at velocities of > 20 % of the maximum one. In conclusion, the coupling between the ATPase and the force-generating attachment-detachment cycles of actomyosin is not rigidly determined in a one-to-one fashion but is variable depending on the load.