Search

This study focused on the problem of projection parameter search in 3D reconstruction using single-particle analysis. We treated the sampling distribution for the parameter search as a prior distribution and designed a probabilistic model for efficient parameter estimation. Using our method, we showed that it is possible to perform 3D reconstruction from synthetic and actual electron microscope images using an initial model and to generate the initial model itself. We also examined whether the optimization function used in the stochastic gradient descent method can be applied with loose constraints to improve the convergence of initial model generation and confirmed the effect. In order to investigate the advantage of generating a smooth sampling distribution from the stochastic model, we compared the distribution of estimated projection directions with the conventional method of performing a global search using spherical gridding. As a result, our method, which is simple in both mathematical model and implementation, showed no algorithmic artifacts.

The difficulty in evaluating the research performance of groups is attributable to the following two factors: 1) difference of population size or discipline of group members and 2) skewed distribution of the research performance of individuals. This study attempts to overcome this difficulty, focusing on the research performance based on publication productivity. We employ the normalized index for the number of papers, in which publication efficiency was considered and disciplinary variation in the publication intensity was corrected by the disciplinary averages, to calculate a new percentile rank score. The score was developed on the basis of the principle that a person who is rare is valuable. The score was also tested with publication data for faculty members of 17 Japanese universities. The employment of the normalized index increased the score of universities with relatively few faculty members working in the disciplines of high productivity, resulting in more plausible university rankings. The rankings show a high correlation with those for a previously established percentile rank score, which was developed for citation analysis, and they are consistent with the judgment by evaluators of several universities under study. The advantage of the new score over the previous one is that it has no room for arbitrariness in determining the scheme of rank classification and the weights given to each rank class.

To clarify the precise subunit composition of the respiratory supercomplex of Corynebacterium glutamicum, several wash conditions were examined. MEGA (9 + 10) wash-buffer (0.5%) was used for this purpose and two-step column chromatography was performed. Almost equal amounts of cytochrome c, b, and a were observed in the purified fraction, estimated by their different absorption spectra. The 833 kDa and 685 kDa bands were observed in the clear native polyacrylamide gel electrophoresis (CN-PAGE) of the purified fraction. Both bands were stained using N,N',N′,N-tetramethyl-p-phenylenediamine (TMPD) oxidase dye, and the 833 kDa band was also stained using NADH oxidase dye. The 3D map reconstructed from the 833 kDa band indicated that the bcc complex and aa3 oxidase are heterodimers. Lastly, electron transfer from NADH to the bcc-aa3 supercomplex was observed. The 833 kDa band is the supercomplex, which includes the heterodimer cytochrome bcc complex and cytochrome aa3 oxidase, as well as the monomer NDH-II. Hence, we termed the 833 kDa band the extended supercomplex (ESC).

Bending of cilia and flagella occurs when axonemal dynein molecules on one side of the axoneme produce force and move toward the microtubule (MT) minus end. These dyneins are then pulled back when the axoneme bends in the other direction, meaning oscillatory back and forth movement of dynein during repetitive bending of cilia/flagella. There are various factors that may regulate the dynein activity, e.g. the nexin-dynein regulatory complex, radial spokes, and central apparatus. In order to understand the basic mechanism of dynein’s oscillatory movement, we constructed a simple model system composed of MTs, outer-arm dyneins, and crosslinks between the MTs made of DNA origami. Electron microscopy (EM) showed pairs of parallel MTs crossbridged by patches of regularly arranged dynein molecules bound in two different orientations, depending on which of the MTs their tails bind to. The oppositely oriented dyneins are expected to produce opposing forces when the pair of MTs have the same polarity. Optical trapping experiments showed that the dynein-MT-DNA-origami complex actually oscillates back and forth after photolysis of caged ATP. Intriguingly, the complex, when held at one end, showed repetitive bending motions. The results show that a simple system composed of ensembles of oppositely oriented dyneins, MTs, and inter-MT crosslinkers, without any additional regulatory structures, has an intrinsic ability to cause oscillation and repetitive bending motions.

