Array Of Filaments Research Articles

Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi

Background Trypanosoma cruzi, the agent of Chagas disease, is a protozoan member of the Kinetoplastidae family characterized for the presence of specific and unique structures that are involved in different cell activities. One of them is the paraflagellar rod (PFR), a complex array of filaments connected to the flagellar axoneme. Although the function played by the PFR is not well established, it has been shown that silencing of the synthesis of its major proteins by either knockout of RNAi impairs and/or modifies the flagellar motility.Methodology/Principal FindingsHere, we present results obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM) of replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. The pattern of organization of the PFR filament network differs between regions of the flagellum in a straight state and those in a bent state. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme as well as of the angles between the intercrossed filaments supported this idea.Conclusions/SignificanceGraphic computation based on the information obtained allowed the proposal of an animated model for the PFR structure during flagellar beating and provided a new way of observing PFR filaments during flagellar beating.

Disrupted zinc-binding sites in structures of pathogenic SOD1 variants D124V and H80R.

Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we present structures of the pathogenic SOD1 variants D124V and H80R, both of which demonstrate compromised zinc-binding sites. The disruption of the zinc-binding sites in H80R SOD1 leads to conformational changes in loop elements, permitting non-native SOD1-SOD1 interactions that mediate the assembly of these proteins into higher-order filamentous arrays. Analytical ultracentrifugation sedimentation velocity experiments indicate that these SOD1 variants are more prone to monomerization than the wild-type enzyme. Although D124V and H80R SOD1 proteins appear to have fully functional copper-binding sites, inductively coupled plasma mass spectrometery (ICP-MS) and anomalous scattering X-ray diffraction analyses reveal that zinc (not copper) occupies the copper-binding sites in these variants. The absence of copper in these proteins, together with the results of covalent thiol modification experiments in yeast strains with and without the gene encoding the copper chaperone for SOD1 (CCS), suggests that CCS may not fully act on newly translated forms of these polypeptides. Overall, these findings lend support to the hypothesis that immature mutant SOD1 species contribute to toxicity in SOD1-linked ALS.

Recent progress in a development of Nb3Sn internal tin strand for fusion application

The layout optimization of ITER-type Nb3Sn internal tin (IT) superconducting strand for magnet systems of future fusion reactors was carried out aiming on the large scaled industrial production. The new approach to a formation of the initial composite billets for fabrication of IT superconducting strand has been proposed. This approach assumes the use of round shaped instead of hexagonal shaped Cu-Nb subelements covered by tin layers. The positive influence of the use of round shaped multifilamentary Cu-Nb subelements on the uniformity of filaments array in the final strands has been shown. The investigation on the use of composite filaments consisting of multiple niobium rods and rods made from NbTi alloy was carried out. It was shown that the use of proposed composite filaments enabled to effectively regulate the grain size of niobium in Cu-Nb subelements before the cold deformation by drawing. The experimental data on the influence of the proposed composite design of the filaments on the kinetics of Nb3Sn layer formation process are given. The relations of microstructure and properties of Nb3Sn IT strand with modified design have been analyzed. The superconducting properties attained met the main ITER requirements for the TF strands with non-copper critical current density higher than 800 A/mm2 (4.2 K; 12 T) and hysteresis losses (±3 T) less than 500 mJ/cm3.

Actin-Bundling Protein TRIOBP Forms Resilient Rootlets of Hair Cell Stereocilia Essential for Hearing

Inner ear hair cells detect sound through deflection of mechanosensory stereocilia. Each stereocilium is supported by a paracrystalline array of parallel actin filaments that are packed more densely at the base, forming a rootlet extending into the cell body. The function of rootlets and the molecules responsible for their formation are unknown. We found that TRIOBP, a cytoskeleton-associated protein mutated in human hereditary deafness DFNB28, is localized to rootlets. In vitro, purified TRIOBP isoform 4 protein organizes actin filaments into uniquely dense bundles reminiscent of rootlets but distinct from bundles formed by espin, an actin crosslinker in stereocilia. We generated mutant Triobp mice (Triobp(Deltaex8/Deltaex8)) that are profoundly deaf. Stereocilia of Triobp(Deltaex8/Deltaex8) mice develop normally but fail to form rootlets and are easier to deflect and damage. Thus, F-actin bundling by TRIOBP provides durability and rigidity for normal mechanosensitivity of stereocilia and may contribute to resilient cytoskeletal structures elsewhere.

