Microtubule dynamics in cell division: exploring living cells with polarized light microscopy.

PubMed

Inoué, Shinya

2008-01-01

This Perspective is an account of my early experience while I studied the dynamic organization and behavior of the mitotic spindle and its submicroscopic filaments using polarized light microscopy. The birefringence of spindle filaments in normally dividing plant and animal cells, and those treated by various agents, revealed (a) the reality of spindle fibers and fibrils in healthy living cells; (b) the labile, dynamic nature of the molecular filaments making up the spindle fibers; (c) the mode of fibrogenesis and action of orienting centers; and (d) force-generating properties based on the disassembly and assembly of the fibrils. These studies, which were carried out directly on living cells using improved polarizing microscopes, in fact predicted the reversible assembly properties of microtubules.

Protein machines and self assembly in muscle organization

NASA Technical Reports Server (NTRS)

Barral, J. M.; Epstein, H. F.

1999-01-01

The remarkable order of striated muscle is the result of a complex series of protein interactions at different levels of organization. Within muscle, the thick filament and its major protein myosin are classical examples of functioning protein machines. Our understanding of the structure and assembly of thick filaments and their organization into the regular arrays of the A-band has recently been enhanced by the application of biochemical, genetic, and structural approaches. Detailed studies of the thick filament backbone have shown that the myosins are organized into a tubular structure. Additional protein machines and specific myosin rod sequences have been identified that play significant roles in thick filament structure, assembly, and organization. These include intrinsic filament components, cross-linking molecules of the M-band and constituents of the membrane-cytoskeleton system. Muscle organization is directed by the multistep actions of protein machines that take advantage of well-established self-assembly relationships. Copyright 1999 John Wiley & Sons, Inc.

Active control of complex, multicomponent self-assembly processes

NASA Astrophysics Data System (ADS)

Schulman, Rebecca

The kinetics of many complex biological self-assembly processes such as cytoskeletal assembly are precisely controlled by cells. Spatiotemporal control over rates of filament nucleation, growth and disassembly determine how self-assembly occurs and how the assembled form changes over time. These reaction rates can be manipulated by changing the concentrations of the components needed for assembly by activating or deactivating them. I will describe how we can use these principles to design driven self-assembly processes in which we assemble and disassemble multiple types of components to create micron-scale networks of semiflexible filaments assembled from DNA. The same set of primitive components can be assembled into many different, structures depending on the concentrations of different components and how designed, DNA-based chemical reaction networks manipulate these concentrations over time. These chemical reaction networks can in turn interpret environmental stimuli to direct complex, multistage response. Such a system is a laboratory for understanding complex active material behaviors, such as metamorphosis, self-healing or adaptation to the environment that are ubiquitous in biological systems but difficult to quantitatively characterize or engineer.

Thioredoxin is required for filamentous phage assembly.

PubMed Central

Russel, M; Model, P

1985-01-01

Sequence comparisons show that the fip gene product of Escherichia coli, which is required for filamentous phage assembly, is thioredoxin. Thioredoxin serves as a cofactor for reductive processes in many cell types and is a constituent of phage T7 DNA polymerase. The fip-1 mutation makes filamentous phage and T7 growth temperature sensitive in cells that carry it. The lesion lies within a highly conserved thioredoxin active site. Thioredoxin reductase (NADPH), as well as thioredoxin, is required for efficient filamentous phage production. Mutant phages defective in phage gene I are particularly sensitive to perturbations in the fip-thioredoxin system. A speculative model is presented in which thioredoxin reductase, thioredoxin, and the gene I protein interact to drive an engine for filamentous phage assembly. Images PMID:3881756

Non-muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.

PubMed

Zhang, Wenwu; Gunst, Susan J

2017-07-01

Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

GTPase activity, structure, and mechanical properties of filaments assembled from bacterial cytoskeleton protein MreB.

PubMed

Esue, Osigwe; Wirtz, Denis; Tseng, Yiider

2006-02-01

MreB, a major component of the recently discovered bacterial cytoskeleton, displays a structure homologous to its eukaryotic counterpart actin. Here, we study the assembly and mechanical properties of Thermotoga maritima MreB in the presence of different nucleotides in vitro. We found that GTP, not ADP or GDP, can mediate MreB assembly into filamentous structures as effectively as ATP. Upon MreB assembly, both GTP and ATP release the gamma phosphate at similar rates. Therefore, MreB is an equally effective ATPase and GTPase. Electron microscopy and quantitative rheology suggest that the morphologies and micromechanical properties of filamentous ATP-MreB and GTP-MreB are similar. In contrast, mammalian actin assembly is favored in the presence of ATP over GTP. These results indicate that, despite high structural homology of their monomers, T. maritima MreB and actin filaments display different assembly, morphology, micromechanics, and nucleotide-binding specificity. Furthermore, the biophysical properties of T. maritima MreB filaments, including high rigidity and propensity to form bundles, suggest a mechanism by which MreB helical structure may be involved in imposing a cylindrical architecture on rod-shaped bacterial cells.

Differential assembly of alpha- and gamma-filagenins into thick filaments in Caenorhabditis elegans

NASA Technical Reports Server (NTRS)

Liu, F.; Ortiz, I.; Hutagalung, A.; Bauer, C. C.; Cook, R. G.; Epstein, H. F.

2000-01-01

Muscle thick filaments are highly organized supramolecular assemblies of myosin and associated proteins with lengths, diameters and flexural rigidities characteristic of their source. The cores of body wall muscle thick filaments of the nematode Caenorhabditis elegans are tubular structures of paramyosin sub-filaments coupled by filagenins and have been proposed to serve as templates for the assembly of native thick filaments. We have characterized alpha- and gamma-filagenins, two novel proteins of the cores with calculated molecular masses of 30,043 and 19,601 and isoelectric points of 10.52 and 11.49, respectively. Western blot and immunoelectron microscopy using affinity-purified antibodies confirmed that the two proteins are core components. Immunoelectron microscopy of the cores revealed that they assemble with different periodicities. Immunofluorescence microscopy showed that alpha-filagenin is localized in the medial regions of the A-bands of body wall muscle cells whereas gamma-filagenin is localized in the flanking regions, and that alpha-filagenin is expressed in 1.5-twofold embryos while gamma-filagenin becomes detectable only in late vermiform embryos. The expression of both proteins continues throughout later stages of development. C. elegans body wall muscle thick filaments of these developmental stages have distinct lengths. Our results suggest that the differential assembly of alpha- and gamma-filagenins into thick filaments of distinct lengths may be developmentally regulated.

The assembly of MreB, a prokaryotic homolog of actin.

PubMed

Esue, Osigwe; Cordero, Maria; Wirtz, Denis; Tseng, Yiider

2005-01-28

MreB, a major component of the bacterial cytoskeleton, exhibits high structural homology to its eukaryotic counterpart actin. Live cell microscopy studies suggest that MreB molecules organize into large filamentous spirals that support the cell membrane and play a key shape-determining function. However, the basic properties of MreB filament assembly remain unknown. Here, we studied the assembly of Thermotoga maritima MreB triggered by ATP in vitro and compared it to the well-studied assembly of actin. These studies show that MreB filament ultrastructure and polymerization depend crucially on temperature as well as the ions present on solution. At the optimal growth temperature of T. maritima, MreB assembly proceeded much faster than that of actin, without nucleation (or nucleation is highly favorable and fast) and with little or no contribution from filament end-to-end annealing. MreB exhibited rates of ATP hydrolysis and phosphate release similar to that of F-actin, however, with a critical concentration of approximately 3 nm, which is approximately 100-fold lower than that of actin. Furthermore, MreB assembled into filamentous bundles that have the ability to spontaneously form ring-like structures without auxiliary proteins. These findings suggest that despite high structural homology, MreB and actin display significantly different assembly properties.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

PubMed Central

Valades Cruz, Cesar Augusto; Shaban, Haitham Ahmed; Kress, Alla; Bertaux, Nicolas; Monneret, Serge; Mavrakis, Manos; Savatier, Julien; Brasselet, Sophie

2016-01-01

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells. PMID:26831082

Self-Assembly of Hierarchical DNA Nanotube Architectures with Well-Defined Geometries.

PubMed

Jorgenson, Tyler D; Mohammed, Abdul M; Agrawal, Deepak K; Schulman, Rebecca

2017-02-28

An essential motif for the assembly of biological materials such as actin at the scale of hundreds of nanometers and beyond is a network of one-dimensional fibers with well-defined geometry. Here, we demonstrate the programmed organization of DNA filaments into micron-scale architectures where component filaments are oriented at preprogrammed angles. We assemble L-, T-, and Y-shaped DNA origami junctions that nucleate two or three micron length DNA nanotubes at high yields. The angles between the nanotubes mirror the angles between the templates on the junctions, demonstrating that nanoscale structures can control precisely how micron-scale architectures form. The ability to precisely program filament orientation could allow the assembly of complex filament architectures in two and three dimensions, including circuit structures, bundles, and extended materials.

Analysis of the function of Spire in actin assembly and its synergy with formin and profilin.

PubMed

Bosch, Montserrat; Le, Kim Ho Diep; Bugyi, Beata; Correia, John J; Renault, Louis; Carlier, Marie-France

2007-11-30

The Spire protein, together with the formin Cappuccino and profilin, plays an important role in actin-based processes that establish oocyte polarity. Spire contains a cluster of four actin-binding WH2 domains. It has been shown to nucleate actin filaments and was proposed to remain bound to their pointed ends. Here we show that the multifunctional character of the WH2 domains allows Spire to sequester four G-actin subunits binding cooperatively in a tight SA(4) complex and to nucleate, sever, and cap filaments at their barbed ends. Binding of Spire to barbed ends does not affect the thermodynamics of actin assembly at barbed ends but blocks barbed end growth from profilin-actin. The resulting Spire-induced increase in profilin-actin concentration enhances processive filament assembly by formin. The synergy between Spire and formin is reconstituted in an in vitro motility assay, which provides a functional basis for the genetic interplay between Spire, formin, and profilin in oogenesis.

Assembly Kinetics Determine the Architecture of α-actinin Crosslinked F-actin Networks

PubMed Central

Falzone, Tobias T.; Lenz, Martin; Kovar, David R.; Gardel, Margaret L.

2013-01-01

The actin cytoskeleton is organized into diverse meshworks and bundles that support many aspects of cell physiology. Understanding the self-assembly of these actin-based structures is essential for developing predictive models of cytoskeletal organization. Here we show that the competing kinetics of bundle formation with the onset of dynamic arrest arising from filament entanglements and cross-linking determine the architecture of reconstituted actin networks formed with α-actinin cross-links. Cross-link mediated bundle formation only occurs in dilute solutions of highly mobile actin filaments. As actin polymerization proceeds, filament mobility and bundle formation are arrested concomitantly. By controlling the onset of dynamic arrest, perturbations to actin assembly kinetics dramatically alter the architecture of biochemically identical samples. Thus, the morphology of reconstituted F-actin networks is a kinetically determined structure similar to those formed by physical gels and glasses. These results establish mechanisms controlling the structure and mechanics in diverse semi-flexible biopolymer networks. PMID:22643888

Probing the Energetics of Dynactin Filament Assembly and the Binding of Cargo Adaptor Proteins Using Molecular Dynamics Simulation and Electrostatics-Based Structural Modeling.

PubMed

Zheng, Wenjun

2017-01-10

Dynactin, a large multiprotein complex, binds with the cytoplasmic dynein-1 motor and various adaptor proteins to allow recruitment and transportation of cellular cargoes toward the minus end of microtubules. The structure of the dynactin complex is built around an actin-like minifilament with a defined length, which has been visualized in a high-resolution structure of the dynactin filament determined by cryo-electron microscopy (cryo-EM). To understand the energetic basis of dynactin filament assembly, we used molecular dynamics simulation to probe the intersubunit interactions among the actin-like proteins, various capping proteins, and four extended regions of the dynactin shoulder. Our simulations revealed stronger intersubunit interactions at the barbed and pointed ends of the filament and involving the extended regions (compared with the interactions within the filament), which may energetically drive filament termination by the capping proteins and recruitment of the actin-like proteins by the extended regions, two key features of the dynactin filament assembly process. Next, we modeled the unknown binding configuration among dynactin, dynein tails, and a number of coiled-coil adaptor proteins (including several Bicaudal-D and related proteins and three HOOK proteins), and predicted a key set of charged residues involved in their electrostatic interactions. Our modeling is consistent with previous findings of conserved regions, functional sites, and disease mutations in the adaptor proteins and will provide a structural framework for future functional and mutational studies of these adaptor proteins. In sum, this study yielded rich structural and energetic information about dynactin and associated adaptor proteins that cannot be directly obtained from the cryo-EM structures with limited resolutions.

Consequences of Molecular-Scale Non-Equilibrium Activity on the Dynamics and Mechanics of Self-Assembled Actin-Based Structures and Materials

NASA Astrophysics Data System (ADS)

Marshall Mccall, Patrick

Living cells are hierarchically self-organized forms of active soft matter: molecules on the nanometer scale form functional structures and organelles on the micron scale, which then compose cells on the scale of 10s of microns. While the biological functions of intracellular organelles are defined by the composition and properties of the structures themselves, how those bulk properties emerge from the properties and interactions of individual molecules remains poorly understood. Actin, a globular protein which self-assembles into dynamic semi-flexible polymers, is the basic structural material of cells and the major component of many functional organelles. In this thesis, I have used purified actin as a model system to explore the interplay between molecular-scale dynamics and organelle-scale functionality, with particular focus on the role of molecular-scale non-equilibrium activity. One of the most canonical forms of molecular-scale non-equilibrium activity is that of mechanoenzymes, also called motor proteins. These proteins utilized the free energy liberated by hydrolysis of ATP to perform mechanical work, thereby introducing non-equilibrium "active" stresses on the molecular scale. Combining experiments with mathematical modeling, we demonstrate in this thesis that non-equilibrium motor activity is sufficient to drive self-organization and pattern formation of the multimeric actin-binding motor protein Myosin II on 1D reconstituted actomyosin bundles. Like myosin, actin is itself an ATPase. However, nono-equilibrium ATP hydrolysis on actin is known to regulate the stability and assembly kinetics of actin filaments rather than generate active stresses per se. At the level of single actin filaments, the inhomogeneous nucleotide composition generated along the filament length by hydrolysis directs binding of regulatory proteins like cofilin, which mediate filament disassembly and thereby accelerate actin filament turnover. The concequences of this non-equilibrium turnover on the steady-state properties of collections of filaments remained unclear. Here, I reconstituted tunable, non-equilibrium actin turnover dynamics in entangled solutions of actin filaments as a model of the actin cortex of living cells. We found that this non-equilibrium turnover decouples solution mechanics from microstructure, enabling structurally indistinguishable materials to behave effectively as either viscous fluids or elastic gels. Additionally, we employed computer simulations to identify the dynamical regime in which actin turnover controls the effective viscosity of 2D cross-linked actin networks in the presence of motors. Additionally, I examine in this thesis the localization and self-assembly of actin filaments in condensed liquid phases called polyelectrolyte coacervates as a model membrane-less organelle. We find that concentration of actin through spontaneous partitioning preferentially to the coacervate phase accelerates the assembly of filaments. These filaments then localize to the coacervate-bulk interface, generating particles with visco-elastic shells surrounding liquid cores. In this case, the properties of the condensed phase enable regulation of actin assembly dynamics.

Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites

PubMed Central

Haas, Kalina T; Lee, MiYoung; Esposito, Alessandro; Venkitaraman, Ashok R

2018-01-01

Abstract RAD51 recombinase assembles on single-stranded (ss)DNA substrates exposed by DNA end-resection to initiate homologous recombination (HR), a process fundamental to genome integrity. RAD51 assembly has been characterized using purified proteins, but its ultrastructural topography in the cell nucleus is unexplored. Here, we combine cell genetics with single-molecule localization microscopy and a palette of bespoke analytical tools, to visualize molecular transactions during RAD51 assembly in the cellular milieu at resolutions approaching 30–40 nm. In several human cell types, RAD51 focalizes in clusters that progressively extend into long filaments, which abut—but do not overlap—with globular bundles of replication protein A (RPA). Extended filaments alter topographically over time, suggestive of succeeding steps in HR. In cells depleted of the tumor suppressor protein BRCA2, or overexpressing its RAD51-binding BRC repeats, RAD51 fails to assemble at damage sites, although RPA accumulates unhindered. By contrast, in cells lacking a BRCA2 carboxyl (C)-terminal region targeted by cancer-causing mutations, damage-induced RAD51 assemblies initiate but do not extend into filaments. We suggest a model wherein RAD51 assembly proceeds concurrently with end-resection at adjacent sites, via an initiation step dependent on the BRC repeats, followed by filament extension through the C-terminal region of BRCA2. PMID:29309696

MIDAS: A Modular DNA Assembly System for Synthetic Biology.

PubMed

van Dolleweerd, Craig J; Kessans, Sarah A; Van de Bittner, Kyle C; Bustamante, Leyla Y; Bundela, Rudranuj; Scott, Barry; Nicholson, Matthew J; Parker, Emily J

2018-04-20

A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements. We describe the detailed design and use of MIDAS, exemplified by the reconstruction, in the filamentous fungus Penicillium paxilli, of the metabolic pathway for production of paspaline and paxilline, key intermediates in the biosynthesis of a range of indole diterpenes-a class of secondary metabolites produced by several species of filamentous fungi. MIDAS was used to efficiently assemble a 25.2 kb plasmid from 21 different modules (seven genes, each composed of three basic parts). By using a parts library-based system for construction of complex assemblies, and a unique set of vectors, MIDAS can provide a flexible route to assembling tailored combinations of genes and other genetic elements, thereby supporting synthetic biology applications in a wide range of expression hosts.

Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

PubMed Central

Carlier, Marie-France; Laurent, Valérie; Santolini, Jérôme; Melki, Ronald; Didry, Dominique; Xia, Gui-Xian; Hong, Yan; Chua, Nam-Hai; Pantaloni, Dominique

1997-01-01

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics. PMID:9087445

Myosin isoform switching during assembly of the Drosophila flight muscle thick filament lattice.

PubMed

Orfanos, Zacharias; Sparrow, John C

2013-01-01

During muscle development myosin molecules form symmetrical thick filaments, which integrate with the thin filaments to produce the regular sarcomeric lattice. In Drosophila indirect flight muscles (IFMs) the details of this process can be studied using genetic approaches. The weeP26 transgenic line has a GFP-encoding exon inserted into the single Drosophila muscle myosin heavy chain gene, Mhc. The weeP26 IFM sarcomeres have a unique MHC-GFP-labelling pattern restricted to the sarcomere core, explained by non-translation of the GFP exon following alternative splicing. Characterisation of wild-type IFM MHC mRNA confirmed the presence of an alternately spliced isoform, expressed earlier than the major IFM-specific isoform. The two wild-type IFM-specific MHC isoforms differ by the presence of a C-terminal 'tailpiece' in the minor isoform. The sequential expression and assembly of these two MHCs into developing thick filaments suggest a role for the tailpiece in initiating A-band formation. The restriction of the MHC-GFP sarcomeric pattern in weeP26 is lifted when the IFM lack the IFM-specific myosin binding protein flightin, suggesting that it limits myosin dissociation from thick filaments. Studies of flightin binding to developing thick filaments reveal a progressive binding at the growing thick filament tips and in a retrograde direction to earlier assembled, proximal filament regions. We propose that this flightin binding restricts myosin molecule incorporation/dissociation during thick filament assembly and explains the location of the early MHC isoform pattern in the IFM A-band.

Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

PubMed Central

Sborgi, Lorenzo; Ravotti, Francesco; Dandey, Venkata P.; Dick, Mathias S.; Mazur, Adam; Reckel, Sina; Chami, Mohamed; Scherer, Sebastian; Huber, Matthias; Böckmann, Anja; Egelman, Edward H.; Stahlberg, Henning; Broz, Petr; Meier, Beat H.; Hiller, Sebastian

2015-01-01

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms. PMID:26464513

Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast.

PubMed

Skau, Colleen T; Courson, David S; Bestul, Andrew J; Winkelman, Jonathan D; Rock, Ronald S; Sirotkin, Vladimir; Kovar, David R

2011-07-29

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.

A silk purse from a sow's ear-bioinspired materials based on α-helical coiled coils.

PubMed

Quinlan, Roy A; Bromley, Elizabeth H; Pohl, Ehmke

2015-02-01

This past few years have heralded remarkable times for intermediate filaments with new revelations of their structural properties that has included the first crystallographic-based model of vimentin to build on the experimental data of intra-filament interactions determined by chemical cross-linking. Now with these and other advances on their assembly, their biomechanical and their cell biological properties outlined in this review, the exploitation of the biomechanical and structural properties of intermediate filaments, their nanocomposites and biomimetic derivatives in the biomedical and private sectors has started. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Using active colloids as machines to weave and braid on the micrometer scale

NASA Astrophysics Data System (ADS)

Goodrich, Carl P.; Brenner, Michael P.

2017-01-01

Controlling motion at the microscopic scale is a fundamental goal in the development of biologically inspired systems. We show that the motion of active, self-propelled colloids can be sufficiently controlled for use as a tool to assemble complex structures such as braids and weaves out of microscopic filaments. Unlike typical self-assembly paradigms, these structures are held together by geometric constraints rather than adhesive bonds. The out-of-equilibrium assembly that we propose involves precisely controlling the 2D motion of active colloids so that their path has a nontrivial topology. We demonstrate with proof-of-principle Brownian dynamics simulations that, when the colloids are attached to long semiflexible filaments, this motion causes the filaments to braid. The ability of the active particles to provide sufficient force necessary to bend the filaments into a braid depends on a number of factors, including the self-propulsion mechanism, the properties of the filament, and the maximum curvature in the braid. Our work demonstrates that nonequilibrium assembly pathways can be designed using active particles.

Using active colloids as machines to weave and braid on the micrometer scale

PubMed Central

Goodrich, Carl P.; Brenner, Michael P.

2017-01-01

Controlling motion at the microscopic scale is a fundamental goal in the development of biologically inspired systems. We show that the motion of active, self-propelled colloids can be sufficiently controlled for use as a tool to assemble complex structures such as braids and weaves out of microscopic filaments. Unlike typical self-assembly paradigms, these structures are held together by geometric constraints rather than adhesive bonds. The out-of-equilibrium assembly that we propose involves precisely controlling the 2D motion of active colloids so that their path has a nontrivial topology. We demonstrate with proof-of-principle Brownian dynamics simulations that, when the colloids are attached to long semiflexible filaments, this motion causes the filaments to braid. The ability of the active particles to provide sufficient force necessary to bend the filaments into a braid depends on a number of factors, including the self-propulsion mechanism, the properties of the filament, and the maximum curvature in the braid. Our work demonstrates that nonequilibrium assembly pathways can be designed using active particles. PMID:28034922

High-resolution Observations of Sympathetic Filament Eruptions by NVST

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Shangwei; Su, Yingna; Zhou, Tuanhui

We investigate two sympathetic filament eruptions observed by the New Vacuum Solar Telescope on 2015 October 15. The full picture of the eruptions is obtained from the corresponding Solar Dynamics Observatory ( SDO )/Atmospheric Imaging Assembly (AIA) observations. The two filaments start from active region NOAA 12434 in the north and end in one large quiescent filament channel in the south. The left filament erupts first, followed by the right filament eruption about 10 minutes later. Clear twist structure and rotating motion are observed in both filaments during the eruption. Both eruptions failed, since the filaments first rise up, thenmore » flow toward the south and merge into the southern large quiescent filament. We also observe repeated activations of mini filaments below the right filament after its eruption. Using magnetic field models constructed based on SDO /HMI magnetograms via the flux rope insertion method, we find that the left filament eruption is likely to be triggered by kink instability, while the weakening of overlying magnetic fields due to magnetic reconnection at an X-point between the two filament systems might play an important role in the onset of the right filament eruption.« less

WAVE binds Ena/VASP for enhanced Arp2/3 complex–based actin assembly

PubMed Central

Havrylenko, Svitlana; Noguera, Philippe; Abou-Ghali, Majdouline; Manzi, John; Faqir, Fahima; Lamora, Audrey; Guérin, Christophe; Blanchoin, Laurent; Plastino, Julie

2015-01-01

The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex–based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly. PMID:25355952

Physical principles of filamentous protein self-assembly kinetics

NASA Astrophysics Data System (ADS)

Michaels, Thomas C. T.; Liu, Lucie X.; Meisl, Georg; Knowles, Tuomas P. J.

2017-04-01

The polymerization of proteins and peptides into filamentous supramolecular structures is an elementary form of self-organization of key importance to the functioning biological systems, as in the case of actin biofilaments that compose the cellular cytoskeleton. Aberrant filamentous protein self-assembly, however, is associated with undesired effects and severe clinical disorders, such as Alzheimer’s and Parkinson’s diseases, which, at the molecular level, are associated with the formation of certain forms of filamentous protein aggregates known as amyloids. Moreover, due to their unique physicochemical properties, protein filaments are finding extensive applications as biomaterials for nanotechnology. With all these different factors at play, the field of filamentous protein self-assembly has experienced tremendous activity in recent years. A key question in this area has been to elucidate the microscopic mechanisms through which filamentous aggregates emerge from dispersed proteins with the goal of uncovering the underlying physical principles. With the latest developments in the mathematical modeling of protein aggregation kinetics as well as the improvement of the available experimental techniques it is now possible to tackle many of these complex systems and carry out detailed analyses of the underlying microscopic steps involved in protein filament formation. In this paper, we review some classical and modern kinetic theories of protein filament formation, highlighting their use as a general strategy for quantifying the molecular-level mechanisms and transition states involved in these processes.

Structural analysis and modeling reveals new mechanisms governing ESCRT-III spiral filament assembly

PubMed Central

Shen, Qing-Tao; Schuh, Amber L.; Zheng, Yuqing; Quinney, Kyle; Wang, Lei; Hanna, Michael; Mitchell, Julie C.; Otegui, Marisa S.; Ahlquist, Paul; Cui, Qiang

2014-01-01

The scission of biological membranes is facilitated by a variety of protein complexes that bind and manipulate lipid bilayers. ESCRT-III (endosomal sorting complex required for transport III) filaments mediate membrane scission during the ostensibly disparate processes of multivesicular endosome biogenesis, cytokinesis, and retroviral budding. However, mechanisms by which ESCRT-III subunits assemble into a polymer remain unknown. Using cryogenic electron microscopy (cryo-EM), we found that the full-length ESCRT-III subunit Vps32/CHMP4B spontaneously forms single-stranded spiral filaments. The resolution afforded by two-dimensional cryo-EM combined with molecular dynamics simulations revealed that individual Vps32/CHMP4B monomers within a filament are flexible and able to accommodate a range of bending angles. In contrast, the interface between monomers is stable and refractory to changes in conformation. We additionally found that the carboxyl terminus of Vps32/CHMP4B plays a key role in restricting the lateral association of filaments. Our findings highlight new mechanisms by which ESCRT-III filaments assemble to generate a unique polymer capable of membrane remodeling in multiple cellular contexts. PMID:25202029

Profilin-Dependent Nucleation and Assembly of Actin Filaments Controls Cell Elongation in Arabidopsis1[OPEN

PubMed Central

Cao, Lingyan; Blanchoin, Laurent; Staiger, Christopher J.

2016-01-01

Actin filaments in plant cells are incredibly dynamic; they undergo incessant remodeling and assembly or disassembly within seconds. These dynamic events are choreographed by a plethora of actin-binding proteins, but the exact mechanisms are poorly understood. Here, we dissect the contribution of Arabidopsis (Arabidopsis thaliana) PROFILIN1 (PRF1), a conserved actin monomer-binding protein, to actin organization and single filament dynamics during axial cell expansion of living epidermal cells. We found that reduced PRF1 levels enhanced cell and organ growth. Surprisingly, we observed that the overall frequency of nucleation events in prf1 mutants was dramatically decreased and that a subpopulation of actin filaments that assemble at high rates was reduced. To test whether profilin cooperates with plant formin proteins to execute actin nucleation and rapid filament elongation in cells, we used a pharmacological approach. Here, we used Small Molecule Inhibitor of Formin FH2 (SMIFH2), after validating its mode of action on a plant formin in vitro, and observed a reduced nucleation frequency of actin filaments in live cells. Treatment of wild-type epidermal cells with SMIFH2 mimicked the phenotype of prf1 mutants, and the nucleation frequency in prf1-2 mutant was completely insensitive to these treatments. Our data provide compelling evidence that PRF1 coordinates the stochastic dynamic properties of actin filaments by modulating formin-mediated actin nucleation and assembly during plant cell expansion. PMID:26574597

Self-assembly of nematic liquid crystal elastomer filaments

NASA Astrophysics Data System (ADS)

Wei, Wei-Shao; Xia, Yu; Yang, Shu; Yodh, A. G.

In this work we investigate the self-assembly of nematic liquid crystal polymer (NLCP) filaments and their corresponding cross-linked elastomer structures. Specifically, by fine-tuning surfactant concentration, prepolymer chain length, and temperature within a background aqueous phase we can generate filaments composed of oligomerized LC monomers. Filaments with narrowly dispersed diameters ranging from one hundred nanometers to a few micrometers can be obtained. Using polarization optical microscopy, we show that the nematic LCs within the filaments have an escaped radial structure. After photo-cross-linking, nematic liquid crystal elastomer filaments are obtained with well-maintained directors and smooth surface structure. Since these materials are elastomers, the size and mechanical and optical response of the filaments can be ''tuned'' near the nematic to isotropic phase transition temperature. This work is supported by NSF DMR16-07378, PENN MRSEC Grant DMR11-20901, and NASA Grant NNX08AO0G.

Drosophila homologues of adenomatous polyposis coli (APC) and the formin diaphanous collaborate by a conserved mechanism to stimulate actin filament assembly.

PubMed

Jaiswal, Richa; Stepanik, Vince; Rankova, Aneliya; Molinar, Olivia; Goode, Bruce L; McCartney, Brooke M

2013-05-10

Vertebrate APC collaborates with Dia through its Basic domain to assemble actin filaments. Despite limited sequence homology between the vertebrate and Drosophila APC Basic domains, Drosophila APC1 collaborates with Dia to stimulate actin assembly in vitro. The mechanism of actin assembly is highly conserved over evolution. APC-Dia collaborations may be crucial in a wide range of animal cells. Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad range of animal phyla.

Understanding nucleotide-regulated FtsZ filament dynamics and the monomer assembly switch with large-scale atomistic simulations.

PubMed

Ramírez-Aportela, Erney; López-Blanco, José Ramón; Andreu, José Manuel; Chacón, Pablo

2014-11-04

Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg(2+) ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.

MinCD cell division proteins form alternating co-polymeric cytomotive filaments

PubMed Central

Ghosal, Debnath; Trambaiolo, Daniel; Amos, Linda A.; Löwe, Jan

2014-01-01

Summary During bacterial cell division, filaments of the tubulin-like protein FtsZ assemble at midcell to form the cytokinetic Z-ring. Its positioning is regulated by the oscillation of MinCDE proteins. MinC is activated by MinD through an unknown mechanism and prevents Z-ring assembly anywhere but midcell. Here, using X-ray crystallography, electron microscopy and in vivo analyses we show that MinD activates MinC by forming a new class of alternating copolymeric filaments that show similarity to eukaryotic septin filaments A non-polymerising mutation in MinD causes aberrant cell division in E. coli. MinCD copolymers bind to membrane, interact with FtsZ, and are disassembled by MinE. Imaging a functional msfGFP-MinC fusion protein in MinE deleted cells reveals filamentous structures. EM imaging of our reconstitution of the MinCD-FtsZ interaction on liposome surfaces reveals a plausible mechanism for regulation of FtsZ ring assembly by MinCD copolymers. PMID:25500731

Investigation of International Space Station Major Constituent Analyzer Anomalous ORU 02 Performance

NASA Technical Reports Server (NTRS)

Gardner, Ben D.; Burchfield, David E.; Pargellis, Andrew; Erwin, Phillip M.; Thoresen, Souzan; Gentry, Grey; Granahan, John; Matty, Chris

2013-01-01

The Major Constituent Analyzer (MCA) is a mass spectrometer based system that measures the major atmospheric constituents on the International Space Station. In 2011, two MCA ORU 02 analyzer assemblies experienced premature on-orbit failures. These failures were determined to be the result of off-nominal ion source filament performance. Recent product improvements to ORU 02 designed to improve the lifetime of the ion pump also constrained the allowable tuning criteria for the ion source filaments. This presentation describes the filament failures as well as the corrective actions implemented to preclude such failures in the future.

Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament

PubMed Central

Ma, Chu Jian; Gibb, Bryan; Kwon, YoungHo; Sung, Patrick; Greene, Eric C.

2017-01-01

Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly. PMID:27903895

A Structural Model for the Single-Stranded DNA Genome of Filamentous Bacteriophage Pf1†

PubMed Central

Tsuboi, Masamichi; Tsunoda, Masaru; Overman, Stacy A.; Benevides, James M.; Thomas, George J.

2010-01-01

The filamentous bacteriophage Pf1, which infects strain PAK of Pseudomonas aeruginosa, is a flexible filament (~2000 × 6.5 nm) consisting of a covalently closed DNA loop of 7349 nucleotides sheathed by 7350 copies of a 46-residue α-helical subunit. The subunit α-helices, which are inclined at a small average angle (~16°) from the virion axis, are arranged compactly around the DNA core. Orientations of the Pf1 DNA nucleotides with respect to the filament axis are not known. In this work we report and interpret the polarized Raman spectra of oriented Pf1 filaments. We demonstrate that the polarizations of DNA Raman band intensities establish that the nucleotide bases of packaged Pf1 DNA are well ordered within the virion and that the base planes are positioned close to parallel to the filament axis. The present results are combined with a previously proposed projection of the intraviral path of Pf1 DNA (1) to develop a novel molecular model for the Pf1 assembly. PMID:20078135

Explicit demonstration of the role of Marangoni effect in the breakup of nanoscale liquid filaments

NASA Astrophysics Data System (ADS)

Seric, Ivana; Mahady, Kyle; Afkhami, Shahriar; Hartnett, Chris; Fowlkes, Jason; Rack, Philip; Kondic, Lou

2016-11-01

We consider a breakup of bi-metal filaments deposited on a solid substrate. These filaments are exposed to laser irradiation and, while in the liquid phase, evolve by a process resembling breakup of a liquid jet governed by the Rayleigh-Plateau instability. The novel element is that the Marangoni effect, resulting from a different surface tension of the two metals from which the filament is built, is crucial in understanding the instability development. In particular, Marangoni effect may lead to the inversion of the breakup process, producing droplets at the locations where according to the Rayleigh-Plateau theory dry spots would be expected. We present experimental results carried out with Cu-Ni filaments, as well as direct numerical simulations based on a novel algorithm that includes variable surface tension in a Volume-of-Fluid based Navier-Stokes solver. These results suggest the possibility of using Marangoni effect for the purpose of self- and directed-assembly on the nanoscale. Supported by the NSF Grant No. CBET-1604351.

Solving a meiotic LEGO puzzle: transverse filaments and the assembly of the synaptonemal complex in Caenorhabditis elegans.

PubMed

Hawley, R Scott

2011-10-01

The structure of the meiosis-specific synaptonemal complex, which is perhaps the central visible characteristic of meiotic prophase, has been a matter of intense interest for decades. Although a general picture of the interactions between the transverse filament proteins that create this structure has emerged from studies in a variety of organisms, a recent analysis of synaptonemal complex structure in Caenorhabditis elegans by Schild-Prüfert et al. (2011) has provided the clearest picture of the structure of the architecture of a synaptonemal complex to date. Although the transverse filaments of the worm synaptonemal complex are assembled differently then those observed in yeast, mammalian, and Drosophila synaptonemal complexes, a comparison of the four assemblies shows that achieving the overall basic structure of the synaptonemal complex is far more crucial than conserving the structures of the individual transverse filaments.

Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament.

PubMed

Ma, Chu Jian; Gibb, Bryan; Kwon, YoungHo; Sung, Patrick; Greene, Eric C

2017-01-25

Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Filament structure, organization, and dynamics in MreB sheets.

PubMed

Popp, David; Narita, Akihiro; Maeda, Kayo; Fujisawa, Tetsuro; Ghoshdastider, Umesh; Iwasa, Mitsusada; Maéda, Yuichiro; Robinson, Robert C

2010-05-21

In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several mum long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions.

Analysis of Septin Reorganization at Cytokinesis Using Polarized Fluorescence Microscopy

PubMed Central

McQuilken, Molly; Jentzsch, Maximilian S.; Verma, Amitabh; Mehta, Shalin B.; Oldenbourg, Rudolf; Gladfelter, Amy S.

2017-01-01

Septins are conserved filament-forming proteins that act in diverse cellular processes. They closely associate with membranes and, in some systems, components of the cytoskeleton. It is not well understood how filaments assemble into higher-order structures in vivo or how they are remodeled throughout the cell cycle. In the budding yeast S. cerevisiae, septins are found through most of the cell cycle in an hourglass organization at the mother-bud neck until cytokinesis when the collar splits into two rings that disassemble prior to the next cell cycle. Experiments using polarized fluorescence microscopy have suggested that septins are arranged in ordered, paired filaments in the hourglass and undergo a coordinated 90° reorientation during splitting at cytokinesis. This apparent reorganization could be due to two orthogonal populations of filaments disassembling and reassembling or being preferentially retained at cytokinesis. In support of this idea, we report a decrease in septin concentration at the mother-bud neck during cytokinesis consistent with other reports and the timing of the decrease depends on known septin regulators including the Gin4 kinase. We took a candidate-based approach to examine what factors control reorientation during splitting and used polarized fluorescence microscopy to screen mutant yeast strains deficient in septin interacting proteins. Using this method, we have linked known septin regulators to different aspects of the assembly, stability, and reorganization of septin assemblies. The data support that ring splitting requires Gin4 activity and an anillin-like protein Bud4, and normal accumulation of septins at the ring requires phosphorylation of Shs1. We found distinct regulatory requirements for septin organization in the hourglass compared to split rings. We propose that septin subpopulations can vary in their localization and assembly/disassembly behavior in a cell-cycle dependent manner at cytokinesis. PMID:28516085

Self-assembling enzymes and the origins of the cytoskeleton

PubMed Central

Barry, Rachael; Gitai, Zemer

2011-01-01

The bacterial cytoskeleton is composed of a complex and diverse group of proteins that self-assemble into linear filaments. These filaments support and organize cellular architecture and provide a dynamic network controlling transport and localization within the cell. Here, we review recent discoveries related to a newly appreciated class of self-assembling proteins that expand our view of the bacterial cytoskeleton and provide potential explanations for its evolutionary origins. Specifically, several types of metabolic enzymes can form structures similar to established cytoskeletal filaments and, in some cases, these structures have been repurposed for structural uses independent of their normal role. The behaviors of these enzymes suggest that some modern cytoskeletal proteins may have evolved from dual-role proteins with catalytic and structural functions. PMID:22014508

Assembly and Function of the Actin Cytoskeleton of Yeast: Relationships between Cables and Patches

PubMed Central

Karpova, Tatiana S.; McNally, James G.; Moltz, Samuel L.; Cooper, John A.

1998-01-01

Actin in eukaryotic cells is found in different pools, with filaments being organized into a variety of supramolecular assemblies. To investigate the assembly and functional relationships between different parts of the actin cytoskeleton in one cell, we studied the morphology and dynamics of cables and patches in yeast. The fine structure of actin cables and the manner in which cables disassemble support a model in which cables are composed of a number of overlapping actin filaments. No evidence for intrinsic polarity of cables was found. To investigate to what extent different parts of the actin cytoskeleton depend on each other, we looked for relationships between cables and patches. Patches and cables were often associated, and their polarized distributions were highly correlated. Therefore, patches and cables do appear to depend on each other for assembly and function. Many cell types show rearrangements of the actin cytoskeleton, which can occur via assembly or movement of actin filaments. In our studies, dramatic changes in actin polarization did not include changes in filamentous actin. In addition, the concentration of actin patches was relatively constant as cells grew. Therefore, cells do not have bursts of activity in which new parts of the actin cytoskeleton are created. PMID:9744880

Amyloid Oligomers and Protofibrils, but Not Filaments, Self-Replicate from Native Lysozyme

PubMed Central

2015-01-01

Self-assembly of amyloid fibrils is the molecular mechanism best known for its connection with debilitating human disorders such as Alzheimer’s disease but is also associated with various functional cellular responses. There is increasing evidence that amyloid formation proceeds along two distinct assembly pathways involving either globular oligomers and protofibrils or rigid monomeric filaments. Oligomers, in particular, have been implicated as the dominant molecular species responsible for pathogenesis. Yet the molecular mechanisms regulating their self-assembly have remained elusive. Here we show that oligomers/protofibrils and monomeric filaments, formed along distinct assembly pathways, display critical differences in their ability to template amyloid growth at physiological vs denaturing temperatures. At physiological temperatures, amyloid filaments remained stable but could not seed growth of native monomers. In contrast, oligomers and protofibrils not only remained intact but were capable of self-replication using native monomers as the substrate. Kinetic data further suggested that this prion-like growth mode of oligomers/protofibrils involved two distinct activities operating orthogonal from each other: autocatalytic self-replication of oligomers from native monomers and nucleated polymerization of oligomers into protofibrils. The environmental changes to stability and templating competence of these different amyloid species in different environments are likely to be important for understanding the molecular mechanisms underlying both pathogenic and functional amyloid self-assembly. PMID:24884889

Amyloid oligomers and protofibrils, but not filaments, self-replicate from native lysozyme.

PubMed

Mulaj, Mentor; Foley, Joseph; Muschol, Martin

2014-06-25

Self-assembly of amyloid fibrils is the molecular mechanism best known for its connection with debilitating human disorders such as Alzheimer's disease but is also associated with various functional cellular responses. There is increasing evidence that amyloid formation proceeds along two distinct assembly pathways involving either globular oligomers and protofibrils or rigid monomeric filaments. Oligomers, in particular, have been implicated as the dominant molecular species responsible for pathogenesis. Yet the molecular mechanisms regulating their self-assembly have remained elusive. Here we show that oligomers/protofibrils and monomeric filaments, formed along distinct assembly pathways, display critical differences in their ability to template amyloid growth at physiological vs denaturing temperatures. At physiological temperatures, amyloid filaments remained stable but could not seed growth of native monomers. In contrast, oligomers and protofibrils not only remained intact but were capable of self-replication using native monomers as the substrate. Kinetic data further suggested that this prion-like growth mode of oligomers/protofibrils involved two distinct activities operating orthogonal from each other: autocatalytic self-replication of oligomers from native monomers and nucleated polymerization of oligomers into protofibrils. The environmental changes to stability and templating competence of these different amyloid species in different environments are likely to be important for understanding the molecular mechanisms underlying both pathogenic and functional amyloid self-assembly.

Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

PubMed Central

Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R.; Drubin, David G.

2016-01-01

Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin–Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

β-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR

PubMed Central

Vasa, Suresh; Lin, Lin; Shi, Chaowei; Habenstein, Birgit; Riedel, Dietmar; Kühn, Juliane; Thanbichler, Martin; Lange, Adam

2015-01-01

Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly β-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified β-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of β-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a β-helical architecture, in which 18 β-strand segments are arranged in six consecutive windings of a β-helix. PMID:25550503

β-Helical architecture of cytoskeletal bactofilin filaments revealed by solid-state NMR.

PubMed

Vasa, Suresh; Lin, Lin; Shi, Chaowei; Habenstein, Birgit; Riedel, Dietmar; Kühn, Juliane; Thanbichler, Martin; Lange, Adam

2015-01-13

Bactofilins are a widespread class of bacterial filament-forming proteins, which serve as cytoskeletal scaffolds in various cellular pathways. They are characterized by a conserved architecture, featuring a central conserved domain (DUF583) that is flanked by variable terminal regions. Here, we present a detailed investigation of bactofilin filaments from Caulobacter crescentus by high-resolution solid-state NMR spectroscopy. De novo sequential resonance assignments were obtained for residues Ala39 to Phe137, spanning the conserved DUF583 domain. Analysis of the secondary chemical shifts shows that this core region adopts predominantly β-sheet secondary structure. Mutational studies of conserved hydrophobic residues located in the identified β-strand segments suggest that bactofilin folding and polymerization is mediated by an extensive and redundant network of hydrophobic interactions, consistent with the high intrinsic stability of bactofilin polymers. Transmission electron microscopy revealed a propensity of bactofilin to form filament bundles as well as sheet-like, 2D crystalline assemblies, which may represent the supramolecular arrangement of bactofilin in the native context. Based on the diffraction pattern of these 2D crystalline assemblies, scanning transmission electron microscopy measurements of the mass per length of BacA filaments, and the distribution of β-strand segments identified by solid-state NMR, we propose that the DUF583 domain adopts a β-helical architecture, in which 18 β-strand segments are arranged in six consecutive windings of a β-helix.

Electrohydrodynamic Printing and Manufacturing

NASA Technical Reports Server (NTRS)

Aksay, Ilhan A. (Inventor); Poon, Hak Fei (Inventor); Korkut, Sibel (Inventor); Saville, Dudley A. (Inventor); Chen, Chuan-hua (Inventor)

2014-01-01

An stable electrohydrodynamic filament is obtained by causing a straight electrohydrodynamic filament formed from a liquid to emerge from a Taylor cone, the filament having a diameter of from 10 nm to 100.mu.m. Such filaments are useful in electrohydrodynamic printing and manufacturing techniques and their application in liquid drop/particle and fiber production, colloidal deployment and assembly, and composite materials processing.