To clarify the precise subunit composition of the respiratory supercomplex of Corynebacterium glutamicum, several wash conditions were examined. MEGA (9 + 10) wash-buffer (0.5%) was used for this purpose and two-step column chromatography was performed. Almost equal amounts of cytochrome c, b, and a were observed in the purified fraction, estimated by their different absorption spectra. The 833 kDa and 685 kDa bands were observed in the clear native polyacrylamide gel electrophoresis (CN-PAGE) of the purified fraction. Both bands were stained using N,N',N',N-tetramethyl-p-phenylenediamine (TMPD) oxidase dye, and the 833 kDa band was also stained using NADH oxidase dye. The 3D map reconstructed from the 833 kDa band indicated that the bcc complex and aa

Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH&ndash, cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (&Delta, TGEE) is tightly bound to heme&ndash, HO-1, whereas the reduction in heme&ndash, HO-1 coupled with &Delta, TGEE is considerably slow because the distance between FAD and FMN in &Delta, TGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme&ndash, HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme&ndash, HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of &Delta, TGEE in complex with heme&ndash, HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme&ndash, HO-1 in solution. We conclude that the &ldquo, open&ndash, close&rdquo, transition of CPR is indispensable for electron transfer from CPR to heme&ndash, HO-1.

Filopodia are finger-like protrusions at the leading edge of migrating cells that play a crucial antennal function during cell motility. It is known that actin filaments are bundled hexagonally and provide rigidity to filopodia by virtue of fascin, which plays a central role in actin filament bundling. However, the molecular mechanisms underlying their formation remain unclear. Here, we observed the filopodia of intact whole cells fixed by rapid freezing and revealed their three-dimensional structure by cryo-electron tomography and image processing; the actin filament bundling structure by fascin was clarified at high resolution under physiological conditions. It was found that actin filaments in vivo were more numerous than in bundles reconstructed in vitro, and each filopodial actin filament had limited variability in helical twisting. In addition, statistical analysis of actin filament bundles unveiled their detailed architecture. In filopodia, actin filaments had highly ordered structures, and the shift between cross-links of each adjacent actin filament was approximately 2.7 nm, similar to the monomer repeat of actin filaments. We then proposed a plausible actin-fascin cross-link model at the amino acid level and identified three fascin binding sites on two adjacent actin filaments: one filament bound fascin at two discrete, widely separated regions and the other bound fascin in a single small region. We propose that these two different binding modalities should confer rigid bundles that retain flexibility and dynamic performance. © 2016 Wiley Periodicals, Inc.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Cytoplasmic dynein is a microtubule-based motor protein that transports intracellular cargo and performs various functions during cell division. We previously reported that Lis1 suppressed dynein motility on microtubules in an idling state. Recently, a model showed that Lis1 prevents the ATPase domain of dynein from transmitting a detachment signal to its microtubule-binding domain. However, conformational information on dynein is limited. We used electron microscopy to investigate the conformation of dynein and nucleotide-induced conformational changes on microtubules. The conformation of dynein differed depending on the presence or absence of a nucleotide. In the presence of the nucleotide ADP-vanadate, dynein displayed an extended form on microtubules (extended form), whereas in the absence of a nucleotide, dynein lay along microtubules (compact form). This conformational change reflects chemomechanical coupling in dynein walking on microtubules. We also found that Lis1 fixed the conformation of dynein in the compact form regardless of the nucleotide condition. Removal of the Lis1 dimerization motif abolished Lis1-dependent fixation of dynein in the compact form. This suggests that the idling state of dynein on microtubules induced by Lis1 occurs through the Lis1-dependent arrest of dynein chemomechanical coupling.

GM130 is a cytoplasmic peripheral membrane protein localized on the cis side of the Golgi apparatus. GM130 is proposed to function as a membrane skeleton, maintaining the structure of the Golgi apparatus, and as a vesicle tether that facilitates vesicle fusion to the Golgi membrane. More than 60% of the GM130 molecule is believed to exist as coiled-coil structures with a probability above 90%, based on its primary amino acid sequence. The predicted coiled-coil region was similar to that of yeast Uso1p and its mammalian homolog, p115, both of which form coiled-coil homodimers. Therefore, GM130 has long been thought to form a homodimer with a rod-like shape. However, our biochemical and electron microscopical analyses revealed that GM130 is a parallel homotetramer with a flexible rod-like structure with I- and Y-shaped conformations. The structure of the N-terminal region may interchange between an open conformation (branched or Y-shaped) and a closed conformation (non-branched or I-shaped), possibly with the help of interacting molecules. This conformational change may alter the oligomeric state of the GM130 molecules and the function of GM130 in the vesicle tethering and the maintenance of the Golgi structure.