Force Generation of Curved Actin Gels Characterized by Combined AFM-Epifluorescence Measurements

Polymerization of actin into branched filaments is the driving force behind active migration of eukaryotic cells and motility of intracellular organelles. The site-directed assembly of a polarized branched array forms an expanding gel that generates the force that pushes the membrane. Here, we use atomic force microscopy to understand the relation between actin polymerization and the produced force. Functionalized spherical colloidal probes of varying size and curvature are attached to the atomic force microscopy cantilever and initiate the formation of a polarized actin gel in a solution mimicking the in vivo context. The gel growth is recorded by epifluorescence microscopy both against the cantilever and in the perpendicular (lateral) nonconstrained direction. In this configuration, the gel growth stops simultaneously in both directions at the stall force, which corresponds to a pressure of 0.15 nN/μm2. The results show that the growth of the gel is limited laterally, in the absence of external force, by internal mechanical stresses resulting from a combination of the curved geometry and the molecular mechanism of site-directed assembly of a cohesive branched filament array.

Three-dimensional mapping of multiple filament arrays

We study experimentally and numerically the spatiotemporal dynamics of the multiple filament arrays excited by self-focusing of intense elliptical laser beams in fused silica. Our results demonstrate that although multiple filament arrays emerge as apparently regular patterns in the space domain, the spatiotemporal dynamics of the individual filaments is governed by the input-beam power and the input-beam ellipticity. In the case of moderate input-beam ellipticity, the individual filaments propagate in curved trajectories arising from skewed (spatiotemporal) coherence. The spatiotemporal propagation dynamics is regularized by increasing the input-beam ellipticity, and in part due to permanent modifications of fused silica that occur under intense irradiation. In this case, strong pulse reshaping and shock-front generation are observed, which yields a regular array of very short ($<5$ fs) superluminally propagating localized peaks in the leading front, followed by the subpulses centered on the input-pulse top, and trailed by subluminally propagating pulses with rather complex transverse intensity distribution.

Morphological Transformation of a Liquid Micropattern on Dynamically Tunable Microwrinkles

When a very thin, hard layer supported by a soft substrate is laterally compressed beyond a critical strain, then buckling of the hard layer occurs, leading to the formation of small sinusoidal surface undulations (microwrinkles). The orientation of the wrinkle grooves can be reversibly altered by simply adding a strain externally. Using this nonlinear microtopological change, we demonstrate that the morphology of an array of liquid filaments formed on microwrinkle grooves is dramatically and reversibly transformed into liquid filaments with a different orientation or a regular array of microdroplets, "dots", depending on the predefined contact angle. The novel liquid transformation at nanomicrometer scales will find unique applications, such as switchable light diffraction grating, laboratories-on-a-chip systems as well as a simple liquid micropatterning technique.

Control of Actin Filament Treadmilling in Cell Motility

Recent advances in structural, biochemical, biophysical, and live cell imaging approaches have furthered our understanding of the molecular mechanisms by which regulated assembly dynamics of actin filaments drive motile processes. Attention is focused on lamellipodium protrusion, powered by the turnover of a branched filament array. ATP hydrolysis on actin is the key reaction that allows filament treadmilling. It regulates barbed-end dynamics and length fluctuations at steady state and specifies the functional interaction of actin with essential regulatory proteins such as profilin and ADF/cofilin. ATP hydrolysis on actin and Arp2/3 acts as a timer, regulating the assembly and disassembly of the branched array to generate tropomyosin-mediated heterogeneity in the structure and dynamics of the lamellipodial network. The detailed molecular mechanisms of ATP hydrolysis/Pi release on F-actin remain elusive, as well as the mechanism of filament branching with Arp2/3 complex or that of the formin-driven processive actin assembly. Novel biophysical methods involving single-molecule measurements should foster progress in these crucial issues.