A filamentous molecular chaperone of the prefoldin family from the deep-sea hyperthermophile Methanocaldococcus jannaschii.

PubMed

Whitehead, Timothy A; Boonyaratanakornkit, Boonchai B; Höllrigl, Volker; Clark, Douglas S

2007-04-01

Prefoldin is a molecular chaperone found in the domains eukarya and archaea that acts in conjunction with Group II chaperonin to correctly fold other nascent proteins. Previously, our group identified a putative single subunit of prefoldin, gamma PFD, that was up-regulated in response to heat stress in the hyperthermophilic archaeon Methanocaldococcus jannaschii. In order to characterize this protein, we subcloned and expressed it and the other two prefoldin subunits from M. jannaschii, alpha and beta PFD, into Eschericia coli and characterized the proteins. Whereas alpha and beta PFD readily assembled into the expected hexamer, gamma PFD would not assemble with either protein. Instead, gamma PFD forms long filaments of defined dimensions measuring 8.5 nm x 1.7-3.5 nm and lengths exceeding 1 microm. Filamentous gamma PFD acts as a molecular chaperone through in vitro assays, in a manner comparable to PFD. A possible molecular model for filament assembly is discussed.

A filamentous molecular chaperone of the prefoldin family from the deep-sea hyperthermophile Methanocaldococcus jannaschii

PubMed Central

Whitehead, Timothy A.; Boonyaratanakornkit, Boonchai B.; Höllrigl, Volker; Clark, Douglas S.

2007-01-01

Prefoldin is a molecular chaperone found in the domains eukarya and archaea that acts in conjunction with Group II chaperonin to correctly fold other nascent proteins. Previously, our group identified a putative single subunit of prefoldin, γ PFD, that was up-regulated in response to heat stress in the hyperthermophilic archaeon Methanocaldococcus jannaschii. In order to characterize this protein, we subcloned and expressed it and the other two prefoldin subunits from M. jannaschii, α and β PFD, into Eschericia coli and characterized the proteins. Whereas α and β PFD readily assembled into the expected hexamer, γ PFD would not assemble with either protein. Instead, γ PFD forms long filaments of defined dimensions measuring 8.5 nm × 1.7–3.5 nm and lengths exceeding 1 μm. Filamentous γ PFD acts as a molecular chaperone through in vitro assays, in a manner comparable to PFD. A possible molecular model for filament assembly is discussed. PMID:17384227

Single-Molecule Studies of Actin Assembly and Disassembly Factors

PubMed Central

Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.

2014-01-01

The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103

Rigid indented cylindrical cathode for X-ray tube

DOEpatents

Hudgens, Claude R.

1985-01-01

A cathode assembly for a vacuum tube includes a wire filament, a straight bular anode parallel to and surrounding the wire filament, and insulating spacers for rigidly fastening the filament with respect to the anode, and with one side of the anode indented or flattened such that only one portion of the anode is heated to emitting temperatures by the filament.

Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly

PubMed Central

Risco, Cristina; Rodríguez, Juan R.; López-Iglesias, Carmen; Carrascosa, José L.; Esteban, Mariano; Rodríguez, Dolores

2002-01-01

Vaccinia virus (VV) has a complex morphogenetic pathway whose first steps are poorly characterized. We have studied the early phase of VV assembly, when viral factories and spherical immature viruses (IVs) form in the cytoplasm of the infected cell. After freeze-substitution numerous cellular elements are detected around assembling viruses: membranes, ribosomes, microtubules, filaments, and unidentified structures. A double membrane is clearly resolved in the VV envelope for the first time, and freeze fracture reveals groups of tubules interacting laterally on the surface of the viroplasm foci. These data strongly support the hypothesis of a cellular tubulovesicular compartment, related to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), as the origin of the first VV envelope. Moreover, the cytoskeletal vimentin intermediate filaments are found around viral factories and inside the viroplasm foci, where vimentin and the VV core protein p39 colocalize in the areas where crescents protrude. Confocal microscopy showed that ERGIC elements and vimentin filaments concentrate in the viral factories. We propose that modified cellular ERGIC membranes and vimentin intermediate filaments act coordinately in the construction of viral factories and the first VV form through a unique mechanism of viral morphogenesis from cellular elements. PMID:11799179

Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics.

PubMed

Appaduray, Mark A; Masedunskas, Andrius; Bryce, Nicole S; Lucas, Christine A; Warren, Sean C; Timpson, Paul; Stear, Jeffrey H; Gunning, Peter W; Hardeman, Edna C

2016-01-01

The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.

Assembly of MreB filaments on liposome membranes: a synthetic biology approach.

PubMed

Maeda, Yusuke T; Nakadai, Tomoyoshi; Shin, Jonghyeon; Uryu, Kunihiro; Noireaux, Vincent; Libchaber, Albert

2012-02-17

The physical interaction between the cytoskeleton and the cell membrane is essential in defining the morphology of living organisms. In this study, we use a synthetic approach to polymerize bacterial MreB filaments inside phospholipid vesicles. When the proteins MreB and MreC are expressed inside the liposomes, the MreB cytoskeleton structure develops at the inner membrane. Furthermore, when purified MreB is used inside the liposomes, MreB filaments form a 4-10 μm rigid bundle structure and deform the lipid vesicles in physical contact with the vesicle inner membrane. These results indicate that the fibrillation of MreB filaments can take place either in close proximity of deformable lipid membrane or in the presence of associated protein. Our finding might be relevant for the self-assembly of cytoskeleton filaments toward the construction of artificial cell systems.

Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers

PubMed Central

Kiuchi, Tai; Ohashi, Kazumasa; Kurita, Souichi; Mizuno, Kensaku

2007-01-01

Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using photoactivatable Dronpa-actin, we show that cofilin is involved in producing more than half of all cytoplasmic actin monomers and that the rate of actin monomer incorporation into the tip of the lamellipodium is dependent on the size of this actin monomer pool. Finally, in cofilin-depleted cells, stimulus-induced actin monomer incorporation at the cell periphery is attenuated, but the incorporation of microinjected actin monomers is not. We propose that cofilin contributes to stimulus-induced actin filament assembly and lamellipodium extension by supplying an abundant pool of cytoplasmic actin monomers. PMID:17470633

ZOMG - III. The effect of halo assembly on the satellite population

NASA Astrophysics Data System (ADS)

Garaldi, Enrico; Romano-Díaz, Emilio; Borzyszkowski, Mikolaj; Porciani, Cristiano

2018-01-01

We use zoom hydrodynamical simulations to investigate the properties of satellites within galaxy-sized dark-matter haloes with different assembly histories. We consider two classes of haloes at redshift z = 0: 'stalled' haloes that assembled at z > 1 and 'accreting' ones that are still forming nowadays. Previously, we showed that the stalled haloes are embedded within thick filaments of the cosmic web, while the accreting ones lie where multiple thin filaments converge. We find that satellites in the two classes have both similar and different properties. Their mass spectra, radial count profiles, baryonic and stellar content, and the amount of material they shed are indistinguishable. However, the mass fraction locked in satellites is substantially larger for the accreting haloes as they experience more mergers at late times. The largest difference is found in the satellite kinematics. Substructures fall towards the accreting haloes along quasi-radial trajectories whereas an important tangential velocity component is developed, before accretion, while orbiting the filament that surrounds the stalled haloes. Thus, the velocity anisotropy parameter of the satellites (β) is positive for the accreting haloes and negative for the stalled ones. This signature enables us to tentatively categorize the Milky Way halo as stalled based on a recent measurement of β. Half of our haloes contain clusters of satellites with aligned orbital angular momenta corresponding to flattened structures in space. These features are not driven by baryonic physics and are only found in haloes hosting grand-design spiral galaxies, independently of their assembly history.

Filament-wound spar shell graphite/epoxy fan blades

NASA Technical Reports Server (NTRS)

Yao, S.

1976-01-01

The methodology for fabrication of wet filament wound spar shell fan blades is presented. All principal structural elements were filament wound, assembled, formed, bonded and co-cured in a female mold. A pair of blades were fabricated as one integral unit and parted into two after curing.

Mechanical force antagonizes the inhibitory effects of RecX on RecA filament formation in Mycobacterium tuberculosis

PubMed Central

Le, Shimin; Chen, Hu; Zhang, Xinghua; Chen, Jin; Patil, K. Neelakanteshwar; Muniyappa, Kalappa; Yan, Jie

2014-01-01

Efficient bacterial recombinational DNA repair involves rapid cycles of RecA filament assembly and disassembly. The RecX protein plays a crucial inhibitory role in RecA filament formation and stability. As the broken ends of DNA are tethered during homologous search, RecA filaments assembled at the ends are likely subject to force. In this work, we investigated the interplay between RecX and force on RecA filament formation and stability. Using magnetic tweezers, at single molecular level, we found that Mycobacterium tuberculosis (Mt) RecX could catalyze stepwise de-polymerization of preformed MtRecA filament in the presence of ATP hydrolysis at low forces (<7 pN). However, applying larger forces antagonized the inhibitory effects of MtRecX, and a partially de-polymerized MtRecA filament could re-polymerize in the presence of MtRecX, which cannot be explained by previous models. Theoretical analysis of force-dependent conformational free energies of naked ssDNA and RecA nucleoprotein filament suggests that mechanical force stabilizes RecA filament, which provides a possible mechanism for the observation. As the antagonizing effect of force on the inhibitory function of RecX takes place in a physiological range; these findings broadly suggest a potential mechanosensitive regulation during homologous recombination. PMID:25294832

STEM Analysis of Caenorhabditis elegans muscle thick filaments: evidence for microdifferentiated substructures

NASA Technical Reports Server (NTRS)

Muller, S. A.; Haner, M.; Ortiz, I.; Aebi, U.; Epstein, H. F.

2001-01-01

In the thick filaments of body muscle in Caenorhabditis elegans, myosin A and myosin B isoforms and a subpopulation of paramyosin, a homologue of myosin heavy chain rods, are organized about a tubular core. As determined by scanning transmission electron microscopy, the thick filaments show a continuous decrease in mass-per-length (MPL) from their central zones to their polar regions. This is consistent with previously reported morphological studies and suggests that both their content and structural organization are microdifferentiated as a function of position. The cores are composed of a second distinct subpopulation of paramyosin in association with the alpha, beta, and gamma-filagenins. MPL measurements suggest that cores are formed from seven subfilaments containing four strands of paramyosin molecules, rather than the two originally proposed. The periodic locations of the filagenins within different regions and the presence of a central zone where myosin A is located, implies that the cores are also microdifferentiated with respect to molecular content and structure. This differentiation may result from a novel "induced strain" assembly mechanism based upon the interaction of the filagenins, paramyosin and myosin A. The cores may then serve as "differentiated templates" for the assembly of myosin B and paramyosin in the tapering, microdifferentiated polar regions of the thick filaments.

Assembly of bipolar microtubule structures by passive cross-linkers and molecular motors

NASA Astrophysics Data System (ADS)

Johann, D.; Goswami, D.; Kruse, K.

2016-06-01

During cell division, sister chromatids are segregated by the mitotic spindle, a bipolar assembly of interdigitating antiparallel polar filaments called microtubules. The spindle contains the midzone, a stable region of overlapping antiparallel microtubules, that is essential for maintaining bipolarity. Although a lot is known about the molecular players involved, the mechanism underlying midzone formation and maintenance is still poorly understood. We study the interaction of polar filaments that are cross-linked by molecular motors moving directionally and by passive cross-linkers diffusing along microtubules. Using a particle-based stochastic model, we find that the interplay of motors and passive cross-linkers can generate a stable finite overlap between a pair of antiparallel polar filaments. We develop a mean-field theory to study this mechanism in detail and investigate the influence of steric interactions between motors and passive cross-linkers on the overlap dynamics. In the presence of interspecies steric interactions, passive cross-linkers mimic the behavior of molecular motors and stable finite overlaps are generated even for non-cross-linking motors. Finally, we develop a mean-field theory for a bundle of aligned polar filaments and show that they can self-organize into a spindlelike pattern. Our work suggests possible ways as to how cells can generate spindle midzones and control their extensions.

Intelligent electrical harness connector assembly using Bell Helicopter Textron's 'Wire Harness Automated Manufacturing System'

NASA Astrophysics Data System (ADS)

Springer, D. W.

Bell Helicopter Textron, Incorporated (BHTI) installed two Digital Equipment Corporation PDP-11 computers and an American Can Inc. Ink Jet printer in 1980 as the cornerstone of the Wire Harness Automated Manufacturing System (WHAMS). WHAMS is based upon the electrical assembly philosophy of continuous filament harness forming. This installation provided BHTI with a 3 to 1 return-on-investment by reducing wire and cable identification cycle time by 80 percent and harness forming, on dedicated layout tooling, by 40 percent. Yet, this improvement in harness forming created a bottle neck in connector assembly. To remove this bottle neck, BHTI has installed a prototype connector assembly cell that integrates the WHAMS' data base and innovative computer technologies to cut harness connector assembly cycle time. This novel connector assembly cell uses voice recognition, laser identification, and animated computer graphics to help the electrician in the correct assembly of harness connectors.

Tracking Filament Evolution in the Low Solar Corona Using Remote Sensing and In Situ Observations

NASA Astrophysics Data System (ADS)

Kocher, Manan; Landi, Enrico; Lepri, Susan. T.

2018-06-01

In the present work, we analyze a filament eruption associated with an interplanetary coronal mass ejection that arrived at L1 on 2011 August 5. In multiwavelength Solar Dynamic Observatory/Advanced Imaging Assembly (AIA) images, three plasma parcels within the filament were tracked at high cadence along the solar corona. A novel absorption diagnostic technique was applied to the filament material traveling along the three chosen trajectories to compute the column density and temperature evolution in time. Kinematics of the filamentary material were estimated using STEREO/Extreme Ultraviolet Imager and STEREO/COR1 observations. The Michigan Ionization Code used inputs of these density, temperature, and speed profiles for the computation of ionization profiles of the filament plasma. Based on these measurements, we conclude that the core plasma was in near ionization equilibrium, and the ionization states were still evolving at the altitudes where they were visible in absorption in AIA images. Additionally, we report that the filament plasma was heterogeneous, and the filamentary material was continuously heated as it expanded in the low solar corona.

Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane

PubMed Central

Gokhin, David S.; Nowak, Roberta B.; Khoory, Joseph A.; de la Piedra, Alfonso; Ghiran, Ionita C.; Fowler, Velia M.

2015-01-01

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (∼25–30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ∼60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane. PMID:25717184

Multi-wavelength Observation of Filament Eruption associated with M-class Flare

NASA Astrophysics Data System (ADS)

Kim, S.; Yurchyshyn, V.; Jiang, C.

2017-12-01

We have investigated a M-class flare associated with filament eruption which developed into a Halo CME. The M-class flare occurred in 2011 August 4. For this study, we used the Nobryama Radioheliograph (NoRH) 17 and 34 GHz, RHESSI Hard X-ray satellite, and Atmo- spheric Imaging Assembly (AIA) and the Heliospheric Magentic Imager(HMI) onboard the Solar Dynamic Observatory (SDO). During the pre-eruption phase, clear nonthermal emission was detected in microwaves of NoRH and hard-X-ray of RHESSI. At the moment that the nonthermal emission start, the nonthermal sources appeared at the one edge of the filament structure on a polarity inversion line, and the slowing rising filament structure in AIA 94A underwent a sudden acceleration on its ascendance. Magnetograms showed converging motion of magnetic elements at the source position of HXR and MW. Based on the results, we conjecture that the plausible trigger of the filament eruption is magnetic reconnections at the HXR source position by converging motion of magnetic elements. In addition, we will discuss on the magnetic flux variation before and after the eruption based on the result of Nonlinear force-free field model.

A disassembly-driven mechanism explains F-actin-mediated chromosome transport in starfish oocytes

PubMed Central

Bun, Philippe; Dmitrieff, Serge; Belmonte, Julio M

2018-01-01

While contraction of sarcomeric actomyosin assemblies is well understood, this is not the case for disordered networks of actin filaments (F-actin) driving diverse essential processes in animal cells. For example, at the onset of meiosis in starfish oocytes a contractile F-actin network forms in the nuclear region transporting embedded chromosomes to the assembling microtubule spindle. Here, we addressed the mechanism driving contraction of this 3D disordered F-actin network by comparing quantitative observations to computational models. We analyzed 3D chromosome trajectories and imaged filament dynamics to monitor network behavior under various physical and chemical perturbations. We found no evidence of myosin activity driving network contractility. Instead, our observations are well explained by models based on a disassembly-driven contractile mechanism. We reconstitute this disassembly-based contractile system in silico revealing a simple architecture that robustly drives chromosome transport to prevent aneuploidy in the large oocyte, a prerequisite for normal embryonic development. PMID:29350616

Formin and capping protein together embrace the actin filament in a ménage à trois

PubMed Central

Shekhar, Shashank; Kerleau, Mikael; Kühn, Sonja; Pernier, Julien; Romet-Lemonne, Guillaume; Jégou, Antoine; Carlier, Marie-France

2015-01-01

Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed. PMID:26564775

Tropomodulin isoforms regulate thin filament pointed-end capping and skeletal muscle physiology

PubMed Central

Gokhin, David S.; Lewis, Raymond A.; McKeown, Caroline R.; Nowak, Roberta B.; Kim, Nancy E.; Littlefield, Ryan S.; Lieber, Richard L.

2010-01-01

During myofibril assembly, thin filament lengths are precisely specified to optimize skeletal muscle function. Tropomodulins (Tmods) are capping proteins that specify thin filament lengths by controlling actin dynamics at pointed ends. In this study, we use a genetic targeting approach to explore the effects of deleting Tmod1 from skeletal muscle. Myofibril assembly, skeletal muscle structure, and thin filament lengths are normal in the absence of Tmod1. Tmod4 localizes to thin filament pointed ends in Tmod1-null embryonic muscle, whereas both Tmod3 and -4 localize to pointed ends in Tmod1-null adult muscle. Substitution by Tmod3 and -4 occurs despite their weaker interactions with striated muscle tropomyosins. However, the absence of Tmod1 results in depressed isometric stress production during muscle contraction, systemic locomotor deficits, and a shift to a faster fiber type distribution. Thus, Tmod3 and -4 compensate for the absence of Tmod1 structurally but not functionally. We conclude that Tmod1 is a novel regulator of skeletal muscle physiology. PMID:20368620

Synthetic inhibitors of bacterial cell division targeting the GTP-binding site of FtsZ.

PubMed

Ruiz-Avila, Laura B; Huecas, Sonia; Artola, Marta; Vergoñós, Albert; Ramírez-Aportela, Erney; Cercenado, Emilia; Barasoain, Isabel; Vázquez-Villa, Henar; Martín-Fontecha, Mar; Chacón, Pablo; López-Rodríguez, María L; Andreu, José M

2013-09-20

Cell division protein FtsZ is the organizer of the cytokinetic Z-ring in most bacteria and a target for new antibiotics. FtsZ assembles with GTP into filaments that hydrolyze the nucleotide at the association interface between monomers and then disassemble. We have replaced FtsZ's GTP with non-nucleotide synthetic inhibitors of bacterial division. We searched for these small molecules among compounds from the literature, from virtual screening (VS), and from our in-house synthetic library (UCM), employing a fluorescence anisotropy primary assay. From these screens we have identified the polyhydroxy aromatic compound UCM05 and its simplified analogue UCM44 that specifically bind to Bacillus subtilis FtsZ monomers with micromolar affinities and perturb normal assembly, as examined with light scattering, polymer sedimentation, and negative stain electron microscopy. On the other hand, these ligands induce the cooperative assembly of nucleotide-devoid archaeal FtsZ into distinct well-ordered polymers, different from GTP-induced filaments. These FtsZ inhibitors impair localization of FtsZ into the Z-ring and inhibit bacterial cell division. The chlorinated analogue UCM53 inhibits the growth of clinical isolates of antibiotic-resistant Staphylococcus aureus and Enterococcus faecalis. We suggest that these interfacial inhibitors recapitulate binding and some assembly-inducing effects of GTP but impair the correct structural dynamics of FtsZ filaments and thus inhibit bacterial division, possibly by binding to a small fraction of the FtsZ molecules in a bacterial cell, which opens a new approach to FtsZ-based antibacterial drug discovery.

Draft Genome Assembly of a Filamentous Euendolithic (True Boring) Cyanobacterium, Mastigocoleus testarum Strain BC008.

PubMed

Guida, Brandon S; Garcia-Pichel, Ferran

2016-01-28

Mastigocoleus testarum strain BC008 is a model organism used to study marine photoautotrophic carbonate dissolution. It is a multicellular, filamentous, diazotrophic, euendolithic cyanobacterium ubiquitously found in marine benthic environments. We present an accurate draft genome assembly of 172 contigs spanning 12,700,239 bp with 9,131 annotated genes with an average G+C% of 37.3. Copyright © 2016 Guida and Garcia-Pichel.

Kinetic signature of fractal-like filament networks formed by orientational linear epitaxy.

PubMed

Hwang, Wonmuk; Eryilmaz, Esma

2014-07-11

We study a broad class of epitaxial assembly of filament networks on lattice surfaces. Over time, a scale-free behavior emerges with a 2.5-3 power-law exponent in filament length distribution. Partitioning between the power-law and exponential behaviors in a network can be used to find the stage and kinetic parameters of the assembly process. To analyze real-world networks, we develop a computer program that measures the network architecture in experimental images. Application to triaxial networks of collagen fibrils shows quantitative agreement with our model. Our unifying approach can be used for characterizing and controlling the network formation that is observed across biological and nonbiological systems.

Direct membrane binding by bacterial actin MreB.

PubMed

Salje, Jeanne; van den Ent, Fusinita; de Boer, Piet; Löwe, Jan

2011-08-05

Bacterial actin MreB is one of the key components of the bacterial cytoskeleton. It assembles into short filaments that lie just underneath the membrane and organize the cell wall synthesis machinery. Here we show that MreB from both T. maritima and E. coli binds directly to cell membranes. This function is essential for cell shape determination in E. coli and is proposed to be a general property of many, if not all, MreBs. We demonstrate that membrane binding is mediated by a membrane insertion loop in TmMreB and by an N-terminal amphipathic helix in EcMreB and show that purified TmMreB assembles into double filaments on a membrane surface that can induce curvature. This, the first example of a membrane-binding actin filament, prompts a fundamental rethink of the structure and dynamics of MreB filaments within cells. Copyright © 2011 Elsevier Inc. All rights reserved.

Bacterial flagella grow through an injection-diffusion mechanism.

PubMed

Renault, Thibaud T; Abraham, Anthony O; Bergmiller, Tobias; Paradis, Guillaume; Rainville, Simon; Charpentier, Emmanuelle; Guet, Călin C; Tu, Yuhai; Namba, Keiichi; Keener, James P; Minamino, Tohru; Erhardt, Marc

2017-03-06

The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially ∼1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell.

ASC filament formation serves as a signal amplification mechanism for inflammasomes

PubMed Central

Dick, Mathias S.; Sborgi, Lorenzo; Rühl, Sebastian; Hiller, Sebastian; Broz, Petr

2016-01-01

A hallmark of inflammasome activation is the ASC speck, a micrometre-sized structure formed by the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), which consists of a pyrin domain (PYD) and a caspase recruitment domain (CARD). Here we show that assembly of the ASC speck involves oligomerization of ASCPYD into filaments and cross-linking of these filaments by ASCCARD. ASC mutants with a non-functional CARD only assemble filaments but not specks, and moreover disrupt endogenous specks in primary macrophages. Systematic site-directed mutagenesis of ASCPYD is used to identify oligomerization-deficient ASC mutants and demonstrate that ASC speck formation is required for efficient processing of IL-1β, but dispensable for gasdermin-D cleavage and pyroptosis induction. Our results suggest that the oligomerization of ASC creates a multitude of potential caspase-1 activation sites, thus serving as a signal amplification mechanism for inflammasome-mediated cytokine production. PMID:27329339

Accessory factors promote AlfA-dependent plasmid segregation by regulating filament nucleation, disassembly, and bundling

PubMed Central

Polka, Jessica K.; Kollman, Justin M.; Mullins, R. Dyche

2014-01-01

In bacteria, some plasmids are partitioned to daughter cells by assembly of actin-like proteins (ALPs). The best understood ALP, ParM, has a core set of biochemical properties that contributes to its function, including dynamic instability, spontaneous nucleation, and bidirectional elongation. AlfA, an ALP that pushes plasmids apart in Bacillus, relies on a different set of underlying properties to segregate DNA. AlfA elongates unidirectionally and is not dynamically unstable; its assembly and disassembly are regulated by a cofactor, AlfB. Free AlfB breaks up AlfA bundles and promotes filament turnover. However, when AlfB is bound to the centromeric DNA sequence, parN, it forms a segrosome complex that nucleates and stabilizes AlfA filaments. When reconstituted in vitro, this system creates polarized, motile comet tails that associate by antiparallel filament bundling to form bipolar, DNA-segregating spindles. PMID:24481252

Strain-stiffening response in organogels assembled using steroidal biomolecules

NASA Astrophysics Data System (ADS)

Tung, Shih-Huang; Raghavan, Srinivasa R.

2007-03-01

The phenomenon of strain-stiffening or strain-hardening refers to an increase in the elastic modulus (stiffness) of a material with increasing strain amplitude. While this response is exhibited by many biological materials, including gels of biopolymers such as actin, it is rarely seen in other types of soft matter. Here, we report strain-stiffening in a new class of self- assembled organogels being studied in our laboratory. These gels are formed in nonpolar organic liquids by combining a lipid (lecithin) or two-tailed surfactant (AOT) with a type of naturally occurring steroidal amphiphile called a bile salt. Based on rheological and scattering data, we deduce that the gel structure comprises a network of semiflexible filaments. Interestingly, gels induced by small organic molecules other than bile salts do not show strain-stiffening. We suggest that the bile salt molecules confer an intrinsic stiffness to the filaments in the gel, which is important for strain-stiffening.

Septins - active GTPases or just GTP-binding proteins?

PubMed

Abbey, Megha; Gaestel, Matthias; Menon, Manoj B

2018-05-10

Septins are conserved cytoskeletal proteins with unique filament forming capabilities and roles in cytokinesis and cell morphogenesis. Septins undergo hetero-oligomerization and assemble into higher order structures including filaments, rings and cages. Hetero- and homotypic interactions of septin isoforms involve alternating GTPase (G)-domain interfaces and those mediated by N- and C-terminal extensions. While most septins bind GTP, display weak GTP-hydrolysis activity and incorporate guanine nucleotides in their interaction interfaces, studies using GTPase-inactivating mutations have failed to conclusively establish a crucial role for GTPase activity in mediating septin functions. In this mini-review, we will critically assess the role of GTP-binding and -hydrolysis on septin assembly and function. The relevance of G-domain activity will also be discussed in the context of human septin mutations as well as the development of specific small-molecules targeting septin polymerization. As structural determinants of septin oligomer interfaces, G-domains are attractive targets for ligand-based inhibition of septin assembly. Whether such an intervention can predictably alter septin function is a major question for future research. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Nucleation of actin polymerization by gelsolin.

PubMed

Ditsch, A; Wegner, A

1994-08-15

The time-course of assembly of actin with gelsolin was measured by the fluorescence increase of a fluorescent label covalently linked to actin. The actin concentrations ranged from values far below the critical concentration to values above the critical concentration of the pointed ends of actin filaments. If the concentration of actin was in the range of the critical monomer concentration (0.64 microM), the time-course of the concentration of actin assembled with gelsolin revealed a sigmoidal shape. At higher actin concentrations the time-course of association of actin with gelsolin approximated an exponential curve. The measured time-courses of assembly were quantitatively interpreted by kinetic rate equations. A poor fit was obtained if two actin molecules were assumed to bind to gelsolin to form a 1:2 gelsolin-actin complex and subsequently further actin molecules were assumed to polymerize onto the 1:2 gelsolin-actin complex toward the pointed end. A considerably better agreement between calculated and measured time-courses was achieved if additional creation of actin filaments by fast fragmentation of newly formed actin filaments by not yet consumed gelsolin was assumed to occur. This suggests that both polymerization of actin onto gelsolin and fragmentation of actin filaments contribute to formation of new actin filaments by gelsolin. Furthermore it could be demonstrated that below the critical monomer concentration appreciable amounts of actin are incorporated into gelsolin-actin oligomers.

High pressure storage vessel

DOEpatents

Liu, Qiang

2013-08-27

Disclosed herein is a composite pressure vessel with a liner having a polar boss and a blind boss a shell is formed around the liner via one or more filament wrappings continuously disposed around at least a substantial portion of the liner assembly combined the liner and filament wrapping have a support profile. To reduce susceptible to rupture a locally disposed filament fiber is added.

Dynamic DNA devices and assemblies formed by shape-complementary, non-base pairing 3D components

NASA Astrophysics Data System (ADS)

Gerling, Thomas; Wagenbauer, Klaus F.; Neuner, Andrea M.; Dietz, Hendrik

2015-03-01

We demonstrate that discrete three-dimensional (3D) DNA components can specifically self-assemble in solution on the basis of shape-complementarity and without base pairing. Using this principle, we produced homo- and heteromultimeric objects, including micrometer-scale one- and two-stranded filaments and lattices, as well as reconfigurable devices, including an actuator, a switchable gear, an unfoldable nanobook, and a nanorobot. These multidomain assemblies were stabilized via short-ranged nucleobase stacking bonds that compete against electrostatic repulsion between the components’ interfaces. Using imaging by electron microscopy, ensemble and single-molecule fluorescence resonance energy transfer spectroscopy, and electrophoretic mobility analysis, we show that the balance between attractive and repulsive interactions, and thus the conformation of the assemblies, may be finely controlled by global parameters such as cation concentration or temperature and by an allosteric mechanism based on strand-displacement reactions.

Loss of Smyhc1 or Hsp90α1 Function Results in Different Effects on Myofibril Organization in Skeletal Muscles of Zebrafish Embryos

PubMed Central

Codina, Marta; Li, Junling; Gutiérrez, Joaquim; Kao, Joseph P. Y.; Du, Shao Jun

2010-01-01

Background Myofibrillogenesis requires the correct folding and assembly of sarcomeric proteins into highly organized sarcomeres. Heat shock protein 90α1 (Hsp90α1) has been implicated as a myosin chaperone that plays a key role in myofibrillogenesis. Knockdown or mutation of hsp90α1 resulted in complete disorganization of thick and thin filaments and M- and Z-line structures. It is not clear whether the disorganization of these sarcomeric structures is due to a direct effect from loss of Hsp90α1 function or indirectly through the disorganization of myosin thick filaments. Methodology/Principal Findings In this study, we carried out a loss-of-function analysis of myosin thick filaments via gene-specific knockdown or using a myosin ATPase inhibitor BTS (N-benzyl-p-toluene sulphonamide) in zebrafish embryos. We demonstrated that knockdown of myosin heavy chain 1 (myhc1) resulted in sarcomeric defects in the thick and thin filaments and defective alignment of Z-lines. Similarly, treating zebrafish embryos with BTS disrupted thick and thin filament organization, with little effect on the M- and Z-lines. In contrast, loss of Hsp90α1 function completely disrupted all sarcomeric structures including both thick and thin filaments as well as the M- and Z-lines. Conclusion/Significance Together, these studies indicate that the hsp90α1 mutant phenotype is not simply due to disruption of myosin folding and assembly, suggesting that Hsp90α1 may play a role in the assembly and organization of other sarcomeric structures. PMID:20049323

Decidable and undecidable arithmetic functions in actin filament networks

NASA Astrophysics Data System (ADS)

Schumann, Andrew

2018-01-01

The plasmodium of Physarum polycephalum is very sensitive to its environment, and reacts to stimuli with appropriate motions. Both the sensory and motor stages of these reactions are explained by hydrodynamic processes, based on fluid dynamics, with the participation of actin filament networks. This paper is devoted to actin filament networks as a computational medium. The point is that actin filaments, with contributions from many other proteins like myosin, are sensitive to extracellular stimuli (attractants as well as repellents), and appear and disappear at different places in the cell to change aspects of the cell structure—e.g. its shape. By assembling and disassembling actin filaments, some unicellular organisms, like Amoeba proteus, can move in response to various stimuli. As a result, these organisms can be considered a simple reversible logic gate—extracellular signals being its inputs and motions its outputs. In this way, we can implement various logic gates on amoeboid behaviours. These networks can embody arithmetic functions within p-adic valued logic. Furthermore, within these networks we can define the so-called diagonalization for deducing undecidable arithmetic functions.

Molecular Precision at Micrometer Length Scales: Hierarchical Assembly of DNA-Protein Nanostructures.

PubMed

Schiffels, Daniel; Szalai, Veronika A; Liddle, J Alexander

2017-07-25

Robust self-assembly across length scales is a ubiquitous feature of biological systems but remains challenging for synthetic structures. Taking a cue from biology-where disparate molecules work together to produce large, functional assemblies-we demonstrate how to engineer microscale structures with nanoscale features: Our self-assembly approach begins by using DNA polymerase to controllably create double-stranded DNA (dsDNA) sections on a single-stranded template. The single-stranded DNA (ssDNA) sections are then folded into a mechanically flexible skeleton by the origami method. This process simultaneously shapes the structure at the nanoscale and directs the large-scale geometry. The DNA skeleton guides the assembly of RecA protein filaments, which provides rigidity at the micrometer scale. We use our modular design strategy to assemble tetrahedral, rectangular, and linear shapes of defined dimensions. This method enables the robust construction of complex assemblies, greatly extending the range of DNA-based self-assembly methods.

Constitutive Models for the Force-Extension Behavior of Biological Filaments

NASA Astrophysics Data System (ADS)

Palmer, J. S.; Castro, C. E.; Arslan, M.; Boyce, M. C.

Biopolymer filaments form the molecular backbone of biological structures throughout the body. The biomechanical response of single filaments yields insight into their individual behavior at the molecular level as well as their concerted networked behavior at the cellular and tissue scales. This paper focuses on modeling approaches for axial force vs. extension behavior of single biopolymer filaments within three stiffness regimes: flexible, semiflexible, and stiff. The end-to-end force-extension behaviors of flexible and semiflexible filaments arise as a result of a reduction in configurational space as the filament is straightened and are captured with entropic models including the freely jointed chain model and the worm-like chain model. As the filament is straightened and the end-to-end distance approaches the filament contour length, the contour length is directly axially extended and an internal energy contribution governs the force-extension behavior in this limiting extension regime. On the other hand, for stiff filaments in originally crimped or kinked configurations, the end-to-end force vs. extension behavior results from the unbending (straightening) of the crimped configuration as governed by an internal energy based elastica approximation which is also complemented by an axial stretching contribution once the end-to-end distance approaches the contour length of the filament. Simplified, analytical force-extension relationships are developed for the worm-like chain model for semiflexible filaments, and for the Euler elastica model for stiffer, wavy fibers. For the case of flexible molecules containing modular folded domains, the influence of force-induced unfolding on the force-extension behavior of single molecules and assemblies of multiple molecules is also presented.

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

PubMed Central

Nowrousian, Minou; Stajich, Jason E.; Chu, Meiling; Engh, Ines; Espagne, Eric; Halliday, Karen; Kamerewerd, Jens; Kempken, Frank; Knab, Birgit; Kuo, Hsiao-Che; Osiewacz, Heinz D.; Pöggeler, Stefanie; Read, Nick D.; Seiler, Stephan; Smith, Kristina M.; Zickler, Denise; Kück, Ulrich; Freitag, Michael

2010-01-01

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology. PMID:20386741

De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.

PubMed

Nowrousian, Minou; Stajich, Jason E; Chu, Meiling; Engh, Ines; Espagne, Eric; Halliday, Karen; Kamerewerd, Jens; Kempken, Frank; Knab, Birgit; Kuo, Hsiao-Che; Osiewacz, Heinz D; Pöggeler, Stefanie; Read, Nick D; Seiler, Stephan; Smith, Kristina M; Zickler, Denise; Kück, Ulrich; Freitag, Michael

2010-04-08

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

Assembly of filopodia by the formin FRL2 (FMNL3)

PubMed Central

Harris, Elizabeth S.; Gauvin, Timothy J.; Heimsath, Ernest G.; Higgs, Henry N.

2010-01-01

Actin-dependent finger-like protrusions such as filopodia and microvilli are widespread in eukaryotes, but their assembly mechanisms are poorly understood. Filopodia assembly requires at least three biochemical activities on actin: actin filament nucleation, prolonged actin filament elongation, and actin filament bundling. These activities are shared by several mammalian formin proteins, including mDia2, FRL1 (also called FMNL1), and FRL2 (FMNL3). In this paper, we compare the abilities of constructs from these three formins to induce filopodia. FH1-FH2 constructs of both FRL2 and mDia2 stimulate potent filopodia assembly in multiple cell types, and enrich strongly at filopodia tips. In contrast, FRL1 FH1-FH2 lacks this activity, despite possessing similar biochemical activities and being highly homologous to FRL2. Chimeric FH1-FH2 experiments between FRL1 and FRL2 show that, while both an FH1 and an FH2 are needed, either FH1 domain supports filopodia assembly but only FRL2’s FH2 domain allows this activity. A mutation that compromises FRL2’s barbed end binding ability abolishes filopodia assembly. FRL2’s ability to stimulate filopodia assembly is not altered by additional domains (GBD, DID, DAD), but is significantly reduced in the full-length construct, suggesting that FRL2 is subject to inhibitory regulation. The data suggest that the FH2 domain of FRL2 possesses properties not shared by FRL1 that allow it to generate filopodia. PMID:20862687

The innate immune sensor LGP2 activates antiviral signaling by regulating MDA5-RNA interaction and filament assembly

PubMed Central

Bruns, Annie M.; Leser, George P.; Lamb, Robert A.; Horvath, Curt M.

2014-01-01

SUMMARY Cytoplasmic pattern recognition receptors detect non-self RNAs during virus infections and initiate antiviral signaling. One receptor, MDA5, possesses essential signaling domains, but weak RNA binding. A second receptor, LGP2, rapidly detects diverse dsRNA species, but lacks signaling domains. Accumulating evidence suggests LGP2 and MDA5 work together to detect viral RNA and generate a complete antiviral response, but the basis for their cooperation has been elusive. Experiments presented here address this gap in antiviral signaling, revealing that LGP2 assists MDA5-RNA interactions leading to enhanced MDA5-mediated antiviral signaling. LGP2 increases the initial rate of MDA5-RNA interaction and regulates MDA5 filament assembly, resulting in the formation of more numerous, shorter MDA5 filaments that are shown to generate equivalent or greater signaling activity in vivo than the longer filaments containing only MDA5. These findings provide a mechanism for LGP2 co-activation of MDA5 and a biological context for MDA5-RNA filaments in antiviral responses. PMID:25127512

The selective autophagy receptor p62 forms a flexible filamentous helical scaffold.

PubMed

Ciuffa, Rodolfo; Lamark, Trond; Tarafder, Abul K; Guesdon, Audrey; Rybina, Sofia; Hagen, Wim J H; Johansen, Terje; Sachse, Carsten

2015-05-05

The scaffold protein p62/SQSTM1 is involved in protein turnover and signaling and is commonly found in dense protein bodies in eukaryotic cells. In autophagy, p62 acts as a selective autophagy receptor that recognizes and shuttles ubiquitinated proteins to the autophagosome for degradation. The structural organization of p62 in cellular bodies and the interplay of these assemblies with ubiquitin and the autophagic marker LC3 remain to be elucidated. Here, we present a cryo-EM structural analysis of p62. Together with structures of assemblies from the PB1 domain, we show that p62 is organized in flexible polymers with the PB1 domain constituting a helical scaffold. Filamentous p62 is capable of binding LC3 and addition of long ubiquitin chains induces disassembly and shortening of filaments. These studies explain how p62 assemblies provide a large molecular scaffold for the nascent autophagosome and reveal how they can bind ubiquitinated cargo. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export

PubMed Central

2018-01-01

The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhAC) forms a nonameric ring structure in the flagellar type III protein export apparatus and coordinates flagellar protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhAC (FlhAL) is required not only for FlhAC ring formation but also for substrate specificity switching of the protein export apparatus from the hook protein to the filament protein upon completion of the hook structure. FlhAL was required for cooperative ring formation of FlhAC. Alanine substitutions of residues involved in FlhAC ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhAC. PMID:29707633

Filter holder assembly having extended collar spacer ring

DOEpatents

Alvin, Mary Anne; Bruck, Gerald J.

2002-01-01

A filter holder assembly is provided that utilizes a fail-safe regenerator unit with an annular spacer ring having an extended metal collar for containment and positioning of a compliant ceramic gasket used in the assembly. The filter holder assembly is disclosed for use with advanced composite, filament wound, and metal candle filters.

Structure determination of helical filaments by solid-state NMR spectroscopy

PubMed Central

Ahmed, Mumdooh; Spehr, Johannes; König, Renate; Lünsdorf, Heinrich; Rand, Ulfert; Lührs, Thorsten; Ritter, Christiane

2016-01-01

The controlled formation of filamentous protein complexes plays a crucial role in many biological systems and represents an emerging paradigm in signal transduction. The mitochondrial antiviral signaling protein (MAVS) is a central signal transduction hub in innate immunity that is activated by a receptor-induced conversion into helical superstructures (filaments) assembled from its globular caspase activation and recruitment domain. Solid-state NMR (ssNMR) spectroscopy has become one of the most powerful techniques for atomic resolution structures of protein fibrils. However, for helical filaments, the determination of the correct symmetry parameters has remained a significant hurdle for any structural technique and could thus far not be precisely derived from ssNMR data. Here, we solved the atomic resolution structure of helical MAVSCARD filaments exclusively from ssNMR data. We present a generally applicable approach that systematically explores the helical symmetry space by efficient modeling of the helical structure restrained by interprotomer ssNMR distance restraints. Together with classical automated NMR structure calculation, this allowed us to faithfully determine the symmetry that defines the entire assembly. To validate our structure, we probed the protomer arrangement by solvent paramagnetic resonance enhancement, analysis of chemical shift differences relative to the solution NMR structure of the monomer, and mutagenesis. We provide detailed information on the atomic contacts that determine filament stability and describe mechanistic details on the formation of signaling-competent MAVS filaments from inactive monomers. PMID:26733681

Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin*

PubMed Central

Courson, David S.; Rock, Ronald S.

2010-01-01

Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and α-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. α-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo. PMID:20551315

β-Filagenin, a Newly Identified Protein Coassembling with Myosin and Paramyosin in Caenorhabditis elegans

PubMed Central

Liu, Feizhou; Bauer, Christopher C.; Ortiz, Irving; Cook, Richard G.; Schmid, Michael F.; Epstein, Henry F.

1998-01-01

Muscle thick filaments are stable assemblies of myosin and associated proteins whose dimensions are precisely regulated. The mechanisms underlying the stability and regulation of the assembly are not understood. As an approach to these problems, we have studied the core proteins that, together with paramyosin, form the core structure of the thick filament backbone in the nematode Caenorhabditis elegans. We obtained partial peptide sequences from one of the core proteins, β-filagenin, and then identified a gene that encodes a novel protein of 201–amino acid residues from databases using these sequences. β-Filagenin has a calculated isoelectric point at 10.61 and a high percentage of aromatic amino acids. Secondary structure algorithms predict that it consists of four β-strands but no α-helices. Western blotting using an affinity-purified antibody showed that β-filagenin was associated with the cores. β-Filagenin was localized by immunofluorescence microscopy to the A bands of body–wall muscles, but not the pharynx. β-filagenin assembled with the myosin homologue paramyosin into the tubular cores of wild-type nematodes at a periodicity matching the 72-nm repeats of paramyosin, as revealed by immunoelectron microscopy. In CB1214 mutants where paramyosin is absent, β-filagenin assembled with myosin to form abnormal tubular filaments with a periodicity identical to wild type. These results verify that β-filagenin is a core protein that coassembles with either myosin or paramyosin in C. elegans to form tubular filaments. PMID:9442110

Filamentous phages as building blocks for reconfigurable and hierarchical self-assembly

NASA Astrophysics Data System (ADS)

Gibaud, Thomas

2017-12-01

Filamentous bacteriophages such as fd-like viruses are monodisperse rod-like colloids that have well defined properties of diameter, length, rigidity, charge and chirality. Engineering these viruses leads to a library of colloidal rods, which can be used as building blocks for reconfigurable and hierarchical self-assembly. Their condensation in an aqueous solution with additive polymers, which act as depletants to induce attraction between the rods, leads to a myriad of fluid-like micronic structures ranging from isotropic/nematic droplets, colloid membranes, achiral membrane seeds, twisted ribbons, π-wall, pores, colloidal skyrmions, Möbius anchors, scallop membranes to membrane rafts. These structures, and the way that they shape-shift, not only shed light on the role of entropy, chiral frustration and topology in soft matter, but also mimic many structures encountered in different fields of science. On the one hand, filamentous phages being an experimental realization of colloidal hard rods, their condensation mediated by depletion interactions constitutes a blueprint for the self-assembly of rod-like particles and provides a fundamental foundation for bio- or material-oriented applications. On the other hand, the chiral properties of the viruses restrict the generalities of some results but vastly broaden the self-assembly possibilities.

Spire and Formin 2 synergize and antagonize in regulating actin assembly in meiosis by a ping-pong mechanism.

