Motion Of Defects Research Articles

Modification of Tobacco rattle virus RNA1 to Serve as a VIGS Vector Reveals That the 29K Movement Protein Is an RNA Silencing Suppressor of the Virus

Tobacco rattle virus (TRV) has a bipartite, positive-sense single-stranded RNA genome and is widely used for virus-induced gene silencing (VIGS) in plants. RNA1 of TRV that lacks the gene for the cysteine-rich 16K silencing-suppression protein infects plants systemically in the absence of RNA2. Here, we attempted to engineer RNA1 for use as a VIGS vector by inserting heterologous gene fragments to replace 16K. The RNA1 vector systemically silenced the phytoene desaturase (PDS) gene, although less efficiently than when the original VIGS vector system was used, which consists of wild-type RNA1 and engineered RNA2 carrying the heterologous gene. Infectious RNA1 mutants with a dysfunctional 16K suppressed silencing and enhanced transgene expression in green fluorescent protein-transgenic Nicotiana benthamiana following inoculation by agroinfiltration, unlike mutants that also lacked 29K, a movement protein (MP) gene. The 30K MP gene of Tobacco mosaic virus complemented in cis the movement defect but not the silencing suppression functions of TRV 29K. Silencing suppression by 29K occurred in the context of RNA1 replication but not in an agroinfiltration assay which tested 29K alone for suppression of sense-mediated silencing. Both 29K and 16K were needed to avoid necrotic symptoms in RNA1-infected N. benthamiana. The results shed new light on virulence factors of TRV.

On conditions leading to crossing of I–V curve in metal1|mixed-ionic–electronic-conductor|metal2 devices

Abstract Many experimental and some theoretical studies on two terminal devices in the form metal1|semiconductor|metal2 report on unusual I–V relations which exhibit hysteresis and crossing of the I–V curve under periodic applied voltage. It is believed that the semiconductors allow also for motion of ionic defects and they are thus, mixed-ionic–electronic-conductors (MIECs). The device simulated here is of the form metal1|MIEC|metal2 where the thin semiconductor layer is n-type and the donors are mobile. The applied voltage is periodic. It is limited so that the temperature is constant. The conduction electrons are coupled to the mobile donors via the Poisson equation thus the redistribution of donors under an applied voltage leads to dramatic changes in the device's resistance. The donors considered are native ones and the electrodes allow exchange of matter with the surroundings which affects the I–V relations. The results suggest that a necessary condition for I–V curve crossing is an asymmetry in the device or in the applied voltage.

Expression of zinc-deficient human superoxide dismutase in Drosophila neurons produces a locomotor defect linked to mitochondrial dysfunction

More than 130 different mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been associated with amyotrophic lateral sclerosis but the mechanism of this toxicity remains controversial. To gain insight into the importance of the zinc site in the pathogenesis of SOD1 in vivo, we generated a Drosophila model with transgenic expression of a zinc-deficient human SOD1. Expression of zinc-deficient SOD1 in Drosophila resulted in a progressive movement defect with associated mitochondrial cristae vacuolization and reductions in adenosine triphosphate (ATP) levels. Furthermore, these flies are sensitized to mitochondrial toxins, paraquat, and zinc. Importantly, we show that the zinc-deficient SOD1-induced motor defect can be ameliorated by supplementing the endogenous fly respiratory chain machinery with the single-subunit NADH–ubiquinone oxidoreductase from yeast (NADH is nicotinamide adenine dinucleotide, reduced form.). These results demonstrate that zinc-deficient SOD1 is neurotoxic in vivo and suggest that mitochondrial dysfunction plays a critical role in this toxicity. The robust behavioral, pathological, and biochemical phenotypes conferred by zinc-deficient SOD1 in Drosophila have general implications for the role of the zinc ion in familial and sporadic amyotrophic lateral sclerosis.