Processing and properties of centimeter-long, in-fiber, crystalline-selenium filaments

We report on the fabrication and characterization of globally ordered crystalline selenium filaments with diameters about 200 nm and aspect ratios upwards of 105. Amorphous Se filaments are fabricated by a recently developed approach in which a thin film evolves into an ordered array of filaments in fiber. Single-crystal and polycrystalline filaments are attained with a postdrawing annealing procedure. Arrays of two-cm-long crystalline nanowires, electrically contacted to external circuitry through the fiber end facets, exhibit a two-orders-of-magnitude change in conductivity between dark and illuminated states. These results hold promise for the fabrication of filament-detector arrays that may be integrated with large-area electronics.

Different Orientation of Two Heads of a Myosin Crossbridge in Full-Filament Overlapped and Overstretched Muscles Obtained by X-Ray Fiber Diffraction

A novel method using the cylindrically averaged difference Patterson function was applied to correct a sampling effect due to the hexagonal filament array on the thick filament-based layer-line intensities from frog skeletal muscles at the full-filament overlap length. Using the corrected intensity data and the mixed structural model of a thick filament with two different axial periodicities of the myosin crossbridges, we performed an optimum search of azimuthal orientation of two heads of a myosin crossbridge and compared the optimum orientation to that from muscles stretched beyond filament overlap reported previously. The result showed that the myosin crossbriges in the regular repeating region had a similar configuration in both muscles. Two heads of a myosin crossbridge formed a windmill-shape when seen from the top of the filament and one head of a myosin crossbridge seemed to be almost in contact with another head in a pair at an adjacent crown level along the filament axis. One head was toward the converter domain of the other head, similar to regulated myosin heads in Tarantula muscles in which the intramolecular head-head interaction occurs. In the perturbed region, however, myosin crossbridges had different configurations in these muscles. In top view, two heads of a myosin crossbridge showed a U-shape structure in the overstreched muscles while a cross-shape structure in muscles with the full-filament overlap. One myosin head seemed to be in contact with the other head at the same axial crown level. The models suggest that the disposition of two-headed myosin crossbridges is stabilized by the head-head interaction at same or different axial crown levels. Probably this would be related to the inhibition mechanism of actomyosin interaction in the relaxed muscles.

Single-step large-scale and template-free electrochemical growth of Ni–Zn alloy filament arrays from a zinc chloride based ionic liquid

For the first time, direct template-free electrodeposition of aligned NiZn nanofilaments was achieved on a tungsten substrate in a quiescent Lewis acidic ZnCl2-EMIC ionic liquid containing NiCl2 at 150 degrees C by applying an extremely large deposition overpotential.

The Association of the Arabidopsis Actin-Related Protein2/3 Complex with Cell Membranes Is Linked to Its Assembly Status But Not Its Activation

In growing plant cells, the combined activities of the cytoskeleton, endomembrane, and cell wall biosynthetic systems organize the cytoplasm and define the architecture and growth properties of the cell. These biosynthetic machineries efficiently synthesize, deliver, and recycle the raw materials that support cell expansion. The precise roles of the actin cytoskeleton in these processes are unclear. Certainly, bundles of actin filaments position organelles and are a substrate for long-distance intracellular transport, but the functional linkages between dynamic actin filament arrays and the cell growth machinery are poorly understood. The Arabidopsis (Arabidopsis thaliana) "distorted group" mutants have defined protein complexes that appear to generate and convert small GTPase signals into an Actin-Related Protein2/3 (ARP2/3)-dependent actin filament nucleation response. However, direct biochemical knowledge about Arabidopsis ARP2/3 and its cellular distribution is lacking. In this paper, we provide biochemical evidence for a plant ARP2/3. The plant complex utilizes a conserved assembly mechanism. ARPC4 is the most critical core subunit that controls the assembly and steady-state levels of the complex. ARP2/3 in other systems is believed to be mostly a soluble complex that is locally recruited and activated. Unexpectedly, we find that Arabidopsis ARP2/3 interacts strongly with cell membranes. Membrane binding is linked to complex assembly status and not to the extent to which it is activated. Mutant analyses implicate ARP2 as an important subunit for membrane association.