PubMed

Montaville, Pierre; Jégou, Antoine; Pernier, Julien; Compper, Christel; Guichard, Bérengère; Mogessie, Binyam; Schuh, Melina; Romet-Lemonne, Guillaume; Carlier, Marie-France

2014-02-01

In mammalian oocytes, three actin binding proteins, Formin 2 (Fmn2), Spire, and profilin, synergistically organize a dynamic cytoplasmic actin meshwork that mediates translocation of the spindle toward the cortex and is required for successful fertilization. Here we characterize Fmn2 and elucidate the molecular mechanism for this synergy, using bulk solution and individual filament kinetic measurements of actin assembly dynamics. We show that by capping filament barbed ends, Spire recruits Fmn2 and facilitates its association with barbed ends, followed by rapid processive assembly and release of Spire. In the presence of actin, profilin, Spire, and Fmn2, filaments display alternating phases of rapid processive assembly and arrested growth, driven by a "ping-pong" mechanism, in which Spire and Fmn2 alternately kick off each other from the barbed ends. The results are validated by the effects of injection of Spire, Fmn2, and their interacting moieties in mouse oocytes. This original mechanism of regulation of a Rho-GTPase-independent formin, recruited by Spire at Rab11a-positive vesicles, supports a model for modulation of a dynamic actin-vesicle meshwork in the oocyte at the origin of asymmetric positioning of the meiotic spindle.

The cell division protein MinD from Pseudomonas aeruginosa dominates the assembly of the MinC-MinD copolymers.

PubMed

Huang, Haiyan; Wang, Ping; Bian, Li; Osawa, Masaki; Erickson, Harold P; Chen, Yaodong

2018-05-18

Cell division of rod-shaped bacteria requires the Z ring, a ring of FtsZ filaments associated with the inner-membrane wall. The MinCDE proteins help localize the Z ring to the center of the Escherichia coli cell. MinC, which inhibits Z-ring assembly, is a passenger on MinD. Previous studies have shown that MinC-MinD from E. coli and Aquifex aeolicus assemble in vitro into extended filaments with a 1:1 stoichiometry. However, a recent study has raised questions about the function of the MinC-MinD copolymer in vivo , because its assembly appears to require a high concentration of these two proteins and has a long lag time, and its blockade does not affect in vivo activities. Here, we found that MinC and MinD from Pseudomonas aeruginosa coassemble into filaments with a 1:1 stoichiometry. We also found that the minimal concentration of ∼4 μm required for assembly applies only to MinD because above 4 μm MinD, even very low MinC concentrations sustained coassembly. As previously reported, the MinC-MinD coassembly exhibited a long lag of ∼100 s when initiated by ATP. Premixing MinD with ATP eliminated this lag, suggesting that it may be due to slow MinD dimerization following ATP activation. We also discovered that MinC-MinD copolymers quickly bound FtsZ filaments and formed huge bundles. Our results resolve previous questions about the low concentration of MinC and the lag time, insights that may inform future investigations into the exact role of the MinC-MinD copolymer in vivo . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments*

PubMed Central

Montaville, Pierre; Kühn, Sonja; Compper, Christel; Carlier, Marie-France

2016-01-01

Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end. PMID:26668326

Self-organization and stability of magnetosome chains—A simulation study

PubMed Central

Faivre, Damien; Klumpp, Stefan

2018-01-01

Magnetotactic bacteria orient in magnetic fields with the help of their magnetosome chain, a linear structure of membrane enclosed magnetic nanoparticles (magnetosomes) anchored to a cytoskeletal filament. Here, we use simulations to study the assembly and the stability of magnetosome chains. We introduce a computational model describing the attachment of the magnetosomes to the filament and their magnetic interactions. We show that the filamentous backbone is crucial for the robust assembly of the magnetic particles into a linear chain, which in turn is key for the functionality of the chain in cellular orientation and magnetically directed swimming. In addition, we simulate the response to an external magnetic field that is rotated away from the axis of the filament, an experimental method used to probe the mechanical stability of the chain. The competition between alignment along the filament and alignment with the external fields leads to the rupture of a chain if the applied field exceeeds a threshold value. These observations are in agreement with previous experiments at the population level. Beyond that, our simulations provide a detailed picture of chain rupture at the single cell level, which is found to happen through two abrupt events, which both depend on the field strength and orientation. The re-formation of the chain structure after such rupture is found to be strongly sped up in the presence of a magnetic field parallel to the filament, an observation that may also be of interest for the design of self-healing materials. Our simulations underline the dynamic nature of the magnetosome chain. More generally, they show the rich complexity of self-assembly in systems with competing driving forces for alignment. PMID:29315342

Myosin storage myopathy mutations yield defective myosin filament assembly in vitro and disrupted myofibrillar structure and function in vivo.

PubMed

Viswanathan, Meera C; Tham, Rick C; Kronert, William A; Sarsoza, Floyd; Trujillo, Adriana S; Cammarato, Anthony; Bernstein, Sanford I

2017-12-15

Myosin storage myopathy (MSM) is a congenital skeletal muscle disorder caused by missense mutations in the β-cardiac/slow skeletal muscle myosin heavy chain rod. It is characterized by subsarcolemmal accumulations of myosin that have a hyaline appearance. MSM mutations map near or within the assembly competence domain known to be crucial for thick filament formation. Drosophila MSM models were generated for comprehensive physiological, structural, and biochemical assessment of the mutations' consequences on muscle and myosin structure and function. L1793P, R1845W, and E1883K MSM mutant myosins were expressed in an indirect flight (IFM) and jump muscle myosin null background to study the effects of these variants without confounding influences from wild-type myosin. Mutant animals displayed highly compromised jump and flight ability, disrupted muscle proteostasis, and severely perturbed IFM structure. Electron microscopy revealed myofibrillar disarray and degeneration with hyaline-like inclusions. In vitro assembly assays demonstrated a decreased ability of mutant myosin to polymerize, with L1793P filaments exhibiting shorter lengths. In addition, limited proteolysis experiments showed a reduced stability of L1793P and E1883K filaments. We conclude that the disrupted hydropathy or charge of residues in the heptad repeat of the mutant myosin rods likely alters interactions that stabilize coiled-coil dimers and thick filaments, causing disruption in ordered myofibrillogenesis and/or myofibrillar integrity, and the consequent myosin aggregation. Our Drosophila models are the first to recapitulate the human MSM phenotype with ultrastructural inclusions, suggesting that the diminished ability of the mutant myosin to form stable thick filaments contributes to the dystrophic phenotype observed in afflicted subjects. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Microtubule-based nanomaterials: Exploiting nature's dynamic biopolymers

DOE PAGES

Bachand, George D.; Stevens, Mark J.; Spoerke, Erik David

2015-04-09

For more than a decade now, biomolecular systems have served as an inspiration for the development of synthetic nanomaterials and systems that are capable of reproducing many of unique and emergent behaviors of living systems. In addition, one intriguing element of such systems may be found in a specialized class of proteins known as biomolecular motors that are capable of performing useful work across multiple length scales through the efficient conversion of chemical energy. Microtubule (MT) filaments may be considered within this context as their dynamic assembly and disassembly dissipate energy, and perform work within the cell. MTs are onemore » of three cytoskeletal filaments in eukaryotic cells, and play critical roles in a range of cellular processes including mitosis and vesicular trafficking. Based on their function, physical attributes, and unique dynamics, MTs also serve as a powerful archetype of a supramolecular filament that underlies and drives multiscale emergent behaviors. In this review, we briefly summarize recent efforts to generate hybrid and composite nanomaterials using MTs as biomolecular scaffolds, as well as computational and synthetic approaches to develop synthetic one-dimensional nanostructures that display the enviable attributes of the natural filaments.« less

Bacterial flagella grow through an injection-diffusion mechanism

PubMed Central

Renault, Thibaud T; Abraham, Anthony O; Bergmiller, Tobias; Paradis, Guillaume; Rainville, Simon; Charpentier, Emmanuelle; Guet, Călin C; Tu, Yuhai; Namba, Keiichi; Keener, James P; Minamino, Tohru; Erhardt, Marc

2017-01-01

The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially ∼1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell. DOI: http://dx.doi.org/10.7554/eLife.23136.001 PMID:28262091

Bacterial flagellar capping proteins adopt diverse oligomeric states

DOE Office of Scientific and Technical Information (OSTI.GOV)

Postel, Sandra; Deredge, Daniel; Bonsor, Daniel A.

2016-09-24

Flagella are crucial for bacterial motility and pathogenesis. The flagellar capping protein (FliD) regulates filament assembly by chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip. In the absence of FliD, flagella are not formed, resulting in impaired motility and infectivity. Here, we report the 2.2 Å resolution X-ray crystal structure of FliD fromPseudomonas aeruginosa, the first high-resolution structure of any FliD protein from any bacterium. Using this evidence in combination with a multitude of biophysical and functional analyses, we find thatPseudomonasFliD exhibits unexpected structural similarity to other flagellar proteins atmore » the domain level, adopts a unique hexameric oligomeric state, and depends on flexible determinants for oligomerization. Considering that the flagellin filaments on which FliD oligomers are affixed vary in protofilament number between bacteria, our results suggest that FliD oligomer stoichiometries vary across bacteria to complement their filament assemblies.« less

Mechanics of Lamellipodia

NASA Astrophysics Data System (ADS)

Quint, D. A.; Schwarz, J. M.

2008-03-01

The actin cytoskeleton is a morphologically-complex assembly of cross-linked F-actin filaments. The cytoskeleton provides rigidity for the cell within appropriate time scales so that it can change its shape to, for example, crawl along surfaces. In addition to cross-linking proteins, many other proteins are involved in the assembly of the actin cytoskeleton such as branching proteins, capping proteins, and severing proteins. Presumably these proteins work cooperatively toward the dynamic formation of rigidity. We will initially focus on the role of branching proteins. The F-actin filaments in lamellipodia---protrusions of the mobile edge of a crawling cell---have some overall orientation due to the branching. Branched filaments emerge at a 70 degree angle from the mother filament's growing end.^1 This overall orientation is modelled as an anisotropy in an effective medium theory determining the cytoskeleton's elasticity in the static regime. The potential for a splay rigid phase, in addition to a rigid phase, is also investigated. ^1T. M. Svitkina and G. G. Borisy, J. Cell Biol. 145, 1009 (1999).

MDA5 cooperatively forms dimers and ATP-sensitive filaments upon binding double-stranded RNA

PubMed Central

Berke, Ian C; Modis, Yorgo

2012-01-01

Melanoma differentiation-associated gene-5 (MDA5) detects viral double-stranded RNA in the cytoplasm. RNA binding induces MDA5 to activate the signalling adaptor MAVS through interactions between the caspase recruitment domains (CARDs) of the two proteins. The molecular mechanism of MDA5 signalling is not well understood. Here, we show that MDA5 cooperatively binds short RNA ligands as a dimer with a 16–18-basepair footprint. A crystal structure of the MDA5 helicase-insert domain demonstrates an evolutionary relationship with the archaeal Hef helicases. In X-ray solution structures, the CARDs in unliganded MDA5 are flexible, and RNA binds on one side of an asymmetric MDA5 dimer, bridging the two subunits. On longer RNA, full-length and CARD-deleted MDA5 constructs assemble into ATP-sensitive filaments. We propose a signalling model in which the CARDs on MDA5–RNA filaments nucleate the assembly of MAVS filaments with the same polymeric geometry. PMID:22314235

The titin A-band rod domain is dispensable for initial thick filament assembly in zebrafish.

PubMed

Myhre, J Layne; Hills, Jordan A; Prill, Kendal; Wohlgemuth, Serene L; Pilgrim, David B

2014-03-01

The sarcomeres of skeletal and cardiac muscle are highly structured protein arrays, consisting of thick and thin filaments aligned precisely to one another and to their surrounding matrix. The contractile mechanisms of sarcomeres are generally well understood, but how the patterning of sarcomeres is initiated during early skeletal muscle and cardiac development remains uncertain. Two of the most widely accepted hypotheses for this process include the "molecular ruler" model, in which the massive protein titin defines the length of the sarcomere and provides a scaffold along which the myosin thick filament is assembled, and the "premyofibril" model, which proposes that thick filament formation does not require titin, but that a "premyofibril" consisting of non-muscle myosin, α-actinin and cytoskeletal actin is used as a template. Each model posits a different order of necessity of the various components, but these have been difficult to test in vivo. Zebrafish motility mutants with developmental defects in sarcomere patterning are useful for the elucidation of such mechanisms, and here we report the analysis of the herzschlag mutant, which shows deficits in both cardiac and skeletal muscle. The herzschlag mutant produces a truncated titin protein, lacking the C-terminal rod domain that is proposed to act as a thick filament scaffold, yet muscle patterning is still initiated, with grossly normal thick and thin filament assembly. Only after embryonic muscle contraction begins is breakdown of sarcomeric myosin patterning observed, consistent with the previously noted role of titin in maintaining the contractile integrity of mature sarcomeres. This conflicts with the "molecular ruler" model of early sarcomere patterning and supports a titin-independent model of thick filament organization during sarcomerogenesis. These findings are also consistent with the symptoms of human titin myopathies that exhibit a late onset, such as tibial muscular dystrophy. Copyright © 2013 Elsevier Inc. All rights reserved.

PHD3-mediated prolyl hydroxylation of nonmuscle actin impairs polymerization and cell motility

PubMed Central

Luo, Weibo; Lin, Benjamin; Wang, Yingfei; Zhong, Jun; O'Meally, Robert; Cole, Robert N.; Pandey, Akhilesh; Levchenko, Andre; Semenza, Gregg L.

2014-01-01

Actin filaments play an essential role in cell movement, and many posttranslational modifications regulate actin filament assembly. Here we report that prolyl hydroxylase 3 (PHD3) interacts with nonmuscle actin in human cells and catalyzes hydroxylation of actin at proline residues 307 and 322. Blocking PHD3 expression or catalytic activity by short hairpin RNA knockdown or pharmacological inhibition, respectively, decreased actin prolyl hydroxylation. PHD3 knockdown increased filamentous F-actin assembly, which was reversed by PHD3 overexpression. PHD3 knockdown increased cell velocity and migration distance. Inhibition of PHD3 prolyl hydroxylase activity by dimethyloxalylglycine also increased actin polymerization and cell migration. These data reveal a novel role for PHD3 as a negative regulator of cell motility through posttranslational modification of nonmuscle actins. PMID:25079693

Filamin A Is a Regulator of Blood-Testis Barrier Assembly during Postnatal Development in the Rat Testis

PubMed Central

Su, Wenhui; Mruk, Dolores D.; Lie, Pearl P. Y.; Lui, Wing-yee

2012-01-01

The blood-testis barrier (BTB) is an important ultrastructure in the testis. A delay in its assembly during postnatal development leads to meiotic arrest. Also, a disruption of the BTB by toxicants in adult rats leads to a failure in spermatogonial differentiation. However, the regulation of BTB assembly remains unknown. Herein, filamin A, an actin filament cross-linker that is known to maintain and regulate cytoskeleton structure and function in other epithelia, was shown to be highly expressed during the assembly of Sertoli cell BTB in vitro and postnatal development of BTB in vivo, perhaps being used to maintain the actin filament network at the BTB. A knockdown of filamin A by RNA interference was found to partially perturb the Sertoli cell tight junction (TJ) permeability barrier both in vitro and in vivo. Interestingly, this down-regulating effect on the TJ barrier function after the knockdown of filamin A was associated with a mis-localization of both TJ and basal ectoplasmic specialization proteins. Filamin A knockdown also induced a disorganization of the actin filament network in Sertoli cells in vitro and in vivo. Collectively, these findings illustrate that filamin A regulates BTB assembly by recruiting these proteins to the microenvironment in the seminiferous epithelium to serve as the building blocks. In short, filamin A participates in BTB assembly by regulating protein recruitment during postnatal development in the rat testis. PMID:22872576

International Space Station Major Constituent Analyzer On-Orbit Performance

NASA Technical Reports Server (NTRS)

Gardner, Ben D.; Erwin, Phillip M.; Cougar, Tamara; Ulrich, BettyLynn

2017-01-01

The Major Constituent Analyzer (MCA) is a mass spectrometer based system that measures the major atmospheric constituents on the International Space Station. A number of limited-life components require periodic change-out, including the ORU 02 analyzer and the ORU 08 Verification Gas Assembly. The most recent ORU 02 and ORU 08 assemblies in the LAB MCA are operating nominally. For ORU 02, the ion source filaments and ion pump lifetime continue to be key determinants of MCA performance. Finally, the Node 3 MCA is being brought to an operational configuration.

Cell type–dependent mechanisms for formin-mediated assembly of filopodia

PubMed Central

Young, Lorna E.; Heimsath, Ernest G.; Higgs, Henry N.

2015-01-01

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex–nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in adherent cells. In adherent cells only, mDia1 and VASP also contribute to filopodial assembly, and filopodia are disproportionately associated with focal adhesions. We propose an extension of the existing models for filopodial assembly in which any cluster of actin filament barbed ends in proximity to the plasma membrane, either Arp2/3 complex dependent or independent, can initiate filopodial assembly by specific formins. PMID:26446836

Contractile-Ring Assembly in Fission Yeast Cytokinesis: Recent Advances and New Perspectives

PubMed Central

Lee, I-Ju; Coffman, Valerie C.; Wu, Jian-Qiu

2017-01-01

The fission yeast Schizosaccharomyces pombe is an excellent model organism to study cytokinesis. Here, we review recent advances on contractile-ring assembly in fission yeast. First, we summarize the assembly of cytokinesis nodes, the precursors of a normal contractile ring. IQGAP Rng2 and myosin essential light chain Cdc4 are recruited by the anillin-like protein Mid1, followed by the addition of other cytokinesis node proteins. Mid1 localization on the plasma membrane is stabilized by interphase node proteins. Second, we discuss proteins and processes that contribute to the search, capture, pull, and release mechanism of contractile-ring assembly. Actin filaments nucleated by formin Cdc12, the motor activity of myosin-II, the stiffness of the actin network, and severing of actin filaments by cofilin all play essential roles in contractile-ring assembly. Finally, we discuss the Mid1-independent pathway for ring assembly, and the possible mechanisms underlying the ring maturation and constriction. Collectively, we provide an overview of the current understanding of contractile-ring assembly and uncover future directions in studying cytokinesis in fission yeast. PMID:22887981

Contractile-ring assembly in fission yeast cytokinesis: Recent advances and new perspectives.

PubMed

Lee, I-Ju; Coffman, Valerie C; Wu, Jian-Qiu

2012-10-01

The fission yeast Schizosaccharomyces pombe is an excellent model organism to study cytokinesis. Here, we review recent advances on contractile-ring assembly in fission yeast. First, we summarize the assembly of cytokinesis nodes, the precursors of a normal contractile ring. IQGAP Rng2 and myosin essential light chain Cdc4 are recruited by the anillin-like protein Mid1, followed by the addition of other cytokinesis node proteins. Mid1 localization on the plasma membrane is stabilized by interphase node proteins. Second, we discuss proteins and processes that contribute to the search, capture, pull, and release mechanism of contractile-ring assembly. Actin filaments nucleated by formin Cdc12, the motor activity of myosin-II, the stiffness of the actin network, and severing of actin filaments by cofilin all play essential roles in contractile-ring assembly. Finally, we discuss the Mid1-independent pathway for ring assembly, and the possible mechanisms underlying the ring maturation and constriction. Collectively, we provide an overview of the current understanding of contractile-ring assembly and uncover future directions in studying cytokinesis in fission yeast. Copyright © 2012 Wiley Periodicals, Inc.

Bacterial Actins.

PubMed

Izoré, Thierry; van den Ent, Fusinita

2017-01-01

A diverse set of protein polymers, structurally related to actin filaments contributes to the organization of bacterial cells as cytomotive or cytoskeletal filaments. This chapter describes actin homologs encoded by bacterial chromosomes. MamK filaments, unique to magnetotactic bacteria, help establishing magnetic biological compasses by interacting with magnetosomes. Magnetosomes are intracellular membrane invaginations containing biomineralized crystals of iron oxide that are positioned by MamK along the long-axis of the cell. FtsA is widespread across bacteria and it is one of the earliest components of the divisome to arrive at midcell, where it anchors the cell division machinery to the membrane. FtsA binds directly to FtsZ filaments and to the membrane through its C-terminus. FtsA shows altered domain architecture when compared to the canonical actin fold. FtsA's subdomain 1C replaces subdomain 1B of other members of the actin family and is located on the opposite side of the molecule. Nevertheless, when FtsA assembles into protofilaments, the protofilament structure is preserved, as subdomain 1C replaces subdomain IB of the following subunit in a canonical actin filament. MreB has an essential role in shape-maintenance of most rod-shaped bacteria. Unusually, MreB filaments assemble from two protofilaments in a flat and antiparallel arrangement. This non-polar architecture implies that both MreB filament ends are structurally identical. MreB filaments bind directly to membranes where they interact with both cytosolic and membrane proteins, thereby forming a key component of the elongasome. MreB filaments in cells are short and dynamic, moving around the long axis of rod-shaped cells, sensing curvature of the membrane and being implicated in peptidoglycan synthesis.

Self-assembly of actin monomers into long filaments: Brownian dynamics simulations

NASA Astrophysics Data System (ADS)

Guo, Kunkun; Shillcock, Julian; Lipowsky, Reinhard

2009-07-01

Brownian dynamics simulations are used to study the dynamical process of self-assembly of actin monomers into long filaments containing up to 1000 actin protomers. In order to overcome the large separation of time scales between the diffusive motion of the free monomers and the relatively slow attachment and detachment processes at the two ends of the filaments, we introduce a novel rescaling procedure by which we speed all dynamical processes related to actin polymerization and depolymerization up by the same factor. In general, the actin protomers within a filament can attain three different states corresponding to a bound adenosine triphosphate (ATP), adenosine diphosphate with inorganic phosphate (ADP/P), and ADP molecule. The simplest situation that has been studied experimentally is provided by the polymerization of ADP-actin, for which all protomers are identical. This case is used to unravel certain relations between the filament's physical properties and the model parameters such as the attachment rate constant and the size of the capture zone, the detachment rate and the probability of the detached event, as well as the growth rate and waiting times between two successive attachment/detachment events. When a single filament is allowed to grow in a bath of constant concentration of free ADP-actin monomers, its growth rate increases linearly with the free monomer concentration in quantitative agreement with in vitro experiments. The results also show that the waiting time is governed by exponential distributions and that the two ends of a filament undergo biased random walks. The filament length fluctuations are described by a length diffusion constant that is found to attain a constant value at low ADP-actin concentration and to increase linearly with this concentration. It is straightforward to apply our simulation code to more complex processes such as polymerization of ATP-actin coupled to ATP hydrolysis, force generation by filaments, formation of filament bundles, and filament-membrane interactions.

Dynamics of in vivo ASC speck formation

PubMed Central

2017-01-01

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC. PMID:28701426

ZOMG - I. How the cosmic web inhibits halo growth and generates assembly bias

NASA Astrophysics Data System (ADS)

Borzyszkowski, Mikolaj; Porciani, Cristiano; Romano-Díaz, Emilio; Garaldi, Enrico

2017-07-01

The clustering of dark matter haloes with fixed mass depends on their formation history, an effect known as assembly bias. We use zoom N-body simulations to investigate the origin of this phenomenon. For each halo at redshift z = 0, we determine the time in which the physical volume containing its final mass becomes stable. We consider five examples for which this happens at z ˜ 1.5 and two that do not stabilize by z = 0. The zoom simulations show that early-collapsing haloes do not grow in mass at z = 0 while late-forming ones show a net inflow. The reason is that 'accreting' haloes are located at the nodes of a network of thin filaments feeding them. Conversely, each 'stalled' halo lies within a prominent filament that is thicker than the halo size. Infalling material from the surroundings becomes part of the filament while matter within it recedes from the halo. We conclude that assembly bias originates from quenching halo growth due to tidal forces following the formation of non-linear structures in the cosmic web, as previously conjectured in the literature. Also the internal dynamics of the haloes change: the velocity anisotropy profile is biased towards radial (tangential) orbits in accreting (stalled) haloes. Our findings reveal the cause of the yet unexplained dependence of halo clustering on the anisotropy. Finally, we extend the excursion-set theory to account for these effects. A simple criterion based on the ellipticity of the linear tidal field combined with the spherical-collapse model provides excellent predictions for both classes of haloes.

Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus.

PubMed

Kim, So Yeon; Gitai, Zemer; Kinkhabwala, Anika; Shapiro, Lucy; Moerner, W E

2006-07-18

The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity.

Spire and Formin 2 Synergize and Antagonize in Regulating Actin Assembly in Meiosis by a Ping-Pong Mechanism

PubMed Central

Montaville, Pierre; Jégou, Antoine; Pernier, Julien; Compper, Christel; Guichard, Bérengère; Mogessie, Binyam; Schuh, Melina; Romet-Lemonne, Guillaume; Carlier, Marie-France

2014-01-01

In mammalian oocytes, three actin binding proteins, Formin 2 (Fmn2), Spire, and profilin, synergistically organize a dynamic cytoplasmic actin meshwork that mediates translocation of the spindle toward the cortex and is required for successful fertilization. Here we characterize Fmn2 and elucidate the molecular mechanism for this synergy, using bulk solution and individual filament kinetic measurements of actin assembly dynamics. We show that by capping filament barbed ends, Spire recruits Fmn2 and facilitates its association with barbed ends, followed by rapid processive assembly and release of Spire. In the presence of actin, profilin, Spire, and Fmn2, filaments display alternating phases of rapid processive assembly and arrested growth, driven by a “ping-pong” mechanism, in which Spire and Fmn2 alternately kick off each other from the barbed ends. The results are validated by the effects of injection of Spire, Fmn2, and their interacting moieties in mouse oocytes. This original mechanism of regulation of a Rho-GTPase–independent formin, recruited by Spire at Rab11a-positive vesicles, supports a model for modulation of a dynamic actin-vesicle meshwork in the oocyte at the origin of asymmetric positioning of the meiotic spindle. PMID:24586110

Alignment between Protostellar Outflows and Filamentary Structure

NASA Astrophysics Data System (ADS)

Stephens, Ian W.; Dunham, Michael M.; Myers, Philip C.; Pokhrel, Riwaj; Sadavoy, Sarah I.; Vorobyov, Eduard I.; Tobin, John J.; Pineda, Jaime E.; Offner, Stella S. R.; Lee, Katherine I.; Kristensen, Lars E.; Jørgensen, Jes K.; Goodman, Alyssa A.; Bourke, Tyler L.; Arce, Héctor G.; Plunkett, Adele L.

2017-09-01

We present new Submillimeter Array (SMA) observations of CO(2-1) outflows toward young, embedded protostars in the Perseus molecular cloud as part of the Mass Assembly of Stellar Systems and their Evolution with the SMA (MASSES) survey. For 57 Perseus protostars, we characterize the orientation of the outflow angles and compare them with the orientation of the local filaments as derived from Herschel observations. We find that the relative angles between outflows and filaments are inconsistent with purely parallel or purely perpendicular distributions. Instead, the observed distribution of outflow-filament angles are more consistent with either randomly aligned angles or a mix of projected parallel and perpendicular angles. A mix of parallel and perpendicular angles requires perpendicular alignment to be more common by a factor of ˜3. Our results show that the observed distributions probably hold regardless of the protostar’s multiplicity, age, or the host core’s opacity. These observations indicate that the angular momentum axis of a protostar may be independent of the large-scale structure. We discuss the significance of independent protostellar rotation axes in the general picture of filament-based star formation.

Twisted ribbon structure of paired helical filaments revealed by atomic force microscopy.

PubMed Central

Pollanen, M. S.; Markiewicz, P.; Bergeron, C.; Goh, M. C.

1994-01-01

Progressive deposition of phosphorylated tau into the paired helical filaments (PHF) that compose neurofibrillary tangles, dystrophic neurites, and neuropil threads is an obligate feature of Alzheimer's disease. The standard model of PHF structure, derived from electron microscopic studies, suggests that two 8- to 10-nm filaments each composed of three to four protofilaments are wound into a helix with a maximal diameter of -20 nm and a half period of 65 to 80 nm. However, recent vertical platinum-carbon replicas of PHF more closely resemble a thin helical ribbon without constitutive protofilaments. Here we report that native PHF imaged with an atomic force microscope appear as twisted ribbons rather than the generally accepted structure derived from electron microscopic studies. These data imply that the assembly of PHF is not due to the twisting of pair-wise filaments but rather the helical winding of self-associated tau molecules arranged into a flattened structure. Future structural models of PHF should be based on quantitative data obtained from imaging techniques, such as scanning probe microscopy, which do not require harsh specimen preparation procedures. Images Figure 1 PMID:8178938

Twisted ribbon structure of paired helical filaments revealed by atomic force microscopy.

PubMed

Pollanen, M S; Markiewicz, P; Bergeron, C; Goh, M C

1994-05-01

Progressive deposition of phosphorylated tau into the paired helical filaments (PHF) that compose neurofibrillary tangles, dystrophic neurites, and neuropil threads is an obligate feature of Alzheimer's disease. The standard model of PHF structure, derived from electron microscopic studies, suggests that two 8- to 10-nm filaments each composed of three to four protofilaments are wound into a helix with a maximal diameter of -20 nm and a half period of 65 to 80 nm. However, recent vertical platinum-carbon replicas of PHF more closely resemble a thin helical ribbon without constitutive protofilaments. Here we report that native PHF imaged with an atomic force microscope appear as twisted ribbons rather than the generally accepted structure derived from electron microscopic studies. These data imply that the assembly of PHF is not due to the twisting of pair-wise filaments but rather the helical winding of self-associated tau molecules arranged into a flattened structure. Future structural models of PHF should be based on quantitative data obtained from imaging techniques, such as scanning probe microscopy, which do not require harsh specimen preparation procedures.

Development and Application of Wood Flour-Filled Polylactic Acid Composite Filament for 3D Printing

PubMed Central

Tao, Yubo; Wang, Honglei; Li, Zelong; Li, Peng; Shi, Sheldon Q.

2017-01-01

This paper presents the development of wood flour (WF)-filled polylactic acid (PLA) composite filaments for a fused deposition modeling (FDM) process with the aim of application to 3D printing. The composite filament consists of wood flour (5 wt %) in a PLA matrix. The detailed formulation and characterization of the composite filament were investigated experimentally, including tensile properties, microstructure, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The feedstock filaments of this composite were produced and used successfully in an assembled FDM 3D printer. The research concludes that compared with pure PLA filament, adding WF changed the microstructure of material fracture surface, the initial deformation resistance of the composite was enhanced, the starting thermal degradation temperature of the composite decreased slightly, and there were no effects on the melting temperature. The WF/PLA composite filament is suitable to be printed by the FDM process. PMID:28772694

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

PubMed Central

Meshram, Chetan D.; Baviskar, Pradyumna S.; Ognibene, Cherie M.

2016-01-01

ABSTRACT Virus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology. IMPORTANCE hRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines. PMID:27654298

Cryo-electron microscopy structure of human peroxiredoxin-3 filament reveals the assembly of a putative chaperone.

PubMed

Radjainia, Mazdak; Venugopal, Hariprasad; Desfosses, Ambroise; Phillips, Amy J; Yewdall, N Amy; Hampton, Mark B; Gerrard, Juliet A; Mitra, Alok K

2015-05-05

Peroxiredoxins (Prxs) are a ubiquitous class of thiol-dependent peroxidases that play an important role in the protection and response of cells to oxidative stress. The catalytic unit of typical 2-Cys Prxs are homodimers, which can self-associate to form complex assemblies that are hypothesized to have signaling and chaperone activity. Mitochondrial Prx3 forms dodecameric toroids, which can further stack to form filaments, the so-called high-molecular-weight (HMW) form that has putative holdase activity. We used single-particle analysis and helical processing of electron cryomicroscopy images of human Prx3 filaments induced by low pH to generate a ∼7-Å resolution 3D structure of the HMW form, the first such structure for a 2-Cys Prx. The pseudo-atomic model reveals interactions that promote the stacking of the toroids and shows that unlike previously reported data, the structure can accommodate a partially folded C terminus. The HMW filament lumen displays hydrophobic patches, which we hypothesize bestow holdase activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Long-range self-organization of cytoskeletal myosin II filament stacks.

PubMed

Hu, Shiqiong; Dasbiswas, Kinjal; Guo, Zhenhuan; Tee, Yee-Han; Thiagarajan, Visalatchi; Hersen, Pascal; Chew, Teng-Leong; Safran, Samuel A; Zaidel-Bar, Ronen; Bershadsky, Alexander D

2017-02-01

Although myosin II filaments are known to exist in non-muscle cells, their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their 'registered' alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and α-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.

3-D Enzymatic Nanomaterial Architectures for Energy Harvesting

DTIC Science & Technology

2016-06-30

Sibbett, D.N. Petsev, S. Babanova, G. Gupta and P. Atanassov, Practical Electricity Generation from a Paper Based Biofuel Cell Powered by Glucose...Arechederra, R.L., Falase, A., Garcia, K., Rincon, R., Minteer, S.D., Banta, S., Gupta, G., Babanova, S. and Atanassov, P. (2015) “ Paper based biofuel cells...control of the self-assembled protein filament with the cysteine-introduced building blocks. Protein Sci. 18:960–969. Wheeldon IR , Gallaway JW, Barton SC

Vimentin filament precursors exchange subunits in an ATP-dependent manner

PubMed Central

Robert, Amélie; Rossow, Molly J.; Hookway, Caroline; Adam, Stephen A.; Gelfand, Vladimir I.

2015-01-01

Intermediate filaments (IFs) are a component of the cytoskeleton capable of profound reorganization in response to specific physiological situations, such as differentiation, cell division, and motility. Various mechanisms were proposed to be responsible for this plasticity depending on the type of IF polymer and the biological context. For example, recent studies suggest that mature vimentin IFs (VIFs) undergo rearrangement by severing and reannealing, but direct subunit exchange within the filament plays little role in filament dynamics at steady state. Here, we studied the dynamics of subunit exchange in VIF precursors, called unit-length filaments (ULFs), formed by the lateral association of eight vimentin tetramers. To block vimentin assembly at the ULF stage, we used the Y117L vimentin mutant (vimentinY117L). By tagging vimentinY117L with a photoconvertible protein mEos3.2 and photoconverting ULFs in a limited area of the cytoplasm, we found that ULFs, unlike mature filaments, were highly dynamic. Subunit exchange among ULFs occurred within seconds and was limited by the diffusion of soluble subunits in the cytoplasm rather than by the association and dissociation of subunits from ULFs. Our data demonstrate that cells expressing vimentinY117L contained a large pool of soluble vimentin tetramers that was in rapid equilibrium with ULFs. Furthermore, vimentin exchange in ULFs required ATP, and ATP depletion caused a dramatic reduction of the soluble tetramer pool. We believe that the dynamic exchange of subunits plays a role in the regulation of ULF assembly and the maintenance of a soluble vimentin pool during the reorganization of filament networks. PMID:26109569

Thermal Unfolding Simulations of Bacterial Flagellin: Insight into its Refolding Before Assembly

PubMed Central

Chng, Choon-Peng; Kitao, Akio

2008-01-01

Flagellin is the subunit of the bacterial filament, the micrometer-long propeller of a bacterial flagellum. The protein is believed to undergo unfolding for transport through the channel of the filament and to refold in a chamber at the end of the channel before being assembled into the growing filament. We report a thermal unfolding simulation study of S. typhimurium flagellin in aqueous solution as an attempt to gain atomic-level insight into the refolding process. Each molecule comprises two filament-core domains {D0, D1} and two hypervariable-region domains {D2, D3}. D2 can be separated into subdomains D2a and D2b. We observed a similar unfolding order of the domains as reported in experimental thermal denaturation. D2a and D3 exhibited high thermal stability and contained persistent three-stranded β-sheets in the denatured state which could serve as folding cores to guide refolding. A recent mutagenesis study on flagellin stability seems to suggest the importance of the folding cores. Using crude size estimates, our data suggests that the chamber might be large enough for either denatured hypervariable-region domains or filament-core domains, but not whole flagellin; this implicates a two-staged refolding process. PMID:18263660

A novel role for WAVE1 in controlling actin network growth rate and architecture

PubMed Central

Sweeney, Meredith O.; Collins, Agnieszka; Padrick, Shae B.; Goode, Bruce L.

2015-01-01

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 (“V”) domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2. PMID:25473116

Self-assembly behaviours of peptide-drug conjugates: influence of multiple factors on aggregate morphology and potential self-assembly mechanism

NASA Astrophysics Data System (ADS)

Fan, Qin; Ji, Yujie; Wang, Jingjing; Wu, Li; Li, Weidong; Chen, Rui; Chen, Zhipeng

2018-04-01

Peptide-drug conjugates (PDCs) as self-assembly prodrugs have the unique and specific features to build one-component nanomedicines. Supramolecular structure based on PDCs could form various morphologies ranging from nanotube, nanofibre, nanobelt to hydrogel. However, the assembly process of PDCs is too complex to predict or control. Herein, we investigated the effects of extrinsic factors on assembly morphology and the possible formation of nanostructures based on PDCs. To this end, we designed a PDC consisting of hydrophobic drug (S)-ketoprofen (Ket) and valine-glutamic acid dimeric repeats peptide (L-VEVE) to study their assembly behaviour. Our results showed that the critical assembly concentration of Ket-L-VEVE was 0.32 mM in water to form various nanostructures which experienced from micelle, nanorod, nanofibre to nanoribbon. The morphology was influenced by multiple factors including molecular design, assembly time, pH and hydrogen bond inhibitor. On the basis of experimental results, we speculated the possible assembly mechanism of Ket-L-VEVE. The π-π stacking interaction between Ket molecules could serve as an anchor, and hydrogen bonded-induced β-sheets and hydrophilic/hydrophobic balance between L-VEVE peptide play structure-directing role in forming filament-like or nanoribbon morphology. This work provides a new sight to rationally design and precisely control the nanostructure of PDCs based on aromatic fragment.

Self-assembly behaviours of peptide-drug conjugates: influence of multiple factors on aggregate morphology and potential self-assembly mechanism.

PubMed

Fan, Qin; Ji, Yujie; Wang, Jingjing; Wu, Li; Li, Weidong; Chen, Rui; Chen, Zhipeng

2018-04-01

Peptide-drug conjugates (PDCs) as self-assembly prodrugs have the unique and specific features to build one-component nanomedicines. Supramolecular structure based on PDCs could form various morphologies ranging from nanotube, nanofibre, nanobelt to hydrogel. However, the assembly process of PDCs is too complex to predict or control. Herein, we investigated the effects of extrinsic factors on assembly morphology and the possible formation of nanostructures based on PDCs. To this end, we designed a PDC consisting of hydrophobic drug ( S )-ketoprofen (Ket) and valine-glutamic acid dimeric repeats peptide (L-VEVE) to study their assembly behaviour. Our results showed that the critical assembly concentration of Ket-L-VEVE was 0.32 mM in water to form various nanostructures which experienced from micelle, nanorod, nanofibre to nanoribbon. The morphology was influenced by multiple factors including molecular design, assembly time, pH and hydrogen bond inhibitor. On the basis of experimental results, we speculated the possible assembly mechanism of Ket-L-VEVE. The π-π stacking interaction between Ket molecules could serve as an anchor, and hydrogen bonded-induced β-sheets and hydrophilic/hydrophobic balance between L-VEVE peptide play structure-directing role in forming filament-like or nanoribbon morphology. This work provides a new sight to rationally design and precisely control the nanostructure of PDCs based on aromatic fragment.

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments.

PubMed

Montaville, Pierre; Kühn, Sonja; Compper, Christel; Carlier, Marie-France

2016-02-12

Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Thermodynamics and kinetics of DNA nanotube polymerization from single-filament measurements† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c3sc53331j Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.

PubMed Central

Hariadi, Rizal F.; Yurke, Bernard

2015-01-01

DNA nanotubes provide a programmable architecture for molecular self-assembly and can serve as model systems for one-dimensional biomolecular assemblies. While a variety of DNA nanotubes have been synthesized and employed as models for natural biopolymers, an extensive investigation of DNA nanotube kinetics and thermodynamics has been lacking. Using total internal reflection microscopy, DNA nanotube polymerization was monitored in real time at the single filament level over a wide range of free monomer concentrations and temperatures. The measured polymerization rates were subjected to a global nonlinear fit based on polymerization theory in order to simultaneously extract kinetic and thermodynamic parameters. For the DNA nanotubes used in this study, the association rate constant is (5.99 ± 0.15) × 105 M–1 s–1, the enthalpy is 87.9 ± 2.0 kcal mol–1, and the entropy is 0.252 ± 0.006 kcal mol–1 K–1. The qualitative and quantitative similarities between the kinetics of DNA nanotubes, actin filaments, and microtubules polymerization highlight the prospect of building complex dynamic systems from DNA molecules inspired by biological architecture. PMID:29308139

Roles of Formin Nodes and Myosin Motor Activity in Mid1p-dependent Contractile-Ring Assembly during Fission Yeast Cytokinesis

PubMed Central

Coffman, Valerie C.; Nile, Aaron H.; Lee, I-Ju; Liu, Huayang

2009-01-01

Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30–50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. α-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis. PMID:19864459

Fabrication of compact electron gun for 6 MeV X-ray source

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghodke, S.R.; Barnwal, Rajesh; Kumar, Mahendra, E-mail: ghodke_barc@yahoo.co.in

The 6 MeV X-Ray source for container cargo scanning application has been designed and developed by the Accelerator and Pulse Power Division, BARC, Mumbai. This compact linac has been designed as a mobile system, to be mounted on a moving container. In linac-based cargo-scanning system, to work electron gun on a movable container, it has to be robust. Electron gun is to work at 10{sup -7} mbar vacuum and 2000 degree Celsius temperature. An effort is made to engineer the gun assembly to make it more robust and aligned. The linac acts as the source of X-rays, which fall onmore » the cargo and are then detected by the detector system. Many components are indigenously developed like grid, insulating ring, Tungsten filament and filament guide, which are made from alumina ceramic and Tantalum which is to work at 1500 degree Celsius. Filament connector is made from Invar to reduce heat loss and to make rigid connection. It was CNC machined and wire cut by EDM. Invar and Copper electrode feed through is shrink fitted with the help of liquid Nitrogen. Shrink fit tolerances of 15 micrometer are achieved by jig boring machining processes. Tantalum cup for LaB6 cathode and heat shield are made from die and punch mechanism. For alignment of electron emitter with beam axis this Tantalum cup is a crucial component. Electron gun is assembled and aligned its components with the help of precision jigs. The whole assembly was Helium leak tested by MSLD up to 4 x 10{sup -10} mbar.l/s vacuum, no leak was found. This paper will describe the machining, Tantalum cup forming, ceramic components development, heat shields, ceramic feed through etc of electron gun. (author)« less

Cell-cycle regulation of formin-mediated actin cable assembly

PubMed Central

Miao, Yansong; Wong, Catherine C. L.; Mennella, Vito; Michelot, Alphée; Agard, David A.; Holt, Liam J.; Yates, John R.; Drubin, David G.

2013-01-01

Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation. PMID:24133141

Drug delivery vectors based on filamentous bacteriophages and phage-mimetic nanoparticles.

PubMed

Ju, Zhigang; Sun, Wei

2017-11-01

With the development of nanomedicine, a mass of nanocarriers have been exploited and utilized for targeted drug delivery, including liposomes, polymers, nanoparticles, viruses, and stem cells. Due to huge surface bearing capacity and flexible genetic engineering property, filamentous bacteriophage and phage-mimetic nanoparticles are attracting more and more attentions. As a rod-like bio-nanofiber without tropism to mammalian cells, filamentous phage can be easily loaded with drugs and directly delivered to the lesion location. In particular, chemical drugs can be conjugated on phage surface by chemical modification, and gene drugs can also be inserted into the genome of phage by recombinant DNA technology. Meanwhile, specific peptides/proteins displayed on the phage surface are able to conjugate with nanoparticles which will endow them specific-targeting and huge drug-loading capacity. Additionally, phage peptides/proteins can directly self-assemble into phage-mimetic nanoparticles which may be applied for self-navigating drug delivery nanovehicles. In this review, we summarize the production of phage particles, the identification of targeting peptides, and the recent applications of filamentous bacteriophages as well as their protein/peptide for targeting drug delivery in vitro and in vivo. The improvement of our understanding of filamentous bacteriophage and phage-mimetic nanoparticles will supply new tools for biotechnological approaches.

Presynaptic Filament Dynamics in Homologous Recombination and DNA Repair

PubMed Central

Liu, Jie; Ehmsen, Kirk T.; Heyer, Wolf-Dietrich; Morrical, Scott W.

2014-01-01

Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity. PMID:21599536

Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging

PubMed Central

Smith, Benjamin A.; Daugherty-Clarke, Karen; Goode, Bruce L.; Gelles, Jeff

2013-01-01

Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region of proteins in the Wiskott-Aldrich syndrome protein (WASp) family, Arp2/3 complex produces new (daughter) filaments as branches from the sides of preexisting (mother) filaments. We visualized individual fluorescently labeled Arp2/3 complexes dynamically interacting with and producing branches on growing actin filaments in vitro. Branch formation was strikingly inefficient, even in the presence of VCA: only ∼1% of filament-bound Arp2/3 complexes yielded a daughter filament. VCA acted at multiple steps, increasing both the association rate of Arp2/3 complexes with mother filament and the fraction of filament-bound complexes that nucleated a daughter. The results lead to a quantitative kinetic mechanism for branched actin assembly, revealing the steps that can be stimulated by additional cellular factors. PMID:23292935

The novel Tau mutation G335S: clinical, neuropathological and molecular characterization.