Although α-synuclein (αSyn) has been linked to Parkinson’s disease (PD), the mechanisms underlying the causative role in PD remain unclear. We previously proposed a model for a transportable microtubule (tMT), in which dynein is anchored to a short tMT by LIS1 followed by the kinesin-dependent anterograde transport; however the mechanisms that produce tMTs have not been determined. Our in vitro investigations of microtubule (MT) dynamics revealed that αSyn facilitates the formation of short MTs and preferentially binds to MTs carrying 14 protofilaments (pfs). Live-cell imaging showed that αSyn co-transported with dynein and mobile βIII-tubulin fragments in the anterograde transport. Furthermore, bi-directional axonal transports are severely affected in αSyn and γSyn depleted dorsal root ganglion neurons. SR-PALM analyses further revealed the fibrous co-localization of αSyn, dynein and βIII-tubulin in axons. More importantly, 14-pfs MTs have been found in rat femoral nerve tissue, and they increased approximately 19 fold the control in quantify upon nerve ligation, indicating the unconventional MTs are mobile. Our findings indicate that αSyn facilitates to form short, mobile tMTs that play an important role in the axonal transport. This unexpected and intriguing discovery related to axonal transport provides new insight on the pathogenesis of PD.

Human mutations in KATNB1 (p80) cause severe congenital cortical malformations, which encompass the clinical features of both microcephaly and lissencephaly. Although p80 plays critical roles during brain development, the underlying mechanisms remain predominately unknown. Here, we demonstrate that p80 regulates microtubule (MT) remodeling in combination with NuMA (nuclear mitotic apparatus protein) and cytoplasmic dynein. We show that p80 shuttles between the nucleus and spindle pole in synchrony with the cell cycle. Interestingly, this striking feature is shared with NuMA. Importantly, p80 is essential for aster formation and maintenance in vitro. siRNA-mediated depletion of p80 and/or NuMA induced abnormal mitotic phenotypes in cultured mouse embryonic fibroblasts and aberrant neurogenesis and neuronal migration in the mouse embryonic brain. Importantly, these results were confirmed in p80-mutant harboring patient-derived induced pluripotent stem cells and brain organoids. Taken together, our findings provide valuable insights into the pathogenesis of severe microlissencephaly, in which p80 and NuMA delineate a common pathway for neurogenesis and neuronal migration via MT organization at the centrosome/spindle pole.

Using molecular dynamics (MD) simulation, we simulate the structural change of a telomeric DNA by β-decay of substituted tritium to helium-3. The configuration of the telomeric DNA is obtained by removing TRF2 protein from the TRF2-Dbd-DNA complex (Protein Data Bank ID is 3SJM). We assume that hydrogens (H) of guanines in the telomeric DNA are replaced to helium-3. Since this replacement of the H atoms to the 3He atoms changes the charge distribution significantly, the charge distribution used in the MD simulation for the modified guanine is obtained by the density functional theory calculations. We adopt, as the MD simulation, nanoscale molecular dynamics code with CHARMM36 force field using Langevin thermostat and Nosé–Hoover Langevin piston to control the temperature and pressure of the system, respectively. Moreover, changing both the number of replaced guanine N and the temperature of the system T, we calculate the root mean square deviation RMSD to quantify the dependence of the durability of the telomeric DNA on the β-decays. From the MD simulation, it is found that as N or T becomes larger, the RMSD of the DNA becomes also larger. Namely, it denotes that as the intensity of the β-decays becomes larger or as the temperature is increased, the DNA structure becomes more fragile.