Magnetoresistance and exchange bias effect of the periodically nanostructured cobalt filament arrays

Periodically nanostructured cobalt filament arrays have been fabricated by the ultrathin layer electrochemical deposition method, where the unique spontaneous oscillation during the electrodeposition introduces periodical corrugations on the filaments. Magnetoresistance (MR) and the exchange bias effect have been detected in the cobalt filament arrays. We suggest that the MR originates from the nucleation and annihilation of domain walls in the periodic junction regions on the filaments. The contribution of spatial magnetization modulation to the MR has been discussed on account of the competition between the local transverse shape anisotropy and the dipolar interaction among the periodic nanostructures on the filaments. By increasing the temperature, the coercivity field and the exchange bias field decrease due to the weakness of Co/CoO exchange coupling. Our results are helpful for understanding the spatial magnetization modulation tailoring the spin-dependent transportation and have potential application in spintronics.

The vertebrate muscle Z-disc: sarcomere anchor for structure and signalling

The Z-disc, appearing as a fine dense line forming sarcomere boundaries in striated muscles, when studied in detail reveals crosslinked filament arrays that transmit tension and house myriads of proteins with diverse functions. At the Z-disc the barbed ends of the antiparallel actin filaments from adjoining sarcomeres interdigitate and are crosslinked primarily by layers of α-actinin. The Z-disc is therefore the site of polarity reversal of the actin filaments, as needed to interact with the bipolar myosin filaments in successive sarcomeres. The layers of α-actinin determine the Z-disc width: fast fibres have narrow (~30–50 nm) Z-discs and slow and cardiac fibres have wide (~100 nm) Z-discs. Comprehensive reviews on the roles of the numerous proteins located at the Z-disc in signalling and disease have been published; the aim here is different, namely to review the advances in structural aspects of the Z-disc.

Dynamin2 GTPase and Cortactin Remodel Actin Filaments

The large GTPase dynamin, best known for its activities that remodel membranes during endocytosis, also regulates F-actin-rich structures, including podosomes, phagocytic cups, actin comet tails, subcortical ruffles, and stress fibers. The mechanisms by which dynamin regulates actin filaments are not known, but an emerging view is that dynamin influences F-actin via its interactions with proteins that interact directly or indirectly with actin filaments. We show here that dynamin2 GTPase activity remodels actin filaments in vitro via a mechanism that depends on the binding partner and F-actin-binding protein, cortactin. Tightly associated actin filaments cross-linked by dynamin2 and cortactin became loosely associated after GTP addition when viewed by transmission electron microscopy. Actin filaments were dynamically unraveled and fragmented after GTP addition when viewed in real time using total internal reflection fluorescence microscopy. Cortactin stimulated the intrinsic GTPase activity of dynamin2 and maintained a stable link between actin filaments and dynamin2, even in the presence of GTP. Filaments remodeled by dynamin2 GTPase in vitro exhibit enhanced sensitivity to severing by the actin depolymerizing factor, cofilin, suggesting that GTPase-dependent remodeling influences the interactions of actin regulatory proteins and F-actin. The global organization of the actomyosin cytoskeleton was perturbed in U2-OS cells depleted of dynamin2, implicating dynamin2 in remodeling actin filaments that comprise supramolecular F-actin arrays in vivo. We conclude that dynamin2 GTPase remodels actin filaments and plays a role in orchestrating the global actomyosin cytoskeleton.