PubMed

Spina, Salvatore; Murrell, Jill R; Yoshida, Hirotaka; Ghetti, Bernardino; Bermingham, Niamh; Sweeney, Brian; Dlouhy, Stephen R; Crowther, R Anthony; Goedert, Michel; Keohane, Catherine

2007-04-01

Mutations in Tau cause the inherited neurodegenerative disease, frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Known coding region mutations cluster in the microtubule-binding region, where they alter the ability of tau to promote microtubule assembly. Depending on the tau isoforms, this region consists of three or four imperfect repeats of 31 or 32 amino acids, each of which contains a characteristic and invariant PGGG motif. Here, we report the novel G335S mutation, which changes the PGGG motif of the third tau repeat to PGGS, in an individual who developed social withdrawal, emotional bluntness and stereotypic behavior at age 22, followed by disinhibition, hyperorality and ideomotor apraxia. Abundant tau-positive inclusions were present in neurons and glia in the frontotemporal cortex, hippocampus and brainstem. Sarkosyl-insoluble tau showed paired helical and straight filaments, as well as more irregular rope-like filaments. The pattern of pathological tau bands was like that of Alzheimer disease. Experimentally, the G335S mutation resulted in a greatly reduced ability of tau to promote microtubule assembly, while having no significant effect on heparin-induced assembly of recombinant tau into filaments.

Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

PubMed

Ruano-Gallego, David; Álvarez, Beatriz; Fernández, Luis Ángel

2015-09-18

Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these "molecular syringes" for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells.

Caveolae provide a specialized membrane environment for respiratory syncytial virus assembly

PubMed Central

Nguyen, Tra Huong; Leong, Daniel; Ravi, Laxmi Iyer; Tan, Boon Huan; Sandin, Sara; Sugrue, Richard J.

2017-01-01

ABSTRACT Respiratory syncytial virus (RSV) is an enveloped virus that assembles into filamentous virus particles on the surface of infected cells. Morphogenesis of RSV is dependent upon cholesterol-rich (lipid raft) membrane microdomains, but the specific role of individual raft molecules in RSV assembly is not well defined. Here, we show that RSV morphogenesis occurs within caveolar membranes and that both caveolin-1 and cavin-1 (also known as PTRF), the two major structural and functional components of caveolae, are actively recruited to and incorporated into the RSV envelope. The recruitment of caveolae occurred just prior to the initiation of RSV filament assembly, and was dependent upon an intact actin network as well as a direct physical interaction between caveolin-1 and the viral G protein. Moreover, cavin-1 protein levels were significantly increased in RSV-infected cells, leading to a virus-induced change in the stoichiometry and biophysical properties of the caveolar coat complex. Our data indicate that RSV exploits caveolae for its assembly, and we propose that the incorporation of caveolae into the virus contributes to defining the biological properties of the RSV envelope. PMID:28154158

Intermediate filament mechanics in vitro and in the cell: from coiled coils to filaments, fibers and networks.

PubMed

Köster, Sarah; Weitz, David A; Goldman, Robert D; Aebi, Ueli; Herrmann, Harald

2015-02-01

Intermediate filament proteins form filaments, fibers and networks both in the cytoplasm and the nucleus of metazoan cells. Their general structural building plan accommodates highly varying amino acid sequences to yield extended dimeric α-helical coiled coils of highly conserved design. These 'rod' particles are the basic building blocks of intrinsically flexible, filamentous structures that are able to resist high mechanical stresses, that is, bending and stretching to a considerable degree, both in vitro and in the cell. Biophysical and computer modeling studies are beginning to unfold detailed structural and mechanical insights into these major supramolecular assemblies of cell architecture, not only in the 'test tube' but also in the cellular and tissue context. Copyright © 2015 Elsevier Ltd. All rights reserved.

Self-assembled bifunctional surface mimics an enzymatic and templating protein for the synthesis of a metal oxide semiconductor

PubMed Central

Kisailus, David; Truong, Quyen; Amemiya, Yosuke; Weaver, James C.; Morse, Daniel E.

2006-01-01

The recent discovery and characterization of silicatein, a mineral-synthesizing enzyme that assembles to form the filamentous organic core of the glassy skeletal elements (spicules) of a marine sponge, has led to the development of new low-temperature synthetic routes to metastable semiconducting metal oxides. These protein filaments were shown in vitro to catalyze the hydrolysis and structurally direct the polycondensation of metal oxides at neutral pH and low temperature. Based on the confirmation of the catalytic mechanism and the essential participation of specific serine and histidine residues (presenting a nucleophilic hydroxyl and a nucleophilicity-enhancing hydrogen-bonding imidazole nitrogen) in silicatein’s catalytic active site, we therefore sought to develop a synthetic mimic that provides both catalysis and the surface determinants necessary to template and structurally direct heterogeneous nucleation through condensation. Using lithographically patterned poly(dimethylsiloxane) stamps, bifunctional self-assembled monolayer surfaces containing the essential catalytic and templating elements were fabricated by using alkane thiols microcontact-printed on gold substrates. The interface between chemically distinct self-assembled monolayer domains provided the necessary juxtaposition of nucleophilic (hydroxyl) and hydrogen-bonding (imidazole) agents to catalyze the hydrolysis of a gallium oxide precursor and template the condensed product to form gallium oxohydroxide (GaOOH) and the defect spinel, gamma-gallium oxide (γ-Ga2O3). Using this approach, the production of patterned substrates for catalytic synthesis and templating of semiconductors for device applications can be envisioned. PMID:16585518

Extension arm for mobile travelers suit case

DOEpatents

Byington, Gerald A.

1999-01-01

The invention is an apparatus for adjusting a luggage handle in relation to a luggage frame utilized to transport luggage by a traveler. The handle is connected to two extendable and retractable slide tube assemblies, the assemblies allow for the telescoping of the luggage handle to multiple positions in relation to a pair of fixed frame tubes connected to a luggage shell with wheels, to accommodate the height and personal stride of traveler. The luggage handle incorporates triggering buttons that allow ambidextrous and single-handed control of the height of the handle and slide tube assembly in relation to the luggage. The handle and slide tube assembly are connected by interior filaments to pulleys and filaments within two concentric light-weight slide tubes, which are inserted respectively into two fixed frame tubes, to allow a multitude of positions for the slide tubes to lock into the fixed frame tubes. The apparatus can be pushed or pulled by the traveler, and the support shell can accommodate multiple pieces of luggage.

The RecF protein antagonizes RecX function via direct interaction

PubMed Central

Lusetti, Shelley L.; Hobbs, Michael D.; Stohl, Elizabeth A.; Chitteni-Pattu, Sindhu; Inman, Ross B.; Seifert, H. Steven; Cox, Michael M.

2014-01-01

Summary The RecX protein inhibits RecA filament extension leading to net filament disassembly. The RecF protein physically interacts with the RecX protein and protects RecA from the inhibitory effects of RecX. In vitro, efficient RecA filament formation onto SSB-coated circular single-stranded DNA in the presence of RecX occurs only when all of the RecFOR proteins are present. The RecOR proteins contribute only to RecA filament nucleation onto SSB-coated single-stranded DNA and are unable to counter the inhibitory effects of RecX on RecA filaments. RecF protein uniquely supports substantial RecA filament extension in the presence of RecX. In vivo, RecF protein counters a RecX-mediated inhibition of plasmid recombination. Thus, a significant positive contribution of RecF to RecA filament assembly is to antagonize the effects of the negative modulator, RecX, specifically during the extension phase. PMID:16387652

The domain organization of the bacterial intermediate filament-like protein crescentin is important for assembly and function

PubMed Central

Cabeen, Matthew T; Herrmann, Harald; Jacobs-Wagner, Christine

2011-01-01

Crescentin is a bacterial filament-forming protein that exhibits domain organization features found in metazoan intermediate filament (IF) proteins. Structure-function studies of eukaryotic IFs have been hindered by a lack of simple genetic systems and easily quantifiable phenotypes. Here we exploit the characteristic localization of the crescentin structure along the inner curvature of Caulobacter crescentus cells and the loss of cell curvature associated with impaired crescentin function to analyze the importance of the domain organization of crescentin. By combining biochemistry and ultrastructural analysis in vitro with cellular localization and functional studies, we show that crescentin requires its distinctive domain organization, and furthermore that different structural elements have distinct structural and functional contributions. The head domain can be functionally subdivided into two subdomains; the first (amino-terminal) is required for function but not assembly, while the second is necessary for structure assembly. The rod domain is similarly required for structure assembly, and the linker L1 appears important to prevent runaway assembly into nonfunctional aggregates. The data also suggest that the stutter and the tail domain have critical functional roles in stabilizing crescentin structures against disassembly by monovalent cations in the cytoplasm. This study suggests that the IF-like behavior of crescentin is a consequence of its domain organization, implying that the IF protein layout is an adaptable cytoskeletal motif, much like the actin and tubulin folds, that is broadly exploited for various functions throughout life from bacteria to humans. © 2011 Wiley-Liss, Inc. PMID:21360832

Unc-45 Mutations in Caenorhabditis elegans Implicate a CRO1/She4p-like Domain in Myosin Assembly

PubMed Central

Barral, José M.; Bauer, Christopher C.; Ortiz, Irving; Epstein, Henry F.

1998-01-01

The Caenorhabditis elegans unc-45 locus has been proposed to encode a protein machine for myosin assembly. The UNC-45 protein is predicted to contain an NH2-terminal domain with three tetratricopeptide repeat motifs, a unique central region, and a COOH-terminal domain homologous to CRO1 and She4p. CRO1 and She4p are fungal proteins required for the segregation of other molecules in budding, endocytosis, and septation. Three mutations that lead to temperature-sensitive (ts) alleles have been localized to conserved residues within the CRO1/She4p-like domain, and two lethal alleles were found to result from stop codon mutations in the central region that would prevent translation of the COOH-terminal domain. Electron microscopy shows that thick filament accumulation in vivo is decreased by ∼50% in the CB286 ts mutant grown at the restrictive temperature. The thick filaments that assemble have abnormal structure. Immunofluorescence and immunoelectron microscopy show that myosins A and B are scrambled, in contrast to their assembly into distinct regions at the permissive temperature and in wild type. This abnormal structure correlates with the high degree of instability of the filaments in vitro as reflected by their extremely low yields and shortened lengths upon isolation. These results implicate the UNC-45 CRO1/She4p-like region in the assembly of myosin isoforms in C. elegans and suggest a possible common mechanism for the function of this UCS (UNC-45/CRO1/She4p) protein family. PMID:9832550

Pure F-actin networks are distorted and branched by steps in the critical-point drying method.

PubMed

Resch, Guenter P; Goldie, Kenneth N; Hoenger, Andreas; Small, J Victor

2002-03-01

Elucidation of the ultrastructural organization of actin networks is crucial for understanding the molecular mechanisms underlying actin-based motility. Results obtained from cytoskeletons and actin comets prepared by the critical-point procedure, followed by rotary shadowing, support recent models incorporating actin filament branching as a main feature of lamellipodia and pathogen propulsion. Since actin branches were not evident in earlier images obtained by negative staining, we explored how these differences arise. Accordingly, we have followed the structural fate of dense networks of pure actin filaments subjected to steps of the critical-point drying protocol. The filament networks have been visualized in parallel by both cryo-electron microscopy and negative staining. Our results demonstrate the selective creation of branches and other artificial structures in pure F-actin networks by the critical-point procedure and challenge the reliability of this method for preserving the detailed organization of actin assemblies that drive motility. (c) 2002 Elsevier Science (USA).

DARK RIBBONS PROPAGATING AND SWEEPING ACROSS EXTREME ULTRAVIOLET STRUCTURES AFTER FILAMENT ERUPTIONS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Junmin; Zhang, Jun; Li, Ting

2015-05-20

With observations from the Atmospheric Imaging Assembly on board the Solar Dynamics Observatory, we first report that dark ribbons (DRs) moved apart from the filament channel and swept across EUV structures after filament eruptions on 2013 June 23 and 2012 February 10 and 24, respectively. In the first event, the DR with a length of 168 Mm appeared at 100 Mm to the northwest of the filament channel, where the filament erupted 15 hr previously. The DR moved toward the northwest with the different sections having different velocities, ranging from 0.3 to 1.6 km s{sup −1}. When the DR’s middlemore » part swept across a strong EUV structure, the motion of this part was blocked, appearing to deflect the DR. With the DR propagation, the connection of the surrounding EUV structures gradually changed. After one day passed, the DR eventually disappeared. In the other two events, the dynamic evolution of the DRs was similar to that in the first event. Based on the observations, we speculate that the reconnection during the filament eruption changes the configuration of the surrounding magnetic fields systematically. During the reconnection process, magnetic fields are deflecting and the former arbitrarily distributed magnetic fields are rearranged along specific directions. The deflection of magnetic fields results in an instantaneous void region where the magnetic strength is smaller and the plasma density is lower. Consequently, the void region is observed as a DR and propagates outward with the reconnection developing.« less

Alignment between Protostellar Outflows and Filamentary Structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephens, Ian W.; Dunham, Michael M.; Myers, Philip C.

2017-09-01

We present new Submillimeter Array (SMA) observations of CO(2–1) outflows toward young, embedded protostars in the Perseus molecular cloud as part of the Mass Assembly of Stellar Systems and their Evolution with the SMA (MASSES) survey. For 57 Perseus protostars, we characterize the orientation of the outflow angles and compare them with the orientation of the local filaments as derived from Herschel observations. We find that the relative angles between outflows and filaments are inconsistent with purely parallel or purely perpendicular distributions. Instead, the observed distribution of outflow-filament angles are more consistent with either randomly aligned angles or a mixmore » of projected parallel and perpendicular angles. A mix of parallel and perpendicular angles requires perpendicular alignment to be more common by a factor of ∼3. Our results show that the observed distributions probably hold regardless of the protostar’s multiplicity, age, or the host core’s opacity. These observations indicate that the angular momentum axis of a protostar may be independent of the large-scale structure. We discuss the significance of independent protostellar rotation axes in the general picture of filament-based star formation.« less

Expansion and concatenation of nonmuscle myosin IIA filaments drive cellular contractile system formation during interphase and mitosis

PubMed Central

Fenix, Aidan M.; Taneja, Nilay; Buttler, Carmen A.; Lewis, John; Van Engelenburg, Schuyler B.; Ohi, Ryoma; Burnette, Dylan T.

2016-01-01

Cell movement and cytokinesis are facilitated by contractile forces generated by the molecular motor, nonmuscle myosin II (NMII). NMII molecules form a filament (NMII-F) through interactions of their C-terminal rod domains, positioning groups of N-terminal motor domains on opposite sides. The NMII motors then bind and pull actin filaments toward the NMII-F, thus driving contraction. Inside of crawling cells, NMIIA-Fs form large macromolecular ensembles (i.e., NMIIA-F stacks), but how this occurs is unknown. Here we show NMIIA-F stacks are formed through two non–mutually exclusive mechanisms: expansion and concatenation. During expansion, NMIIA molecules within the NMIIA-F spread out concurrent with addition of new NMIIA molecules. Concatenation occurs when multiple NMIIA-Fs/NMIIA-F stacks move together and align. We found that NMIIA-F stack formation was regulated by both motor activity and the availability of surrounding actin filaments. Furthermore, our data showed expansion and concatenation also formed the contractile ring in dividing cells. Thus interphase and mitotic cells share similar mechanisms for creating large contractile units, and these are likely to underlie how other myosin II–based contractile systems are assembled. PMID:26960797

International Space Station Major Constituent Analyzer On-orbit Performance

NASA Technical Reports Server (NTRS)

Gardner, Ben D.; Erwin, Phillip M.; Wiedemann, Rachel; Matty, Chris

2016-01-01

The Major Constituent Analyzer (MCA) is a mass spectrometer based system that measures the major atmospheric constituents on the International Space Station. A number of limited-life components require periodic change-out, including the ORU 02 analyzer and the ORU 08 Verification Gas Assembly. The most recent ORU 02 and ORU 08 assemblies are operating nominally. For ORU 02, the ion source filaments and ion pump lifetime continue to be key determinants of MCA performance. Additionally, testing is underway to evaluate the capacity of the MCA to analyze ammonia. Finally, plans are being made to bring the second MCA on ISS to an operational configuration.

Structure and dynamics of human vimentin intermediate filament dimer and tetramer in explicit and implicit solvent models.

PubMed

Qin, Zhao; Buehler, Markus J

2011-01-01

Intermediate filaments, in addition to microtubules and microfilaments, are one of the three major components of the cytoskeleton in eukaryotic cells, and play an important role in mechanotransduction as well as in providing mechanical stability to cells at large stretch. The molecular structures, mechanical and dynamical properties of the intermediate filament basic building blocks, the dimer and the tetramer, however, have remained elusive due to persistent experimental challenges owing to the large size and fibrillar geometry of this protein. We have recently reported an atomistic-level model of the human vimentin dimer and tetramer, obtained through a bottom-up approach based on structural optimization via molecular simulation based on an implicit solvent model (Qin et al. in PLoS ONE 2009 4(10):e7294, 9). Here we present extensive simulations and structural analyses of the model based on ultra large-scale atomistic-level simulations in an explicit solvent model, with system sizes exceeding 500,000 atoms and simulations carried out at 20 ns time-scales. We report a detailed comparison of the structural and dynamical behavior of this large biomolecular model with implicit and explicit solvent models. Our simulations confirm the stability of the molecular model and provide insight into the dynamical properties of the dimer and tetramer. Specifically, our simulations reveal a heterogeneous distribution of the bending stiffness along the molecular axis with the formation of rather soft and highly flexible hinge-like regions defined by non-alpha-helical linker domains. We report a comparison of Ramachandran maps and the solvent accessible surface area between implicit and explicit solvent models, and compute the persistence length of the dimer and tetramer structure of vimentin intermediate filaments for various subdomains of the protein. Our simulations provide detailed insight into the dynamical properties of the vimentin dimer and tetramer intermediate filament building blocks, which may guide the development of novel coarse-grained models of intermediate filaments, and could also help in understanding assembly mechanisms.

The core of tau-paired helical filaments studied by scanning transmission electron microscopy and limited proteolysis.

PubMed

von Bergen, Martin; Barghorn, Stefan; Müller, Shirley A; Pickhardt, Marcus; Biernat, Jacek; Mandelkow, Eva-Maria; Davies, Peter; Aebi, Ueli; Mandelkow, Eckhard

2006-05-23

In Alzheimer's disease and frontotemporal dementias the microtubule-associated protein tau forms intracellular paired helical filaments (PHFs). The filaments formed in vivo consist mainly of full-length molecules of the six different isoforms present in adult brain. The substructure of the PHF core is still elusive. Here we applied scanning transmission electron microscopy (STEM) and limited proteolysis to probe the mass distribution of PHFs and their surface exposure. Tau filaments assembled from the three repeat domain have a mass per length (MPL) of approximately 60 kDa/nm and filaments from full-length tau (htau40DeltaK280 mutant) have approximately 160 kDa/nm, compared with approximately 130 kDa/nm for PHFs from Alzheimer's brain. Polyanionic cofactors such as heparin accelerate assembly but are not incorporated into PHFs. Limited proteolysis combined with N-terminal sequencing and mass spectrometry of fragments reveals a protease-sensitive N-terminal half and semiresistant PHF core starting in the first repeat and reaching to the C-terminus of tau. Continued proteolysis leads to a fragment starting at the end of the first repeat and ending in the fourth repeat. PHFs from tau isoforms with four repeats revealed an additional cleavage site within the middle of the second repeat. Probing the PHFs with antibodies detecting epitopes either over longer stretches in the C-terminal half of tau or in the fourth repeat revealed that they grow in a polar manner. These data describe the physical parameters of the PHFs and enabled us to build a model of the molecular arrangement within the filamentous structures.

An atomic model of the tropomyosin cable on F-actin.

PubMed

Orzechowski, Marek; Li, Xiaochuan Edward; Fischer, Stefan; Lehman, William

2014-08-05

Tropomyosin regulates a wide variety of actin filament functions and is best known for the role that it plays together with troponin in controlling muscle activity. For effective performance on actin filaments, adjacent 42-nm-long tropomyosin molecules are joined together by a 9- to 10-residue head-to-tail overlapping domain to form a continuous cable that wraps around the F-actin helix. Yet, despite the apparent simplicity of tropomyosin's coiled-coil structure and its well-known periodic association with successive actin subunits along F-actin, the structure of the tropomyosin cable on actin is uncertain. This is because the conformation of the overlap region that joins neighboring molecules is poorly understood, thus leaving a significant gap in our understanding of thin-filament structure and regulation. However, recent molecular-dynamics simulations of overlap segments defined their overall shape and provided unique and sufficient cues to model the whole actin-tropomyosin filament assembly in atomic detail. In this study, we show that these MD structures merge seamlessly onto the ends of tropomyosin coiled-coils. Adjacent tropomyosin molecules can then be joined together to provide a comprehensive model of the tropomyosin cable running continuously on F-actin. The resulting complete model presented here describes for the first time (to our knowledge) an atomic-level structure of αα-striated muscle tropomyosin bound to an actin filament that includes the critical overlap domain. Thus, the model provides a structural correlate to evaluate thin-filament mechanics, self-assembly mechanisms, and the effect of disease-causing mutations. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Interactions regulating the head-to-tail directed assembly of biological Janus rods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greene, A. C.; Bachand, M.; Gomez, A.

We can generalize the directed, head-to-tail self-assembly of microtubule filaments in the context of Janus colloidal rods. Specifically, their assembly at the tens of micron-length scale involves a careful balance between long-range electrostatic repulsion and short-range attractive forces. We show that the addition of counterion salts increases the rate of directed assembly by screening the electrostatic forces and enhancing the effectiveness of short-range interactions at the microtubule ends.

Interactions regulating the head-to-tail directed assembly of biological Janus rods

DOE PAGES

Greene, A. C.; Bachand, M.; Gomez, A.; ...

2017-03-31

We can generalize the directed, head-to-tail self-assembly of microtubule filaments in the context of Janus colloidal rods. Specifically, their assembly at the tens of micron-length scale involves a careful balance between long-range electrostatic repulsion and short-range attractive forces. We show that the addition of counterion salts increases the rate of directed assembly by screening the electrostatic forces and enhancing the effectiveness of short-range interactions at the microtubule ends.

Deinococcus radiodurans RecA nucleoprotein filaments characterized at the single-molecule level with optical tweezers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pobegalov, Georgii, E-mail: george.pobegalov@nanobio.spbstu.ru; Cherevatenko, Galina; Alekseev, Aleksandr

2015-10-23

Deinococcus radiodurans can survive extreme doses of ionizing radiation due to the very efficient DNA repair mechanisms that are able to cope even with hundreds of double-strand breaks. RecA, the critical protein of homologous recombination in bacteria, is one of the key components of the DNA-repair system. Repair of double-strand breaks requires RecA binding to DNA and assembly of the RecA nucleoprotein helical filaments. The Escherichia coli RecA protein (EcRecA) and its interactions with DNA have been extensively studied using various approaches including single-molecule techniques, while the D. radiodurans RecA (DrRecA) remains much less characterized. However, DrRecA shows some remarkable differencesmore » from E. coli homolog. Here we combine microfluidics and single-molecule DNA manipulation with optical tweezers to follow the binding of DrRecA to long double-stranded DNA molecules and probe the mechanical properties of DrRecA nucleoprotein filaments at physiological pH. Our data provide a direct comparison of DrRecA and EcRecA binding to double-stranded DNA under identical conditions. We report a significantly faster filaments assembly as well as lower values of persistence length and contour length for DrRecA nucleoprotein filaments compared to EcRecA. Our results support the existing model of DrRecA forming more frequent and less continuous filaments relative to those of EcRecA. - Highlights: • We investigate Deinococcus radiodurans RecA interactions with long double-stranded DNA at the single-molecule level. • At physiological pH D. radiodurans RecA forms nucleoprotein filaments significantly faster relative to Escherichia coli RecA. • D. radiodurans RecA-dsDNA nucleoprotein filaments are more flexible and slightly shorter compared to those of E. coli RecA.« less

Colloidal transport by active filaments

NASA Astrophysics Data System (ADS)

Manna, Raj Kumar; Kumar, P. B. Sunil; Adhikari, R.

2017-01-01

Enhanced colloidal transport beyond the limit imposed by diffusion is usually achieved through external fields. Here, we demonstrate the ballistic transport of a colloidal sphere using internal sources of energy provided by an attached active filament. The latter is modeled as a chain of chemo-mechanically active beads connected by potentials that enforce semi-flexibility and self-avoidance. The fluid flow produced by the active beads and the forces they mediate are explicitly taken into account in the overdamped equations of motion describing the colloid-filament assembly. The speed and efficiency of transport depend on the dynamical conformational states of the filament. We characterize these states using filament writhe as an order parameter and identify ones yielding maxima in speed and efficiency of transport. The transport mechanism reported here has a remarkable resemblance to the flagellar propulsion of microorganisms which suggests its utility in biomimetic systems.

Toward the Structure of Dynamic Membrane-Anchored Actin Networks

PubMed Central

Weber, Igor

2007-01-01

In the cortex of a motile cell, membrane-anchored actin filaments assemble into structures of varying shape and function. Filopodia are distinguished by a core of bundled actin filaments within finger-like extensions of the membrane. In a recent paper by Medalia et al1 cryo-electron tomography has been used to reconstruct, from filopodia of Dictyostelium cells, the 3-dimensional organization of actin filaments in connection with the plasma membrane. A special arrangement of short filaments converging toward the filopod's tip has been called a “terminal cone”. In this region force is applied for protrusion of the membrane. Here we discuss actin organization in the filopodia of Dictyostelium in the light of current views on forces that are generated by polymerizing actin filaments, and on the resistance of membranes against deformation that counteracts these forces. PMID:19262130

Molecular engineering of chiral colloidal liquid crystals using DNA origami

NASA Astrophysics Data System (ADS)

Siavashpouri, Mahsa; Wachauf, Christian H.; Zakhary, Mark J.; Praetorius, Florian; Dietz, Hendrik; Dogic, Zvonimir

2017-08-01

Establishing precise control over the shape and the interactions of the microscopic building blocks is essential for design of macroscopic soft materials with novel structural, optical and mechanical properties. Here, we demonstrate robust assembly of DNA origami filaments into cholesteric liquid crystals, one-dimensional supramolecular twisted ribbons and two-dimensional colloidal membranes. The exquisite control afforded by the DNA origami technology establishes a quantitative relationship between the microscopic filament structure and the macroscopic cholesteric pitch. Furthermore, it also enables robust assembly of one-dimensional twisted ribbons, which behave as effective supramolecular polymers whose structure and elastic properties can be precisely tuned by controlling the geometry of the elemental building blocks. Our results demonstrate the potential synergy between DNA origami technology and colloidal science, in which the former allows for rapid and robust synthesis of complex particles, and the latter can be used to assemble such particles into bulk materials.

Molecular engineering of chiral colloidal liquid crystals using DNA origami.

PubMed

Siavashpouri, Mahsa; Wachauf, Christian H; Zakhary, Mark J; Praetorius, Florian; Dietz, Hendrik; Dogic, Zvonimir

2017-08-01

Establishing precise control over the shape and the interactions of the microscopic building blocks is essential for design of macroscopic soft materials with novel structural, optical and mechanical properties. Here, we demonstrate robust assembly of DNA origami filaments into cholesteric liquid crystals, one-dimensional supramolecular twisted ribbons and two-dimensional colloidal membranes. The exquisite control afforded by the DNA origami technology establishes a quantitative relationship between the microscopic filament structure and the macroscopic cholesteric pitch. Furthermore, it also enables robust assembly of one-dimensional twisted ribbons, which behave as effective supramolecular polymers whose structure and elastic properties can be precisely tuned by controlling the geometry of the elemental building blocks. Our results demonstrate the potential synergy between DNA origami technology and colloidal science, in which the former allows for rapid and robust synthesis of complex particles, and the latter can be used to assemble such particles into bulk materials.

Nano-functionalized filamentous fungus hyphae with fast reversible macroscopic assembly & disassembly features.

PubMed

Wang, Haiying; Li, Xiaorui; Chai, Liyuan; Zhang, Liyuan

2015-05-18

A uniform decoration of hyphae by polyaniline nanoparticles (PANI NPs) was achieved here. This novel hybrid structure can be effectively assembled into a film by filtration and disassembled in water by shaking. This reversible process is very fast, which promises applications in nanomaterials including adsorption.

Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF.

PubMed

Barillà, Daniela; Rosenberg, Mark F; Nobbmann, Ulf; Hayes, Finbarr

2005-04-06

Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins.

Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF

PubMed Central

Barillà, Daniela; Rosenberg, Mark F; Nobbmann, Ulf; Hayes, Finbarr

2005-01-01

Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins. PMID:15775965

Molecular architecture of the Spire-actin nucleus and its implication for actin filament assembly.

PubMed

Sitar, Tomasz; Gallinger, Julia; Ducka, Anna M; Ikonen, Teemu P; Wohlhoefler, Michael; Schmoller, Kurt M; Bausch, Andreas R; Joel, Peteranne; Trybus, Kathleen M; Noegel, Angelika A; Schleicher, Michael; Huber, Robert; Holak, Tad A

2011-12-06

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs--even a single WH2 repeat--sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.

The small heat shock protein Hsp27 affects assembly dynamics and structure of keratin intermediate filament networks.

PubMed

Kayser, Jona; Haslbeck, Martin; Dempfle, Lisa; Krause, Maike; Grashoff, Carsten; Buchner, Johannes; Herrmann, Harald; Bausch, Andreas R

2013-10-15

The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance. Our knowledge about the molecular and physical mechanisms defining the network structure remains scarce, especially for the intermediate filament cytoskeleton. Here, we investigate the effect of small heat shock proteins on the keratin 8/18 intermediate filament cytoskeleton using a well-controlled model system of reconstituted keratin networks. We demonstrate that Hsp27 severely alters the structure of such networks by changing their assembly dynamics. Furthermore, the C-terminal tail domain of keratin 8 is shown to be essential for this effect. Combining results from fluorescence and electron microscopy with data from analytical ultracentrifugation reveals the crucial role of kinetic trapping in keratin network formation. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Advances in the mechanical modeling of filamentous actin and its cross-linked networks on multiple scales.

PubMed

Unterberger, Michael J; Holzapfel, Gerhard A

2014-11-01

The protein actin is a part of the cytoskeleton and, therefore, responsible for the mechanical properties of the cells. Starting with the single molecule up to the final structure, actin creates a hierarchical structure of several levels exhibiting a remarkable behavior. The hierarchy spans several length scales and limitations in computational power; therefore, there is a call for different mechanical modeling approaches for the different scales. On the molecular level, we may consider each atom in molecular dynamics simulations. Actin forms filaments by combining the molecules into a double helix. In a model, we replace molecular subdomains using coarse-graining methods, allowing the investigation of larger systems of several atoms. These models on the nanoscale inform continuum mechanical models of large filaments, which are based on worm-like chain models for polymers. Assemblies of actin filaments are connected with cross-linker proteins. Models with discrete filaments, so-called Mikado models, allow us to investigate the dependence of the properties of networks on the parameters of the constituents. Microstructurally motivated continuum models of the networks provide insights into larger systems containing cross-linked actin networks. Modeling of such systems helps to gain insight into the processes on such small scales. On the other hand, they call for verification and hence trigger the improvement of established experiments and the development of new methods.

THE STRUCTURE, ORIGIN, ISOLATION, AND COMPOSITION OF THE TUBULAR MASTIGONEMES OF THE OCHROMONAS FLAGELLUM

PubMed Central

Bouck, G. Benjamin

1971-01-01

The structure, assembly, and composition of the extracellular hairs (mastigonemes) of Ochromonas are detailed in this report. These mastigonemes form two lateral unbalanced rows, each row on opposite sides of the long anterior flagellum. Each mastigoneme consists of lateral filaments of two distinct sizes attached to a tubular shaft. The shaft is further differentiated into a basal region at one end and a group of from one to three terminal filaments at the free end. Mastigoneme ontogeny as revealed especially in deflagellated and regenerating cells appears to begin by assembly of the basal region and shaft within the perinuclear continuum. However, addition of lateral filaments to the shaft and extrusion of the mastigonemes to the cell surface is mediated by the Golgi complex. The ultimate distribution of mastigonemes on the flagellar surface seems to be the result of extrusion of mastigonemes near the base of the flagellum, and it is suggested that mastigonemes are then pulled up the flagellum as the axoneme elongates. Efforts to characterize mastigonemes biochemically after isolation and purification on cesium chloride (CsCl) followed by electrophoresis on acrylamide gels have demonstrated what appear to be a single major polypeptide and several differentially migrating carbohydrates. The polypeptide is not homologous with microtuble protein. The functionally anomalous role of mastigonemes in reversing flagellar thrust is discussed in relation to their distribution relative to flagellar anatomy and to the plane of flagellar undulations. PMID:5123323

Filamentous actin organization in the unfertilized sea urchin egg cortex.

PubMed

Henson, J H; Begg, D A

1988-06-01

We have investigated the organization of filamentous actin in the cortex of unfertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus. Rhodamine phalloidin and anti-actin immunofluorescent staining of isolated cortices reveal a punctate pattern of fluorescent sources. Comparison of this pattern with SEM images of microvillar morphology and distribution indicates that filamentous actin in the cortex is predominantly localized in the microvilli. Thin-section TEM and quick-freeze deep-etch ultrastructure of isolated cortices demonstrates that this microvillar-associated actin is in a novel organizational state composed of very short filaments arranged in a tight network and that these filament networks form mounds that extend beyond the plane of the plasma membrane. Actin filaments within the networks do not exhibit free ends and make end-on attachments with the membrane only within the region of the evaginating microvilli. Myosin S-1 dissociable crosslinks, 2-3 nm in diameter, are observed between network filaments and between network filaments and the membrane. A second population of long, individual actin filaments is observed in close lateral association with the plasma membrane and frequently complexes with the microvillar actin networks. The filamentous actin of the unfertilized egg cortex may participate in establishing the mechanical properties of the egg surface and may function in nucleating the assembly of cortical actin following fertilization.

Optical and Calorimetric Studies of Cholesterol-Rich Filamentous, Helical Ribbon and Crystal Microstructures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miroshnikova, Y. A.; Elsenbeck, M.; Zastavker, Y. V.

2009-04-19

Formation of biological self-assemblies at all scales is a focus of studies in fields ranging from biology to physics to biomimetics. Understanding the physico-chemical properties of these self-assemblies may lead to the design of bio-inspired structures and technological applications. Here we examine self-assembled filamentous, helical ribbon, and crystal microstructures formed in chemically defined lipid concentrate (CDLC), a model system for cholesterol crystallization in gallbladder bile. CDLC consists of cholesterol, bilayer-forming amphiphiles, micelle-forming amphiphiles, and water. Phase contrast and differential interference contrast (DIC) microscopy indicate the presence of three microstructure types in all samples studied, and allow for an investigation ofmore » the structures' unique geometries. Additionally, confocal microscopy is used for qualitative assessment of surface and internal composition. To complement optical observations, calorimetric (differential-scanning and modulation) experiments, provide the basis for an in-depth understanding of collective and individual thermal behavior. Observed ''transition'' features indicate clustering and ''straightening'' of helical ribbons into short, increasingly thickening, filaments that dissolve with increasing temperature. These results suggest that all microstructures formed in CDLC may coexist in a metastable chemical equilibrium. Further investigation of the CDLC thermal profile should uncover the process of cholesterol crystallization as well as the unique design and function of microstructures formed in this system.« less

Dynamic gradients of an intermediate filament-like cytoskeleton are recruited by a polarity landmark during apical growth.

PubMed

Fuchino, Katsuya; Bagchi, Sonchita; Cantlay, Stuart; Sandblad, Linda; Wu, Di; Bergman, Jessica; Kamali-Moghaddam, Masood; Flärdh, Klas; Ausmees, Nora

2013-05-21

Intermediate filament (IF)-like cytoskeleton emerges as a versatile tool for cellular organization in all kingdoms of life, underscoring the importance of mechanistically understanding its diverse manifestations. We showed previously that, in Streptomyces (a bacterium with a mycelial lifestyle similar to that of filamentous fungi, including extreme cell and growth polarity), the IF protein FilP confers rigidity to the hyphae by an unknown mechanism. Here, we provide a possible explanation for the IF-like function of FilP by demonstrating its ability to self-assemble into a cis-interconnected regular network in vitro and its localization into structures consistent with a cytoskeletal network in vivo. Furthermore, we reveal that a spatially restricted interaction between FilP and DivIVA, the main component of the Streptomyces polarisome complex, leads to formation of apical gradients of FilP in hyphae undergoing active tip extension. We propose that the coupling between the mechanism driving polar growth and the assembly of an IF cytoskeleton provides each new hypha with an additional stress-bearing structure at its tip, where the nascent cell wall is inevitably more flexible and compliant while it is being assembled and matured. Our data suggest that recruitment of cytoskeleton around a cell polarity landmark is a broadly conserved strategy in tip-growing cells.

Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy

PubMed Central

Goldsbury, Claire; Baxa, Ulrich; Simon, Martha N.; Steven, Alasdair C.; Engel, Andreas; Wall, Joseph S.; Aebi, Ueli; Müller, Shirley A.

2010-01-01

Amyloid fibrils are filamentous protein aggregates implicated in several common diseases like Alzheimer’s disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies like Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). PMID:20868754

Process for producing fine and ultrafine filament superconductor wire

DOEpatents

Kanithi, H.C.

1992-02-18

A process for producing a superconductor wire made up of a large number of round monofilament rods is provided for, comprising assembling a multiplicity of round monofilaments inside each of a multiplicity of thin wall hexagonal tubes and then assembling a number of said thin wall hexagonal tubes within an extrusion can and subsequently consolidating, extruding and drawing the entire assembly down to the desired wire size. 8 figs.

Process for producing fine and ultrafine filament superconductor wire

DOEpatents

Kanithi, Hem C.

1992-01-01

A process for producing a superconductor wire made up of a large number of round monofilament rods is provided for, comprising assembling a multiplicity of round monofilaments inside each of a multiplicity of thin wall hexagonal tubes and then assembling a number of said thin wall hexagonal tubes within an extrusion can and subsequently consolidating, extruding and drawing the entire assembly down to the desired wire size.

Carbon composites in space vehicle structures

NASA Technical Reports Server (NTRS)

Mayer, N. J.

1974-01-01

Recent developments in the technology of carbon or graphite filaments now provide the designer with greatly improved materials offering high specific strength and modulus. Besides these advantages are properties which are distinctly useful for space applications and which provide feasibility for missions not obtainable by other means. Current applications include major and secondary structures of communications satellites. A number of R & D projects are exploring carbon-fiber application to rocket engine motor cases, advanced antenna systems, and space shuttle components. Future system studies are being made, based on the successful application of carbon fibers for orbiting space telescope assemblies, orbital transfer vehicles, and very large deployable energy generation systems. Continued technology development is needed in analysis, material standards, and advanced structural concepts to exploit the full potential of carbon filaments in composite materials.

Sarcomeric Pattern Formation by Actin Cluster Coalescence

PubMed Central

Friedrich, Benjamin M.; Fischer-Friedrich, Elisabeth; Gov, Nir S.; Safran, Samuel A.

2012-01-01

Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells. PMID:22685394

Corrosion Performance of Friction Stir Linear Lap Welded AM60B Joints

NASA Astrophysics Data System (ADS)

Kish, J. R.; Birbilis, N.; McNally, E. M.; Glover, C. F.; Zhang, X.; McDermid, J. R.; Williams, G.

2017-11-01

A corrosion investigation of friction stir linear lap welded AM60B joints used to fabricate an Mg alloy-intensive automotive front end sub-assembly was performed. The stir zone exhibited a slightly refined grain size and significant break-up and re-distribution of the divorced Mg17Al12 (β-phase) relative to the base material. Exposures in NaCl (aq) environments revealed that the stir zone was more susceptible to localized corrosion than the base material. Scanning vibrating electrode technique measurements revealed differential galvanic activity across the joint. Anodic activity was confined to the stir zone surface and involved initiation and lateral propagation of localized filaments. Cathodic activity was initially confined to the base material surface, but was rapidly modified to include the cathodically-activated corrosion products in the filament wake. Site-specific surface analyses revealed that the corrosion observed across the welded joint was likely linked to variations in Al distribution across the surface film/metal interface.

Actin kinetics shapes cortical network structure and mechanics

PubMed Central

Fritzsche, Marco; Erlenkämper, Christoph; Moeendarbary, Emad; Charras, Guillaume; Kruse, Karsten

2016-01-01

The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs. PMID:27152338

Actin kinetics shapes cortical network structure and mechanics.

PubMed

Fritzsche, Marco; Erlenkämper, Christoph; Moeendarbary, Emad; Charras, Guillaume; Kruse, Karsten

2016-04-01

The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs.

Role of filament annealing in the kinetics and thermodynamics of nucleated polymerization.

PubMed

Michaels, Thomas C T; Knowles, Tuomas P J

2014-06-07

The formation of nanoscale protein filaments from soluble precursor molecules through nucleated polymerization is a common form of supra-molecular assembly phenomenon. This process underlies the generation of a range of both functional and pathological structures in nature. Filament breakage has emerged as a key process controlling the kinetics of the growth reaction since it increases the number of filament ends in the system that can act as growth sites. In order to ensure microscopic reversibility, however, the inverse process of fragmentation, end-to-end annealing of filaments, is a necessary component of a consistent description of such systems. Here, we combine Smoluchowski kinetics with nucleated polymerization models to generate a master equation description of protein fibrillization, where filamentous structures can undergo end-to-end association, in addition to elongation, fragmentation, and nucleation processes. We obtain self-consistent closed-form expressions for the growth kinetics and discuss the key physics that emerges from considering filament fusion relative to current fragmentation only models. Furthermore, we study the key time scales that describe relaxation to equilibrium.

Plasma Brightenings in a Failed Solar Filament Eruption

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Y.; Ding, M. D., E-mail: yingli@nju.edu.cn

Failed filament eruptions are solar eruptions that are not associated with coronal mass ejections. In a failed filament eruption, the filament materials usually show some ascending and falling motions as well as generating bright EUV emissions. Here we report a failed filament eruption (SOL2016-07-22) that occurred in a quiet-Sun region observed by the Atmospheric Imaging Assembly on board the Solar Dynamics Observatory . In this event, the filament spreads out but gets confined by the surrounding magnetic field. When interacting with the ambient magnetic field, the filament material brightens up and flows along the magnetic field lines through the coronamore » to the chromosphere. We find that some materials slide down along the lifting magnetic structure containing the filament and impact the chromosphere, and through kinetic energy dissipation, cause two ribbon-like brightenings in a wide temperature range. There is evidence suggesting that magnetic reconnection occurs between the filament magnetic structure and the surrounding magnetic fields where filament plasma is heated to coronal temperatures. In addition, thread-like brightenings show up on top of the erupting magnetic fields at low temperatures, which might be produced by an energy imbalance from a fast drop of radiative cooling due to plasma rarefaction. Thus, this single event of a failed filament eruption shows the existence of a variety of plasma brightenings that may be caused by completely different heating mechanisms.« less

Complete Structure of an Epithelial Keratin Dimer: Implications for Intermediate Filament Assembly.

PubMed

Bray, David J; Walsh, Tiffany R; Noro, Massimo G; Notman, Rebecca

2015-01-01

Keratins are cytoskeletal proteins that hierarchically arrange into filaments, starting with the dimer sub-unit. They are integral to the structural support of cells, in skin, hair and nails. In skin, keratin is thought to play a critical role in conferring the barrier properties and elasticity of skin. In general, the keratin dimer is broadly described by a tri-domain structure: a head, a central rod and a tail. As yet, no atomistic-scale picture of the entire dimer structure exists; this information is pivotal for establishing molecular-level connections between structure and function in intermediate filament proteins. The roles of the head and tail domains in facilitating keratin filament assembly and function remain as open questions. To address these, we report results of molecular dynamics simulations of the entire epithelial human K1/K10 keratin dimer. Our findings comprise: (1) the first three-dimensional structural models of the complete dimer unit, comprising of the head, rod and tail domains; (2) new insights into the chirality of the rod-domain twist gained from analysis of the full domain structure; (3) evidence for tri-subdomain partitioning in the head and tail domains; and, (4) identification of the residue characteristics that mediate non-covalent contact between the chains in the dimer. Our findings are immediately applicable to other epithelial keratins, such as K8/K18 and K5/K14, and to intermediate filament proteins in general.

Does Thioflavin-T Detect Oligomers Formed During Amyloid Fibril Assembly

NASA Astrophysics Data System (ADS)

Persichilli, Christopher; Hill, Shannon E.; Mast, Jason; Muschol, Martin

2011-03-01

Recent results have shown that oligomeric intermediates of amyloid fibril assembly represent the main toxic species in disorders such as Alzheimer's disease and type II diabetes. Thioflavin-T (ThT) is among the most commonly used indicator dyes for mature amyloid fibrils in vitro. We used ThT to monitor amyloid fibril formation of lysozyme (HEWL), and correlated ThT fluorescence to concurrent dynamic light scattering and atomic force microscopy measurements. Specifically, we tested the ability of ThT to discern among oligomer-free vs. oligomeric fibril assembly pathways. We found that ThT fluorescence did not detect oligomer growth; however, fluorescence increases did coincide with the formation of monomeric filaments in the oligomer-free assembly pathway. This implies that ThT fluorescence is not generally suitable for the detection of oligomeric intermediates. The results further suggest different internal structures for oligomeric vs. monomeric filaments. This research was supported, in part, by funding through the Byrd Alzheimer's Institute (ARG-2007-22) and the BITT-Florida Center of Excellence for M.M., an NSF-REU grant (DMR-1004873) for C. P. and an NSF-IGERT fellowship for S.H.