Characterization of Axon Innervation to the Extraocular Muscles in Wild Type Mice

Certain forms of human strabismus result from abnormal ocular cranial nerve development, such that the nerves do not reach appropriate eye muscle targets, causing stereotyped defects in eye movement. Human eye movement disorders are now being modeled in mice. Little is known about the three‐dimensional innervation pattern of ocular cranial nerves onto extraocular muscles during mouse development. This research aims to gain an understanding of the normal developmental growth and innervation of ocular cranial nerves III, IV, and VI from their brainstem nuclei to the extraocular muscles in wild type mice to form a database from which to compare innervation patterns of normal and mutant mouse models. ISLMN:GFP reporter mice on a mixed background were used to visualize ocular cranial nerve input onto immunostained extraocular muscles during embryonic development. Histological sections were reconstructed into 3D images at multiple embryonic time points between E11.5 and E17.5 to determine innervation patterns. Characterization of normal extraocular muscle innervation during mouse development will facilitate the identification of innervation defects in mouse models of human eye movement disorders.

Frataxin Deficiency Leads to Defects in Expression of Antioxidants and Nrf2 Expression in Dorsal Root Ganglia of the Friedreich's Ataxia YG8R Mouse Model

Oxidative stress is thought to be involved in Friedreich's ataxia (FRDA), yet it has not been demonstrated in the target neurons that are first to degenerate. Using the YG8R mouse model of FRDA, microarray and neuritic growth experiments were carried out in the dorsal root ganglion (DRG), the primary site of neurodegeneration in this disease. YG8R hemizygous mice exhibited defects in movement, and DRG neurites had growth defects. Microarray of DRG tissue identified decreased transcripts encoding the antioxidants, including peroxiredoxins, glutaredoxins, and glutathione S-transferase, and these were confirmed by immunoblots and quantitative real-time PCR. Because the decreased gene transcripts are the known targets of the antioxidant transcription factor nuclear factor-E2-related factor-2 (Nrf2), Nrf2 expression was measured; it was significantly decreased at the transcript and protein level in both the DRG and the cerebella of the YG8R hemizygous mouse; further, frataxin expression was significantly correlated with Nrf2 expression. Functionally, in YG8R hemizygous DRG, the total glutathione levels were reduced and explanted cells were more sensitive to the thioredoxin reductase (TxnRD) inhibitor auranofin, a thiol oxidant. In cell models of FRDA, including Schwann and the DRG, frataxin deficiency caused a decreased expression of the Nrf2 protein level in the nucleus, but not a defect in its translocation from the cytosol. Further, frataxin-deficient cells had decreased enzyme activity and expression of TxnRD, which is regulated by Nrf2, and were sensitive the TxnRD inhibitor auranofin. These results support a mechanistic hypothesis in which frataxin deficiency decreases Nrf2 expression in vivo, causing the sensitivity to oxidative stress in target tissues the DRG and the cerebella, which contributes to the process of neurodegeneration.

MiR-153 Regulates SNAP-25, Synaptic Transmission, and Neuronal Development

SNAP-25 is a core component of the trimeric SNARE complex mediating vesicle exocytosis during membrane addition for neuronal growth, neuropeptide/growth factor secretion, and neurotransmitter release during synaptic transmission. Here, we report a novel microRNA mechanism of SNAP-25 regulation controlling motor neuron development, neurosecretion, synaptic activity, and movement in zebrafish. Loss of miR-153 causes overexpression of SNAP-25 and consequent hyperactive movement in early zebrafish embryos. Conversely, overexpression of miR-153 causes SNAP-25 down regulation resulting in near complete paralysis, mimicking the effects of treatment with Botulinum neurotoxin. miR-153-dependent changes in synaptic activity at the neuromuscular junction are consistent with the observed movement defects. Underlying the movement defects, perturbation of miR-153 function causes dramatic developmental changes in motor neuron patterning and branching. Together, our results indicate that precise control of SNAP-25 expression by miR-153 is critically important for proper neuronal patterning as well as neurotransmission.