The molecular mechanism through which how beta decays in tritium-substituted species damage DNA and polymeric materials is still unknown. Molecular dynamics simulations of hydrogen-removed polyethylene were performed to predict the structural change of the polyethylene chain after the substituted tritium decays. We calculated the potential energy, the global orientational order parameter, and the average number of consecutive trans bonds. The results are that, the greater the number of removed hydrogen atoms, the higher the potential energy and the lower the value of the global orientational order parameter and the average number of consecutive trans bonds. Thus, after losing hydrogen, polyethylene becomes poorer in terms of both thermal and structural stabilities.

A cadmium-binding, genetically encoded protein tag, consisting of three repeats of metallothionein (3MT), can be used in electron microscopy for the visualization of multimeric- but not monomeric-tagged proteins due to insufficient electron density in monomeric proteins. Here, we present a technique for detecting monomeric 3MT-tagged green fluorescent protein (GFP-3MT) using a platinum compound to intensify the electron density. Substitution of cadmium by platinum as a result of incubating purified cadmium-binding 3MT-tagged GFP (GFP-Cd-3MT) with cis-diamminedichloroplatinum(II) (cisDDP) was assessed by a UV absorption band centered at 284 nm thereby indicating platinum-sulfhydryl bonds. The incubation time and the concentration of cisDDP to reach maximal absorption were 2 h and 36-fold molar equivalent of cisDDP, respectively. GFP-Pt-3MT isolated by gel filtration chromatography contained 29 platinum atoms per single GFP-3MT molecule. Electron-dense particles were observed in a GFP-Pt-3MT sample by electron microscopy without negative staining. Further image processing and image analysis demonstrated that particles with higher density relative to their surroundings were detectable in both GFP-Cd-3MT and GFP-Pt-3MT samples. These results demonstrate that replacement of cadmium with platinum, together with proper image analyses, improve detection efficiency and enable the visualization of 3MT-tagged monomeric protein by electron microscopy.

The small heat shock protein (sHsp), categorized into a class of molecular chaperones, binds and stabilizes denatured proteins for the purpose of preventing aggregation. The sHsps undergo transition between different oligomeric states to control their nature. We have been studying the function of sHsp of Sulfolobus tokodaii, StHsp14.0. StHsp14.0 exists as 24meric oligomer, and exhibits oligomer dissociation and molecular chaperone activity over 80°C. We constructed and characterized StHsp14.0 mutants with replacement of the C-terminal IKI to WKW, IKF, FKI and FKF. All mutant complexes dissociated into dimers at 50°C. Among them, StHsp14.0FKF is almost completely dissociated, probably to dimers. All mutants protected citrate synthase (CS) from thermal aggregation at 50°C. But, the activity of StHsp14.0FKF was the lowest. Then, we examined the complexes of StHsp14.0 mutants with denatured CS by SAXS. StHsp14.0WKW protects denatured CS by forming the globular complexes of 24 subunits and a substrate. StHsp14.0FKF also formed similar complex but the number of subunits in the complex is a little smaller. These results suggest that the dimer itself exhibits low chaperone activity, and a partially dissociated oligomer of StHsp14.0 protects a denatured protein from interacting with other molecules by surrounding it.

1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC) is one of the major lipids constituting cellular membranes. When DLPC itself is dispersed in aqueous solutions, multilamellar vesicles are formed, a...

SummaryAssembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. ATP hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. Here, we present the cryo-electron microscopic (cryo-EM) structure of filamentous actin (F-actin) in the presence of phosphate, with the visualization of some α-helical backbones and large side chains. A complete atomic model based on the EM map identified intermolecular interactions mediated by bound magnesium and phosphate ions. Comparison of the F-actin model with G-actin monomer crystal structures reveals a critical role for bending of the conserved proline-rich loop in triggering phosphate release following ATP hydrolysis. Crystal structures of G-actin show that mutations in this loop trap the catalytic site in two intermediate states of the ATPase cycle. The combined structural information allows us to propose a detailed molecular mechanism for the biochemical events, including actin polymerization and ATPase activation, critical for actin filament dynamics.