Assembly Mechanism of the Contractile Ring for Cytokinesis by Fission Yeast

NASA Astrophysics Data System (ADS)

Vavylonis, Dimitrios; Wu, Jian-Qiu; Huang, Xiaolei; O'Shaughnessy, Ben; Pollard, Thomas

2008-03-01

Animals and fungi assemble a contractile ring of actin filaments and the motor protein myosin to separate into individual daughter cells during cytokinesis. We studied the mechanism of contractile ring assembly in fission yeast with high time resolution confocal microscopy, computational image analysis methods, and numerical simulations. Approximately 63 nodes containing myosin, broadly distributed around the cell equator, assembled into a ring through stochastic motions, making many starts, stops, and changes of direction as they condense into a ring. Estimates of node friction coefficients from the mean square displacement of stationary nodes imply forces for node movement are greater than ˜ 4 pN, similarly to forces by a few molecular motors. Skeletonization and topology analysis of images of cells expressing fluorescent actin filament markers showed transient linear elements extending in all directions from myosin nodes and establishing connections among them. We propose a model with traction between nodes depending on transient connections established by stochastic search and capture (``search, capture, pull and release''). Numerical simulations of the model using parameter values obtained from experiment succesfully condense nodes into a continuous ring.

Daam1 regulates fascin for actin assembly in mouse oocyte meiosis.

PubMed

Lu, Yujie; Zhang, Yu; Pan, Meng-Hao; Kim, Nam-Hyung; Sun, Shao-Chen; Cui, Xiang-Shun

2017-07-18

As a formin protein, Daam1 (Dishevelled-associated activator of morphogenesis 1) is reported to regulate series of cell processes like endocytosis, cell morphology and migration via its effects on actin assembly in mitosis. However, whether Daam1 plays roles in female meiosis remains uncertain. In this study, we investigated the expression and functions of Daam1 during mouse oocyte meiosis. Our results indicated that Daam1 localized at the cortex of oocytes, which was similar with actin filaments. After Daam1 morpholino (MO) microinjection, the expression of Daam1 significantly decreased, which resulted in the failure of oocyte polar body extrusion. These results might be due to the defects of actin assembly, since the decreased fluorescence intensity of actin filaments in oocyte cortex and cytoplasm were observed. However, Daam1 knockdown seemed not to affect the meiotic spindle movement. In addition, we found that fascin might be the down effector of Daam1, since the protein expression of fascin decreased after Daam1 knockdown. Thus, our data suggested that Daam1 affected actin assembly during oocyte meiotic division via the regulation of fascin expression.

Quantitative Analysis of Statics and Dynamics of Actin Cables in Fission Yeast

NASA Astrophysics Data System (ADS)

Yusuf, Eddy; Wu, Jian-Qiu; Vavylonis, Dimitrios

2010-03-01

The assembly of actin and tubulin proteins into long filaments and bundles, i.e. closely-packed filaments, underlies important cellular processes such as cell motility, intracellular transport, and cell division. Recent theoretical and experimental work has addressed the nonequilibrium dynamics of single microtubules within live cells [1]. Actin filaments usually form dense networks that prevents microscopic imaging of individual filaments or bundles. Here, we studied actin dynamics using fission yeast that has low-density actin cytoskeleton consisting of actin cables (actin bundles aligned along the long axis of the cell) and ``actin patches.'' Yeast cells expressing GFP-CHD were imaged by 3D confocal microscopy. Stretching open active contours [2] were used to segment and track individual actin cables. We analyzed their curvature distribution, the tangent correlation, and the temporal bending amplitude fluctuations. We contrast our findings to equilibrium fluctuating semiflexible polymers and to microtubules in cells. We calculate the important time and length scales for the actin cables. We also discuss our findings within the broad context of understanding actin assembly in cells. [1] C. P. Brangwynne et. al., Phys. Rev. Lett. 100, 118104 (2008) [2] H. Li et. al., Proc. of the IEEE Int'l Symposium on Biomedical Imaging: From Nano to Macro, ISBI'09

Ecophysiology of an uncultivated lineage of Aigarchaeota from an oxic, hot spring filamentous ‘streamer’ community

DOE PAGES

Beam, Jacob P.; Jay, Zackary J.; Schmid, Markus C.; ...

2015-07-03

In this study, the candidate archaeal phylum ‘Aigarchaeota’ contains microorganisms from terrestrial and subsurface geothermal ecosystems. The phylogeny and metabolic potential of Aigarchaeota has been deduced from several recent single-cell amplified genomes; however, a detailed description of their metabolic potential and in situ transcriptional activity is absent. Here, we report a comprehensive metatranscriptome-based reconstruction of the in situ metabolism of Aigarchaeota in an oxic, hot spring filamentous ‘streamer’ community. Fluorescence in situ hybridization showed that these newly discovered Aigarchaeota are filamentous, which is consistent with the presence and transcription of an actin-encoding gene. Aigarchaeota filaments are intricately associated with othermore » community members, which include both bacteria (for example, filamentous Thermocrinis spp.) and archaea. Metabolic reconstruction of genomic and metatranscriptomic data suggests that this aigarchaeon is an aerobic, chemoorganoheterotroph with autotrophic potential. A heme copper oxidase complex was identified in the environmental genome assembly and highly transcribed in situ. Potential electron donors include acetate, fatty acids, amino acids, sugars and aromatic compounds, which may originate from extracellular polymeric substances produced by other microorganisms shown to exist in close proximity and/or autochthonous dissolved organic carbon (OC). Transcripts related to genes specific to each of these potential electron donors were identified, indicating that this aigarchaeon likely utilizes several OC substrates. Characterized members of this lineage cannot synthesize heme, and other cofactors and vitamins de novo, which suggests auxotrophy. We propose the name Candidatus ‘Calditenuis aerorheumensis’ for this aigarchaeon, which describes its filamentous morphology and its primary electron acceptor, oxygen.« less

Ecophysiology of an uncultivated lineage of Aigarchaeota from an oxic, hot spring filamentous ‘streamer’ community

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beam, Jacob P.; Jay, Zackary J.; Schmid, Markus C.

In this study, the candidate archaeal phylum ‘Aigarchaeota’ contains microorganisms from terrestrial and subsurface geothermal ecosystems. The phylogeny and metabolic potential of Aigarchaeota has been deduced from several recent single-cell amplified genomes; however, a detailed description of their metabolic potential and in situ transcriptional activity is absent. Here, we report a comprehensive metatranscriptome-based reconstruction of the in situ metabolism of Aigarchaeota in an oxic, hot spring filamentous ‘streamer’ community. Fluorescence in situ hybridization showed that these newly discovered Aigarchaeota are filamentous, which is consistent with the presence and transcription of an actin-encoding gene. Aigarchaeota filaments are intricately associated with othermore » community members, which include both bacteria (for example, filamentous Thermocrinis spp.) and archaea. Metabolic reconstruction of genomic and metatranscriptomic data suggests that this aigarchaeon is an aerobic, chemoorganoheterotroph with autotrophic potential. A heme copper oxidase complex was identified in the environmental genome assembly and highly transcribed in situ. Potential electron donors include acetate, fatty acids, amino acids, sugars and aromatic compounds, which may originate from extracellular polymeric substances produced by other microorganisms shown to exist in close proximity and/or autochthonous dissolved organic carbon (OC). Transcripts related to genes specific to each of these potential electron donors were identified, indicating that this aigarchaeon likely utilizes several OC substrates. Characterized members of this lineage cannot synthesize heme, and other cofactors and vitamins de novo, which suggests auxotrophy. We propose the name Candidatus ‘Calditenuis aerorheumensis’ for this aigarchaeon, which describes its filamentous morphology and its primary electron acceptor, oxygen.« less

Preparation and Characterization of Monomodal Grapevine Virus A Capsid Protein.

PubMed

Santana, Vinícius S; Mariutti, Ricardo B; Eberle, Raphael J; Ullah, Anwar; Caruso, Icaro P; Arni, Raghuvir K

2015-01-01

Grapevine virus A (GVA), a flexible filament of approximately 800 nm in length is composed of capsid subunits that spontaneously assembles around a positive sense genomic RNA. In addition to encapsidation, plant viruses capsid proteins (CPs) participate in other processes throughout infection and GVA CP is involved in cell-to-cell translocation of the virus. A protocol was developed to obtain low-molecular weight GVA-CP that is not prone to aggregation and spontaneous assembly and this was characterized by circular dichroism and dynamic light scattering. These results indicate the suitably of GVA-CP for X-ray crystallographic and NMR studies that should lead to the elucidation of the first three-dimensional structure of a flexible filamentous virus from the Betaflexiviridae family.

The Antibacterial Cell Division Inhibitor PC190723 Is an FtsZ Polymer-stabilizing Agent That Induces Filament Assembly and Condensation*

PubMed Central

Andreu, José M.; Schaffner-Barbero, Claudia; Huecas, Sonia; Alonso, Dulce; Lopez-Rodriguez, María L.; Ruiz-Avila, Laura B.; Núñez-Ramírez, Rafael; Llorca, Oscar; Martín-Galiano, Antonio J.

2010-01-01

Cell division protein FtsZ can form single-stranded filaments with a cooperative behavior by self-switching assembly. Subsequent condensation and bending of FtsZ filaments are important for the formation and constriction of the cytokinetic ring. PC190723 is an effective bactericidal cell division inhibitor that targets FtsZ in the pathogen Staphylococcus aureus and Bacillus subtilis and does not affect Escherichia coli cells, which apparently binds to a zone equivalent to the binding site of the antitumor drug taxol in tubulin (Haydon, D. J., Stokes, N. R., Ure, R., Galbraith, G., Bennett, J. M., Brown, D. R., Baker, P. J., Barynin, V. V., Rice, D. W., Sedelnikova, S. E., Heal, J. R., Sheridan, J. M., Aiwale, S. T., Chauhan, P. K., Srivastava, A., Taneja, A., Collins, I., Errington, J., and Czaplewski, L. G. (2008) Science 312, 1673–1675). We have found that the benzamide derivative PC190723 is an FtsZ polymer-stabilizing agent. PC190723 induced nucleated assembly of Bs-FtsZ into single-stranded coiled protofilaments and polymorphic condensates, including bundles, coils, and toroids, whose formation could be modulated with different solution conditions. Under conditions for reversible assembly of Bs-FtsZ, PC190723 binding reduced the GTPase activity and induced the formation of straight bundles and ribbons, which was also observed with Sa-FtsZ but not with nonsusceptible Ec-FtsZ. The fragment 2,6-difluoro-3-methoxybenzamide also induced Bs-FtsZ bundling. We propose that polymer stabilization by PC190723 suppresses in vivo FtsZ polymer dynamics and bacterial division. The biochemical action of PC190723 on FtsZ parallels that of the microtubule-stabilizing agent taxol on the eukaryotic structural homologue tubulin. Both taxol and PC190723 stabilize polymers against disassembly by preferential binding to each assembled protein. It is yet to be investigated whether both ligands target structurally related assembly switches. PMID:20212044

PRE-ERUPTION OSCILLATIONS IN THIN AND LONG FEATURES IN A QUIESCENT FILAMENT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Joshi, Anand D.; Hanaoka, Yoichiro; Suematsu, Yoshinori

We investigate the eruption of a quiescent filament located close to an active region. Large-scale activation was observed in only half of the filament in the form of pre-eruption oscillations. Consequently only this half erupted nearly 30 hr after the oscillations commenced. Time-slice diagrams of 171 Å images from the Atmospheric Imaging Assembly were used to study the oscillations. These were observed in several thin and long features connecting the filament spine to the chromosphere below. This study traces the origin of such features and proposes their possible interpretation. Small-scale magnetic flux cancellation accompanied by a brightening was observed atmore » the footpoint of the features shortly before their appearance, in images recorded by the Helioseismic and Magnetic Imager. A slow rise of the filament was detected in addition to the oscillations, indicating a gradual loss of equilibrium. Our analysis indicates that a change in magnetic field connectivity between two neighbouring active regions and the quiescent filament resulted in a weakening of the overlying arcade of the filament, leading to its eruption. It is also suggested that the oscillating features are filament barbs, and the oscillations are a manifestation during the pre-eruption phase of the filaments.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, X. L.; Xue, Z. K.; Ma, L.

Kink instability is a possible mechanism for solar filament eruption. However, it is very difficult to directly measure the twist of the solar filament from observation. In this paper, we measured the twist of a solar filament by analyzing its leg rotation. An inverse S-shaped filament in the active region NOAA 11485 was observed by the Atmospheric Imaging Assembly of the Solar Dynamics Observatory on 2012 May 22. During its eruption, the leg of the filament exhibited a significant rotation motion. The 304 Å images were used to uncurl the circles, the centers of which are the axis of themore » filament's leg. The result shows that the leg of the filament rotated up to about 510° (about 2.83π) around the axis of the filament within 23 minutes. The maximal rotation speed reached 100 degrees/minute (about 379.9 km s{sup –1} at radius 18''), which is the fastest rotation speed reported. We also calculated the decay index along the polarity inversion line in this active region and found that the decline of the overlying field with height is not fast enough to trigger the torus instability. According to the kink instability condition, this indicates that the kink instability is the trigger mechanism for the solar filament eruption.« less

Design and manufacturing of the CFRP lightweight telescope structure

NASA Astrophysics Data System (ADS)

Stoeffler, Guenter; Kaindl, Rainer

2000-06-01

Design of earthbound telescopes is normally based on conventional steel constructions. Several years ago thermostable CFRP Telescope and reflector structures were developed and manufacturing for harsh terrestrial environments. The airborne SOFIA TA requires beyond thermostability an excessive stiffness to mass ratio for the structure fulfilling performance and not to exceed mass limitations by the aircraft Boeing 747 SP. Additional integration into A/C drives design of structure subassemblies. Thickness of CFRP Laminates, either filament wound or prepreg manufactured need special attention and techniques to gain high material quality according to aerospace requirements. Sequential shop assembly of the structure subassemblies minimizes risk for assembling TA. Design goals, optimization of layout and manufacturing techniques and results are presented.

International Space Station Major Constituent Analyzer On-Orbit Performance

NASA Technical Reports Server (NTRS)

Gardner, Ben D.; Erwin, Phillip M.; Thoresen, Souzan; Wiedemann, Rachel; Matty, Chris

2015-01-01

The Major Constituent Analyzer is a mass spectrometer based system that measures the major atmospheric constituents on the International Space Station. A number of limited-life components require periodic change-out, including the ORU 02 analyzer and the ORU 08 Verification Gas Assembly. Improvements to ion pump operation and ion source tuning have improved lifetime performance of the current ORU 02 design. The most recent ORU 02 analyzer assemblies, as well as ORU 08, have operated nominally. For ORU 02, the ion source filaments and ion pump lifetime continue to be key determinants of MCA performance and logistical support. Monitoring several key parameters provides the capacity to monitor ORU health and properly anticipate end of life.

Single Molecule Visualization of Protein-DNA Complexes: Watching Machines at Work

NASA Astrophysics Data System (ADS)

Kowalczykowski, Stephen

2013-03-01

We can now watch individual proteins acting on single molecules of DNA. Such imaging provides unprecedented interrogation of fundamental biophysical processes. Visualization is achieved through the application of two complementary procedures. In one, single DNA molecules are attached to a polystyrene bead and are then captured by an optical trap. The DNA, a worm-like coil, is extended either by the force of solution flow in a micro-fabricated channel, or by capturing the opposite DNA end in a second optical trap. In the second procedure, DNA is attached by one end to a glass surface. The coiled DNA is elongated either by continuous solution flow or by subsequently tethering the opposite end to the surface. Protein action is visualized by fluorescent reporters: fluorescent dyes that bind double-stranded DNA (dsDNA), fluorescent biosensors for single-stranded DNA (ssDNA), or fluorescently-tagged proteins. Individual molecules are imaged using either epifluorescence microscopy or total internal reflection fluorescence (TIRF) microscopy. Using these approaches, we imaged the search for DNA sequence homology conducted by the RecA-ssDNA filament. The manner by which RecA protein finds a single homologous sequence in the genome had remained undefined for almost 30 years. Single-molecule imaging revealed that the search occurs through a mechanism termed ``intersegmental contact sampling,'' in which the randomly coiled structure of DNA is essential for reiterative sampling of DNA sequence identity: an example of parallel processing. In addition, the assembly of RecA filaments on single molecules of single-stranded DNA was visualized. Filament assembly requires nucleation of a protein dimer on DNA, and subsequent growth occurs via monomer addition. Furthermore, we discovered a class of proteins that catalyzed both nucleation and growth of filaments, revealing how the cell controls assembly of this protein-DNA complex.

Atomic structure of the vimentin central α-helical domain and its implications for intermediate filament assembly.

PubMed

Chernyatina, Anastasia A; Nicolet, Stefan; Aebi, Ueli; Herrmann, Harald; Strelkov, Sergei V

2012-08-21

Together with actin filaments and microtubules, intermediate filaments (IFs) are the basic cytoskeletal components of metazoan cells. Over 80 human diseases have been linked to mutations in various IF proteins to date. However, the filament structure is far from being resolved at the atomic level, which hampers rational understanding of IF pathologies. The elementary building block of all IF proteins is a dimer consisting of an α-helical coiled-coil (CC) "rod" domain flanked by the flexible head and tail domains. Here we present three crystal structures of overlapping human vimentin fragments that comprise the first half of its rod domain. Given the previously solved fragments, a nearly complete atomic structure of the vimentin rod has become available. It consists of three α-helical segments (coils 1A, 1B, and 2) interconnected by linkers (L1 and L12). Most of the CC structure has a left-handed twist with heptad repeats, but both coil 1B and coil 2 also exhibit untwisted, parallel stretches with hendecad repeats. In the crystal structure, linker L1 was found to be α-helical without being involved in the CC formation. The available data allow us to construct an atomic model of the antiparallel tetramer representing the second level of vimentin assembly. Although the presence of the nonhelical head domains is essential for proper tetramer stabilization, the precise alignment of the dimers forming the tetramer appears to depend on the complementarity of their surface charge distribution patterns, while the structural plasticity of linker L1 and coil 1A plays a role in the subsequent IF assembly process.

Effect of catalyst type on field emission properties of nanostructured carbon films grown by a modified hot-filament chemical vapor deposition technique

NASA Astrophysics Data System (ADS)

Kang, Sukill; Lowndes, Douglas H.; Ellis, Darren

2001-03-01

Nanostructured carbon films have been grown on uncatalysed n-type Si using a modified HF-CVD process and catalytic decomposition of ethylene (C_2H_4). Various metal catalyst wires such as Ni, Co, Fe and a NiFe composite were placed within the windings of a tungsten filament and the assembly was placed in close proximity ( ~7 mm) to the unheated substrate. Radiative heating of the substrate by the filament results in a substrate temperature of ~ 500^oC after 7 min. Films grown using the Ni catalyst showed a field emission turn-on field that varied from 9 to 15 V/μm and was stable for 30-50 hours (1-10 A/cm^2 emission current density), a result that is comparable to carbon nanotube- and carbon nanofiber-based structures. In this contribution, we present results from field emission scanning electron microscopy, transmission electron microscopy, and electron field emission measurements that elucidate the relationship between field emission properties, film morphology, and type of catalyst.

Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

PubMed Central

Mueller, Jan; Pfanzelter, Julia; Winkler, Christoph; Narita, Akihiro; Le Clainche, Christophe; Nemethova, Maria; Carlier, Marie-France; Maeda, Yuichiro; Welch, Matthew D.; Ohkawa, Taro; Schmeiser, Christian; Resch, Guenter P.; Small, J. Victor

2014-01-01

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion. PMID:24453943

Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.

PubMed

Mueller, Jan; Pfanzelter, Julia; Winkler, Christoph; Narita, Akihiro; Le Clainche, Christophe; Nemethova, Maria; Carlier, Marie-France; Maeda, Yuichiro; Welch, Matthew D; Ohkawa, Taro; Schmeiser, Christian; Resch, Guenter P; Small, J Victor

2014-01-01

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.

SHG-specificity of cellular Rootletin filaments enables naïve imaging with universal conservation

NASA Astrophysics Data System (ADS)

Akiyama, Toshihiro; Inoko, Akihito; Kaji, Yuichi; Yonemura, Shigenobu; Kakiguchi, Kisa; Segawa, Hiroki; Ishitsuka, Kei; Yoshida, Masaki; Numata, Osamu; Leproux, Philippe; Couderc, Vincent; Oshika, Tetsuro; Kano, Hideaki

2017-01-01

Despite growing demand for truly naïve imaging, label-free observation of cilium-related structure remains challenging, and validation of the pertinent molecules is correspondingly difficult. In this study, in retinas and cultured cells, we distinctively visualized Rootletin filaments in rootlets in the second harmonic generation (SHG) channel, integrated in custom coherent nonlinear optical microscopy (CNOM) with a simple, compact, and ultra-broadband supercontinuum light source. This SHG signal was primarily detected on rootlets of connecting cilia in the retinal photoreceptor and was validated by colocalization with anti-Rootletin staining. Transfection of cells with Rootletin fragments revealed that the SHG signal can be ascribed to filaments assembled from the R234 domain, but not to cross-striations assembled from the R123 domain. Consistent with this, Rootletin-depleted cells lacked SHG signal expected as centrosome linker. As a proof of concept, we confirmed that similar fibrous SHG was observed even in unicellular ciliates. These findings have potential for broad applications in clinical diagnosis and biophysical experiments with various organisms.

Molecular architecture of the Spire–actin nucleus and its implication for actin filament assembly

PubMed Central

Sitar, Tomasz; Gallinger, Julia; Ducka, Anna M.; Ikonen, Teemu P.; Wohlhoefler, Michael; Schmoller, Kurt M.; Bausch, Andreas R.; Joel, Peteranne; Trybus, Kathleen M.; Noegel, Angelika A.; Schleicher, Michael; Huber, Robert; Holak, Tad A.

2011-01-01

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire–actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire–actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott–Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire–actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire–actin module. In addition, we find that preformed, isolated Spire–actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs—even a single WH2 repeat—sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem. PMID:22106272

Crystal Structure of FadA Adhesin from Fusobacterium nucleatum Reveals a Novel Oligomerization Motif, the Leucine Chain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nithianantham, Stanley; Xu, Minghua; Yamada, Mitsunori

2009-04-07

Many bacterial appendages have filamentous structures, often composed of repeating monomers assembled in a head-to-tail manner. The mechanisms of such linkages vary. We report here a novel protein oligomerization motif identified in the FadA adhesin from the Gram-negative bacterium Fusobacterium nucleatum. The 2.0 {angstrom} crystal structure of the secreted form of FadA (mFadA) reveals two antiparallel {alpha}-helices connected by an intervening 8-residue hairpin loop. Leucine-leucine contacts play a prominent dual intra- and intermolecular role in the structure and function of FadA. First, they comprise the main association between the two helical arms of the monomer; second, they mediate the head-to-tailmore » association of monomers to form the elongated polymers. This leucine-mediated filamentous assembly of FadA molecules constitutes a novel structural motif termed the 'leucine chain.' The essential role of these residues in FadA is corroborated by mutagenesis of selected leucine residues, which leads to the abrogation of oligomerization, filament formation, and binding to host cells.« less

Dynamics of myosin II organization into cortical contractile networks and fibers

NASA Astrophysics Data System (ADS)

Nie, Wei; Wei, Ming-Tzo; Ou-Yang, Daniel; Jedlicka, Sabrina; Vavylonis, Dimitrios

2014-03-01

The morphology of adhered cells critically depends on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin which disrupts actomyosin stress fibers. Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared them to studies by other groups. This analysis suggested that the following processes contribute to the assembly of cortical actomyosin into fibers: random myosin mini-filament assembly and disassembly along the cortex; myosin mini-filament aligning and contraction; stabilization of cortical myosin upon increasing contractile tension. We developed simple numerical simulations that include those processes. The results of simulations of cells at steady state and in response to blebbistatin capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness.

Skip residues modulate the structural properties of the myosin rod and guide thick filament assembly

DOE PAGES

Taylor, Keenan C.; Buvoli, Massimo; Korkmaz, Elif Nihal; ...

2015-07-06

The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report in this paper that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility.more » Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. Finally, in addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.« less

Molecular Motor-Induced Instabilities and Cross Linkers Determine Biopolymer Organization

PubMed Central

Smith, D.; Ziebert, F.; Humphrey, D.; Duggan, C.; Steinbeck, M.; Zimmermann, W.; Käs, J.

2007-01-01

All eukaryotic cells rely on the active self-organization of protein filaments to form a responsive intracellular cytoskeleton. The necessity of motility and reaction to stimuli additionally requires pathways that quickly and reversibly change cytoskeletal organization. While thermally driven order-disorder transitions are, from the viewpoint of physics, the most obvious method for controlling states of organization, the timescales necessary for effective cellular dynamics would require temperatures exceeding the physiologically viable temperature range. We report a mechanism whereby the molecular motor myosin II can cause near-instantaneous order-disorder transitions in reconstituted cytoskeletal actin solutions. When motor-induced filament sliding diminishes, the actin network structure rapidly and reversibly self-organizes into various assemblies. Addition of stable cross linkers was found to alter the architectures of ordered assemblies. These isothermal transitions between dynamic disorder and self-assembled ordered states illustrate that the interplay between passive crosslinking and molecular motor activity plays a substantial role in dynamic cellular organization. PMID:17604319

Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly

PubMed Central

Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

2015-01-01

Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells. DOI: http://dx.doi.org/10.7554/eLife.06126.001 PMID:26652273

Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly.

PubMed

Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

2015-12-10

Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells.

Bacterial flagellar microhydrodynamics: Laminar flow over complex flagellar filaments, analog archimedean screws and cylinders, and its perturbations.

PubMed

Trachtenberg, Shlomo; Fishelov, Dalia; Ben-Artzi, Matania

2003-09-01

The flagellar filament, the bacterial organelle of motility, is the smallest rotary propeller known. It consists of 1), a basal body (part of which is the proton driven rotary motor), 2), a hook (universal joint-allowing for off-axial transmission of rotary motion), and 3), a filament (propeller-a long, rigid, supercoiled helical assembly allowing for the conversion of rotary motion into linear thrust). Helically perturbed (so-called "complex") filaments have a coarse surface composed of deep grooves and ridges following the three-start helical lines. These surface structures, reminiscent of a turbine or Archimedean screw, originate from symmetry reduction along the six-start helical lines due to dimerization of the flagellin monomers from which the filament self assembles. Using high-resolution electron microscopy and helical image reconstruction methods, we calculated three-dimensional density maps of the complex filament of Rhizobium lupini H13-3 and determined its surface pattern and boundaries. The helical symmetry of the filament allows viewing it as a stack of identical slices spaced axially and rotated by constant increments. Here we use the closed outlines of these slices to explore, in two dimensions, the hydrodynamic effect of the turbine-like boundaries of the flagellar filament. In particular, we try to determine if, and under what conditions, transitions from laminar to turbulent flow (or perturbations of the laminar flow) may occur on or near the surface of the bacterial propeller. To address these questions, we apply the boundary element method in a manner allowing the handling of convoluted boundaries. We tested the method on several simple, well-characterized cylindrical structures before applying it to real, highly convoluted biological surfaces and to simplified mechanical analogs. Our results indicate that under extreme structural and functional conditions, and at low Reynolds numbers, a deviation from laminar flow might occur on the flagellar surface. These transitions, and the conditions enabling them, may affect flagellar polymorphism and the formation and dispersion of flagellar bundles-factors important in the chemotactic response.

Metal-free current collectors based on graphene materials for supecapacitors produced by 3D printing

NASA Astrophysics Data System (ADS)

Baskakov, S. A.; Baskakova, Yu. V.; Lyskov, N. V.; Dremova, N. N.; Shul'ga, Yu. M.

2017-10-01

Supercapacitor (SC) current collectors with electrodes made of graphite oxide reduced during microwave exfoliation are produced from a commercial filament with a graphene component via layer-by-layer fusing with a 3D printer. The separator is made of a graphene oxide film. The current collectors are investigated by means of IR spectroscopy. Electrochemical tests are performed for the assembled SC that include tests of its cyclic stability up to 1000 cycles.

Amyloid-like assembly of the low complexity domain of yeast Nab3.

PubMed

O'Rourke, Thomas W; Loya, Travis J; Head, PamelaSara E; Horton, John R; Reines, Daniel

2015-01-01

Termination of transcription of short non-coding RNAs is carried out in yeast by the Nab3-Nrd1-Sen1 complex. Nab3 and Nrd1 are hnRNP-like proteins that dimerize and bind RNA with sequence specificity. We show here that an essential region of Nab3 that is predicted to be prion-like based upon its sequence bias, formed amyloid-like filaments. A similar region from Nrd1 also assembled into filaments in vitro. The purified Nab3 domain formed a macroscopic gel whose lattice organization was observed by X-ray fiber diffraction. Filaments were resistant to dissociation in anionic detergent, bound the fluorescent dye thioflavin T, and showed a β-sheet rich structure by circular dichroism spectroscopy, similar to human amyloid β which served as a reference amyloid. A version of the Nab3 domain with a mutation that impairs its termination function, also formed fibers as observed by electron microscopy. Using a protein fragment interaction assay, the purified Nab3 domain was seen to interact with itself in living yeast. A similar observation was made for full length Nab3. These results suggest that the Nab3 and Nrd1 RNA-binding proteins can attain a complex polymeric form and raise the possibility that this property is important for organizing their functional state during termination. These findings are congruent with recent work showing that RNA binding proteins with low complexity domains form a dynamic subcellular matrix in which RNA metabolism takes place but can also aberrantly yield pathological aggregated particles.

Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope

PubMed Central

Otsuka, Shotaro; Bui, Khanh Huy; Schorb, Martin; Hossain, M Julius; Politi, Antonio Z; Koch, Birgit; Eltsov, Mikhail; Beck, Martin; Ellenberg, Jan

2016-01-01

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM. DOI: http://dx.doi.org/10.7554/eLife.19071.001 PMID:27630123

Aerodynamic flail for a spinning projectile

DOEpatents

Cole, James K.

1990-05-01

A flail is provided which reduces the spin of a projectile in a recovery system which includes a parachute, a cable connected to the parachute, a swivel, and means for connecting the swivel to the projectile. The flail includes a plurality of flexible filaments and a rotor for attaching the filaments to the front end of the projectile. The rotor is located radially with respect to the spinning axis of the projectile. In one embodiment, the projectile includes a first nose cone section housing a deployable spin damping assembly; a second nose cone section, housing a deployable parachute assembly; a shell section, supporting the first and second nose cone sections during flight of the projectile; a mechanism for releasing the first nose cone section from the second cone section; and a mechanism for releasing the second nose cone section from the shell section. In operation of this embodiment, the deployable spin damping assembly deploys during flight of the projectile when the mechanism for releasing the first nose cone section from the second nose cone section are actuated. Then, upon actuation of the mechanism for releasing the second nose cone section from the shell section, two things happen: the spin damping assembly separates from the projectile; and the deployable parachute assembly is deployed.

Aerodynamic flail for a spinning projectile

DOEpatents

Cole, James K.

1990-01-01

A flail is provided which reduces the spin of a projectile in a recovery system which includes a parachute, a cable connected to the parachute, a swivel, and means for connecting the swivel to the projectile. The flail includes a plurality of flexible filaments and a rotor for attaching the filaments to the front end of the projectile. The rotor is located radially with respect to the spinning axis of the projectile. In one embodiment, the projectile includes a first nose cone section housing a deployable spin damping assembly; a second nose cone section, housing a deployable parachute assembly; a shell section, supporting the first and second nose cone sections during flight of the projectile; a mechanism for releasing the first nose cone section from the second cone section; and a mechanism for releasing the second nose cone section from the shell section. In operation of this embodiment, the deployable spin damping assembly deploys during flight of the projectile when the mechanism for releasing the first nose cone section from the second nose cone section are actuated. Then, upon actuation of the mechanism for releasing the second nose cone section from the shell section, two things happen: the spin damping assembly separates from the projectile; and the deployable parachute assembly is deployed.

Relation between halo spin and cosmic-web filaments at z ≃ 3

NASA Astrophysics Data System (ADS)

González, Roberto E.; Prieto, Joaquin; Padilla, Nelson; Jimenez, Raul

2017-02-01

We investigate the spin evolution of dark matter haloes and their dependence on the number of connected filaments from the cosmic web at high redshift (spin-filament relation hereafter). To this purpose, we have simulated 5000 haloes in the mass range 5 × 109 h-1 M⊙ to 5 × 1011 h-1 M⊙ at z = 3 in cosmological N-body simulations. We confirm the relation found by Prieto et al. (2015) where haloes with fewer filaments have larger spin. We also found that this relation is more significant for higher halo masses, and for haloes with a passive (no major mergers) assembly history. Another finding is that haloes with larger spin or with fewer filaments have their filaments more perpendicularly aligned with the spin vector. Our results point to a picture in which the initial spin of haloes is well described by tidal torque theory and then gets subsequently modified in a predictable way because of the topology of the cosmic web, which in turn is given by the currently favoured Lambda cold dark matter (LCDM) model. Our spin-filament relation is a prediction from LCDM that could be tested with observations.

Galaxy evolution in the metric of the cosmic web

NASA Astrophysics Data System (ADS)

Kraljic, K.; Arnouts, S.; Pichon, C.; Laigle, C.; de la Torre, S.; Vibert, D.; Cadiou, C.; Dubois, Y.; Treyer, M.; Schimd, C.; Codis, S.; de Lapparent, V.; Devriendt, J.; Hwang, H. S.; Le Borgne, D.; Malavasi, N.; Milliard, B.; Musso, M.; Pogosyan, D.; Alpaslan, M.; Bland-Hawthorn, J.; Wright, A. H.

2018-02-01

The role of the cosmic web in shaping galaxy properties is investigated in the Galaxy And Mass Assembly (GAMA) spectroscopic survey in the redshift range 0.03 ≤ z ≤ 0.25. The stellar mass, u - r dust corrected colour and specific star formation rate (sSFR) of galaxies are analysed as a function of their distances to the 3D cosmic web features, such as nodes, filaments and walls, as reconstructed by DisPerSE. Significant mass and type/colour gradients are found for the whole population, with more massive and/or passive galaxies being located closer to the filament and wall than their less massive and/or star-forming counterparts. Mass segregation persists among the star-forming population alone. The red fraction of galaxies increases when closing in on nodes, and on filaments regardless of the distance to nodes. Similarly, the star-forming population reddens (or lowers its sSFR) at fixed mass when closing in on filament, implying that some quenching takes place. These trends are also found in the state-of-the-art hydrodynamical simulation HORIZON-AGN. These results suggest that on top of stellar mass and large-scale density, the traceless component of the tides from the anisotropic large-scale environment also shapes galactic properties. An extension of excursion theory accounting for filamentary tides provides a qualitative explanation in terms of anisotropic assembly bias: at a given mass, the accretion rate varies with the orientation and distance to filaments. It also explains the absence of type/colour gradients in the data on smaller, non-linear scales.

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

PubMed

Smith, Alyson S; Nowak, Roberta B; Zhou, Sitong; Giannetto, Michael; Gokhin, David S; Papoin, Julien; Ghiran, Ionita C; Blanc, Lionel; Wan, Jiandi; Fowler, Velia M

2018-05-08

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths

PubMed Central

Zimmermann, Dennis; Santos, Alicja; Kovar, David R.; Rock, Ronald S.

2015-01-01

Summary Unlike a static and immobile skeleton, the actin cytoskeleton is a highly dynamic network of filamentous actin (F-actin) polymers that continuously turn over. In addition to generating mechanical forces and sensing mechanical deformation, dynamic F-actin networks serve as cellular tracks for myosin motor traffic. However, much of our mechanistic understanding of processive myosins comes from in vitro studies where motility was studied on pre-assembled and artificially stabilized, static F-actin tracks. In this work, we examine the role of actin dynamics in single-molecule myosin motility using assembling F-actin and the two highly processive motors, myosin-5 and myosin-6. These two myosins have distinct functions in the cell and travel in opposite directions along actin filaments [1–3]. Myosin-5 walks towards the barbed ends of F-actin, traveling to sites of actin polymerization at the cell periphery [4]. Myosin-6 walks towards the pointed end of F-actin [5], traveling towards the cell center along older segments of the actin filament. We find that myosin-5 takes 1.3 to 1.5-fold longer runs on ADP•Pi (young) F-actin, while myosin-6 takes 1.7 to 3.6-fold longer runs along ADP (old) F-actin. These results suggest that conformational differences between ADP•Pi and ADP F-actin tailor these myosins to walk farther toward their preferred actin filament end. Taken together, these experiments define a new mechanism by which myosin traffic may sort to different F-actin networks depending on filament age. PMID:26190073

A novel role for WAVE1 in controlling actin network growth rate and architecture.

PubMed

Sweeney, Meredith O; Collins, Agnieszka; Padrick, Shae B; Goode, Bruce L

2015-02-01

Branched actin filament networks in cells are assembled through the combined activities of Arp2/3 complex and different WASP/WAVE proteins. Here we used TIRF and electron microscopy to directly compare for the first time the assembly kinetics and architectures of actin filament networks produced by Arp2/3 complex and dimerized VCA regions of WAVE1, WAVE2, or N-WASP. WAVE1 produced strikingly different networks from WAVE2 or N-WASP, which comprised unexpectedly short filaments. Further analysis showed that the WAVE1-specific activity stemmed from an inhibitory effect on filament elongation both in the presence and absence of Arp2/3 complex, which was observed even at low stoichiometries of WAVE1 to actin monomers, precluding an effect from monomer sequestration. Using a series of VCA chimeras, we mapped the elongation inhibitory effects of WAVE1 to its WH2 ("V") domain. Further, mutating a single conserved lysine residue potently disrupted WAVE1's inhibitory effects. Taken together, our results show that WAVE1 has unique activities independent of Arp2/3 complex that can govern both the growth rates and architectures of actin filament networks. Such activities may underlie previously observed differences between the cellular functions of WAVE1 and WAVE2. © 2015 Sweeney et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Engineering emergent multicellular behavior through synthetic adhesion

NASA Astrophysics Data System (ADS)

Glass, David; Riedel-Kruse, Ingmar

In over a decade, synthetic biology has developed increasingly robust gene networks within single cells, but constructed very few systems that demonstrate multicellular spatio-temporal dynamics. We are filling this gap in synthetic biology's toolbox by developing an E. coli self-assembly platform based on modular cell-cell adhesion. We developed a system in which adhesive selectivity is provided by a library of outer membrane-displayed peptides with intra-library specificities, while affinity is provided by consistent expression across the entire library. We further provide a biophysical model to help understand the parameter regimes in which this tool can be used to self-assemble into cellular clusters, filaments, or meshes. The combined platform will enable future development of synthetic multicellular systems for use in consortia-based metabolic engineering, in living materials, and in controlled study of minimal multicellular systems. Stanford Bio-X Bowes Fellowship.

Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits

PubMed Central

2016-01-01

Ions regulate the assembly and mechanical properties of actin filaments. Recent work using structural bioinformatics and site-specific mutagenesis favors the existence of two discrete and specific divalent cation binding sites on actin filaments, positioned in the long axis between actin subunits. Cation binding at one site drives polymerization, while the other modulates filament stiffness and plays a role in filament severing by the regulatory protein, cofilin. Existing structural methods have not been able to resolve filament-associated cations, and so in this work we turn to molecular dynamics simulations to suggest a candidate binding pocket geometry for each site and to elucidate the mechanism by which occupancy of the “stiffness site” affects filament mechanical properties. Incorporating a magnesium ion in the “polymerization site” does not seem to require any large-scale change to an actin subunit’s conformation. Binding of a magnesium ion in the “stiffness site” adheres the actin DNase-binding loop (D-loop) to its long-axis neighbor, which increases the filament torsional stiffness and bending persistence length. Our analysis shows that bound D-loops occupy a smaller region of accessible conformational space. Cation occupancy buries key conserved residues of the D-loop, restricting accessibility to regulatory proteins and enzymes that target these amino acids. PMID:27146246

Tension modulates actin filament polymerization mediated by formin and profilin

PubMed Central

Courtemanche, Naomi; Lee, Ja Yil; Pollard, Thomas D.; Greene, Eric C.

2013-01-01

Formins promote processive elongation of actin filaments for cytokinetic contractile rings and other cellular structures. In vivo, these structures are exposed to tension, but the effect of tension on these processes was unknown. Here we used single-molecule imaging to investigate the effects of tension on actin polymerization mediated by yeast formin Bni1p. Small forces on the filaments dramatically slowed formin-mediated polymerization in the absence of profilin, but resulted in faster polymerization in the presence of profilin. We propose that force shifts the conformational equilibrium of the end of a filament associated with formin homology 2 domains toward the closed state that precludes polymerization, but that profilin–actin associated with formin homology 1 domains reverses this effect. Thus, physical forces strongly influence actin assembly by formin Bni1p. PMID:23716666

Eukaryotic chaperonin containing T-complex polypeptide 1 interacts with filamentous actin and reduces the initial rate of actin polymerization in vitro

PubMed Central

Grantham, Julie; Ruddock, Lloyd W.; Roobol, Anne; Carden, Martin J.

2002-01-01

We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament “seeds” and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament (+) ends, beyond its already well-established role in producing new actin monomers. PMID:12482199

Structural analysis of vimentin and keratin intermediate filaments by cryo-electron tomography.

PubMed

Norlén, Lars; Masich, Sergej; Goldie, Kenneth N; Hoenger, Andreas

2007-06-10

Intermediate filaments are a large and structurally diverse group of cellular filaments that are classified into five different groups. They are referred to as intermediate filaments (IFs) because they are intermediate in diameter between the two other cytoskeletal filament systems that is filamentous actin and microtubules. The basic building block of IFs is a predominantly alpha-helical rod with variable length globular N- and C-terminal domains. On the ultra-structural level there are two major differences between IFs and microtubules or actin filaments: IFs are non-polar, and they do not exhibit large globular domains. IF molecules associate via a coiled-coil interaction into dimers and higher oligomers. Structural investigations into the molecular building plan of IFs have been performed with a variety of biophysical and imaging methods such as negative staining and metal-shadowing electron microscopy (EM), mass determination by scanning transmission EM, X-ray crystallography on fragments of the IF stalk and low-angle X-ray scattering. The actual packing of IF dimers into a long filament varies between the different families. Typically the dimers form so called protofibrils that further assemble into a filament. Here we introduce new cryo-imaging methods for structural investigations of IFs in vitro and in vivo, i.e., cryo-electron microscopy and cryo-electron tomography, as well as associated techniques such as the preparation and handling of vitrified sections of cellular specimens.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin {beta}4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helixmore » of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin {beta}4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.« less

PEM/SPE fuel cell

DOEpatents

Grot, Stephen Andreas

1998-01-01

A PEM/SPE fuel cell including a membrane-electrode assembly (MEA) having a plurality of oriented filament embedded the face thereof for supporting the MEA and conducting current therefrom to contiguous electrode plates.

Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments

PubMed Central

Miller, Ona K; Potter, Jane A; Vijayakrishnan, Swetha; Bhella, David; Naismith, James H; Elliott, Richard M

2017-01-01

Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83–248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence. PMID:28915104

Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments.

PubMed

Barski, Michal; Brennan, Benjamin; Miller, Ona K; Potter, Jane A; Vijayakrishnan, Swetha; Bhella, David; Naismith, James H; Elliott, Richard M; Schwarz-Linek, Ulrich

2017-09-15

Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83-248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence.

Dynamic assembly of polymer nanotube networks via kinesin powered microtubule filaments

DOE PAGES

Paxton, Walter F.; Bachand, George D.; Gomez, Andrew; ...

2015-04-24

In this study, we describe for the first time how biological nanomotors may be used to actively self-assemble mesoscale networks composed of diblock copolymer nanotubes. The collective force generated by multiple kinesin nanomotors acting on a microtubule filament is large enough to overcome the energy barrier required to extract nanotubes from polymer vesicles comprised of poly(ethylene oxide-b-butadiene) in spite of the higher force requirements relative to extracting nanotubes from lipid vesicles. Nevertheless, large-scale polymer networks were dynamically assembled by the motors. These networks displayed enhanced robustness, persisting more than 24 h post-assembly (compared to 4–5 h for corresponding lipid networks).more » The transport of materials in and on the polymer membranes differs substantially from the transport on analogous lipid networks. Specifically, our data suggest that polymer mobility in nanotubular structures is considerably different from planar or 3D structures, and is stunted by 1D confinement of the polymer subunits. Moreover, quantum dots adsorbed onto polymer nanotubes are completely immobile, which is related to this 1D confinement effect and is in stark contrast to the highly fluid transport observed on lipid tubules.« less

Safety assessment of continuous glass filaments used in eclipse.

PubMed

Swauger, J E; Foy, J W

2000-11-01

Eclipse is a cigarette that produces smoke by primarily heating, rather than burning, tobacco. The Eclipse heat source assembly employs a continuous filament glass mat jacket to insulate the heat source. The glass mat insulator is composed of continuous glass filaments and a binder. The purpose of this article is to address the potential toxicological significance of the continuous glass filaments under the conditions of intended use. Transfer data and the unique physical characteristics of the filaments demonstrate that significant exposure of the smoker will not occur. The available environmental survey data clearly demonstrate that Eclipse smokers are extremely unlikely to be exposed to continuous glass filaments at a level that represents a biologically significant increase over background exposure to glass fibers. The chemical composition of the continuous glass filaments used in Eclipse is generally similar to C-glass fiber compositions such as MMVF 11 that have failed to produce either tumors or fibrosis in chronic inhalation studies conducted in rats. In vitro dissolution data demonstrate that the continuous glass filaments used in Eclipse are more soluble than biologically active fibers such as rock wool (MMVF 21) or asbestos. However, the continuous glass filaments used in Eclipse were not as soluble in simulated extracellular lung fluid as representative C-glass fibers (MMVF 10 and MMVF 11). In brief, exposure of Eclipse smokers to continuous glass filaments is extremely unlikely to occur at a level that may be construed to be of biological significance.