Morphology transition in lipid vesicles due to in-plane order and topological defects

Complex morphologies in lipid membranes typically arise due to chemical heterogeneity, but in the tilted gel phase, complex shapes can form spontaneously even in a membrane containing only a single lipid component. We explore this phenomenon via experiments and coarse-grained simulations on giant unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. When cooled from the untilted L(α) liquid-crystalline phase into the tilted gel phase, vesicles deform from smooth spheres to disordered, highly crumpled shapes. We propose that this shape evolution is driven by nucleation of complex membrane microstructure with topological defects in the tilt orientation that induce nonuniform membrane curvature. Coarse-grained simulations demonstrate this mechanism and show that kinetic competition between curvature change and defect motion can trap vesicles in deeply metastable, defect-rich structures.

Modelling the sputtering of Au surfaces using a multi time-scale technique

We present results from an atomistic computer simulation model of the sputtering of gold crystal surfaces under 500 eV ion bombardment by Au and Ar ions for doses up to 10 14 ions cm −2 . The multi time-scale technique uses molecular dynamics to calculate the fast ballistic collision processes in the early stages of the cascade, whereas an on-the fly kinetic Monte Carlo technique is used to model the relaxation and diffusion processes between successive ion impacts when the defect motion has begun to be dominated by rare events. The results indicate a large amount of crystalline recovery between impacts, some facetting of the crystal surfaces but no large sub-surface defect accumulation. Because of this recovery process, sputtering yields and energy distributions are in good agreement with those obtained assuming a perfect crystal surface and also with those experimentally measured.

SNX15 links clathrin endocytosis to the PtdIns(3)P early endosome independent of the APPL1 endosome

Sorting nexins (SNXs) are key regulators of the endosomal network. In designing an RNAi-mediated loss-of-function screen, we establish that of 30 human SNXs only SNX3, SNX5, SNX9, SNX15 and SNX21 appear to regulate EGF receptor degradative sorting. Suppression of SNX15 results in a delay in receptor degradation arising from a defect in movement of newly internalised EGF-receptor-labelled vesicles into early endosomes. Besides a phosphatidylinositol 3-phosphate- and PX-domain-dependent association to early endosomes, SNX15 also associates with clathrin-coated pits and clathrin-coated vesicles by direct binding to clathrin through a non-canonical clathrin-binding box. From live-cell imaging, it was identified that the activated EGF receptor enters distinct sub-populations of SNX15- and APPL1-labelled peripheral endocytic vesicles, which do not undergo heterotypic fusion. The SNX15-decorated receptor-containing sub-population does, however, undergo direct fusion with the Rab5-labelled early endosome. Our data are consistent with a model in which the EGF receptor enters the early endosome following clathrin-mediated endocytosis through at least two parallel pathways: maturation through an APPL1-intermediate compartment and an alternative more direct fusion between SNX15-decorated endocytic vesicles and the Rab5-positive early endosome.

Morphology Transition in Lipid Vesicles: Interaction of In-Plane Order and Topological Defects

The biological membrane can exhibit complex morphologies driven by compositional heterogeneity, however membrane curvature can also be induced by a defect-driven mechanism. We have found that in the tilted gel phase, complex shapes can form spontaneously even in a membrane containing only a single lipid component as a result of membrane defect formation. To explore this phenomenon we have carried out both experimental observations and coarse-grained computer simulations.In our study, fluorescence microscopy on giant uni-lamellar vesicles (GUVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) reveals that vesicles can crumple dramatically when cooled from the untilted Lα liquid-crystalline phase into the Lβ' tilted gel phase. Similar behavior is observed in simulation studies of the system.To explain the phenomenon, we propose that the observed shape evolution is driven by the nucleation of a complex membrane micro-structure that includes topological defects in the tilt orientation. These defects induce non-uniform membrane curvature resulting in a crumpled morphology. Furthermore, we show that competition between curvature change and defect motion can trap vesicles in deeply metastable, defect-rich structures.