Non-invasive real-time spindle visualization under DIC optics and its predictability of oocyte development

OBJECTIVE: The presence of meiotic spindle predicts the fertilizability and developmental capability of ICSI oocytes. Real-time spindle imaging is believed to require a designated polarized microscope. Here, we attempted to identify meiotic spindles in living oocytes under one of the conventional settings for ICSI, and assessed the developmental ability of oocytes depending on the spindle detection. DESIGN: Meiotic spindle was visualized in living oocytes prior to ICSI. Fertilization and embryonic cleavage were compared between oocytes with or without spindles. MATERIALS AND METHODS: In this study, 103 infertile couples undergoing 115 ICSI cycles were involved with written informed consent. Visualization of the spindle was performed immediately before ICSI on a glass-bottomed dish. Oocytes with the first polar body (PBI) were examined at 400 or 600x magnification under an inverted microscope equipped with differential interference contrast optics and a heated stage. Embryological outcome were compared between the two groups with and without spindle. RESULTS: Total of 427 oocytes with PBI were assessed and subjected to ICSI. Spindle was distinctly identified as a barrel shaped bipolar array of filaments and occasionally even with chromosomes aligned on the equatorial plane, and detected in 30 seconds. The spindle was visualized in 82.0% of oocytes (350/427). The fertilization rate was 65.4% of spindle positive oocytes and similar to that negative (66.2%). However, the incidence of normally fertilized oocytes developed to good quality embryos on day 3 as well as to the blastocyst stage on day 5 was significantly higher in the positive than the negative group (55.5 vs. 31.4%, 61.8 vs. 36.1%). CONCLUSIONS: It was possible to detect the spindles with an expected incidence. The spindle visualization enabled safer injections. The presence of the spindle was associated with higher developmental potential. This system is immediately applicable to the current conventional ICSI setting without introducing expensive devices.

Cytosolic SYT/SS18 Isoforms Are Actin-Associated Proteins that Function in Matrix-Specific Adhesion

SYT (SYnovial sarcoma Translocated gene or SS18) is widely produced as two isoforms, SYT/L and SYT/S, that are thought to function in the nucleus as transcriptional coactivators. Using isoform-specific antibodies, we detected a sizable pool of SYT isoforms in the cytosol where the proteins were organized into filamentous arrays. Actin and actin-associated proteins co-immunoprecipitated with SYT isoforms, which also co-sedimented and co-localized with the actin cytoskeleton in cultured cells and tissues. The association of SYT with actin bundles was extensive yet stopped short of the distal ends at focal adhesions. Disruption of the actin cytoskeleton also led to a breakdown of the filamentous organization of SYT isoforms in the cytosol. RNAi ablation of SYT/L alone or both isoforms markedly impaired formation of stress fibers and focal adhesions but did not affect formation of cortical actin bundles. Furthermore, ablation of SYT led to markedly impaired adhesion and spreading on fibronectin and laminin-111 but not on collagen types I or IV. These findings indicate that cytoplasmic SYT isoforms interact with actin filaments and function in the ability cells to bind and react to specific extracellular matrices.

Helical Nanofilament Phases

In the formation of chiral crystals, the tendency for twist in the orientation of neighboring molecules is incompatible with ordering into a lattice: Twist is expelled from planar layers at the expense of local strain. We report the ordered state of a neat material in which a local chiral structure is expressed as twisted layers, a state made possible by spatial limitation of layering to a periodic array of nanoscale filaments. Although made of achiral molecules, the layers in these filaments are twisted and rigorously homochiral--a broken symmetry. The precise structural definition achieved in filament self-assembly enables collective organization into arrays in which an additional broken symmetry--the appearance of macroscopic coherence of the filament twist--produces a liquid crystal phase of helically precessing layers.

Self-Organization of Periodically Nanostructured Metal Filaments Array by Electrodeposition

not Available.

The Effect of Filament Diameter on ${\rm J}_{\rm e}$ in High Filament Count Bi2212/Ag Round Wire

In this study, we investigated the effect of filament size on J <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">e</sub> for high filament count Bi2212 round wire conductors partial melt processed with T <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">max</sub> between 882 and 890degC in pure oxygen. The filament size that produced maximum J <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">e</sub> was between 12 and 15 mum depending on conductor design and T <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">max</sub> . When the filament size was below 12 mum the filaments were unstable during partial melt processing leading to a break-up in the structure of the filament array and low J <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">e</sub> . This effect is attributed the formation of interconnects between filaments which leads to coarsening driven by a reduction in interfacial surface energy.