The Physics of the Metaphase Spindle.

PubMed

Oriola, David; Needleman, Daniel J; Brugués, Jan

2018-05-20

The assembly of the mitotic spindle and the subsequent segregation of sister chromatids are based on the self-organized action of microtubule filaments, motor proteins, and other microtubule-associated proteins, which constitute the fundamental force-generating elements in the system. Many of the components in the spindle have been identified, but until recently it remained unclear how their collective behaviors resulted in such a robust bipolar structure. Here, we review the current understanding of the physics of the metaphase spindle that is only now starting to emerge.

Linkage of Recognition and Replication Functions by Assembling Combinatorial Antibody Fab Libraries Along Phage Surfaces

NASA Astrophysics Data System (ADS)

Kang, Angray S.; Barbas, Carlos F.; Janda, Kim D.; Benkovic, Stephen J.; Lerner, Richard A.

1991-05-01

We describe a method based on a phagemid vector with helper phage rescue for the construction and rapid analysis of combinatorial antibody Fab libraries. This approach should allow the generation and selection of many monoclonal antibodies. Antibody genes are expressed in concert with phage morphogenesis, thereby allowing incorporation of functional Fab molecules along the surface of filamentous phage. The power of the method depends upon the linkage of recognition and replication functions and is not limited to antibody molecules.

The Bacterial Actin MamK

PubMed Central

Ozyamak, Ertan; Kollman, Justin; Agard, David A.; Komeili, Arash

2013-01-01

It is now recognized that actin-like proteins are widespread in bacteria and, in contrast to eukaryotic actins, are highly diverse in sequence and function. The bacterial actin, MamK, represents a clade, primarily found in magnetotactic bacteria, that is involved in the proper organization of subcellular organelles, termed magnetosomes. We have previously shown that MamK from Magnetospirillum magneticum AMB-1 (AMB-1) forms dynamic filaments in vivo. To gain further insights into the molecular mechanisms that underlie MamK dynamics and function, we have now studied the in vitro properties of MamK. We demonstrate that MamK is an ATPase that, in the presence of ATP, assembles rapidly into filaments that disassemble once ATP is depleted. The mutation of a conserved active site residue (E143A) abolishes ATPase activity of MamK but not its ability to form filaments. Filament disassembly depends on both ATPase activity and potassium levels, the latter of which results in the organization of MamK filaments into bundles. These data are consistent with observations indicating that accessory factors are required to promote filament disassembly and for spatial organization of filaments in vivo. We also used cryo-electron microscopy to obtain a high resolution structure of MamK filaments. MamK adopts a two-stranded helical filament architecture, but unlike eukaryotic actin and other actin-like filaments, subunits in MamK strands are unstaggered giving rise to a unique filament architecture. Beyond extending our knowledge of the properties and function of MamK in magnetotactic bacteria, this study emphasizes the functional and structural diversity of bacterial actins in general. PMID:23204522

Spindles and active vortices in a model of confined filament-motor mixtures.

PubMed

Head, David A; Briels, Wj; Gompper, Gerhard

2011-11-16

Robust self-organization of subcellular structures is a key principle governing the dynamics and evolution of cellular life. In fission yeast cells undergoing division, the mitotic spindle spontaneously emerges from the interaction of microtubules, motor proteins and the confining cell walls, and asters and vortices have been observed to self-assemble in quasi-two dimensional microtubule-kinesin assays. There is no clear microscopic picture of the role of the active motors driving this pattern formation, and the relevance of continuum modeling to filament-scale structures remains uncertain. Here we present results of numerical simulations of a discrete filament-motor protein model confined to a pressurised cylindrical box. Stable spindles, nematic configurations, asters and high-density semi-asters spontaneously emerge, the latter pair having also been observed in cytosol confined within emulsion droplets. State diagrams are presented delineating each stationary state as the pressure, motor speed and motor density are varied. We further highlight a parameter regime where vortices form exhibiting collective rotation of all filaments, but have a finite life-time before contracting to a semi-aster. Quantifying the distribution of life-times suggests this contraction is a Poisson process. Equivalent systems with fixed volume exhibit persistent vortices with stochastic switching in the direction of rotation, with switching times obeying similar statistics to contraction times in pressurised systems. Furthermore, we show that increasing the detachment rate of motors from filament plus-ends can both destroy vortices and turn some asters into vortices. We have shown that discrete filament-motor protein models provide new insights into the stationary and dynamical behavior of active gels and subcellular structures, because many phenomena occur on the length-scale of single filaments. Based on our findings, we argue the need for a deeper understanding of the microscopic activities underpinning macroscopic self-organization in active gels and urge further experiments to help bridge these lengths.

Hydrodynamic alignment and assembly of nanofibrils resulting in strong cellulose filaments

PubMed Central

Håkansson, Karl M. O.; Fall, Andreas B.; Lundell, Fredrik; Yu, Shun; Krywka, Christina; Roth, Stephan V.; Santoro, Gonzalo; Kvick, Mathias; Prahl Wittberg, Lisa; Wågberg, Lars; Söderberg, L. Daniel

2014-01-01

Cellulose nanofibrils can be obtained from trees and have considerable potential as a building block for biobased materials. In order to achieve good properties of these materials, the nanostructure must be controlled. Here we present a process combining hydrodynamic alignment with a dispersion–gel transition that produces homogeneous and smooth filaments from a low-concentration dispersion of cellulose nanofibrils in water. The preferential fibril orientation along the filament direction can be controlled by the process parameters. The specific ultimate strength is considerably higher than previously reported filaments made of cellulose nanofibrils. The strength is even in line with the strongest cellulose pulp fibres extracted from wood with the same degree of fibril alignment. Successful nanoscale alignment before gelation demands a proper separation of the timescales involved. Somewhat surprisingly, the device must not be too small if this is to be achieved. PMID:24887005

Shape effects of filaments versus spherical particles in flow and drug delivery

PubMed Central

GENG, YAN; DALHAIMER, PAUL; CAI, SHENSHEN; TSAI, RICHARD; TEWARI, MANORAMA; MINKO, TAMARA; DISCHER, DENNIS E.

2009-01-01

Interaction of spherical particles with cells and within animals has been studied extensively, but the effects of shape have received little attention. Here we use highly stable, polymer micelle assemblies known as filomicelles to compare the transport and trafficking of flexible filaments with spheres of similar chemistry. In rodents, filomicelles persisted in the circulation up to one week after intravenous injection. This is about ten times longer than their spherical counterparts and is more persistent than any known synthetic nanoparticle. Under fluid flow conditions, spheres and short filomicelles are taken up by cells more readily than longer filaments because the latter are extended by the flow. Preliminary results further demonstrate that filomicelles can effectively deliver the anticancer drug paclitaxel and shrink human-derived tumours in mice. Although these findings show that long-circulating vehicles need not be nanospheres, they also lend insight into possible shape effects of natural filamentous viruses. PMID:18654271

Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

PubMed Central

Tang, Haosu; Bidone, Tamara C.

2015-01-01

The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

Shape effects of filaments versus spherical particles in flow and drug delivery

NASA Astrophysics Data System (ADS)

Geng, Yan; Dalhaimer, Paul; Cai, Shenshen; Tsai, Richard; Tewari, Manorama; Minko, Tamara; Discher, Dennis E.

2007-04-01

Interaction of spherical particles with cells and within animals has been studied extensively, but the effects of shape have received little attention. Here we use highly stable, polymer micelle assemblies known as filomicelles to compare the transport and trafficking of flexible filaments with spheres of similar chemistry. In rodents, filomicelles persisted in the circulation up to one week after intravenous injection. This is about ten times longer than their spherical counterparts and is more persistent than any known synthetic nanoparticle. Under fluid flow conditions, spheres and short filomicelles are taken up by cells more readily than longer filaments because the latter are extended by the flow. Preliminary results further demonstrate that filomicelles can effectively deliver the anticancer drug paclitaxel and shrink human-derived tumours in mice. Although these findings show that long-circulating vehicles need not be nanospheres, they also lend insight into possible shape effects of natural filamentous viruses.

PEM/SPE fuel cell

DOEpatents

Grot, S.A.

1998-01-13

A PEM/SPE fuel cell is described including a membrane-electrode assembly (MEA) having a plurality of oriented filament embedded the face thereof for supporting the MEA and conducting current therefrom to contiguous electrode plates. 4 figs.

Sensor assembly

DOEpatents

Bennett, Thomas E.; Nelson, Drew V.

2004-04-13

A ribbon-like sensor assembly is described wherein a length of an optical fiber embedded within a similar lengths of a prepreg tow. The fiber is ""sandwiched"" by two layers of the prepreg tow which are merged to form a single consolidated ribbon. The consolidated ribbon achieving a generally uniform distribution of composite filaments near the embedded fiber such that excess resin does not ""pool"" around the periphery of the embedded fiber.

Role of the Dc domain of the bacterial hook protein FlgE in hook assembly and function

PubMed Central

Moriya, Nao; Minamino, Tohru; Ferris, Hedda U.; Morimoto, Yusuke V.; Ashihara, Masamichi; Kato, Takayuki; Namba, Keiichi

2013-01-01

The bacterial flagellar hook acts as a universal joint to smoothly transmit torque produced by the motor to the filament. The hook protein FlgE assembles into a 55 nm tubular structure with the help of the hook cap (FlgD). FlgE consists of four domains, D0, Dc, D1 and D2, arranged from the inner to the outer part of the tubular structure of the hook. The Dc domain contributes to the structural stability of the hook, but it is unclear how this Dc domain is responsible for the universal joint mechanism. Here, we carried out a deletion analysis of the FlgE Dc domain. FlgEΔ4/5 with deletion of residues 30 to 49 was not secreted into the culture media. FlgEΔ5 and FlgEΔ6 with deletions of residues 40 to 49 and 50 to 59, respectively, still formed hooks, allowing the export apparatus to export the hook-filament junction proteins FlgK and FlgL and flagellin FliC. However, these deletions inhibited the replacement of the FlgD hook cap by FlgK at the hook tip, thereby abolishing filament formation. Deletion of residues 50 to 59 significantly affected hook morphology. These results suggest that the Dc domain is responsible not only for hook assembly but also for FlgE export, the interaction with FlgK, and the polymorphic supercoiling mechanism of the hook. PMID:27493542

A Novel Tau Mutation in Exon 12, p.Q336H, Causes Hereditary Pick Disease

PubMed Central

Tacik, Pawel; DeTure, Michael; Hinkle, Kelly M.; Lin, Wen-Lang; Sanchez-Contreras, Monica; Carlomagno, Yari; Pedraza, Otto; Rademakers, Rosa; Ross, Owen A.; Wszolek, Zbigniew K.; Dickson, Dennis W.

2015-01-01

Pick disease (PiD) is a frontotemporal lobar degeneration with distinctive neuronal inclusions (Pick bodies) that are enriched in 3-repeat (3R) tau. Although mostly sporadic, mutations in the tau gene (MAPT) have been reported. We screened 24 cases of neuropathologically confirmed PiD for MAPT mutations and found a novel mutation (c.1008G>C, p.Q336H) in one patient. Pathogenicity was confirmed on microtubule assembly and tau filament formation assays. The patient was compared to sporadic PiD and PiD associated with MAPT mutations from a review of the literature. The patient had behavioral changes at 55 years of age, followed by reduced verbal fluency, parkinsonism and death at 63 years of age. His mother and maternal uncle had similar symptoms. Recombinant tau with p.Q336H mutation formed filaments faster than wild type tau, especially with 3R tau. It also promoted more microtubule assembly than wild type tau. We conclude that mutations in MAPT, including p.Q336H, can be associated with clinical, pathologic, and biochemical features that are similar to those in sporadic PiD. The pathomechanism of p.Q336H, and another previously reported variant at the same codon (p.Q336R), appears to be unique to MAPT mutations in that they not only predispose to abnormal tau filament formation but also facilitate microtubule assembly in a 3R tau-dependent manner. PMID:26426266

The Great Solar Active Region NOAA 12192: Helicity Transport, Filament Formation, and Impact on the Polar Field

DOE Office of Scientific and Technical Information (OSTI.GOV)

McMaken, Tyler C.; Petrie, Gordon J. D., E-mail: tmcmaken@gmail.com, E-mail: gpetrie@noao.edu

The solar active region (AR), NOAA 12192, appeared in 2014 October as the largest AR in 24 years. Here we examine the counterintuitive nature of two diffusion-driven processes in the region: the role of helicity buildup in the formation of a major filament, and the relationship between the effects of supergranular diffusion and meridional flow on the AR and on the polar field. Quantitatively, calculations of current helicity and magnetic twist from Helioseismic and Magnetic Imager (HMI) vector magnetograms indicate that, though AR 12192 emerged with negative helicity, positive helicity from subsequent flux emergence, consistent with the hemispheric sign-preference ofmore » helicity, increased over time within large-scale, weak-field regions such as those near the polarity inversion line (PIL). Morphologically, Atmospheric Imaging Assembly observations of filament barbs, sigmoidal patterns, and bases of Fe xii stalks initially exhibited signatures of negative helicity, and the long filament that subsequently formed had a strong positive helicity consistent with the helicity buildup along the PIL. We find from full-disk HMI magnetograms that AR 12192's leading positive flux was initially closer to the equator but, owing either to the region’s magnetic surroundings or to its asymmetric flux density distribution, was transported poleward more quickly on average than its trailing negative flux, contrary to the canonical pattern of bipole flux transport. This behavior caused the AR to have a smaller effect on the polar fields than expected and enabled the formation of the very long neutral line where the filament formed.« less

Plastin 1 Binds to Keratin and Is Required for Terminal Web Assembly in the Intestinal Epithelium

PubMed Central

Grimm-Günter, Eva-Maria S.; Revenu, Céline; Ramos, Sonia; Hurbain, Ilse; Smyth, Neil; Ferrary, Evelyne; Louvard, Daniel; Robine, Sylvie

2009-01-01

Plastin 1 (I-plastin, fimbrin) along with villin and espin is a prominent actin-bundling protein of the intestinal brush border microvilli. We demonstrate here that plastin 1 accumulates in the terminal web and interacts with keratin 19, possibly contributing to anchoring the rootlets to the keratin network. This prompted us to investigate the importance of plastin 1 in brush border assembly. Although in vivo neither villin nor espin is required for brush border structure, plastin 1-deficient mice have conspicuous ultrastructural alterations: microvilli are shorter and constricted at their base, and, strikingly, their core actin bundles lack true rootlets. The composition of the microvilli themselves is apparently normal, whereas that of the terminal web is profoundly altered. Although the plastin 1 knockout mice do not show any overt gross phenotype and present a normal intestinal microanatomy, the alterations result in increased fragility of the epithelium. This is seen as an increased sensitivity of the brush border to biochemical manipulations, decreased transepithelial resistance, and increased sensitivity to dextran sodium sulfate-induced colitis. Plastin 1 thus emerges as an important regulator of brush border morphology and stability through a novel role in the organization of the terminal web, possibly by connecting actin filaments to the underlying intermediate filament network. PMID:19321664

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

PubMed Central

Bjerke, Maureen A.; Dzamba, Bette; Wang, Chong; DeSimone, Douglas W.

2014-01-01

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity. PMID:25127991

Structures of actin-bound Wiskott-Aldrich syndrome protein homology 2 (WH2) domains of Spire and the implication for filament nucleation.

PubMed

Ducka, Anna M; Joel, Peteranne; Popowicz, Grzegorz M; Trybus, Kathleen M; Schleicher, Michael; Noegel, Angelika A; Huber, Robert; Holak, Tad A; Sitar, Tomasz

2010-06-29

Three classes of proteins are known to nucleate new filaments: the Arp2/3 complex, formins, and the third group of proteins that contain ca. 25 amino acid long actin-binding Wiskott-Aldrich syndrome protein homology 2 domains, called the WH2 repeats. Crystal structures of the complexes between the actin-binding WH2 repeats of the Spire protein and actin were determined for the Spire single WH2 domain D, the double (SpirCD), triple (SpirBCD), quadruple (SpirABCD) domains, and an artificial Spire WH2 construct comprising three identical D repeats (SpirDDD). SpirCD represents the minimal functional core of Spire that can nucleate actin filaments. Packing in the crystals of the actin complexes with SpirCD, SpirBCD, SpirABCD, and SpirDDD shows the presence of two types of assemblies, "side-to-side" and "straight-longitudinal," which can serve as actin filament nuclei. The principal feature of these structures is their loose, open conformations, in which the sides of actins that normally constitute the inner interface core of a filament are flipped inside out. These Spire structures are distant from those seen in the filamentous nuclei of Arp2/3, formins, and in the F-actin filament.

Structures of actin-bound Wiskott-Aldrich syndrome protein homology 2 (WH2) domains of Spire and the implication for filament nucleation

PubMed Central

Ducka, Anna M.; Joel, Peteranne; Popowicz, Grzegorz M.; Trybus, Kathleen M.; Schleicher, Michael; Noegel, Angelika A.; Huber, Robert; Holak, Tad A.; Sitar, Tomasz

2010-01-01

Three classes of proteins are known to nucleate new filaments: the Arp2/3 complex, formins, and the third group of proteins that contain ca. 25 amino acid long actin-binding Wiskott-Aldrich syndrome protein homology 2 domains, called the WH2 repeats. Crystal structures of the complexes between the actin-binding WH2 repeats of the Spire protein and actin were determined for the Spire single WH2 domain D, the double (SpirCD), triple (SpirBCD), quadruple (SpirABCD) domains, and an artificial Spire WH2 construct comprising three identical D repeats (SpirDDD). SpirCD represents the minimal functional core of Spire that can nucleate actin filaments. Packing in the crystals of the actin complexes with SpirCD, SpirBCD, SpirABCD, and SpirDDD shows the presence of two types of assemblies, “side-to-side” and “straight-longitudinal,” which can serve as actin filament nuclei. The principal feature of these structures is their loose, open conformations, in which the sides of actins that normally constitute the inner interface core of a filament are flipped inside out. These Spire structures are distant from those seen in the filamentous nuclei of Arp2/3, formins, and in the F-actin filament. PMID:20538977

Protein self-assembly onto nanodots leads to formation of conductive bio-based hybrids

PubMed Central

Hu, Xiao; Dong, Chenbo; Su, Rigu; Xu, Quan; Dinu, Cerasela Zoica

2016-01-01

The next generation of nanowires that could advance the integration of functional nanosystems into synthetic applications from photocatalysis to optical devices need to demonstrate increased ability to promote electron transfer at their interfaces while ensuring optimum quantum confinement. Herein we used the biological recognition and the self-assembly properties of tubulin, a protein involved in building the filaments of cellular microtubules, to create stable, free standing and conductive sulfur-doped carbon nanodots-based conductive bio-hybrids. The physical and chemical properties (e.g., composition, morphology, diameter etc.) of such user-synthesized hybrids were investigated using atomic and spectroscopic techniques, while the electron transfer rate was estimated using peak currents formed during voltammetry scanning. Our results demonstrate the ability to create individually hybrid nanowires capable to reduce energy losses; such hybrids could possibly be used in the future for the advancement and implementation into nanometer-scale functional devices. PMID:27922059

Crystal Structure of the Minor Pilin CofB, the Initiator of CFA/III Pilus Assembly in Enterotoxigenic Escherichia coli*

PubMed Central

Kolappan, Subramania; Ng, Dixon; Yang, Guixiang; Harn, Tony; Craig, Lisa

2015-01-01

Type IV pili are extracellular polymers of the major pilin subunit. These subunits are held together in the pilus filament by hydrophobic interactions among their N-terminal α-helices, which also anchor the pilin subunits in the inner membrane prior to pilus assembly. Type IV pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major pilins but are much less abundant. Minor pilins influence pilus assembly and retraction, but their precise functions are not well defined. The Type IV pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV pilus systems and possess only a single minor pilin. Here we show that the enterotoxigenic E. coli minor pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction. We solved the 2.0 Å crystal structure of N-terminally truncated CofB, revealing a pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate pilus assembly. We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system. PMID:26324721

Crystal Structure of the Minor Pilin CofB, the Initiator of CFA/III Pilus Assembly in Enterotoxigenic Escherichia coli.

PubMed

Kolappan, Subramania; Ng, Dixon; Yang, Guixiang; Harn, Tony; Craig, Lisa

2015-10-23

Type IV pili are extracellular polymers of the major pilin subunit. These subunits are held together in the pilus filament by hydrophobic interactions among their N-terminal α-helices, which also anchor the pilin subunits in the inner membrane prior to pilus assembly. Type IV pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major pilins but are much less abundant. Minor pilins influence pilus assembly and retraction, but their precise functions are not well defined. The Type IV pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV pilus systems and possess only a single minor pilin. Here we show that the enterotoxigenic E. coli minor pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction. We solved the 2.0 Å crystal structure of N-terminally truncated CofB, revealing a pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate pilus assembly. We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure

NASA Technical Reports Server (NTRS)

Maniotis, A. J.; Chen, C. S.; Ingber, D. E.

1997-01-01

We report here that living cells and nuclei are hard-wired such that a mechanical tug on cell surface receptors can immediately change the organization of molecular assemblies in the cytoplasm and nucleus. When integrins were pulled by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoskeleton and nucleus. Actin microfilaments mediated force transfer to the nucleus at low strain; however, tearing of the actin gel resulted with greater distortion. In contrast, intermediate filaments effectively mediated force transfer to the nucleus under both conditions. These filament systems also acted as molecular guy wires to mechanically stiffen the nucleus and anchor it in place, whereas microtubules acted to hold open the intermediate filament lattice and to stabilize the nucleus against lateral compression. Molecular connections between integrins, cytoskeletal filaments, and nuclear scaffolds may therefore provide a discrete path for mechanical signal transfer through cells as well as a mechanism for producing integrated changes in cell and nuclear structure in response to changes in extracellular matrix adhesivity or mechanics.

Overlying extreme-ultraviolet arcades preventing eruption of a filament observed by AIA/SDO

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Huadong; Ma, Suli; Zhang, Jun, E-mail: hdchen@upc.edu.cn

2013-11-20

Using the multi-wavelength data from the Atmospheric Imaging Assembly/Solar Dynamic Observatory (AIA/SDO) and the Sun Earth Connection Coronal and Heliospheric Investigation/Solar Terrestrial Relations Observatory (SECCHI/STEREO), we report a failed filament eruption in NOAA AR 11339 on 2011 November 3. The eruption was associated with an X1.9 flare, but without any coronal mass ejection (CME), coronal dimming, or extreme ultraviolet (EUV) waves. Some magnetic arcades above the filament were observed distinctly in EUV channels, especially in the AIA 94 Å and 131 Å wavebands, before and during the filament eruption process. Our results show that the overlying arcades expanded along withmore » the ascent of the filament at first until they reached a projected height of about 49 Mm above the Sun's surface, where they stopped. The following filament material was observed to be confined by the stopped EUV arcades and not to escape from the Sun. After the flare, a new filament formed at the low corona where part of the former filament remained before its eruption. These results support that the overlying arcades play an important role in preventing the filament from successfully erupting outward. We also discuss in this paper the EUV emission of the overlying arcades during the flare. It is rare for a failed filament eruption to be associated with an X1.9 class flare, but not with a CME or EUV waves. Therefore, this study also provides valuable insight into the triggering mechanism of the initiation of CMEs and EUV waves.« less

Confined partial filament eruption and its reformation within a stable magnetic flux rope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Joshi, Navin Chandra; Kayshap, Pradeep; Uddin, Wahab

2014-05-20

We present observations of a confined partial eruption of a filament on 2012 August 4, which restores its initial shape within ≈2 hr after eruption. From the Global Oscillation Network Group Hα observations, we find that the filament plasma turns into dynamic motion at around 11:20 UT from the middle part of the filament toward the northwest direction with an average speed of ≈105 km s{sup –1}. A little brightening underneath the filament possibly shows the signature of low-altitude reconnection below the filament eruptive part. In Solar Dynamics Observatory/Atmospheric Imaging Assembly 171 Å images, we observe an activation of right-handedmore » helically twisted magnetic flux rope that contains the filament material and confines it during its dynamical motion. The motion of cool filament plasma stops after traveling a distance of ≈215 Mm toward the northwest from the point of eruption. The plasma moves partly toward the right foot point of the flux rope, while most of the plasma returns after 12:20 UT toward the left foot point with an average speed of ≈60 km s{sup –1} to reform the filament within the same stable magnetic structure. On the basis of the filament internal fine structure and its position relative to the photospheric magnetic fields, we find filament chirality to be sinistral, while the activated enveloping flux rope shows a clear right-handed twist. Thus, this dynamic event is an apparent example of one-to-one correspondence between the filament chirality (sinistral) and the enveloping flux rope helicity (positive). From the coronal magnetic field decay index, n, calculation near the flux rope axis, it is evident that the whole filament axis lies within the domain of stability (i.e., n < 1), which provides the filament stability despite strong disturbances at its eastern foot point.« less

Compressive force generation by a bundle of living biofilaments

NASA Astrophysics Data System (ADS)

Ramachandran, Sanoop; Ryckaert, Jean-Paul

2013-08-01

To study the compressional forces exerted by a bundle of living stiff filaments pressing on a surface, akin to the case of an actin bundle in filopodia structures, we have performed particulate molecular dynamics simulations of a grafted bundle of parallel living (self-assembling) filaments, in chemical equilibrium with a solution of their constitutive monomers. Equilibrium is established as these filaments, grafted at one end to a wall of the simulation box, grow at their chemically active free end, and encounter the opposite confining wall of the simulation box. Further growth of filaments requires bending and thus energy, which automatically limit the populations of longer filaments. The resulting filament sizes distribution and the force exerted by the bundle on the obstacle are analyzed for different grafting densities and different sub- or supercritical conditions, these properties being compared with the predictions of the corresponding ideal confined bundle model. In this analysis, non-ideal effects due to interactions between filaments and confinement effects are singled out. For all state points considered at the same temperature and at the same gap width between the two surfaces, the force per filament exerted on the opposite wall appears to be a function of a rescaled free monomer density hat{ρ }_1^eff. This quantity can be estimated directly from the characteristic length of the exponential filament size distribution P observed in the size domain where these grafted filaments are not in direct contact with the wall. We also analyze the dynamics of the filament contour length fluctuations in terms of effective polymerization (U) and depolymerization (W) rates, where again it is possible to disentangle non-ideal and confinement effects.

Large-amplitude Longitudinal Oscillations in a Solar Filament

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Q. M.; Su, Y. N.; Ji, H. S.

In this paper, we report our multiwavelength observations of the large-amplitude longitudinal oscillations of a filament observed on 2015 May 3. Located next to active region 12335, the sigmoidal filament was observed by the ground-based H α telescopes from the Global Oscillation Network Group and by the Atmospheric Imaging Assembly instrument on board the Solar Dynamics Observatory . The filament oscillations were most probably triggered by the magnetic reconnection in the filament channel, which is characterized by the bidirectional flows, brightenings in EUV and soft X-ray, and magnetic cancellation in the photosphere. The directions of oscillations have angles of 4°–36°more » with respect to the filament axis. The whole filament did not oscillate in phase as a rigid body. Meanwhile, the oscillation periods (3100–4400 s) have a spatial dependence, implying that the curvature radii ( R ) of the magnetic dips are different at different positions. The values of R are estimated to be 69.4–133.9 Mm, and the minimum transverse magnetic field of the dips is estimated to be 15 G. The amplitudes of S5-S8 grew with time, while the amplitudes of S9-S14 damped with time. The oscillation amplitudes range from a few to ten Mm, and the maximum velocity can reach 30 km s{sup −1}. Interestingly, the filament experienced mass drainage southward at a speed of ∼27 km s{sup −1}. The oscillations continued after the mass drainage and lasted for more than 11 hr. After the mass drainage, the oscillation phases did not change much. The periods of S5-S8 decreased, while the periods of S9-S14 increased. The amplitudes of S5-S8 damped with time, while the amplitudes of S9-S14 grew. Most of the damping (growing) ratios are between −9 and 14. We offer a schematic cartoon to explain the complex behaviors of oscillations by introducing thread-thread interaction.« less

Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges

PubMed Central

Fujiwara, Ikuko; Remmert, Kirsten; Piszczek, Grzegorz; Hammer, John A.

2014-01-01

Although capping protein (CP) terminates actin filament elongation, it promotes Arp2/3-dependent actin network assembly and accelerates actin-based motility both in vitro and in vivo. In vitro, capping protein Arp2/3 myosin I linker (CARMIL) antagonizes CP by reducing its affinity for the barbed end and by uncapping CP-capped filaments, whereas the protein V-1/myotrophin sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP:V-1 complex, thereby converting inactive CP into a version with moderate affinity for the barbed end. Here we further clarify the mechanism of this exchange reaction, and we demonstrate that the CP:CARMIL complex created by complex exchange slows the rate of barbed-end elongation by rapidly associating with, and dissociating from, the barbed end. Importantly, the cellular concentrations of V-1 and CP determined here argue that most CP is sequestered by V-1 at steady state in vivo. Finally, we show that CARMIL is recruited to the plasma membrane and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of freely diffusing, inactive CP (CP:V-1) feeds, via CARMIL-driven complex exchange, the formation of weak-capping complexes (CP:CARMIL) at the plasma membrane of protruding edges. In vivo, therefore, CARMIL should promote Arp2/3-dependent actin network assembly at the leading edge by promoting barbed-end capping there. PMID:24778263

Galactic cold cores. VIII. Filament formation and evolution: Filament properties in context with evolutionary models

NASA Astrophysics Data System (ADS)

Rivera-Ingraham, A.; Ristorcelli, I.; Juvela, M.; Montillaud, J.; Men'shchikov, A.; Malinen, J.; Pelkonen, V.-M.; Marston, A.; Martin, P. G.; Pagani, L.; Paladini, R.; Paradis, D.; Ysard, N.; Ward-Thompson, D.; Bernard, J.-P.; Marshall, D. J.; Montier, L.; Tóth, L. V.

2017-05-01

Context. The onset of star formation is intimately linked with the presence of massive unstable filamentary structures. These filaments are therefore key for theoretical models that aim to reproduce the observed characteristics of the star formation process in the Galaxy. Aims: As part of the filament study carried out by the Herschel Galactic Cold Cores Key Programme, here we study and discuss the filament properties presented in GCC VII (Paper I) in context with theoretical models of filament formation and evolution. Methods: A conservatively selected sample of filaments located at a distance D< 500 pc was extracted from the GCC fields with the getfilaments algorithm. The physical structure of the filaments was quantified according to two main components: the central (Gaussian) region of the filament (core component), and the power-law-like region dominating the filament column density profile at larger radii (wing component). The properties and behaviour of these components relative to the total linear mass density of the filament and the column density of its environment were compared with the predictions from theoretical models describing the evolution of filaments under gravity-dominated conditions. Results: The feasibility of a transition from a subcritical to supercritical state by accretion at any given time is dependent on the combined effect of filament intrinsic properties and environmental conditions. Reasonably self-gravitating (high Mline,core) filaments in dense environments (AV≳ 3 mag) can become supercritical on timescales of t 1 Myr by accreting mass at constant or decreasing width. The trend of increasing Mline,tot (Mline,core and Mline,wing) and ridge AV with background for the filament population also indicates that the precursors of star-forming filaments evolve coevally with their environment. The simultaneous increase of environment and filament AV explains the observed association between dense environments and high Mline,core values, and it argues against filaments remaining in constant single-pressure equilibrium states. The simultaneous growth of filament and background in locations with efficient mass assembly, predicted in numerical models of filaments in collapsing clouds, presents a suitable scenario for the fulfillment of the combined filament mass-environment criterium that is in quantitative agreement with Herschel observations. Herschel is an ESA space observatory with science instruments provided by European-led Principal Investigator consortia and with important participation from NASA.

Self-assembly of chlorophenols in water

PubMed Central

Rogalska, Ewa; Rogalski, Marek; Gulik-Krzywicki, Tadeusz; Gulik, Annette; Chipot, Christophe

1999-01-01

In saturated solutions of some di- and trichlorophenols, structures with complex morphologies, consisting of thin, transparent sheets often coiling into helices and ultimately twisting into filaments, were observed under the optical microscope. Freeze-fracture electron microscopy, x-ray diffraction, phase diagrams, and molecular modeling were performed to elucidate the observed phenomena. Here, we present evidence that the chlorophenols studied, when interacting with water, self-assemble into bilayers. The fact that some chlorophenols form the same supramolecular structures as those described previously for structurally nonrelated surfactants sheds light on the mechanisms of self-assembly. PMID:10359753

Self assembling proteins

DOEpatents

Yeates, Todd O.; Padilla, Jennifer; Colovos, Chris

2004-06-29

Novel fusion proteins capable of self-assembling into regular structures, as well as nucleic acids encoding the same, are provided. The subject fusion proteins comprise at least two oligomerization domains rigidly linked together, e.g. through an alpha helical linking group. Also provided are regular structures comprising a plurality of self-assembled fusion proteins of the subject invention, and methods for producing the same. The subject fusion proteins find use in the preparation of a variety of nanostructures, where such structures include: cages, shells, double-layer rings, two-dimensional layers, three-dimensional crystals, filaments, and tubes.

Growing Actin Networks Form Lamellipodium and Lamellum by Self-Assembly

PubMed Central

Huber, Florian; Käs, Josef; Stuhrmann, Björn

2008-01-01

Many different cell types are able to migrate by formation of a thin actin-based cytoskeletal extension. Recently, it became evident that this extension consists of two distinct substructures, designated lamellipodium and lamellum, which differ significantly in their kinetic and kinematic properties as well as their biochemical composition. We developed a stochastic two-dimensional computer simulation that includes chemical reaction kinetics, G-actin diffusion, and filament transport to investigate the formation of growing actin networks in migrating cells. Model parameters were chosen based on experimental data or theoretical considerations. In this work, we demonstrate the system's ability to form two distinct networks by self-organization. We found a characteristic transition in mean filament length as well as a distinct maximum in depolymerization flux, both within the first 1–2 μm. The separation into two distinct substructures was found to be extremely robust with respect to initial conditions and variation of model parameters. We quantitatively investigated the complex interplay between ADF/cofilin and tropomyosin and propose a plausible mechanism that leads to spatial separation of, respectively, ADF/cofilin- or tropomyosin-dominated compartments. Tropomyosin was found to play an important role in stabilizing the lamellar actin network. Furthermore, the influence of filament severing and annealing on the network properties is explored, and simulation data are compared to existing experimental data. PMID:18708450

Viral-templated nanocrystalline Pd nanowires for chemiresistive hydrogen (H2) sensors

NASA Astrophysics Data System (ADS)

Moon, Chung Hee; Yan, Yiran; Zhang, Miluo; Myung, Nosang V.; Haberer, Elaine D.

2014-08-01

A palladium (Pd) nanowire-based hydrogen (H2) sensor has been fabricated with a novel viral-templated assembly route. A filamentous M13 bacteriophage was used as the viral-template for assembly of Pd nanowires at ambient conditions. Scanning electron microscopy determined Pd nanowire distribution and morphology with the devices. The phage template concentration controlled the number of physical and electrical nanowire connections across the device. A greater phage concentration resulted in a higher connection density and thicker Pd deposition. A lower phage concentration generated devices which formed chain-like nanowires of Pd nanocrystals, whereas a higher phage concentration formed devices with a continuous mesh-like structure. The lower concentration devices showed 51-78% instantaneous response to 2000 ppm H2 and response time less than 30 s.

Targeting cell division: Small-molecule inhibitors of FtsZ GTPase perturb cytokinetic ring assembly and induce bacterial lethality

PubMed Central

Margalit, Danielle N.; Romberg, Laura; Mets, Rebecca B.; Hebert, Alan M.; Mitchison, Timothy J.; Kirschner, Marc W.; RayChaudhuri, Debabrata

2004-01-01

FtsZ, the ancestral homolog of eukaryotic tubulins, is a GTPase that assembles into a cytokinetic ring structure essential for cell division in prokaryotic cells. Similar to tubulin, purified FtsZ polymerizes into dynamic protofilaments in the presence of GTP; polymer assembly is accompanied by GTP hydrolysis. We used a high-throughput protein-based chemical screen to identify small molecules that target assembly-dependent GTPase activity of FtsZ. Here, we report the identification of five structurally diverse compounds, named Zantrins, which inhibit FtsZ GTPase either by destabilizing the FtsZ protofilaments or by inducing filament hyperstability through increased lateral association. These two classes of FtsZ inhibitors are reminiscent of the antitubulin drugs colchicine and Taxol, respectively. We also show that Zantrins perturb FtsZ ring assembly in Escherichia coli cells and cause lethality to a variety of bacteria in broth cultures, indicating that FtsZ antagonists may serve as chemical leads for the development of new broad-spectrum antibacterial agents. Our results illustrate the utility of small-molecule chemical probes to study FtsZ polymerization dynamics and the feasibility of FtsZ as a novel therapeutic target. PMID:15289600

The sarcomeric cytoskeleton: from molecules to motion.

PubMed

Gautel, Mathias; Djinović-Carugo, Kristina

2016-01-01

Highly ordered organisation of striated muscle is the prerequisite for the fast and unidirectional development of force and motion during heart and skeletal muscle contraction. A group of proteins, summarised as the sarcomeric cytoskeleton, is essential for the ordered assembly of actin and myosin filaments into sarcomeres, by combining architectural, mechanical and signalling functions. This review discusses recent cell biological, biophysical and structural insight into the regulated assembly of sarcomeric cytoskeleton proteins and their roles in dissipating mechanical forces in order to maintain sarcomere integrity during passive extension and active contraction. α-Actinin crosslinks in the Z-disk show a pivot-and-rod structure that anchors both titin and actin filaments. In contrast, the myosin crosslinks formed by myomesin in the M-band are of a ball-and-spring type and may be crucial in providing stable yet elastic connections during active contractions, especially eccentric exercise. © 2016. Published by The Company of Biologists Ltd.

Structure of a Bacterial Dynamin-like Protein Lipid Tube Provides a Mechanism For Assembly and Membrane Curving

PubMed Central

Low, Harry H.; Sachse, Carsten; Amos, Linda A.; Löwe, Jan

2009-01-01

Summary Proteins of the dynamin superfamily mediate membrane fission, fusion, and restructuring events by polymerizing upon lipid bilayers and forcing regions of high curvature. In this work, we show the electron cryomicroscopy reconstruction of a bacterial dynamin-like protein (BDLP) helical filament decorating a lipid tube at ∼11 Å resolution. We fitted the BDLP crystal structure and produced a molecular model for the entire filament. The BDLP GTPase domain dimerizes and forms the tube surface, the GTPase effector domain (GED) mediates self-assembly, and the paddle region contacts the lipids and promotes curvature. Association of BDLP with GMPPNP and lipid induces radical, large-scale conformational changes affecting polymerization. Nucleotide hydrolysis seems therefore to be coupled to polymer disassembly and dissociation from lipid, rather than membrane restructuring. Observed structural similarities with rat dynamin 1 suggest that our results have broad implication for other dynamin family members. PMID:20064379

The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization.

PubMed

He, Qiuxia; Liu, Kechun; Tian, Zhenjun; Du, Shao Jun

2015-01-01

Heat shock protein 90α plays a key role in myosin folding and thick filament assembly in muscle cells. To assess the structure and function of Hsp90α and its potential regulation by post-translational modification, we developed a combined knockdown and rescue assay in zebrafish embryos to systematically analyze the effects of various mutations on Hsp90α function in myosin thick filament organization. DNA constructs expressing the Hsp90α1 mutants with altered putative ATP binding, phosphorylation, acetylation or methylation sites were co-injected with Hsp90α1 specific morpholino into zebrafish embryos. Myosin thick filament organization was analyzed in skeletal muscles of the injected embryos by immunostaining. The results showed that mutating the conserved D90 residue in the Hsp90α1 ATP binding domain abolished its function in thick filament organization. In addition, phosphorylation mimicking mutations of T33D, T33E and T87E compromised Hsp90α1 function in myosin thick filament organization. Similarly, K287Q acetylation mimicking mutation repressed Hsp90α1 function in myosin thick filament organization. In contrast, K206R and K608R hypomethylation mimicking mutations had not effect on Hsp90α1 function in thick filament organization. Given that T33 and T87 are highly conserved residues involved post-translational modification (PTM) in yeast, mouse and human Hsp90 proteins, data from this study could indicate that Hsp90α1 function in myosin thick filament organization is potentially regulated by PTMs involving phosphorylation and acetylation.

The Effects of Hsp90α1 Mutations on Myosin Thick Filament Organization

PubMed Central

He, Qiuxia; Liu, Kechun; Tian, Zhenjun; Du, Shao Jun

2015-01-01

Heat shock protein 90α plays a key role in myosin folding and thick filament assembly in muscle cells. To assess the structure and function of Hsp90α and its potential regulation by post-translational modification, we developed a combined knockdown and rescue assay in zebrafish embryos to systematically analyze the effects of various mutations on Hsp90α function in myosin thick filament organization. DNA constructs expressing the Hsp90α1 mutants with altered putative ATP binding, phosphorylation, acetylation or methylation sites were co-injected with Hsp90α1 specific morpholino into zebrafish embryos. Myosin thick filament organization was analyzed in skeletal muscles of the injected embryos by immunostaining. The results showed that mutating the conserved D90 residue in the Hsp90α1 ATP binding domain abolished its function in thick filament organization. In addition, phosphorylation mimicking mutations of T33D, T33E and T87E compromised Hsp90α1 function in myosin thick filament organization. Similarly, K287Q acetylation mimicking mutation repressed Hsp90α1 function in myosin thick filament organization. In contrast, K206R and K608R hypomethylation mimicking mutations had not effect on Hsp90α1 function in thick filament organization. Given that T33 and T87 are highly conserved residues involved post-translational modification (PTM) in yeast, mouse and human Hsp90 proteins, data from this study could indicate that Hsp90α1 function in myosin thick filament organization is potentially regulated by PTMs involving phosphorylation and acetylation. PMID:26562659

Assembly and Turnover of Short Actin Filaments by the Formin INF2 and Profilin*

PubMed Central

Gurel, Pinar S.; A, Mu; Guo, Bingqian; Shu, Rui; Mierke, Dale F.; Higgs, Henry N.

2015-01-01

INF2 (inverted formin 2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. Although we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (<60 nm) that continuously turn over actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover 6-fold by accelerating nucleotide exchange and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions of INF2 with actin can work in concert to increase the ATP turnover rate of actin. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin but through a different mechanism from that of INF2. PMID:26124273

Birefringence of single and bundled microtubules.

PubMed

Oldenbourg, R; Salmon, E D; Tran, P T

1998-01-01

We have measured the birefringence of microtubules (MTs) and of MT-based macromolecular assemblies in vitro and in living cells by using the new Pol-Scope. A single microtubule in aqueous suspension and imaged with a numerical aperture of 1.4 had a peak retardance of 0.07 nm. The peak retardance of a small bundle increased linearly with the number of MTs in the bundle. Axonemes (prepared from sea urchin sperm) had a peak retardance 20 times higher than that of single MTs, in accordance with the nine doublets and two singlets arrangement of parallel MTs in the axoneme. Measured filament retardance decreased when the filament was defocused or the numerical aperture of the imaging system was decreased. However, the retardance "area," which we defined as the image retardance integrated along a line perpendicular to the filament axis, proved to be independent of focus and of numerical aperture. These results are in good agreement with a theory that we developed for measuring retardances with imaging optics. Our theoretical concept is based on Wiener's theory of mixed dielectrics, which is well established for nonimaging applications. We extend its use to imaging systems by considering the coherence region defined by the optical set-up. Light scattered from within that region interferes coherently in the image point. The presence of a filament in the coherence region leads to a polarization dependent scattering cross section and to a finite retardance measured in the image point. Similar to resolution measurements, the linear dimension of the coherence region for retardance measurements is on the order lambda/(2 NA), where lambda is the wavelength of light and NA is the numerical aperture of the illumination and imaging lenses.

Birefringence of single and bundled microtubules.

PubMed Central

Oldenbourg, R; Salmon, E D; Tran, P T

1998-01-01

We have measured the birefringence of microtubules (MTs) and of MT-based macromolecular assemblies in vitro and in living cells by using the new Pol-Scope. A single microtubule in aqueous suspension and imaged with a numerical aperture of 1.4 had a peak retardance of 0.07 nm. The peak retardance of a small bundle increased linearly with the number of MTs in the bundle. Axonemes (prepared from sea urchin sperm) had a peak retardance 20 times higher than that of single MTs, in accordance with the nine doublets and two singlets arrangement of parallel MTs in the axoneme. Measured filament retardance decreased when the filament was defocused or the numerical aperture of the imaging system was decreased. However, the retardance "area," which we defined as the image retardance integrated along a line perpendicular to the filament axis, proved to be independent of focus and of numerical aperture. These results are in good agreement with a theory that we developed for measuring retardances with imaging optics. Our theoretical concept is based on Wiener's theory of mixed dielectrics, which is well established for nonimaging applications. We extend its use to imaging systems by considering the coherence region defined by the optical set-up. Light scattered from within that region interferes coherently in the image point. The presence of a filament in the coherence region leads to a polarization dependent scattering cross section and to a finite retardance measured in the image point. Similar to resolution measurements, the linear dimension of the coherence region for retardance measurements is on the order lambda/(2 NA), where lambda is the wavelength of light and NA is the numerical aperture of the illumination and imaging lenses. PMID:9449366

Diamond-Like Carbon Nanorods and Fabrication Thereof

NASA Technical Reports Server (NTRS)

Varshney, Deepak (Inventor); Makarov, Vladimir (Inventor); Morell, Gerardo (Inventor); Saxena, Puja (Inventor); Weiner, Brad (Inventor)

2017-01-01

Novel sp. (sup 3) rich diamond-like carbon (DLC) nanorod films were fabricated by hot filament chemical vapor deposition technique. The results are indicative of a bottom-up self-assembly synthesis process, which results in a hierarchical structure that consists of microscale papillae comprising numerous nanorods. The papillae have diameters ranging from 2 to 4 microns and the nanorods have diameters in the 35-45 nanometer range. A growth mechanism based on the vapor liquid-solid mechanism is proposed that accounts for the morphological aspects in the micro- and nano-scales.