Small-angle water reorientations in KOH doped hexagonal ice and clathrate hydrates

Using deuteron nuclear magnetic resonance and dielectric spectroscopy KOH doped tetrahydrofuran clathrate hydrates and KOH doped hexagonal ice are studied at temperatures above 60 and 72 K, respectively. Below these temperatures proton order is established on the lattice formed by the water molecules. In the clathrate hydrate a new type of small-angle motion is discovered using deuteron spin-spin relaxation, line-shape analysis, and stimulated-echo experiments. Based on the latter results a model is developed for the local proton motion that could successfully be tested using random-walk simulations. It is argued that the newly identified small-angle motion, obviously absent in undoped samples, is an important feature of the mechanism which accompanies the establishment of proton order not only in doped clathrate hydrates but also in doped hexagonal ice. Specific motions of OH(-) defects are demonstrated to explain the experimentally observed behavior. The relative importance of localized versus delocalized OH(-) defect motions is discussed.

Identification of a Novel Site in the Tail of Dynein Heavy Chain Important for Dynein Function in Vivo

The minus end-directed microtubule motor cytoplasmic dynein is responsible for the intracellular movements of many organelles, including nuclei and endosomes. The dynein heavy chain contains a C-terminal motor domain and an N-terminal tail domain. The tail binds other dynein subunits and the cargo-interacting dynactin complex but is dispensable for movement of single dynein molecules in vitro. Here, we identified a mutation in the Aspergillus nidulans heavy chain tail domain, nudA(F208V), which causes obvious defects in dynein-mediated nuclear positioning and early endosome movement. Astonishingly, the nudA(F208I) mutation in the same position does not cause the same defects, suggesting that a subtle difference in the size of the amino acid side chain at this position has a significant consequence. Importantly, our biochemical analyses indicate that the nudA(F208V) mutation does not affect dynein subunit interactions and the mutant dynein is also able to bind dynactin and another dynein regulator, NUDF/LIS1. The mutant dynein is able to physically interact with the early endosome cargo, but dynein-mediated early endosome movement away from the hyphal tip occurs at a significantly reduced frequency. Within the small group of early endosomes that move away from the hyphal tip in the mutant, the average speed of movement is lower than that in the wild type. Given the dispensability of the dynein tail in dynein motility in vitro, our results support the notion that the structural integrity of the dynein tail is critical in vivo for the coordination of dynein force production and movement when the motor is heavily loaded.

A novel 9 kDa phosphoprotein is a component of the primary cilium and interacts with polycystin-1

Polycystin-1 (PKD1) forms a mechanosensitive cation channel complex with polycystin-2 (PKD2) in the primary cilium. Functional defects in this complex caused by mutation of PKD1 and PKD2 result in autosomal dominant polycystic kidney disease (ADPKD). The mechanisms by which this complex regulates normal cell physiology remain elusive. In particular the proteins that interact directly with polycystin-1 remain poorly characterised. A Y-2H screen using the C-terminus of polycystin-1 as bait identified a novel binding partner that we have called PIP9 (polycystin interacting protein 9). PIP9 is evolutionary conserved, ubiquitously expressed, and predicted to be a 9KDa phosphoprotein containing a single coiled-coil domain that interacts with the coiled-coil domain of PC-1. Specific anti-PIP9 antisera confirmed the widespread and developmentally regulated expression of PIP9 and identified phosphorylated isoforms. PIP9 co-localised to the renal primary cilium with PC1 and immuno-EM studies revealed localisation to IFT particles. Knockdown of zPip9, using morpholino oligonucleotides, resulted in developmental defects in gastrulation movements. In addition, evidence of cell detachment was observed, common to the phenotype described for double bbs4;bbs10 morphant zebrafish which suggests pip9 may act in the same genetic pathway. zpip9 morphants also displayed variable head and eye degeneration, including coloboma and hydrocephalus, pronephric cysts and shortening of the body axis. Loss of Pip9 function in mice is lethal before embryonic day E11.5. This preliminary analysis suggests that PIP9 plays a central role in mediating polycystin-1 dependant signalling.