The effects of ultrafine colloidal silica particles adsorbed on the surfaces of large silica particles in slurries used for the chemical mechanical polishing of sapphire were studied. Sapphire wafers were polished using hybrid silica particles that were composed of a mixture of ultrafine (4 nm) colloidal silica particles and large (20, 55, and 105 nm) silica particles. Dynamic light scattering results showed that the hybrid particles became larger after mixing. Transmission electron microscopy images revealed that the ultrafine particles adhered to the surfaces of the large particles and that the surface shape of the hybrid particles were changed by the mixing process. Atomic force microscopy and polishing results showed that the surface roughnesses and material removal rates of sapphire substrates were improved by using the hybrid particles. The coefficient of friction between the surface of the sapphire substrate and the hybrid particles was higher than that obtained using single-sized particles. These results confirm that the hybrid silica particles enhanced the performance of sapphire polishing.

Outer arm dynein (OAD) in cilia and flagella contains two to three nonidentical heavy chains (HCs) that possess motor activity. In Chlamydomonas, flagellar OAD contains three HCs, alpha-, beta-, and gamma-HCs, each appearing to have a distinct role. To determine the precise molecular mechanism of their function, cross-sectional electron micrographs of wild-type and single HC-disruption mutants were compared and statistically analyzed. While the alpha-HC mutant displayed an OAD of lower density, which was attributed to a lack of alpha-HC, the OAD of beta- and gamma-HC mutants not only lacked the corresponding HC, but was also significantly affected in its structure, particularly with respect to the localization of alpha-HC. The lack of beta-HC induced mislocalization of alpha-HC, while a disruption of the gamma-HC gene resulted in the synchronized movement of alpha-HC and beta-HC in the manners for stacking. Interestingly, using cryo-electron microscopy, purified OADs were typically observed consisting of two stacked heads and an independent single head, which presumably corresponded to gamma-HC. This conformation is different from previous reports in which the three HCs displayed a stacked form in flagella observed by cryo-electron tomography and a bouquet structure on mica in deep-etch replica images. These results suggest that gamma-HC supports the tight stacking arrangement of inter or intra alpha-/beta-HC to facilitate the proper functioning of OAD.

Background Cellular signal transduction is initiated by the binding of extracellular ligands to membrane receptors. Receptors are often expressed in excess, and cells are activated when a small number of receptors bind ligands. Intracellular signal proteins are activated at a high level soon after ligand binding, and the activation level decreases in a negative feedback manner without ligand clearance. Why are excess receptors required? What is the physiological significance of the negative feedback regulation? Results To answer these questions, we developed a Monte Carlo simulation program to kinetically analyze signal pathways using the model in which ligands are bound to receptors and then membrane complexes with other membrane proteins are formed. Our simulation results showed that excess receptors are not required for cell activation when the dissociation constant (Kd) of the ligand-receptor complex is 10-10 M or less. However, such low Kd values cause delayed signal shutdown after ligand clearance from the extracellular space. In contrast, when the Kd was 10-8 M and the ligand level was less than 1 μM, excess receptors were required for prompt signal propagation and rapid signal cessation after ligand clearance. An initial increase in active cytosolic signal proteins to a high level is required for rapid activation of cellular signal pathways, and a low level of active signal proteins is essential for the rapid shutdown of signal pathways after ligand clearance. Conclusion The present kinetic analysis revealed that excess receptors and negative feedback regulation promote activation and cessation of signal transduction with a low amount of extracellular ligand.

The outer dynein arm of Chlamydomonas flagella contains three heavy chains (α, β, and γ), each of which exhibits motor activity. How they assemble and cooperate is of considerable interest. Here we report the isolation of a novel mutant, oda2 - t , whose γ heavy chain is truncated at about 30% of the sequence. While the previously isolated γ chain mutant oda2 lacks the entire outer arm, oda2 - t retains outer arms that contain α and β heavy chains, suggesting that the N-terminal sequence (corresponding to the tail region) is necessary and sufficient for stable outer-arm assembly. Thin-section electron microscopy and image analysis localize the γ heavy chain to a basal region of the outer-arm image in the axonemal cross section. The motility of oda2 - t is lower than that of the wild type and oda11 (lacking the α heavy chain) but higher than that of oda2 and oda4 - s7 (lacking the motor domain of the β heavy chain). Thus, the outer-arm dynein lacking the γ heavy-chain motor domain is partially functional. The availability of mutants lacking individual heavy chains should greatly facilitate studies on the structure and function of the outer-arm dynein.