Covalent and non-covalent chemical engineering of actin for biotechnological applications.

PubMed

Kumar, Saroj; Mansson, Alf

2017-11-15

The cytoskeletal filaments are self-assembled protein polymers with 8-25nm diameters and up to several tens of micrometres length. They have a range of pivotal roles in eukaryotic cells, including transportation of intracellular cargoes (primarily microtubules with dynein and kinesin motors) and cell motility (primarily actin and myosin) where muscle contraction is one example. For two decades, the cytoskeletal filaments and their associated motor systems have been explored for nanotechnological applications including miniaturized sensor systems and lab-on-a-chip devices. Several developments have also revolved around possible exploitation of the filaments alone without their motor partners. Efforts to use the cytoskeletal filaments for applications often require chemical or genetic engineering of the filaments such as specific conjugation with fluorophores, antibodies, oligonucleotides or various macromolecular complexes e.g. nanoparticles. Similar conjugation methods are also instrumental for a range of fundamental biophysical studies. Here we review methods for non-covalent and covalent chemical modifications of actin filaments with focus on critical advantages and challenges of different methods as well as critical steps in the conjugation procedures. We also review potential uses of the engineered actin filaments in nanotechnological applications and in some key fundamental studies of actin and myosin function. Finally, we consider possible future lines of investigation that may be addressed by applying chemical conjugation of actin in new ways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Structure and Self-Assembly of the Calcium Binding Matrix Protein of Human Metapneumovirus

PubMed Central

Leyrat, Cedric; Renner, Max; Harlos, Karl; Huiskonen, Juha T.; Grimes, Jonathan M.

2014-01-01

Summary The matrix protein (M) of paramyxoviruses plays a key role in determining virion morphology by directing viral assembly and budding. Here, we report the crystal structure of the human metapneumovirus M at 2.8 Å resolution in its native dimeric state. The structure reveals the presence of a high-affinity Ca2+ binding site. Molecular dynamics simulations (MDS) predict a secondary lower-affinity site that correlates well with data from fluorescence-based thermal shift assays. By combining small-angle X-ray scattering with MDS and ensemble analysis, we captured the structure and dynamics of M in solution. Our analysis reveals a large positively charged patch on the protein surface that is involved in membrane interaction. Structural analysis of DOPC-induced polymerization of M into helical filaments using electron microscopy leads to a model of M self-assembly. The conservation of the Ca2+ binding sites suggests a role for calcium in the replication and morphogenesis of pneumoviruses. PMID:24316400

The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in Vitro*

PubMed Central

Haldeman, Brian D.; Brizendine, Richard K.; Facemyer, Kevin C.; Baker, Josh E.; Cremo, Christine R.

2014-01-01

Actin-myosin interactions are well studied using soluble myosin fragments, but little is known about effects of myosin filament structure on mechanochemistry. We stabilized unphosphorylated smooth muscle myosin (SMM) and phosphorylated smooth muscle myosin (pSMM) filaments against ATP-induced depolymerization using a cross-linker and attached fluorescent rhodamine (XL-Rh-SMM). Electron micrographs showed that these side polar filaments are very similar to unmodified filaments. They are ∼0.63 μm long and contain ∼176 molecules. Rate constants for ATP-induced dissociation and ADP release from acto-myosin for filaments and S1 heads were similar. Actin-activated ATPases of SMM and XL-Rh-SMM were similarly regulated. XL-Rh-pSMM filaments moved processively on F-actin that was bound to a PEG brush surface. ATP dependence of filament velocities was similar to that for solution ATPases at high [actin], suggesting that both processes are limited by the same kinetic step (weak to strong transition) and therefore are attachment-limited. This differs from actin sliding over myosin monomers, which is primarily detachment-limited. Fitting filament data to an attachment-limited model showed that approximately half of the heads are available to move the filament, consistent with a side polar structure. We suggest the low stiffness subfragment 2 (S2) domain remains unhindered during filament motion in our assay. Actin-bound negatively displaced heads will impart minimal drag force because of S2 buckling. Given the ADP release rate, the velocity, and the length of S2, these heads will detach from actin before slack is taken up into a backwardly displaced high stiffness position. This mechanism explains the lack of detachment-limited kinetics at physiological [ATP]. These findings address how nonlinear elasticity in assemblies of motors leads to efficient collective force generation. PMID:24907276

Regulation of CTP Synthase Filament Formation During DNA Endoreplication in Drosophila.

PubMed

Wang, Pei-Yu; Lin, Wei-Cheng; Tsai, Yi-Cheng; Cheng, Mei-Ling; Lin, Yu-Hung; Tseng, Shu-Heng; Chakraborty, Archan; Pai, Li-Mei

2015-12-01

CTP synthase (CTPsyn) plays an essential role in DNA, RNA, and lipid synthesis. Recent studies in bacteria, yeast, and Drosophila all reveal a polymeric CTPsyn structure, which dynamically regulates its enzymatic activity. However, the molecular mechanism underlying the formation of CTPsyn polymers is not completely understood. In this study, we found that reversible ubiquitination regulates the dynamic assembly of the filamentous structures of Drosophila CTPsyn. We further determined that the proto-oncogene Cbl, an E3 ubiquitin ligase, controls CTPsyn filament formation in endocycles. While the E3 ligase activity of Cbl is required for CTPsyn filament formation, Cbl does not affect the protein levels of CTPsyn. It remains unclear whether the regulation of CTPsyn filaments by Cbl is through direct ubiquitination of CTPsyn. In the absence of Cbl or with knockdown of CTPsyn, the progression of the endocycle-associated S phase was impaired. Furthermore, overexpression of wild-type, but not enzymatically inactive CTPsyn, rescued the endocycle defect in Cbl mutant cells. Together, these results suggest that Cbl influences the nucleotide pool balance and controls CTPsyn filament formation in endocycles. This study links Cbl-mediated ubiquitination to the polymerization of a metabolic enzyme and reveals a role for Cbl in endocycles during Drosophila development. Copyright © 2015 by the Genetics Society of America.

“Dandelion” Filament Eruption and Coronal Waves Associated with a Solar Flare on 2011 February 16

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cabezas, Denis P.; Ishitsuka, Mutsumi; Ishitsuka, José K.

Coronal disturbances associated with solar flares, such as H α Moreton waves, X-ray waves, and extreme ultraviolet (EUV) coronal waves, are discussed herein in relation to magnetohydrodynamic fast-mode waves or shocks in the corona. To understand the mechanism of coronal disturbances, full-disk solar observations with high spatial and temporal resolution over multiple wavelengths are of crucial importance. We observed a filament eruption, whose shape is like a “dandelion,” associated with the M1.6 flare that occurred on 2011 February 16 in H α images taken by the Flare Monitoring Telescope at Ica University, Peru. We derive the three-dimensional velocity field ofmore » the erupting filament. We also identify winking filaments that are located far from the flare site in the H α images, whereas no Moreton wave is observed. By comparing the temporal evolution of the winking filaments with those of the coronal wave seen in the EUV images data taken by the Atmospheric Imaging Assembly on board the Solar Dynamics Observatory and by the Extreme Ultraviolet Imager on board the Solar Terrestrial Relations Observatory-Ahead , we confirm that the winking filaments were activated by the EUV coronal wave.« less

Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells

PubMed Central

2012-01-01

Background We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments. Results K14 IBs were electroporated into SW13 cells grown in culture together with a “reporter” plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5. Conclusions Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells. PMID:22624805

Liquid droplets of cross-linked actin filaments

NASA Astrophysics Data System (ADS)

Weirich, Kimberly; Banerjee, Shiladitya; Dasbiswas, Kinjal; Vaikuntanathan, Suriyanarayan; Gardel, Margaret

Soft materials constructed from biomolecules self-assemble into a myriad of structures that work in concert to support cell physiology. One critical soft material is the actin cytoskeleton, a viscoelastic gel composed of cross-linked actin filaments. Although actin networks are primarily known for their elastic properties, which are crucial to regulating cell mechanics, the viscous behavior has been theorized to enable shape changes and flows. We experimentally demonstrate a fluid phase of cross-linked actin, where cross-linker condenses dilute short actin filaments into spindle-shaped droplets, or tactoids. Tactoids have shape dynamics consistent with a continuum model of liquid crystal droplets. The cross-linker, which acts as a long range attractive interaction, analogous to molecular cohesion, controls the tactoid shape and dynamics, which reports on the liquid's interfacial tension and viscosity. We investigate how the cross-linker properties and filament length influence the liquid properties. These results demonstrate a novel mechanism to control organization of the actin cytoskeleton and provide insight into design principles for complex, macromolecular liquid phases.

The Fysics of Filopodia (or The Physics of Philopodia)

NASA Astrophysics Data System (ADS)

Schwarz, Jen; Gopinathan, Ajay; Lee, Kun-Chun; Liu, Andrea; Yang, Louise

2006-03-01

Cell motility is driven by the dynamic reorganization of the cellular cytoskeleton which is composed of actin. Monomeric actin assembles into filaments that grow, shrink, branch and bundle. Branching generates new filaments that form a mesh-like structure that protrudes outward allowing the cell to move somewhere. But how does it know where to move? It has been proposed that filopodia serve as scouts for the cell. Filopodia are bundles of actin filaments that extend out ahead of the rest of the cell to probe its upcoming environment. Recent in vitro experiments [Vignjevic et al., J. Ce ll Bio. 160, 951 (2003)] determine the minimal ingredients required for such a process. We model these experiments analytically and via Monte Carlo simulations to estimate the typical bundle size and length. We also estimate the size of the mesh-like structure from which the filopodia emerge and explain the observed nonmonotonicity of this size as a function of capping protein concentration, which inhibits filament growth.

The C Terminus of Formin FMNL3 Accelerates Actin Polymerization and Contains a WH2 Domain-like Sequence That Binds Both Monomers and Filament Barbed Ends*

PubMed Central

Heimsath, Ernest G.; Higgs, Henry N.

2012-01-01

Formin proteins are actin assembly factors that accelerate filament nucleation then remain on the elongating barbed end and modulate filament elongation. The formin homology 2 (FH2) domain is central to these activities, but recent work has suggested that additional sequences enhance FH2 domain function. Here we show that the C-terminal 76 amino acids of the formin FMNL3 have a dramatic effect on the ability of the FH2 domain to accelerate actin assembly. This C-terminal region contains a WASp homology 2 (WH2)-like sequence that binds actin monomers in a manner that is competitive with other WH2 domains and with profilin. In addition, the C terminus binds filament barbed ends. As a monomer, the FMNL3 C terminus inhibits actin polymerization and slows barbed end elongation with moderate affinity. As a dimer, the C terminus accelerates actin polymerization from monomers and displays high affinity inhibition of barbed end elongation. These properties are not common to all formin C termini, as those of mDia1 and INF2 do not behave similarly. Interestingly, mutation of two aliphatic residues, which blocks high affinity actin binding by the WH2-like sequence, has no effect on the ability of the C terminus to enhance FH2-mediated polymerization. However, mutation of three successive basic residues at the C terminus of the WH2-like sequence compromises polymerization enhancement. These results illustrate that the C termini of formins are highly diverse in their interactions with actin. PMID:22094460

Myosin Storage Myopathy in C. elegans and Human Cultured Muscle Cells

PubMed Central

Dahl-Halvarsson, Martin; Pokrzywa, Malgorzata; Rauthan, Manish; Pilon, Marc

2017-01-01

Myosin storage myopathy is a protein aggregate myopathy associated with the characteristic subsarcolemmal accumulation of myosin heavy chain in muscle fibers. Despite similar histological findings, the clinical severity and age of onset are highly variable, ranging from no weakness to severe impairment of ambulation, and usually childhood-onset to onset later in life. Mutations located in the distal end of the tail of slow/ß-cardiac myosin heavy chain are associated with myosin storage myopathy. Four missense mutations (L1793P, R1845W, E1883K and H1901L), two of which have been reported in several unrelated families, are located within or closed to the assembly competence domain. This location is critical for the proper assembly of sarcomeric myosin rod filaments. To assess the mechanisms leading to protein aggregation in myosin storage myopathy and to evaluate the impact of these mutations on myosin assembly and muscle function, we expressed mutated myosin proteins in cultured human muscle cells and in the nematode Caenorhabditis elegans. While L1793P mutant myosin protein efficiently incorporated into the sarcomeric thick filaments, R1845W and H1901L mutants were prone to formation of myosin aggregates without assembly into striated sarcomeric thick filaments in cultured muscle cells. In C. elegans, mutant alleles of the myosin heavy chain gene unc-54 corresponding to R1845W, E1883K and H1901L, were as effective as the wild-type myosin gene in rescuing the null mutant worms, indicating that they retain functionality. Taken together, our results suggest that the basis for the pathogenic effect of the R1845W and H1901L mutations are primarily structural rather than functional. Further analyses are needed to identify the primary trigger for the histological changes seen in muscle biopsies of patients with L1793P and E1883K mutations. PMID:28125727

Myosin Storage Myopathy in C. elegans and Human Cultured Muscle Cells.

PubMed

Dahl-Halvarsson, Martin; Pokrzywa, Malgorzata; Rauthan, Manish; Pilon, Marc; Tajsharghi, Homa

2017-01-01

Myosin storage myopathy is a protein aggregate myopathy associated with the characteristic subsarcolemmal accumulation of myosin heavy chain in muscle fibers. Despite similar histological findings, the clinical severity and age of onset are highly variable, ranging from no weakness to severe impairment of ambulation, and usually childhood-onset to onset later in life. Mutations located in the distal end of the tail of slow/ß-cardiac myosin heavy chain are associated with myosin storage myopathy. Four missense mutations (L1793P, R1845W, E1883K and H1901L), two of which have been reported in several unrelated families, are located within or closed to the assembly competence domain. This location is critical for the proper assembly of sarcomeric myosin rod filaments. To assess the mechanisms leading to protein aggregation in myosin storage myopathy and to evaluate the impact of these mutations on myosin assembly and muscle function, we expressed mutated myosin proteins in cultured human muscle cells and in the nematode Caenorhabditis elegans. While L1793P mutant myosin protein efficiently incorporated into the sarcomeric thick filaments, R1845W and H1901L mutants were prone to formation of myosin aggregates without assembly into striated sarcomeric thick filaments in cultured muscle cells. In C. elegans, mutant alleles of the myosin heavy chain gene unc-54 corresponding to R1845W, E1883K and H1901L, were as effective as the wild-type myosin gene in rescuing the null mutant worms, indicating that they retain functionality. Taken together, our results suggest that the basis for the pathogenic effect of the R1845W and H1901L mutations are primarily structural rather than functional. Further analyses are needed to identify the primary trigger for the histological changes seen in muscle biopsies of patients with L1793P and E1883K mutations.

Cryo-EM reconstruction of AlfA from Bacillus subtilis reveals the structure of a simplified actin-like filament at 3.4-Å resolution.

PubMed

Szewczak-Harris, Andrzej; Löwe, Jan

2018-03-27

Low copy-number plasmid pLS32 of Bacillus subtilis subsp. natto contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequence parN Similar to the ParMRC partitioning system from Escherichia coli plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB and parN Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system's biological raison d'être.

The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus.

PubMed

Ng, Dixon; Harn, Tony; Altindal, Tuba; Kolappan, Subramania; Marles, Jarrad M; Lala, Rajan; Spielman, Ingrid; Gao, Yang; Hauke, Caitlyn A; Kovacikova, Gabriela; Verjee, Zia; Taylor, Ronald K; Biais, Nicolas; Craig, Lisa

2016-12-01

Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system.

DIRECT SPATIAL ASSOCIATION OF AN X-RAY FLARE WITH THE ERUPTION OF A SOLAR QUIESCENT FILAMENT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holman, Gordon D.; Foord, Adi, E-mail: gordon.d.holman@nasa.gov

Solar flares primarily occur in active regions. Hard X-ray flares have been found to occur only in active regions. They are often associated with the eruption of active region filaments and coronal mass ejections (CMEs). CMEs can also be associated with the eruption of quiescent filaments, not located in active regions. Here we report the first identification of a solar X-ray flare outside an active region observed by the Ramaty High Energy Solar Spectroscopic Imager (RHESSI). The X-ray emission was directly associated with the eruption of a long, quiescent filament and fast CME. Images from RHESSI show this flare emissionmore » to be located along a section of the western ribbon of the expanding, post-eruption arcade. EUV images from the Solar Dynamics Observatory Atmospheric Imaging Assembly show no connection between this location and nearby active regions. Therefore the flare emission is found not to be located in or associated with an active region. However, a nearby, small, magnetically strong dipolar region provides a likely explanation for the existence and location of the flare X-ray emission. This emerging dipolar region may have also triggered the filament eruption.« less

Characterization of gamma-tubulin filaments in mammalian cells.

PubMed

Lindström, Lisa; Alvarado-Kristensson, Maria

2018-01-01

Overexpression of γ-tubulin leads to the formation of filaments, but nothing is known about such filaments with regard to possible presence in cells, structure and probable dynamics. Here, we used mammalian cell lines to investigate the ability of γ-tubulin to form filaments. We found that γ-tubulin produces fibers called γ-tubules in a GTP-dependent manner and that γ-tubules are made up of pericentrin and the γ-tubulin complex proteins 2, 3, 5 and 6. Furthermore, we noted that the number of cells with cytosolic γ-tubules is increased in non-dividing cells. Our experiments showed that γ-tubules are polar structures that have a low regrowth rate compared to microtubules. Also, we observed that γ-tubules were disassembled by treatment with cold, colcemid, citral dimethyl acetal, dimethyl fumarate or mutation of γ-tubulin GTPase domain, but were increased in number by treatment with taxol or by stable expression of the γ-tubulin 1-333 GTPase domain. Our results demonstrate that γ-tubulin forms filaments, and such assembly is facilitated by the GTPase domain of γ-tubulin. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Extracellular matrix dynamics during vertebrate axis formation.

PubMed

Czirók, András; Rongish, Brenda J; Little, Charles D

2004-04-01

The first evidence for the dynamics of in vivo extracellular matrix (ECM) pattern formation during embryogenesis is presented below. Fibrillin 2 filaments were tracked for 12 h throughout the avian intraembryonic mesoderm using automated light microscopy and algorithms of our design. The data show that these ECM filaments have a reproducible morphogenic destiny that is characterized by directed transport. Fibrillin 2 particles initially deposited in the segmental plate mesoderm are translocated along an unexpected trajectory where they eventually polymerize into an intricate scaffold of cables parallel to the anterior-posterior axis. The cables coalesce near the midline before the appearance of the next-formed somite. Moreover, the ECM filaments define global tissue movements with high precision because the filaments act as passive motion tracers. Quantification of individual and collective filament "behaviors" establish fate maps, trajectories, and velocities. These data reveal a caudally propagating traveling wave pattern in the morphogenetic movements of early axis formation. We conjecture that within vertebrate embryos, long-range mechanical tension fields are coupled to both large-scale patterning and local organization of the ECM. Thus, physical forces or stress fields are essential requirements for executing an emergent developmental pattern-in this case, paraxial fibrillin cable assembly.

ERUPTION OF A SOLAR FILAMENT CONSISTING OF TWO THREADS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bi Yi; Jiang Yunchun; Li Haidong

The trigger and driving mechanism for the eruption of a filament consisting of two dark threads was studied with unprecedented high cadence and resolution of He II 304 A observations made by the Atmospheric Imagining Assembly (AIA) on board the Solar Dynamics Observatory (SDO) and the observations made by the Solar Magnetic Activity Research Telescope and the Extreme Ultraviolet Imager (EUVI) telescope on board the Solar Terrestrial Relations Observatory Ahead (STEREO-A). The filament was located at the periphery of the active region NOAA 11228 and erupted on 2011 June 6. At the onset of the eruption, a turbulent filament threadmore » was found to be heated and to elongate in stride over a second one. After it rose slowly, most interestingly, the elongating thread was driven to contact and interact with the second one, and it then erupted with its southern leg being wrapped by a newly formed thread produced by the magnetic reconnection between fields carried by the two threads. Combining the observations from STEREO-A/EUVI and SDO/AIA 304 A images, the three-dimensional shape of the axis of the filament was obtained and it was found that only the southern leg of the eruptive filament underwent rotation. We suggest that the eruption was triggered by the reconnection of the turbulent filament thread and the surrounding magnetic field, and that it was mainly driven by the kink instability of the southern leg of the eruptive filament that possessed a more twisted field introduced by the reconnection-produced thread.« less

Structural properties of the tubular appendage spinae from marine bacterium Roseobacter sp. strain YSCB

PubMed Central

Bernadac, A.; Wu, L.-F.; Santini, C.-L.; Vidaud, C.; Sturgis, J. N.; Menguy, N.; Bergam, P.; Nicoletti, C.; Xiao, T.

2012-01-01

Spinae are tubular surface appendages broadly found in Gram-negative bacteria. Little is known about their architecture, function or origin. Here, we report structural characterization of the spinae from marine bacteria Roseobacter sp. YSCB. Electron cryo-tomography revealed that a single filament winds into a hollow flared base with progressive change to a cylinder. Proteinase K unwound the spinae into proteolysis-resistant filaments. Thermal treatment ripped the spinae into ribbons that were melted with prolonged heating. Circular dichroism spectroscopy revealed a dominant beta-structure of the spinae. Differential scanning calorimetry analyses showed three endothermic transformations at 50–85°C, 98°C and 123°C, respectively. The heating almost completely disintegrated the spinae, abolished the 98°C transition and destroyed the beta-structure. Infrared spectroscopy identified the amide I spectrum maximum at a position similar to that of amyloid fibrils. Therefore, the spinae distinguish from other bacterial appendages, e.g. flagella and stalks, in both the structure and mechanism of assembly. PMID:23230515

Biological role and structural mechanism of twinfilin–capping protein interaction

PubMed Central

Falck, Sandra; Paavilainen, Ville O; Wear, Martin A; Grossmann, J Günter; Cooper, John A; Lappalainen, Pekka

2004-01-01

Twinfilin and capping protein (CP) are highly conserved actin-binding proteins that regulate cytoskeletal dynamics in organisms from yeast to mammals. Twinfilin binds actin monomer, while CP binds the barbed end of the actin filament. Remarkably, twinfilin and CP also bind directly to each other, but the mechanism and role of this interaction in actin dynamics are not defined. Here, we found that the binding of twinfilin to CP does not affect the binding of either protein to actin. Furthermore, site-directed mutagenesis studies revealed that the CP-binding site resides in the conserved C-terminal tail region of twinfilin. The solution structure of the twinfilin–CP complex supports these conclusions. In vivo, twinfilin's binding to both CP and actin monomer was found to be necessary for twinfilin's role in actin assembly dynamics, based on genetic studies with mutants that have defined biochemical functions. Our results support a novel model for how sequential interactions between actin monomers, twinfilin, CP, and actin filaments promote cytoskeletal dynamics. PMID:15282541

Polycation-induced assembly of purified tubulin.

PubMed Central

Erickson, H P; Voter, W A

1976-01-01

Several different polycations have been found that can substitute for the microtubule-associated proteins, or tau factor, in facilitating assembly of tubulin that has been purified by ion exchange chromatography. In low concentrations of the polycation diethylaminoethyl-dextran, 7 mg of tubulin is pelleted per 1 mg of polycation added. Under conditions favorable to microtubule assembly the entire pellet is seen by electron microscopy to consist of "double wall microtubules", which are essentially identical to normal microtubules in subunit structure and arrangement. When assembly is inhibited approximately the same amount of tubulin is pelleted, but it is in the form of clusters of curved sheets or filaments apparently related to tubulin rings. When conditions are changed to favor assembly, the tubulin within these clusters appears to reassemble to form the double wall microtubules. Images PMID:1066692

Mind the gap; seven reasons to close fragmented genome assemblies

USDA-ARS?s Scientific Manuscript database

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including tho...

Internal epithelia in Drosophila display rudimentary competence to form cytoplasmic networks of transgenic human vimentin.

PubMed

Gullmets, Josef; Torvaldson, Elin; Lindqvist, Julia; Imanishi, Susumu Y; Taimen, Pekka; Meinander, Annika; Eriksson, John E

2017-12-01

Cytoplasmic intermediate filaments (cIFs) are found in all eumetazoans, except arthropods. To investigate the compatibility of cIFs in arthropods, we expressed human vimentin (hVim), a cIF with filament-forming capacity in vertebrate cells and tissues, transgenically in Drosophila Transgenic hVim could be recovered from whole-fly lysates by using a standard procedure for intermediate filament (IF) extraction. When this procedure was used to test for the possible presence of IF-like proteins in flies, only lamins and tropomyosin were observed in IF-enriched extracts, thereby providing biochemical reinforcement to the paradigm that arthropods lack cIFs. In Drosophila , transgenic hVim was unable to form filament networks in S2 cells and mesenchymal tissues; however, cage-like vimentin structures could be observed around the nuclei in internal epithelia, which suggests that Drosophila retains selective competence for filament formation. Taken together, our results imply that although the filament network formation competence is partially lost in Drosophila , a rudimentary filament network formation ability remains in epithelial cells. As a result of the observed selective competence for cIF assembly in Drosophila , we hypothesize that internal epithelial cIFs were the last cIFs to disappear from arthropods.-Gullmets, J., Torvaldson, E., Lindqvist, J., Imanishi, S. Y., Taimen, P., Meinander, A., Eriksson, J. E. Internal epithelia in Drosophila display rudimentary competence to form cytoplasmic networks of transgenic human vimentin. © FASEB.

Actin Out: Regulation of the Synaptic Cytoskeleton

PubMed Central

Spence, Erin F.; Soderling, Scott H.

2015-01-01

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during spinogenesis and experience-dependent plasticity. Multiple mutations associated with human neurodevelopmental and psychiatric disorders involve genes that encode regulators of the synaptic cytoskeleton. A major, unresolved question is how the disruption of specific actin filament structures leads to the onset and progression of complex synaptic and behavioral phenotypes. This review will cover established and emerging mechanisms of actin cytoskeletal remodeling and how this influences specific aspects of spine biology that are implicated in disease. PMID:26453304

Filamentous Phage: Structure and Biology.

PubMed

Rakonjac, Jasna; Russel, Marjorie; Khanum, Sofia; Brooke, Sam J; Rajič, Marina

2017-01-01

Ff filamentous phage (fd, M13 and f1) of Escherichia coli have been the workhorse of phage display technology for the past 30 years. Dominance of Ff over other bacteriophage in display technology stems from the titres that are about 100-fold higher than any other known phage, efficacious transformation ensuring large library size and superior stability of the virion at high temperatures, detergents and pH extremes, allowing broad range of biopanning conditions in screening phage display libraries. Due to the excellent understanding of infection and assembly requirements, Ff phage have also been at the core of phage-assisted continual protein evolution strategies (PACE). This chapter will give an overview of the Ff filamentous phage structure and biology, emphasizing those properties of the Ff phage life cycle and virion that are pertinent to phage display applications.

Simulation of stationary glow patterns in dielectric barrier discharges at atmospheric pressure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Fucheng, E-mail: hdlfc@hbu.cn; He, Yafeng; Dong, Lifang

2014-12-15

Self-organized stationary patterns in dielectric barrier discharges operating in glow regime at atmospheric pressure are investigated by a self-consistent two-dimensional fluid model. The simulation results show that two different modes, namely, the diffuse mode and the static patterned mode, can be formed in different ranges of the driving frequency. The discharge operates in Townsend regime in the diffuse mode, while it operates in a glow regime inside the filaments and in a Townsend regime outside the filaments in the stable pattered mode. The forming process of the stationary filaments can be divided into three stages, namely, destabilizing stage, self-assembling stage,more » and stable stage. The space charge associated with residual electron density and surface charge is responsible for the formation of these stationary glow patterns.« less

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

PubMed Central

Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen-Ling; Bishop, Douglas K.; Haber, James E.

2015-01-01

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies.

PubMed

Tardivel, Meryem; Bégard, Séverine; Bousset, Luc; Dujardin, Simon; Coens, Audrey; Melki, Ronald; Buée, Luc; Colin, Morvane

2016-11-04

A given cell makes exchanges with its neighbors through a variety of means ranging from diffusible factors to vesicles. Cells use also tunneling nanotubes (TNTs), filamentous-actin-containing membranous structures that bridge and connect cells. First described in immune cells, TNTs facilitate HIV-1 transfer and are found in various cell types, including neurons. We show that the microtubule-associated protein Tau, a key player in Alzheimer's disease, is a bona fide constituent of TNTs. This is important because Tau appears beside filamentous actin and myosin 10 as a specific marker of these fine protrusions of membranes and cytosol that are difficult to visualize. Furthermore, we observed that exogenous Tau species increase the number of TNTs established between primary neurons, thereby facilitating the intercellular transfer of Tau fibrils. In conclusion, Tau may contribute to the formation and function of the highly dynamic TNTs that may be involved in the prion-like propagation of Tau assemblies.

Atomic structure and hierarchical assembly of a cross-β amyloid fibril

PubMed Central

Fitzpatrick, Anthony W. P.; Debelouchina, Galia T.; Bayro, Marvin J.; Clare, Daniel K.; Caporini, Marc A.; Bajaj, Vikram S.; Jaroniec, Christopher P.; Wang, Luchun; Ladizhansky, Vladimir; Müller, Shirley A.; MacPhee, Cait E.; Waudby, Christopher A.; Mott, Helen R.; De Simone, Alfonso; Knowles, Tuomas P. J.; Saibil, Helen R.; Vendruscolo, Michele; Orlova, Elena V.; Griffin, Robert G.; Dobson, Christopher M.

2013-01-01

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale—including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy—we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils. PMID:23513222

A stochastic reaction-diffusion model for protein aggregation on DNA

NASA Astrophysics Data System (ADS)

Voulgarakis, Nikolaos K.

Vital functions of DNA, such as transcription and packaging, depend on the proper clustering of proteins on the double strand. The present study investigates how the interplay between DNA allostery and electrostatic interactions affects protein clustering. The statistical analysis of a simple but transparent computational model reveals two major consequences of this interplay. First, depending on the protein and salt concentration, protein filaments exhibit a bimodal DNA stiffening and softening behavior. Second, within a certain domain of the control parameters, electrostatic interactions can cause energetic frustration that forces proteins to assemble in rigid spiral configurations. Such spiral filaments might trigger both positive and negative supercoiling, which can ultimately promote gene compaction and regulate the promoter. It has been experimentally shown that bacterial histone-like proteins assemble in similar spiral patterns and/or exhibit the same bimodal behavior. The proposed model can, thus, provide computational insights into the physical mechanisms used by proteins to control the mechanical properties of the DNA.

Microgravity

NASA Image and Video Library

1998-10-10

High magnification view of human primary breast tumor cells after 56 days of culture in a NASA Bioreactor. The arrow points to bead surface indicating breast cancer cells (as noted by the staining of tumor cell intermediate filaments). NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Jearne Becker, University of South Florida

ngs (notochord granular surface) gene encodes a novel type of intermediate filament family protein essential for notochord maintenance in zebrafish.

PubMed

Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

2013-01-25

The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins.

ngs (Notochord Granular Surface) Gene Encodes a Novel Type of Intermediate Filament Family Protein Essential for Notochord Maintenance in Zebrafish*

PubMed Central

Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

2013-01-01

The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins. PMID:23132861

Structural characterization of CFA/III and Longus type IVb pili from enterotoxigenic Escherichia coli.

PubMed

Kolappan, Subramaniapillai; Roos, Justin; Yuen, Alex S W; Pierce, Owen M; Craig, Lisa

2012-05-01

The type IV pili are helical filaments found on many Gram-negative pathogenic bacteria, with multiple diverse roles in pathogenesis, including microcolony formation, adhesion, and twitching motility. Many pathogenic enterotoxigenic Escherichia coli (ETEC) isolates express one of two type IV pili belonging to the type IVb subclass: CFA/III or Longus. Here we show a direct correlation between CFA/III expression and ETEC aggregation, suggesting that these pili, like the Vibrio cholerae toxin-coregulated pili (TCP), mediate microcolony formation. We report a 1.26-Å resolution crystal structure of CofA, the major pilin subunit from CFA/III. CofA is very similar in structure to V. cholerae TcpA but possesses a 10-amino-acid insertion that replaces part of the α2-helix with an irregular loop containing a 3(10)-helix. Homology modeling suggests a very similar structure for the Longus LngA pilin. A model for the CFA/III pilus filament was generated using the TCP electron microscopy reconstruction as a template. The unique 3(10)-helix insert fits perfectly within the gap between CofA globular domains. This insert, together with differences in surface-exposed residues, produces a filament that is smoother and more negatively charged than TCP. To explore the specificity of the type IV pilus assembly apparatus, CofA was expressed heterologously in V. cholerae by replacing the tcpA gene with that of cofA within the tcp operon. Although CofA was synthesized and processed by V. cholerae, no CFA/III filaments were detected, suggesting that the components of the type IVb pilus assembly system are highly specific to their pilin substrates.

COSMOS2015 photometric redshifts probe the impact of filaments on galaxy properties

NASA Astrophysics Data System (ADS)

Laigle, C.; Pichon, C.; Arnouts, S.; McCracken, H. J.; Dubois, Y.; Devriendt, J.; Slyz, A.; Le Borgne, D.; Benoit-Lévy, A.; Hwang, Ho Seong; Ilbert, O.; Kraljic, K.; Malavasi, N.; Park, Changbom; Vibert, D.

2018-03-01

The variation of galaxy stellar masses and colour types with the distance to projected cosmic filaments are quantified using the precise photometric redshifts of the COSMOS2015 catalogue extracted from Cosmological Evolution Survey (COSMOS) field (2 deg2). Realistic mock catalogues are also extracted from the lightcone of the cosmological hydrodynamical simulation HORIZON-AGN. They show that the photometric redshift accuracy of the observed catalogue (σz < 0.015 at M* > 1010M⊙ and z < 0.9) is sufficient to provide two-dimensional (2D) filaments that closely match their projected three-dimensional (3D) counterparts. Transverse stellar mass gradients are measured in projected slices of thickness 75 Mpc between 0.5 < z < 0.9, showing that the most massive galaxies are statistically closer to their neighbouring filament. At fixed stellar mass, passive galaxies are also found closer to their filament, while active star-forming galaxies statistically lie further away. The contributions of nodes and local density are removed from these gradients to highlight the specific role played by the geometry of the filaments. We find that the measured signal does persist after this removal, clearly demonstrating that proximity to a filament is not equivalent to proximity to an overdensity. These findings are in agreement with gradients measured in both 2D and 3D in the HORIZON-AGN simulation and those observed in the spectroscopic surveys VIPERS and GAMA (which both rely on the identification of 3D filaments). They are consistent with a picture in which the influence of the geometry of the large-scale environment drives anisotropic tides that impact the assembly history of galaxies, and hence their observed properties.

Negative Stains Containing Trehalose: Application to Tubular and Filamentous Structures

NASA Astrophysics Data System (ADS)

Harris, J. Robin; Gerber, Max; Gebauer, Wolfgang; Wernicke, Wolfgang; Markl, Jürgen

1996-02-01

Several examples are presented that show the successful application of uranyl acetate and ammonium molybdate negative staining in the presence of trehalose for TEM studies of filamentous and tubular structures. The principal benefit to be gained from the inclusion of trehalose stems from the considerably reduced flattening of the large tubular structures and the greater orientational freedom of single molecules due to an increased depth of the negative stain in the presence of trehalose. Trehalose is likely to provide considerable protection to protein molecules and their assemblies during the drying of negatively stained specimens. Some reduction in the excessive density imparted by uranyl acetate around large assemblies is also achieved. Nevertheless, in the presence of 1% (w/v) trehalose, it is desirable to increase the concentration of negative stain to 5% (w/v) for ammonium molybdate and to 4% for uranyl acetate to produce satisfactory image contrast. In general, the ammonium molybdate-trehalose negative stain is more satisfactory than the uranyl acetate-trehalose combination, because of the greater electron beam sensitivity of the uranyl negative stain. Reassembled taxol-stabilized pig brain microtubules, together with collagen fibrils, sperm tails, helical filaments, and reassociated hemocyanin (KLH2), all from the giant keyhole limpet Megathura crenulata, have been studied by negative staining in the presence of trehalose. In all cases satisfactory TEM imaging conditions were readily obtained on the specimens, as long as regions of excessively deep stain were avoided.

Generalization of Filament Braiding Model for Amyloid Fibril Assembly

NASA Astrophysics Data System (ADS)

Pope, Maighdlin; Ionescu-Zanetti, Cristian; Khurana, Ritu; Carter, Sue

2001-03-01

Research into the formation of amyloid fibrils is motivated by their association with several prominent diseases, among these Alzheimer's Disease, Parkinson's Disease and amyloidosis. Previous work in monitering the aggregation of immunoglobulin light chains to form amyloid fibrils suggests a braided structure where filaments and protofibrils wind together to form Type I and Type II fibrils. Non-contact atomic force microscopy is used to image and explore the kinetics of several other amyloid fibril forming proteins in an effort to generalize the filament braiding model. Included in this study are insulin and the B1 domain of G. Both of these have been shown to form fibrils in vitro. Alpha-synuclein is also included in this study. It is involved in the formation of Lewy bodies in Parkinson's Disease. The fourth protein used in this comparitive study is human amylin that is the cause of a systemic amyloidosis. Results from these four proteins and their associated fibrils are compared to the Ig light chain fibril structure in an effort to show the universality of the filament braiding model.

MASS ESTIMATES OF RAPIDLY MOVING PROMINENCE MATERIAL FROM HIGH-CADENCE EUV IMAGES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, David R.; Baker, Deborah; Van Driel-Gesztelyi, Lidia, E-mail: d.r.williams@ucl.ac.uk

We present a new method for determining the column density of erupting filament material using state-of-the-art multi-wavelength imaging data. Much of the prior work on filament/prominence structure can be divided between studies that use a polychromatic approach with targeted campaign observations and those that use synoptic observations, frequently in only one or two wavelengths. The superior time resolution, sensitivity, and near-synchronicity of data from the Solar Dynamics Observatory's Advanced Imaging Assembly allow us to combine these two techniques using photoionization continuum opacity to determine the spatial distribution of hydrogen in filament material. We apply the combined techniques to SDO/AIA observationsmore » of a filament that erupted during the spectacular coronal mass ejection on 2011 June 7. The resulting 'polychromatic opacity imaging' method offers a powerful way to track partially ionized gas as it erupts through the solar atmosphere on a regular basis, without the need for coordinated observations, thereby readily offering regular, realistic mass-distribution estimates for models of these erupting structures.« less

Programmable assembly of nanoarchitectures using genetically engineered viruses.

PubMed

Huang, Yu; Chiang, Chung-Yi; Lee, Soo Kwan; Gao, Yan; Hu, Evelyn L; De Yoreo, James; Belcher, Angela M

2005-07-01

Biological systems possess inherent molecular recognition and self-assembly capabilities and are attractive templates for constructing complex material structures with molecular precision. Here we report the assembly of various nanoachitectures including nanoparticle arrays, hetero-nanoparticle architectures, and nanowires utilizing highly engineered M13 bacteriophage as templates. The genome of M13 phage can be rationally engineered to produce viral particles with distinct substrate-specific peptides expressed on the filamentous capsid and the ends, providing a generic template for programmable assembly of complex nanostructures. Phage clones with gold-binding motifs on the capsid and streptavidin-binding motifs at one end are created and used to assemble Au and CdSe nanocrytals into ordered one-dimensional arrays and more complex geometries. Initial studies show such nanoparticle arrays can further function as templates to nucleate highly conductive nanowires that are important for addressing/interconnecting individual nanostructures.

Actin motility: formin a SCAry tail.

PubMed

Alberts, Art; Way, Michael

2011-01-11

A new biochemical analysis has revealed that the Rickettsia bacterial protein Sca2--recently shown to be essential for virulence and actin-dependent motility--assembles actin filaments using a mechanism that functionally resembles the processive elongation tactics used by formins. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bacterial cytoskeleton and implications for new antibiotic targets.

PubMed

Wang, Huan; Xie, Longxiang; Luo, Hongping; Xie, Jianping

2016-01-01

Traditionally eukaryotes exclusive cytoskeleton has been found in bacteria and other prokaryotes. FtsZ, MreB and CreS are bacterial counterpart of eukaryotic tubulin, actin filaments and intermediate filaments, respectively. FtsZ can assemble to a Z-ring at the cell division site, regulate bacterial cell division; MreB can form helical structure, and involve in maintaining cell shape, regulating chromosome segregation; CreS, found in Caulobacter crescentus (C. crescentus), can form curve or helical filaments in intracellular membrane. CreS is crucial for cell morphology maintenance. There are also some prokaryotic unique cytoskeleton components playing crucial roles in cell division, chromosome segregation and cell morphology. The cytoskeleton components of Mycobacterium tuberculosis (M. tuberculosis), together with their dynamics during exposure to antibiotics are summarized in this article to provide insights into the unique organization of this formidable pathogen and druggable targets for new antibiotics.

Pili and flagella biology, structure, and biotechnological applications.

PubMed

Van Gerven, Nani; Waksman, Gabriel; Remaut, Han

2011-01-01

Bacteria and Archaea expose on their outer surfaces a variety of thread-like proteinaceous organelles with which they interact with their environments. These structures are repetitive assemblies of covalently or non-covalently linked protein subunits, organized into filamentous polymers known as pili ("hair"), flagella ("whips") or injectisomes ("needles"). They serve different roles in cell motility, adhesion and host invasion, protein and DNA secretion and uptake, conductance, or cellular encapsulation. Here we describe the functional, morphological and genetic diversity of these bacterial filamentous protein structures. The organized, multi-copy build-up and/or the natural function of pili and flagella have lead to their biotechnological application as display and secretion tools, as therapeutic targets or as molecular motors. We review the documented and potential technological exploitation of bacterial surface filaments in light of their structural and functional traits. Copyright © 2011 Elsevier Inc. All rights reserved.

“Panta rhei”

PubMed Central

Moch, Marcin; Kölsch, Anne; Windoffer, Reinhard

2011-01-01

The filamentous cytoskeletal systems fulfil seemingly incompatible functions by maintaining a stable scaffolding to ensure tissue integrity and simultaneously facilitating rapid adaptation to intracellular processes and environmental stimuli. This paradox is particularly obvious for the abundant keratin intermediate filaments in epithelial tissues. The epidermal keratin cytoskeleton, for example, supports the protective and selective barrier function of the skin while enabling rapid growth and remodelling in response to physical, chemical and microbial challenges. We propose that these dynamic properties are linked to the perpetual re-cycling of keratin intermediate filaments that we observe in cultured cells. This cycle of assembly and disassembly is independent of protein biosynthesis and consists of distinct, temporally and spatially defined steps. In this way, the keratin cytoskeleton remains in constant motion but stays intact and is also able to restructure rapidly in response to specific regulatory cues as is needed, e.g., during division, differentiation and wound healing. PMID:21866261

An Overview of the Molecular Mechanisms of Recombinational DNA Repair

PubMed Central

Kowalczykowski, Stephen C.

2015-01-01

Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of the RecA/RAD51 filament, which promotes the homology search. On finding homology, the nucleoprotein filament catalyzes exchange of DNA strands to form a joint molecule. Recombination is controlled by regulating the fate of both RecA/RAD51 filaments and DNA pairing intermediates. Finally, intermediates that mature into Holliday structures are disjoined by either nucleolytic resolution or topological dissolution. PMID:26525148

"Panta rhei": Perpetual cycling of the keratin cytoskeleton.

PubMed

Leube, Rudolf E; Moch, Marcin; Kölsch, Anne; Windoffer, Reinhard

2011-01-01

The filamentous cytoskeletal systems fulfil seemingly incompatible functions by maintaining a stable scaffolding to ensure tissue integrity and simultaneously facilitating rapid adaptation to intracellular processes and environmental stimuli. This paradox is particularly obvious for the abundant keratin intermediate filaments in epithelial tissues. The epidermal keratin cytoskeleton, for example, supports the protective and selective barrier function of the skin while enabling rapid growth and remodelling in response to physical, chemical and microbial challenges. We propose that these dynamic properties are linked to the perpetual re-cycling of keratin intermediate filaments that we observe in cultured cells. This cycle of assembly and disassembly is independent of protein biosynthesis and consists of distinct, temporally and spatially defined steps. In this way, the keratin cytoskeleton remains in constant motion but stays intact and is also able to restructure rapidly in response to specific regulatory cues as is needed, e.g., during division, differentiation and wound healing.

PECULIAR STATIONARY EUV WAVE FRONTS IN THE ERUPTION ON 2011 MAY 11

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chandra, R.; Fulara, A.; Chen, P. F.