Molecular Analysis of Arp2/3 Complex Activation in Cells

Many forms of cellular motility are driven by the growth of branched networks of actin filaments, which push against a membrane. In the dendritic nucleation model, Arp2/3 complex is critical, binding to the side of an existing mother filament, nucleating a new daughter filament, and thus creating a branch. Spatial and temporal regulation of Arp2/3 activity is critical for efficient generation of force and movement. A diverse collection of Arp2/3 regulatory proteins has been identified. They bind to and/or activate Arp2/3 complex via an acidic motif with a conserved tryptophan residue. We tested this model for Arp2/3 regulator function in vivo, by examining the roles of multiple Arp2/3 regulators in endocytosis in living yeast cells. We measured the molecular composition of the actin network in cells with mutations that removed the acidic motifs of the four Arp2/3 regulators previously shown to influence the proper function of the actin network. Unexpectedly, we did not find a simple or direct correlation between defects in patch assembly and movement and changes in the composition and dynamics of dendritic nucleation proteins. Taken together our data does not support the simple hypothesis that the primary role for Arp2/3 regulators is to recruit and activate Arp2/3. Rather our data suggests that these regulators may be playing more subtle roles in establishing functional networks in vivo.

Low-Frequency Diffusion Noise in Resistive-Switching Memories Based on Metal–Oxide Polymer Structure

Low-frequency noise is studied in resistive-switching memories based on metal–oxide polymer diodes. The noise spectral power follows a $\hbox{1}/f^{\gamma}$ behavior, with $\gamma = \hbox{1}$ in the ohmic region and with $\gamma = \hbox{3/2}$ at high bias beyond the ohmic region. The exponent $\gamma = \hbox{3/2}$ is explained as noise caused by Brownian motion or diffusion of defects which induce fluctuations in diode current. The figure of merit to classify $\hbox{1}/f$ noise in thin films has an estimated value of $\hbox{10}^{-21} \hbox{cm}^{2}/\Omega$ , which is typical for metals or doped semiconductors. This value in combination with the low diode current indicates that the $\hbox{1}/f$ noise is generated in the narrow localized regions in the polymer between the contacts. The analysis unambiguously shows that the current in bistable nonvolatile memories is filamentary.

Hysteresis and return-point memory in colloidal artificial spin ice systems

Using computer simulations, we investigate hysteresis loops and return-point memory for artificial square and kagome spin ice systems by cycling an applied bias force and comparing microscopic effective spin configurations throughout the hysteresis cycle. Return-point memory loss is caused by motion of individual defects in kagome ice or of grain boundaries in square ice. In successive cycles, return-point memory is recovered rapidly in kagome ice. Memory is recovered more gradually in square ice due to the extended nature of the grain boundaries. Increasing the amount of quenched disorder increases the defect density but also enhances the return-point memory since the defects become trapped more easily.

Identification and characterization of petiolule-like pulvinus mutants with abolished nyctinastic leaf movement in the model legume Medicago truncatula.

Leaves of many plant species open during the day and fold at night. Diurnal leaf movement, named nyctinasty, has been of great interest to researchers since Darwin's time. Nyctinastic leaf movement is generated by the pulvinus, which is a specialized motor organ located at the base of leaf and leaflet. The molecular basis and functional reason behind nyctinasty are unknown.In a forward screening of a retrotransposon-tagged mutant population of Medicago truncatula, four petiolule-like pulvinus (plp) mutant lines with defects in leaf movement were identified and characterized.Loss of function of PLP results in the change of pulvini to petiolules. PLP is specifically expressed in the pulvinus, as demonstrated by quantitative reverse-transcription polymerase chain reaction analysis, expression analysis of a PLP promoter-β-glucuronidase construct in transgenic plants and in situ hybridization. Microarray analysis revealed that the expression levels of many genes were altered in the mutant during the day and at night. Crosses between the plp mutant and several leaf pattern mutants showed that the developmental mechanisms of pulvini and leaf patterns are likely independent.Our results demonstrated that PLP plays a crucial role in the determination of pulvinus development. Leaf movement generated by pulvini may have an impact on plant vegetative growth.