Zero-loss imaging of frozen-hydrated specimens requires a detector with high sensitivity, a low noise level and high spatial resolution, because more electrons are scattered inelastically than elastically by cryo-specimens and the number of electrons detected is approximately 1/4 of incident electrons after energy filtering. Cameras using charge-coupled devices (CCDs) are good candidates due to their high sensitivity. They have been used mainly to record electron diffraction patterns for electron crystallography due to their limited spatial resolution but recently used for acquiring direct images due to their convenience. The spatial resolution has been limited by the characteristics of a phosphor that is necessary to convert high-energy electrons to photons and the coupling. We adopted a CsI scintillator with good modulation transfer function (MTF), which was epitaxially grown from each of optical fibres. The stripes of carbon graphite with 3.4 A spacing and 1.4 A stripes of gold thin crystals could be recorded with a magnification of 240,000x and 560,000x at 200 kV, respectively. A computed Fourier transform of an image of a frozen-hydrated crystal of catalase containing about 1000 units showed diffraction spots at spatial frequencies of 1/9.6 A(-1) up to 1/8 A(-1) without background subtraction, when the image was recorded at 140,000x. These results show that the resolution of the developed camera was good enough to record images. Our used test method for MTF determination may be useful for others.

Naturally occurring mutations are useful in identifying domains that are important for protein function. We studied a mutation in the elastin gene, 800−3G>C, a common disease allele for SVAS (supravalvular aortic stenosis). We showed in primary skin fibroblasts from two different SVAS families that this mutation causes skipping of exons 16–17 and results in a stable mRNA. Tropoelastin lacking domains 16–17 (Δ16–17) was synthesized efficiently and secreted by transfected retinal pigment epithelium cells, but showed the deficient deposition into the extracellular matrix compared with normal as demonstrated by immunofluorescent staining and desmosine assays. Solid-phase binding assays indicated normal molecular interaction of Δ16–17 with fibrillin-1 and fibulin-5. However, self-association of Δ16–17 was diminished as shown by an elevated coacervation temperature. Moreover, negative staining electron microscopy confirmed that Δ16–17 was deficient in forming fibrillar polymers. Domain 16 has high homology with domain 30, which can form a β-sheet structure facilitating fibre formation. Taken together, we conclude that domains 16–17 are important for self-association of tropoelastin and elastic fibre formation. This study is the first to discover that domains of elastin play an essential role in elastic fibre formation by facilitating homotypic interactions.

Filopodia are finger-like protrusions at the leading edge of migrating cells that play a crucial antennal function during cell motility. It is known that actin filaments are bundled hexagonally and provide rigidity to filopodia by virtue of fascin, which plays a central role in actin filament bundling. However, the molecular mechanisms underlying their formation remain unclear. Here, we observed the filopodia of intact whole cells fixed by rapid freezing and revealed their three-dimensional structure by cryo-electron tomography and image processing; the actin filament bundling structure by fascin was clarified at high resolution under physiological conditions. It was found that actin filaments in vivo were more numerous than in bundles reconstructed in vitro, and each filopodial actin filament had limited variability in helical twisting. In addition, statistical analysis of actin filament bundles unveiled their detailed architecture. In filopodia, actin filaments had highly ordered structures, and the shift between cross-links of each adjacent actin filament was approximately 2.7 nm, similar to the monomer repeat of actin filaments. We then proposed a plausible actin-fascin cross-link model at the amino acid level and identified three fascin binding sites on two adjacent actin filaments: one filament bound fascin at two discrete, widely separated regions and the other bound fascin in a single small region. We propose that these two different binding modalities should confer rigid bundles that retain flexibility and dynamic performance. © 2016 Wiley Periodicals, Inc.

We conducted a comparative analysis of the effects of beryllium fluoride (BeFx) on protein folding mediated by the alpha- and beta-subunit homooligomers (alpha16mer or beta16mer) from the hyperthermophilic archaeum Thermococcus strain KS-1. BeFx inhibited the ATPase activities of both alpha16mer and beta16mer with equal efficiency. This indicated that BeFx replaces the gamma-phosphate of chaperonin-bound ATP, thereby forming a stable chaperonin-ADP-BeFx complex. In the presence of ATP and BeFx, both of the two chaperonin subunits mediated green fluorescent protein (GFP) folding. Gel filtration chromatography revealed that the refolded GFP was retained by both chaperonins. Protease digestion and electron microscopic analyses showed that both chaperonin-ADP-BeFx complexes of the two subunits adopted a symmetric closed conformation with the built-in lids of both rings closed and that protein folding took place in their central cavities. These data indicated that basic protein folding mechanisms of alpha16mer and beta16mer are likely similar although there were some apparent differences. While beta16mer-mediated GFP refolding in the presence of ATP-BeFx that proceeded more rapidly than in the presence of ATP alone and reached a twofold higher plateau than that achieved with AMP-PNP, the folding mediated by the alpha16mer that proceeded with much lower yields. A mutant of alpha16mer, trapalpha, which traps the unfolded and partially folded substrate protein, did not affect the ATP-BeFx-dependent GFP folding by beta16mer but it suppressed that mediated by alpha16mer to the level of spontaneous folding. These results suggested that beta16mer differed from the alpha16mer in nucleotide binding affinity or the rate of nucleotide hydrolysis.

Ciliary and flagellar axonemes contain multiple inner arm dyneins of which the functional difference is largely unknown. In this study, a Chlamydomonas mutant, ida9, lacking inner arm dynein c was isolated and shown to carry a mutation in the DHC9 dynein heavy chain gene. The cDNA sequence of DHC9 was determined, and its information was used to show that >80% of it is lost in the mutant. Electron microscopy and image analysis showed that the ida9 axoneme lacked electron density near the base of the S2 radial spoke, indicating that dynein c localizes to this site. The mutant ida9 swam only slightly slower than the wild type in normal media. However, swimming velocity was greatly reduced when medium viscosity was modestly increased. Thus, dynein c in wild type axonemes must produce a significant force when flagella are beating in viscous media. Because motility analyses in vitro have shown that dynein c is the fastest among all the inner arm dyneins, we can regard this dynein as a fast yet powerful motor.

Filopodia are finger-like protrusions at the leading edge of migrating cells and are crucial for cell motilities as antennae. It is known that actin filaments are bundled hexagonally and provide rigidity to filopodia by fascin, which is an actin filament bundling protein and plays a central role of actin bundling. However, molecular mechanisms of their formation have been still unclear. We, here, observed the filopodia of intact whole cells fixed by rapid freezing and revealed their three-dimensional maps by cryo-electron tomography and image processing; the actin filaments’ bundling structure was clarified at high resolutions under close-to-life conditions. It is found that the actin filaments in vivo were more numerous than those in the bundles reconstructed in vitro, whereas actin filaments and fascin possess dynamic properties in filopodia. In additions, each actin filament in the filopodia showed a slightly loose twist similar to those bundled by fascin in vitro. The cross-linking structures between actin filaments were also observed clearly and were determined to be fascin by comparison with actin and fascin atomic models. Then we identified three actin binding sites of fascin against two adjacent actin filaments: one bound fascin in two binding regions with wide surfaces and the other did in a tiny region. We propose that these two different binding manners between actin and fascin should provide rigid bundles but flexible and dynamic performance.

Group II chaperonins of archaea and eukaryotes are distinct from group I chaperonins of bacteria. Whereas group I chaperonins require the co-chaperonin Cpn-10 or GroES for protein folding, no co-chaperonin has been known for group II. The protein folding mechanism of group II chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant α or β-subunit chaperonin homo-oligomer (α16mer and β16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The α16mer and β16mer captured the non-native GFP and promoted its refolding without any co-chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by β16mer but not by the α16mer. A mutant α-subunit chaperonin homo-oligomer (trap-α) could capture the non-native protein but lacked the ability to refold it. Although trap-α suppressed ATP-dependent refolding of GFP mediated by α16mer or β16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by β16mer with AMP-PNP was not accessible to the trap-α. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with β16mer. After the completion of GFP refolding mediated by β16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the β16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-chaperonin.