Loss of Mfn2 results in progressive, retrograde degeneration of dopaminergic neurons in the nigrostriatal circuit

Mitochondria continually undergo fusion and fission, and these dynamic processes play a major role in regulating mitochondrial function. Studies of several genes associated with familial Parkinson's disease (PD) have implicated aberrant mitochondrial dynamics in the disease pathology, but the importance of these processes in dopaminergic neurons remains poorly understood. Because the mitofusins Mfn1 and Mfn2 are essential for mitochondrial fusion, we deleted these genes from a subset of dopaminergic neurons in mice. Loss of Mfn2 results in a movement defect characterized by reduced activity and rearing. In open field tests, Mfn2 mutants show severe, age-dependent motor deficits that can be rescued with L-3,4 dihydroxyphenylalanine. These motor deficits are preceded by the loss of dopaminergic terminals in the striatum. However, the loss of dopaminergic neurons in the midbrain occurs weeks after the onset of these motor and striatal deficits, suggesting a retrograde mode of neurodegeneration. In our conditional knockout strategy, we incorporated a mitochondrially targeted fluorescent reporter to facilitate tracking of mitochondria in the affected neurons. Using an organotypic slice culture system, we detected fragmented mitochondria in the soma and proximal processes of these neurons. In addition, we found markedly reduced mitochondrial mass and transport, which may contribute to the neuronal loss. These effects are specific for Mfn2, as the loss of Mfn1 yielded no corresponding defects in the nigrostriatal circuit. Our findings indicate that perturbations of mitochondrial dynamics can cause nigrostriatal defects and may be a risk factor for the neurodegeneration in PD.

Dislocation-Driven Deformations in Graphene

The movement of dislocations in a crystal is the key mechanism for plastic deformation in all materials. Studies of dislocations have focused on three-dimensional materials, and there is little experimental evidence regarding the dynamics of dislocations and their impact at the atomic level on the lattice structure of graphene. We studied the dynamics of dislocation pairs in graphene, recorded with single-atom sensitivity. We examined stepwise dislocation movement along the zig-zag lattice direction mediated either by a single bond rotation or through the loss of two carbon atoms. The strain fields were determined, showing how dislocations deform graphene by elongation and compression of C-C bonds, shear, and lattice rotations.

Defective motion processing in children with cerebral visual impairment due to periventricular white matter damage

Aim We sought to characterize visual motion processing in children with cerebral visual impairment (CVI) due to periventricular white matter damage caused by either hydrocephalus (eight individuals) or periventricular leukomalacia (PVL) associated with prematurity (11 individuals).Method Using steady‐state visually evoked potentials (ssVEP), we measured cortical activity related to motion processing for two distinct types of visual stimuli: ‘local’ motion patterns thought to activate mainly primary visual cortex (V1), and ‘global’ or coherent patterns thought to activate higher cortical visual association areas (V3, V5, etc.). We studied three groups of children: (1) 19 children with CVI (mean age 9y 6mo [SD 3y 8mo]; 9 male; 10 female); (2) 40 neurologically and visually normal comparison children (mean age 9y 6mo [SD 3y 1mo]; 18 male; 22 female); and (3) because strabismus and amblyopia are common in children with CVI, a group of 41 children without neurological problems who had visual deficits due to amblyopia and/or strabismus (mean age 7y 8mo [SD 2y 8mo]; 28 male; 13 female).Results We found that the processing of global as opposed to local motion was preferentially impaired in individuals with CVI, especially for slower target velocities (p=0.028).Interpretation Motion processing is impaired in children with CVI. ssVEP may provide useful and objective information about the development of higher visual function in children at risk for CVI.