Eos (Extensible object-oriented system) is one of the powerful applications for image processing of electron micrographs. In usual cases, Eos works with only character user interfaces (CUI) under the operating systems (OS) such as OS-X or Linux, not user-friendly. Thus, users of Eos need to be expert at image processing of electron micrographs, and have a little knowledge of computer science, as well. However, all the persons who require Eos does not an expert for CUI. Thus we extended Eos to a web system independent of OS with graphical user interfaces (GUI) by integrating web browser.Advantage to use web browser is not only to extend Eos with GUI, but also extend Eos to work under distributed computational environment. Using Ajax (Asynchronous JavaScript and XML) technology, we implemented more comfortable user-interface on web browser. Eos has more than 400 commands related to image processing for electron microscopy, and the usage of each command is different from each other. Since the beginning of development, Eos has managed their user-interface by using the interface definition file of "OptionControlFile" written in CSV (Comma-Separated Value) format, i.e., Each command has "OptionControlFile", which notes information for interface and its usage generation. Developed GUI system called "Zephyr" (Zone for Easy Processing of HYpermedia Resources) also accessed "OptionControlFIle" and produced a web user-interface automatically, because its mechanism is mature and convenient,The basic actions of client side system was implemented properly and can supply auto-generation of web-form, which has functions of execution, image preview, file-uploading to a web server. Thus the system can execute Eos commands with unique options for each commands, and process image analysis. There remain problems of image file format for visualization and workspace for analysis: The image file format information is useful to check whether the input/output file is correct and we also need to provide common workspace for analysis because the client is physically separated from a server. We solved the file format problem by extension of rules of OptionControlFile of Eos. Furthermore, to solve workspace problems, we have developed two type of system. The first system is to use only local environments. The user runs a web server provided by Eos, access to a web client through a web browser, and manipulate the local files with GUI on the web browser. The second system is employing PIONE (Process-rule for Input/Output Negotiation Environment), which is our developing platform that works under heterogenic distributed environment. The users can put their resources, such as microscopic images, text files and so on, into the server-side environment supported by PIONE, and so experts can write PIONE rule definition, which defines a workflow of image processing. PIONE run each image processing on suitable computers, following the defined rule. PIONE has the ability of interactive manipulation, and user is able to try a command with various setting values. In this situation, we contribute to auto-generation of GUI for a PIONE workflow.As advanced functions, we have developed a module to log user actions. The logs include information such as setting values in image processing, procedure of commands and so on. If we use the logs effectively, we can get a lot of advantages. For example, when an expert may discover some know-how of image processing, other users can also share logs including his know-hows and so we may obtain recommendation workflow of image analysis, if we analyze logs. To implement social platform of image processing for electron microscopists, we have developed system infrastructure, as well.

Muscle contraction is regulated by the intracellular Ca(2+ )concentration. In vertebrate striated muscle, troponin and tropomyosin on actin filaments comprise a Ca(2+)-sensitive switch that controls contraction. Ca(2+ )binds to troponin and triggers a series of changes in actin-containing filaments that lead to cyclic interactions with myosin that generate contraction. However, the precise location of troponin relative to actin and tropomyosin and how its structure changes with Ca(2+ )have been not determined. To understand the regulatory mechanism, we visualized the location of troponin by determining the three-dimensional structure of thin filaments from electron cryo-micrographs without imposing helical symmetry to approximately 35 A resolution. With Ca(2+), the globular domain of troponin was gourd-shaped and was located over the inner domain of actin. Without Ca(2+), the main body of troponin was shifted by approximately 30 A towards the outer domain and bifurcated, with a horizontal branch (troponin arm) covering the N and C-terminal regions of actin. The C-terminal one-third of tropomyosin shifted towards the outer domain of actin by approximately 35 A supporting the steric blocking model, however it is surprising that the N-terminal half of tropomyosin shifted less than approximately 12 A. Therefore tropomyosin shifted differentially without Ca(2+). With Ca(2+), tropomyosin was located entirely over the inner domain thereby allowing greater access of myosin for force generation. The interpretation of three-dimensional maps was facilitated by determining the three-dimensional positions of fluorophores labelled on specific sites of troponin or tropomyosin by applying probabilistic distance geometry to data from fluorescence resonance energy transfer measurements.