We present and interpret the observations of extreme ultraviolet (EUV) waves associated with a filament eruption on 2011 May 11. The filament eruption also produces a small B-class two ribbon flare and a coronal mass ejection. The event is observed by the Solar Dynamic Observatory with high spatio-temporal resolution data recorded by the Atmospheric Imaging Assembly. As the filament erupts, we observe two types of EUV waves (slow and fast) propagating outwards. The faster EUV wave has a propagation velocity of ∼500 km s{sup −1} and the slower EUV wave has an initial velocity of ∼120 km s{sup −1}. Wemore » report, for the first time, that not only does the slower EUV wave stop at a magnetic separatrix to form bright stationary fronts, but also the faster EUV wave transits a magnetic separatrix, leaving another stationary EUV front behind.« less

Nuclear networking.

PubMed

Xie, Wei; Burke, Brian

2017-07-04

Nuclear lamins are intermediate filament proteins that represent important structural components of metazoan nuclear envelopes (NEs). By combining proteomics and superresolution microscopy, we recently reported that both A- and B-type nuclear lamins form spatially distinct filament networks at the nuclear periphery of mouse fibroblasts. In particular, A-type lamins exhibit differential association with nuclear pore complexes (NPCs). Our studies reveal that the nuclear lamina network in mammalian somatic cells is less ordered and more complex than that of amphibian oocytes, the only other system in which the lamina has been visualized at high resolution. In addition, the NPC component Tpr likely links NPCs to the A-type lamin network, an association that appears to be regulated by C-terminal modification of various A-type lamin isoforms. Many questions remain, however, concerning the structure and assembly of lamin filaments, as well as with their mode of association with other nuclear components such as peripheral chromatin.

Structure and self-assembly of the calcium binding matrix protein of human metapneumovirus.

PubMed

Leyrat, Cedric; Renner, Max; Harlos, Karl; Huiskonen, Juha T; Grimes, Jonathan M

2014-01-07

The matrix protein (M) of paramyxoviruses plays a key role in determining virion morphology by directing viral assembly and budding. Here, we report the crystal structure of the human metapneumovirus M at 2.8 Å resolution in its native dimeric state. The structure reveals the presence of a high-affinity Ca²⁺ binding site. Molecular dynamics simulations (MDS) predict a secondary lower-affinity site that correlates well with data from fluorescence-based thermal shift assays. By combining small-angle X-ray scattering with MDS and ensemble analysis, we captured the structure and dynamics of M in solution. Our analysis reveals a large positively charged patch on the protein surface that is involved in membrane interaction. Structural analysis of DOPC-induced polymerization of M into helical filaments using electron microscopy leads to a model of M self-assembly. The conservation of the Ca²⁺ binding sites suggests a role for calcium in the replication and morphogenesis of pneumoviruses. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

3D Model of Cytokinetic Contractile Ring Assembly: Node-Mediated and Backup Pathways

NASA Astrophysics Data System (ADS)

Bidone, Tamara; Vavylonis, Dimitrios

Cytokinetic ring assembly in model organism fission yeast is a dynamic process, involving condensation of a network of actin filaments and myosin motors bound to the cell membrane through cortical nodes. A 3D computational model of ring assembly illustrates how the combined activities of myosin motors, filament crosslinkers and actin turnover lead to robust ring formation [Bidone et al. Biophys. J, 2014]. We modeled the importance of the physical properties of node movement along the cell membrane and of myosin recruitment to nodes. Experiments by D. Zhang (Temasek Life Sciences) show that tethering of the cortical endoplasmic reticulum (ER) to the plasma membrane modulates the speed of node condensation and the degree of node clumping. We captured the trend observed in these experiments by changes in the node drag coefficient and initial node distribution in simulations PM. The model predicted that reducing crosslinking activities in ER tethering mutants with faster node speed enhances actomyosin clumping. We developed a model of how tilted and/or misplaced rings assemble in cells that lack the node structural component anillin-like Mid1 and thus fail to recruit myosin II to nodes independently of actin. If actin-dependent binding of diffusive myosin to the cortex is incorporated into the model, it generates progressively elongating cortical actomyosin strands with fluctuating actin bundles at the tails. These stands often close into a ring, similar to observations by the group of J.Q. Wu (The Ohio State University). NIH R01GM098430.

The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus

PubMed Central

Harn, Tony; Spielman, Ingrid; Gao, Yang; Kovacikova, Gabriela; Biais, Nicolas

2016-01-01

Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system. PMID:27992883

Cytosolic chaperones mediate quality control of higher-order septin assembly in budding yeast.

PubMed

Johnson, Courtney R; Weems, Andrew D; Brewer, Jennifer M; Thorner, Jeremy; McMurray, Michael A

2015-04-01

Septin hetero-oligomers polymerize into cytoskeletal filaments with essential functions in many eukaryotic cell types. Mutations within the oligomerization interface that encompasses the GTP-binding pocket of a septin (its "G interface") cause thermoinstability of yeast septin hetero-oligomer assembly, and human disease. When coexpressed with its wild-type counterpart, a G interface mutant is excluded from septin filaments, even at moderate temperatures. We show that this quality control mechanism is specific to G interface mutants, operates during de novo septin hetero-oligomer assembly, and requires specific cytosolic chaperones. Chaperone overexpression lowers the temperature permissive for proliferation of cells expressing a G interface mutant as the sole source of a given septin. Mutations that perturb the septin G interface retard release from these chaperones, imposing a kinetic delay on the availability of nascent septin molecules for higher-order assembly. Un-expectedly, the disaggregase Hsp104 contributes to this delay in a manner that does not require its "unfoldase" activity, indicating a latent "holdase" activity toward mutant septins. These findings provide new roles for chaperone-mediated kinetic partitioning of non-native proteins and may help explain the etiology of septin-linked human diseases. © 2015 Johnson et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells

PubMed Central

McQuilken, Molly; La Riviere, Patrick J.; Occhipinti, Patricia; Verma, Amitabh; Oldenbourg, Rudolf; Gladfelter, Amy S.; Tani, Tomomi

2016-01-01

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells. PMID:27679846

Micro-rheological behaviour and nonlinear rheology of networks assembled from polysaccharides from the plant cell wall

NASA Astrophysics Data System (ADS)

Vincent, R. R. R.; Mansel, B. W.; Kramer, A.; Kroy, K.; Williams, M. A. K.

2013-03-01

The same fundamental questions that have driven enquiry into cytoskeletal mechanics can be asked of the considerably less-studied, yet arguably just as important, biopolymer matrix in the plant cell wall. In this case, it is well-known that polysaccharides, rather than filamentous and tubular protein assemblies, play a major role in satisfying the mechanical requirements of a successful cell wall, but developing a clear structure-function understanding has been exacerbated by the familiar issue of biological complexity. Herein, in the spirit of the mesoscopic approaches that have proved so illuminating in the study of cytoskeletal networks, the linear microrheological and strain-stiffening responses of biopolymeric networks reconstituted from pectin, a crucial cell wall polysaccharide, are examined. These are found to be well-captured by the glassy worm-like chain (GWLC) model of self-assembled semi-flexible filaments. Strikingly, the nonlinear mechanical response of these pectin networks is found to be much more sensitive to temperature changes than their linear response, a property that is also observed in F-actin networks, and is well reproduced by the GWLC model. Additionally, microrheological measurements suggest that over long timescales (>10 s) internal stresses continue to redistribute facilitating low frequency motions of tracer particles.

A general insert label for peptide display on chimeric filamentous bacteriophages.

PubMed

Kaplan, Gilad; Gershoni, Jonathan M

2012-01-01

The foreign insert intended to be displayed via recombinant phage proteins can have a negative effect on protein expression and phage assembly. A typical example is the case of display of peptides longer than 6 amino acid residues on the major coat protein, protein VIII of the filamentous bacteriophages M13 and fd. A solution to this problem has been the use of "two-gene systems" generating chimeric phages that concomitantly express wild-type protein VIII along with recombinant protein VIII. Although the two-gene systems are much more permissive in regard to insert length and composition, some cases can still adversely affect phage assembly. Although these phages genotypically contain the desired DNA of the insert, they appear to be phenotypically wild type. To avoid false-negative results when using chimeric phages in binding studies, it is necessary to confirm that the observed lack of phage recognition is not due to faulty assembly and display of the intended insert. Here we describe a strategy for generating antibodies that specifically recognize recombinant protein VIII regardless of the nature of its foreign insert. These antibodies can be used as a general monitor of the display of recombinant protein VIII into phage particles. Copyright © 2011 Elsevier Inc. All rights reserved.

Cytoskeletal mechanics: Structure and Dynamics

NASA Astrophysics Data System (ADS)

Bausch, Andreas

2008-03-01

The actin cytoskeleton, a dynamic network of semiflexible filaments and associated regulatory proteins, is responsible for the extraordinary viscoelastic properties of cells. Especially for cellular motility the controlled self assembly to defined structures and the dynamic reorganization on different time scales are of outstanding importance. A prominent example for the controlled self assembly are actin bundles: in many cytoskeletal processes cells rely on the tight control of the structural and mechanical properties of the actin bundles. Using an in vitro model system we show that size control relies on a mismatch between the helical structure of individual actin filaments and the packing symmetry within bundles. While such self assembled structure may evoke the picture of a static network the contrary is the case: the cytoskeleton is highly dynamic and a constant remodeling takes place in vivo. Such dynamic reorganization of the cytoskeleton relies on the non-static nature of single actin/ABP bonds. Here, we study the thermal and forced unbinding events of individual ABP in such in vitro networks. The binding kinetics of the transient crosslinkers determines the mechanical response of such networks -- in the linear as well in the non-linear regime. These effects are important prerequisites for the high adaptability of cells and at the same time might be the molecular mechanism employed by them for mechanosensing.

Characterization of the Biochemical Properties and Biological Function of the Formin Homology Domains of Drosophila DAAM*

PubMed Central

Barkó, Szilvia; Bugyi, Beáta; Carlier, Marie-France; Gombos, Rita; Matusek, Tamás; Mihály, József; Nyitrai, Miklós

2010-01-01

We characterized the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. The results show that although the DAAM-FH2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the DAAM-FH1-FH2 fragment, a profilin-dependent increase in the formation of actin structures is observed. The trachea-specific expression of DAAM-FH1-FH2 also induces phenotypic effects, leading to the collapse of the tracheal tube and lethality in the larval stages. In vitro, both DAAM fragments catalyze actin nucleation but severely decrease both the elongation and depolymerization rate of the filaments. Profilin acts as a molecular switch in DAAM function. DAAM-FH1-FH2, remaining bound to barbed ends, drives processive assembly of profilin-actin, whereas DAAM-FH2 forms an abortive complex with barbed ends that does not support profilin-actin assembly. Both DAAM fragments also bind to the sides of the actin filaments and induce actin bundling. These observations show that the D. melanogaster DAAM formin represents an extreme class of barbed end regulators gated by profilin. PMID:20177055

Interaction between Flavivirus and Cytoskeleton during Virus Replication

PubMed Central

Foo, Kar Yue; Chee, Hui-Yee

2015-01-01

Flaviviruses are potentially human pathogens that cause major epidemics worldwide. Flavivirus interacts with host cell factors to form a favourable virus replication site. Cell cytoskeletons have been observed to have close contact with flaviviruses, which expands the understanding of cytoskeleton functions during virus replication, although many detailed mechanisms are still unclear. The interactions between the virus and host cytoskeletons such as actin filaments, microtubules, and intermediate filaments have provided insight into molecular alterations during the virus infection, such as viral entry, in-cell transport, scaffold assembly, and egress. This review article focuses on the utilization of cytoskeleton by Flavivirus and the respective functions during virus replication. PMID:26347881

FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology.

PubMed

Hernanz-Koers, Miguel; Gandía, Mónica; Garrigues, Sandra; Manzanares, Paloma; Yenush, Lynne; Orzaez, Diego; Marcos, Jose F

2018-07-01

Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for Hyg R or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3α/pDGB3Ω destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community. Copyright © 2018 Elsevier Inc. All rights reserved.

Checkpoint kinase 1-induced phosphorylation of O-linked β-N-acetylglucosamine transferase regulates the intermediate filament network during cytokinesis.

PubMed

Li, Zhe; Li, Xueyan; Nai, Shanshan; Geng, Qizhi; Liao, Ji; Xu, Xingzhi; Li, Jing

2017-12-01

Checkpoint kinase 1 (Chk1) is a kinase instrumental for orchestrating DNA replication, DNA damage checkpoints, the spindle assembly checkpoint, and cytokinesis. Despite Chk1's pivotal role in multiple cellular processes, many of its substrates remain elusive. Here, we identified O- linked β- N -acetylglucosamine ( O -GlcNAc)-transferase (OGT) as one of Chk1's substrates. We found that Chk1 interacts with and phosphorylates OGT at Ser-20, which not only stabilizes OGT, but also is required for cytokinesis. Phospho-specific antibodies of OGT-pSer-20 exhibited specific signals at the midbody of the cell, consistent with midbody localization of OGT as reported previously. Moreover, phospho-deficient OGT (S20A) cells attenuated cellular O -GlcNAcylation levels and also reduced phosphorylation of Ser-71 in the cytoskeletal protein vimentin, a modification critical for severing vimentin filament during cytokinesis. Consequently, elongated vimentin bridges were observed in cells depleted of OGT via an si OGT- based approach. Lastly, expression of plasmids resistant to si OGT efficiently rescued the vimentin bridge phenotype, but the OGT-S20A rescue plasmids did not. Our results suggest a Chk1-OGT-vimentin pathway that regulates the intermediate filament network during cytokinesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Flux Cancellation Leading to Solar Filament Eruptions

NASA Astrophysics Data System (ADS)

Popescu, R. M.; Panesar, N. K.; Sterling, A. C.; Moore, R. L.

2016-12-01

Solar filaments are strands of relatively cool, dense plasma magnetically suspended in the lower density hotter solar corona. They trace magnetic polarity inversion lines (PILs) in the photosphere below, and are supported against gravity at heights of up to 100 Mm above the chromosphere by the magnetic field in and around them. This field erupts when it is rendered unstable by either magnetic flux cancellation or emergence at or near the PIL. We have studied the evolution of photospheric magnetic flux leading to ten observed filament eruptions. Specifically, we look for gradual magnetic changes in the neighborhood of the PIL prior to and during eruption. We use Extreme Ultraviolet (EUV) images from the Atmospheric Imaging Assembly (AIA), and magnetograms from the Helioseismic and Magnetic Imager (HMI), both onboard the Solar Dynamics Observatory (SDO), to study filament eruptions and their photospheric magnetic fields. We examine whether flux cancellation or/and emergence leads to filament eruptions and find that continuous flux cancellation was present at the PIL for many hours prior to each eruption. We present two events in detail and find the following: (a) the pre-eruption filament-holding core field is highly sheared and appears in the shape of a sigmoid above the PIL; (b) at the start of the eruption the opposite arms of the sigmoid reconnect in the middle above the site of (tether-cutting) flux cancellation at the PIL; (c) the filaments first show a slow-rise, followed by a fast-rise as they erupt. We conclude that these two filament eruptions result from flux cancellation in the middle of the sheared field and are in agreement with the standard model for a CME/flare filament eruption from a closed bipolar magnetic field [flux cancellation (van Ballegooijen and Martens 1989 and Moore and Roumelrotis 1992) and runaway tether-cutting (Moore et. al 2001)].

Flux Cancellation Leading to CME Filament Eruptions

NASA Technical Reports Server (NTRS)

Popescu, Roxana M.; Panesar, Navdeep K.; Sterling, Alphonse C.; Moore, Ronald L.

2016-01-01

Solar filaments are strands of relatively cool, dense plasma magnetically suspended in the lower density hotter solar corona. They trace magnetic polarity inversion lines (PILs) in the photosphere below, and are supported against gravity at heights of up to approx.100 Mm above the chromosphere by the magnetic field in and around them. This field erupts when it is rendered unstable, often by magnetic flux cancellation or emergence at or near the PIL. We have studied the evolution of photospheric magnetic flux leading to ten observed filament eruptions. Specifically, we look for gradual magnetic changes in the neighborhood of the PIL prior to and during eruption. We use Extreme Ultraviolet (EUV) images from the Atmospheric Imaging Assembly (AIA), and magnetograms from the Helioseismic and Magnetic Imager (HMI), both on board the Solar Dynamics Observatory (SDO), to study filament eruptions and their photospheric magnetic fields. We examine whether flux cancellation or/and emergence leads to filament eruptions. We find that continuous flux cancellation was present at the PIL for many hours prior to each eruption. We present two CME-producing eruptions in detail and find the following: (a) the pre-eruption filament-holding core field is highly sheared and appears in the shape of a sigmoid above the PIL; (b) at the start of the eruption the opposite arms of the sigmoid reconnect in the middle above the site of (tether-cutting) flux cancellation at the PIL; (c) the filaments first show a slow-rise, followed by a fast-rise as they erupt. We conclude that these two filament eruptions result from flux cancellation in the middle of the sheared field, and thereafter evolve in agreement with the standard model for a CME/flare filament eruption from a closed bipolar magnetic field [flux cancellation (van Ballegooijen and Martens 1989 and Moore and Roumelrotis 1992) and runaway tether-cutting (Moore et. al 2001)].

Halo assembly bias and the tidal anisotropy of the local halo environment

NASA Astrophysics Data System (ADS)

Paranjape, Aseem; Hahn, Oliver; Sheth, Ravi K.

2018-05-01

We study the role of the local tidal environment in determining the assembly bias of dark matter haloes. Previous results suggest that the anisotropy of a halo's environment (i.e. whether it lies in a filament or in a more isotropic region) can play a significant role in determining the eventual mass and age of the halo. We statistically isolate this effect, using correlations between the large-scale and small-scale environments of simulated haloes at z = 0 with masses between 1011.6 ≲ (m/h-1 M⊙) ≲ 1014.9. We probe the large-scale environment, using a novel halo-by-halo estimator of linear bias. For the small-scale environment, we identify a variable αR that captures the tidal anisotropy in a region of radius R = 4R200b around the halo and correlates strongly with halo bias at fixed mass. Segregating haloes by αR reveals two distinct populations. Haloes in highly isotropic local environments (αR ≲ 0.2) behave as expected from the simplest, spherically averaged analytical models of structure formation, showing a negative correlation between their concentration and large-scale bias at all masses. In contrast, haloes in anisotropic, filament-like environments (αR ≳ 0.5) tend to show a positive correlation between bias and concentration at any mass. Our multiscale analysis cleanly demonstrates how the overall assembly bias trend across halo mass emerges as an average over these different halo populations, and provides valuable insights towards building analytical models that correctly incorporate assembly bias. We also discuss potential implications for the nature and detectability of galaxy assembly bias.

Structural Determination of Biomolecules in Microfluidic Systems

NASA Astrophysics Data System (ADS)

Butler, John C.; Menard, Etienne; Rogers, John A.; Wong, Gerard C. L.

2004-03-01

Supramolecular biological complexes are often too large to be crystallized for structural studies. Here, we explore the use of microfluidic arrays to order a model self-assembled cytoskeletal system. Filamentous actin (F-actin) is a negatively charged protein rod and is a key structural component in the eukaryotic cytoskeleton. In this context, F-actin can self-assemble with actin binding proteins (ABP) in a highly regulated manner to dynamically form structures for a wide range of biomechanical functions. In this work, we will systematically study the action of 3 types of actin binding proteins (a-actinin, fimbrin, cofilin) on the self-assembled structures of F-actin that have been aligned in microfluidic arrays.

Morphology manipulation of M13 bacteriophage template for nanostructure assembly

NASA Astrophysics Data System (ADS)

Ngo-Duc, Tam-Triet; Zaman, Mohammed S.; Moon, Chung-Hee; Haberer, Elaine D.

2014-08-01

A gold-binding M13 bacteriophage was used as a model system to explore templating of inorganic material on geometrically transformed viruses . Gold-binding filamentous phage were converted to spheroid form with a short chloroform treatment, and the resulting morphology was investigated with electron microscopy. Binding studies revealed that spheroid-shaped gold-binding phage preserved its affinity for gold. Spheroids adhered to a planar substrate assembled clusters or rings of gold nanoparticles. This gold-binding phage served as a demonstration of a highly shape-modifiable viral-template for inorganic materials.

Counter-streaming flows in a giant quiet-Sun filament observed in the extreme ultraviolet

NASA Astrophysics Data System (ADS)

Diercke, A.; Kuckein, C.; Verma, M.; Denker, C.

2018-03-01

Aim. The giant solar filament was visible on the solar surface from 2011 November 8-23. Multiwavelength data from the Solar Dynamics Observatory (SDO) were used to examine counter-streaming flows within the spine of the filament. Methods: We use data from two SDO instruments, the Atmospheric Imaging Assembly (AIA) and the Helioseismic and Magnetic Imager (HMI), covering the whole filament, which stretched over more than half a solar diameter. Hα images from the Kanzelhöhe Solar Observatory (KSO) provide context information of where the spine of the filament is defined and the barbs are located. We apply local correlation tracking (LCT) to a two-hour time series on 2011 November 16 of the AIA images to derive horizontal flow velocities of the filament. To enhance the contrast of the AIA images, noise adaptive fuzzy equalization (NAFE) is employed, which allows us to identify and quantify counter-streaming flows in the filament. We observe the same cool filament plasma in absorption in both Hα and EUV images. Hence, the counter-streaming flows are directly related to this filament material in the spine. In addition, we use directional flow maps to highlight the counter-streaming flows. Results: We detect counter-streaming flows in the filament, which are visible in the time-lapse movies in all four examined AIA wavelength bands (λ171 Å, λ193 Å, λ304 Å, and λ211 Å). In the time-lapse movies we see that these persistent flows lasted for at least two hours, although they became less prominent towards the end of the time series. Furthermore, by applying LCT to the images we clearly determine counter-streaming flows in time series of λ171 Å and λ193 Å images. In the λ304 Å wavelength band, we only see minor indications for counter-streaming flows with LCT, while in the λ211 Å wavelength band the counter-streaming flows are not detectable with this method. The diverse morphology of the filament in Hα and EUV images is caused by different absorption processes, i.e., spectral line absorption and absorption by hydrogen and helium continua, respectively. The horizontal flows reach mean flow speeds of about 0.5 km s-1 for all wavelength bands. The highest horizontal flow speeds are identified in the λ171 Å band with flow speeds of up to 2.5 km s-1. The results are averaged over a time series of 90 minutes. Because the LCT sampling window has finite width, a spatial degradation cannot be avoided leading to lower estimates of the flow velocities as compared to feature tracking or Doppler measurements. The counter-streaming flows cover about 15-20% of the whole area of the EUV filament channel and are located in the central part of the spine. Conclusions: Compared to the ground-based observations, the absence of seeing effects in AIA observations reveal counter-streaming flows in the filament even with a moderate image scale of 0. ''6 pixel-1. Using a contrast enhancement technique, these flows can be detected and quantified with LCT in different wavelengths. We confirm the omnipresence of counter-streaming flows also in giant quiet-Sun filaments. A movie associated to Fig. 6 is available at http://https://www.aanda.org

Intracellular Peptide Self-Assembly: A Biomimetic Approach for in Situ Nanodrug Preparation.

PubMed

Du, Wei; Hu, Xiaomu; Wei, Weichen; Liang, Gaolin

2018-04-18

Most nanodrugs are preprepared by encapsulating or loading the drugs with nanocarriers (e.g., dendrimers, liposomes, micelles, and polymeric nanoparticles). However, besides the low bioavailability and fast excretion of the nanodrugs in vivo, nanocarriers often exhibit in vitro and in vivo cytotoxicity, oxidative stress, and inflammation. Self-assembly is a ubiquitous process in biology where it plays important roles and underlies the formation of a wide variety of complex biological structures. Inspired by some cellular nanostructures (e.g., actin filaments, microtubules, vesicles, and micelles) in biological systems which are formed via molecular self-assembly, in recent decades, scientists have utilized self-assembly of oligomeric peptide under specific physiological or pathological environments to in situ construct nanodrugs for lesion-targeted therapies. On one hand, peptide-based nanodrugs always have some excellent intrinsic chemical (specificity, intrinsic bioactivity, biodegradability) and physical (small size, conformation) properties. On the other hand, stimuli-regulated intracellular self-assembly of nanodrugs is quite an efficient way to accumulate the drugs in lesion location and can realize an in situ slow release of the drugs. In this review article, we provided an overview on recent design principles for intracellular peptide self-assembly and illustrate how these principles have been applied for the in situ preparation of nanodrugs at the lesion location. In the last part, we list some challenges underlying this strategy and their possible solutions. Moreover, we envision the future possible theranostic applications of this strategy.

Evolution of Mass and Velocity Field in the Cosmic Web: Comparison between Baryonic and Dark Matter

NASA Astrophysics Data System (ADS)

Zhu, Weishan; Feng, Long-Long

2017-03-01

We investigate the evolution of the cosmic web since z = 5 in grid-based cosmological hydrodynamical simulations, focusing on the mass and velocity fields of both baryonic and cold dark matter. The tidal tensor of density is used as the main method for web identification, with λ th = 0.2-1.2. The evolution trends in baryonic and dark matter are similar, although moderate differences are observed. Sheets appear early, and their large-scale pattern may have been set up by z = 3. In terms of mass, filaments supersede sheets as the primary collapsing structures from z ˜ 2-3. Tenuous filaments assembled with each other to form prominent ones at z < 2. In accordance with the construction of the frame of the sheets, the cosmic divergence velocity, v div, was already well-developed above 2-3 Mpc by z = 3. Afterwards, the curl velocity, v curl, grew dramatically along with the rising of filaments, becoming comparable to v div, for <2-3 Mpc at z = 0. The scaling of v curl can be described by the hierarchical turbulence model. The alignment between the vorticity and the eigenvectors of the shear tensor in the baryonic matter field resembles that in the dark matter field, and is even moderately stronger between {\\boldsymbol{ω }} and {{\\boldsymbol{e}}}1, and ω and {{\\boldsymbol{e}}}3. Compared with dark matter, there is slightly less baryonic matter found residing in filaments and clusters, and its vorticity developed more significantly below 2-3 Mpc. These differences may be underestimated because of the limited resolution and lack of star formation in our simulation. The impact of the change of dominant structures in overdense regions at z ˜ 2-3 on galaxy formation and evolution is shortly discussed.

Human somatic cells acquire the plasticity to generate embryoid-like metamorphosis via the actin cytoskeleton in injured tissues.

PubMed

Diaz, Jairo A; Murillo, Mauricio F; Mendoza, Jhonan A; Barreto, Ana M; Poveda, Lina S; Sanchez, Lina K; Poveda, Laura C; Mora, Katherine T

2016-01-01

Emergent biological responses develop via unknown processes dependent on physical collision. In hypoxia, when the tissue architecture collapses but the geometric core is stable, actin cytoskeleton filament components emerge, revealing a hidden internal order that identifies how each molecule is reassembled into the original mold, using one common connection, i.e., a fractal self-similarity that guides the system from the beginning in reverse metamorphosis, with spontaneous self-assembly of past forms that mimics an embryoid phenotype. We captured this hidden collective filamentous assemblage in progress: Hypoxic deformed cells enter into intercellular collisions, generate migratory ejected filaments, and produce self-assembly of triangular chiral hexagon complexes; this dynamic geometry guides the microenvironment scaffold in which this biological process is incubated, recapitulating embryonic morphogenesis. In all injured tissues, especially in damaged skeletal (striated) muscle cells, visibly hypertrophic intercalated actin-myosin filaments are organized in zebra stripe pattern along the anterior-posterior axis in the interior of the cell, generating cephalic-caudal polarity segmentation, with a high selective level of immunopositivity for Actin, Alpha Skeletal Muscle antibody and for Neuron-Specific Enolase expression of ectodermal differentiation. The function of actin filaments in emergent responses to tissue injury is to reconstitute, reactivate and orchestrate cellular metamorphosis, involving the re-expression of fetal genes, providing evidence of the reverse flow of genetic information within a biological system. The resultant embryoid phenotype emerges as a microscopic fractal template copy of the organization of the whole body, likely allowing the modification and reprogramming of the phenotype of the tumor in which these structures develop, as well as establishing a reverse primordial microscopic mold to collectively re-form cellular building blocks to regenerate injured tissues. Tumorigenesis mimics a self-organizing process of early embryo development. All malignant tumors produce fetal proteins, we now know from which these proteins proceed. Embryoid-like metamorphosis phenomena would represent the anatomical and functional entity of the injury stem cell niche. The sufficiently fast identification, isolation, culture, and expansion of these self-organized structures or genetically derived products could, in our opinion, be used to develop new therapeutic strategies against cancer and in regenerative medicine.

Human somatic cells acquire the plasticity to generate embryoid-like metamorphosis via the actin cytoskeleton in injured tissues

PubMed Central

Diaz, Jairo A; Murillo, Mauricio F; Mendoza, Jhonan A; Barreto, Ana M; Poveda, Lina S; Sanchez, Lina K; Poveda, Laura C; Mora, Katherine T

2016-01-01

Emergent biological responses develop via unknown processes dependent on physical collision. In hypoxia, when the tissue architecture collapses but the geometric core is stable, actin cytoskeleton filament components emerge, revealing a hidden internal order that identifies how each molecule is reassembled into the original mold, using one common connection, i.e., a fractal self-similarity that guides the system from the beginning in reverse metamorphosis, with spontaneous self-assembly of past forms that mimics an embryoid phenotype. We captured this hidden collective filamentous assemblage in progress: Hypoxic deformed cells enter into intercellular collisions, generate migratory ejected filaments, and produce self-assembly of triangular chiral hexagon complexes; this dynamic geometry guides the microenvironment scaffold in which this biological process is incubated, recapitulating embryonic morphogenesis. In all injured tissues, especially in damaged skeletal (striated) muscle cells, visibly hypertrophic intercalated actin-myosin filaments are organized in zebra stripe pattern along the anterior-posterior axis in the interior of the cell, generating cephalic-caudal polarity segmentation, with a high selective level of immunopositivity for Actin, Alpha Skeletal Muscle antibody and for Neuron-Specific Enolase expression of ectodermal differentiation. The function of actin filaments in emergent responses to tissue injury is to reconstitute, reactivate and orchestrate cellular metamorphosis, involving the re-expression of fetal genes, providing evidence of the reverse flow of genetic information within a biological system. The resultant embryoid phenotype emerges as a microscopic fractal template copy of the organization of the whole body, likely allowing the modification and reprogramming of the phenotype of the tumor in which these structures develop, as well as establishing a reverse primordial microscopic mold to collectively re-form cellular building blocks to regenerate injured tissues. Tumorigenesis mimics a self-organizing process of early embryo development. All malignant tumors produce fetal proteins, we now know from which these proteins proceed. Embryoid-like metamorphosis phenomena would represent the anatomical and functional entity of the injury stem cell niche. The sufficiently fast identification, isolation, culture, and expansion of these self-organized structures or genetically derived products could, in our opinion, be used to develop new therapeutic strategies against cancer and in regenerative medicine. PMID:27725917

Breast Cancer Research at NASA

NASA Technical Reports Server (NTRS)

1998-01-01

High magnification view of human primary breast tumor cells after 56 days of culture in a NASA Bioreactor. The arrow points to bead surface indicating breast cancer cells (as noted by the staining of tumor cell intermediate filaments). NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Jearne Becker, University of South Florida

Assembly and mechanosensory function of focal adhesions: experiments and models.

PubMed

Bershadsky, Alexander D; Ballestrem, Christoph; Carramusa, Letizia; Zilberman, Yuliya; Gilquin, Benoit; Khochbin, Saadi; Alexandrova, Antonina Y; Verkhovsky, Alexander B; Shemesh, Tom; Kozlov, Michael M

2006-04-01

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.

Plant villin, lily P-135-ABP, possesses G-actin binding activity and accelerates the polymerization and depolymerization of actin in a Ca2+-sensitive manner.

PubMed

Yokota, Etsuo; Tominaga, Motoki; Mabuchi, Issei; Tsuji, Yasunori; Staiger, Christopher J; Oiwa, Kazuhiro; Shimmen, Teruo

2005-10-01

From germinating pollen of lily, two types of villins, P-115-ABP and P-135-ABP, have been identified biochemically. Ca(2+)-CaM-dependent actin-filament binding and bundling activities have been demonstrated for both villins previously. Here, we examined the effects of lily villins on the polymerization and depolymerization of actin. P-115-ABP and P-135-ABP present in a crude protein extract prepared from germinating pollen bound to a DNase I affinity column in a Ca(2+)-dependent manner. Purified P-135-ABP reduced the lag period that precedes actin filament polymerization from monomers in the presence of either Ca(2+) or Ca(2+)-CaM. These results indicated that P-135-ABP can form a complex with G-actin in the presence of Ca(2+) and this complex acts as a nucleus for polymerization of actin filaments. However, the nucleation activity of P-135-ABP is probably not relevant in vivo because the assembly of G-actin saturated with profilin, a situation that mimics conditions found in pollen, was not accelerated in the presence of P-135-ABP. P-135-ABP also enhanced the depolymerization of actin filaments during dilution-mediated disassembly. Growth from filament barbed ends in the presence of Ca(2+)-CaM was also prevented, consistent with filament capping activity. These results suggested that lily villin is involved not only in the arrangement of actin filaments into bundles in the basal and shank region of the pollen tube, but also in regulating and modulating actin dynamics through its capping and depolymerization (or fragmentation) activities in the apical region of the pollen tube, where there is a relatively high concentration of Ca(2+).

New textile composite materials development, production, application

NASA Technical Reports Server (NTRS)

Mikhailov, Petr Y.

1993-01-01

New textile composite materials development, production, and application are discussed. Topics covered include: super-high-strength, super-high-modulus fibers, filaments, and materials manufactured on their basis; heat-resistant and nonflammable fibers, filaments, and textile fabrics; fibers and textile fabrics based on fluorocarbon poylmers; antifriction textile fabrics based on polyfen filaments; development of new types of textile combines and composite materials; and carbon filament-based fabrics.

Self-assembling of calcium salt of the new DNA base 5-carboxylcytosine

NASA Astrophysics Data System (ADS)

Irrera, Simona; Ruiz-Hernandez, Sergio E.; Reggente, Melania; Passeri, Daniele; Natali, Marco; Gala, Fabrizio; Zollo, Giuseppe; Rossi, Marco; Portalone, Gustavo

2017-06-01

Supramolecular architectures involving DNA bases can have a strong impact in several fields such as nanomedicine and nanodevice manufacturing. To date, in addition to the four canonical nucleobases (adenine, thymine, guanine and cytosine), four other forms of cytosine modified at the 5 position have been identified in DNA. Among these four new cytosine derivatives, 5-carboxylcytosine has been recently discovered in mammalian stem cell DNA, and proposed as the final product of the oxidative epigenetic demethylation pathway on the 5 position of cytosine. In this work, a calcium salt of 5-carboxylcytosine has been synthesized and deposited on graphite surface, where it forms self-assembled features as long range monolayers and up to one micron long filaments. These structures have been analyzed in details combining different theoretical and experimental approaches: X-ray single-crystal diffraction data were used to simulate the molecule-graphite interaction, first using molecular dynamics and then refining the results using density functional theory (DFT); finally, data obtained with DFT were used to rationalize atomic force microscopy (AFM) results.

Lattice model for self-assembly with application to the formation of cytoskeletal-like structures

NASA Astrophysics Data System (ADS)

Stewman, Shannon F.; Dinner, Aaron R.

2007-07-01

We introduce a stochastic approach for self-assembly in systems far from equilibrium. The building blocks are represented by a lattice of discrete variables (Potts-like spins), and physically meaningful mechanisms are obtained by restricting transitions through spatially local rules based on experimental data. We use the method to study nucleation of filopodia-like bundles in a system consisting of purified actin, fascin, actin-related protein 2/3 , and beads coated with Wiskott-Aldrich syndrome protein. Consistent with previous speculation based on static experimental images, we find that bundles derive from Λ -precursor-like patterns of spins on the lattice. The ratcheting of the actin network relative to the surface that represents beads plays an important role in determining the number and orientation of bundles due to the fact that branching is the primary means for generating barbed ends pointed in directions that allow rapid filament growth. By enabling the de novo formation of coexisting morphologies without the computational cost of explicit representation of proteins, the approach introduced complements earlier models of cytoskeletal behavior in vitro and in vivo.

Actin-myosin network is required for proper assembly of influenza virus particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumakura, Michiko; Kawaguchi, Atsushi, E-mail: ats-kawaguchi@md.tsukuba.ac.jp; Nagata, Kyosuke, E-mail: knagata@md.tsukuba.ac.jp

Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregatedmore » on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.« less

Chirality of the cytoskeleton in the origins of cellular asymmetry

PubMed Central

2016-01-01

Self-assembly of two important components of the cytoskeleton of eukaryotic cells, actin microfilaments and microtubules (MTs) results in polar filaments of one chirality. As is true for bacterial flagella, in actin microfilaments, screw direction is important for assembly processes and motility. For MTs, polar orientation within the cell is paramount. The alignment of these elements in the cell cytoplasm gives rise to emergent properties, including the potential for cell differentiation and specialization. Complex MTs with a characteristic chirality are found in basal bodies and centrioles; this chirality is preserved in cilia. In motile cilia, it is reflected in the direction of the effective stroke. The positioning of the basal body or cilia on the cell surface depends on polarity proteins. In evolution, survival depends on global polarity information relayed to the cell in part by orientation of the MT and actin filament cytoskeletons and the chirality of the basal body to determine left and right coordinates within a defined anterior–posterior cell and tissue axis. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821520

Large-scale filament formation inhibits the activity of CTP synthetase

PubMed Central

Barry, Rachael M; Bitbol, Anne-Florence; Lorestani, Alexander; Charles, Emeric J; Habrian, Chris H; Hansen, Jesse M; Li, Hsin-Jung; Baldwin, Enoch P; Wingreen, Ned S; Kollman, Justin M; Gitai, Zemer

2014-01-01

CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable. DOI: http://dx.doi.org/10.7554/eLife.03638.001 PMID:25030911

Chevrons, filaments, spinning clusters and phase coexistence: emergent dynamics of 2- and 3-d particle suspensions driven by multiaxial magnetic fields

DOE PAGES

Solis, Kyle J.; Martin, James E.

2017-07-06

In recent years a rich variety of emergent phenomena have been observed when suspensions of magnetic particles are subjected to alternating magnetic fields. These particle assemblies often exhibit vigorous dynamics due to the injection of energy from the field. These include surface and interface phenomena, such as highly organized, segmented “snakes” that can be induced to swim by structural symmetry breaking, and “asters” and “anti-asters,” particle assemblies that can be manipulated to capture and transport cargo. In bulk suspensions of magnetic platelets subjected to multiaxial alternating fields, advection lattices and even vortex lattices have been created, and a variety ofmore » biomimetic dynamics – serpents, bees and amoebas – have been discovered in magnetic fluids suspended in an immiscible liquid. In this paper several new driven phases are presented, including flying chevrons, dense spinning clusters, filaments, and examples of phase coexistence in driven phases. These observations broaden the growing field of driven magnetic suspensions and present new challenges to those interested in simulating the dynamics of these complex systems.« less

Human β-Synuclein Rendered Fibrillogenic by Designed Mutations

PubMed Central

Zibaee, Shahin; Fraser, Graham; Jakes, Ross; Owen, David; Serpell, Louise C.; Crowther, R. Anthony; Goedert, Michel

2010-01-01

Filamentous inclusions made of α-synuclein are found in nerve cells and glial cells in a number of human neurodegenerative diseases, including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. The assembly and spreading of these inclusions are likely to play an important role in the etiology of common dementias and movement disorders. Both α-synuclein and the homologous β-synuclein are abundantly expressed in the central nervous system; however, β-synuclein is not present in the pathological inclusions. Previously, we observed a poor correlation between filament formation and the presence of residues 73–83 of α-synuclein, which are absent in β-synuclein. Instead, filament formation correlated with the mean β-sheet propensity, charge, and hydrophilicity of the protein (global physicochemical properties) and β-strand contiguity calculated by a simple algorithm of sliding averages (local physicochemical property). In the present study, we rendered β-synuclein fibrillogenic via one set of point mutations engineered to enhance global properties and a second set engineered to enhance predominantly β-strand contiguity. Our findings show that the intrinsic physicochemical properties of synucleins influence their fibrillogenic propensity via two distinct but overlapping modalities. The implications for filament formation and the pathogenesis of neurodegenerative diseases are discussed. PMID:20833719

Miniature x-ray source

DOEpatents

Trebes, James E.; Bell, Perry M.; Robinson, Ronald B.

2000-01-01

A miniature x-ray source utilizing a hot filament cathode. The source has a millimeter scale size and is capable of producing broad spectrum x-ray emission over a wide range of x-ray energies. The miniature source consists of a compact vacuum tube assembly containing the hot filament cathode, an anode, a high voltage feedthru for delivering high voltage to the cathode, a getter for maintaining high vacuum, a connector for initial vacuum pump down and crimp-off, and a high voltage connection for attaching a compact high voltage cable to the high voltage feedthru. At least a portion of the vacuum tube wall is fabricated from highly x-ray transparent materials, such as sapphire, diamond, or boron nitride.

Exploring the Secretomes of Microbes and Microbial Communities Using Filamentous Phage Display

PubMed Central

Gagic, Dragana; Ciric, Milica; Wen, Wesley X.; Ng, Filomena; Rakonjac, Jasna

2016-01-01

Microbial surface and secreted proteins (the secretome) contain a large number of proteins that interact with other microbes, host and/or environment. These proteins are exported by the coordinated activities of the protein secretion machinery present in the cell. A group of bacteriophage, called filamentous phage, have the ability to hijack bacterial protein secretion machinery in order to amplify and assemble via a secretion-like process. This ability has been harnessed in the use of filamentous phage of Escherichia coli in biotechnology applications, including screening large libraries of variants for binding to “bait” of interest, from tissues in vivo to pure proteins or even inorganic substrates. In this review we discuss the roles of secretome proteins in pathogenic and non-pathogenic bacteria and corresponding secretion pathways. We describe the basics of phage display technology and its variants applied to discovery of bacterial proteins that are implicated in colonization of host tissues and pathogenesis, as well as vaccine candidates through filamentous phage display library screening. Secretome selection aided by next-generation sequence analysis was successfully applied for selective display of the secretome at a microbial community scale, the latter revealing the richness of secretome functions of interest and surprising versatility in filamentous phage display of secretome proteins from large number of Gram-negative as well as Gram-positive bacteria and archaea. PMID:27092113

Tropomyosin 4 defines novel filaments in skeletal muscle associated with muscle remodelling/regeneration in normal and diseased muscle.

PubMed

Vlahovich, Nicole; Schevzov, Galina; Nair-Shaliker, Visalini; Ilkovski, Biljana; Artap, Stanley T; Joya, Josephine E; Kee, Anthony J; North, Kathryn N; Gunning, Peter W; Hardeman, Edna C

2008-01-01

The organisation of structural proteins in muscle into highly ordered sarcomeres occurs during development, regeneration and focal repair of skeletal muscle fibers. The involvement of cytoskeletal proteins in this process has been documented, with nonmuscle gamma-actin found to play a role in sarcomere assembly during muscle differentiation and also shown to be up-regulated in dystrophic muscles which undergo regeneration and repair [Lloyd et al.,2004; Hanft et al.,2006]. Here, we show that a cytoskeletal tropomyosin (Tm), Tm4, defines actin filaments in two novel compartments in muscle fibers: a Z-line associated cytoskeleton (Z-LAC), similar to a structure we have reported previously [Kee et al.,2004], and longitudinal filaments that are orientated parallel to the sarcomeric apparatus, present during myofiber growth and repair/regeneration. Tm4 is upregulated in paradigms of muscle repair including induced regeneration and focal repair and in muscle diseases with repair/regeneration features, muscular dystrophy and nemaline myopathy. Longitudinal Tm4-defined filaments also are present in diseased muscle. Transition of the Tm4-defined filaments from a longitudinal to a Z-LAC orientation is observed during the course of muscle regeneration. This Tm4-defined cytoskeleton is a marker of growth and repair/regeneration in response to injury, disease state and stress in skeletal muscle.

Well-observed dynamics of flaring and peripheral coronal magnetic loops during an M-class limb flare

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Jinhua; Zhou, Tuanhui; Ji, Haisheng

2014-08-20

In this paper, we present a variety of well-observed dynamic behaviors for the flaring and peripheral magnetic loops of the M6.6 class extreme limb flare that occurred on 2011 February 24 (SOL2011-02-24T07:20) from EUV observations by the Atmospheric Imaging Assembly on the Solar Dynamics Observatory and X-ray observations by RHESSI. The flaring loop motion confirms the earlier contraction-expansion picture. We find that the U-shaped trajectory delineated by the X-ray corona source of the flare roughly follows the direction of a filament eruption associated with the flare. Different temperature structures of the coronal source during the contraction and expansion phases stronglymore » suggest different kinds of magnetic reconnection processes. For some peripheral loops, we discover that their dynamics are closely correlated with the filament eruption. During the slow rising to abrupt, fast rising of the filament, overlying peripheral magnetic loops display different responses. Two magnetic loops on the elbow of the active region had a slow descending motion followed by an abrupt successive fast contraction, while magnetic loops on the top of the filament were pushed outward, slowly being inflated for a while and then erupting as a moving front. We show that the filament activation and eruption play a dominant role in determining the dynamics of the overlying peripheral coronal magnetic loops.« less

Reconstitution of actin-based motility of Listeria and Shigella using pure proteins

NASA Astrophysics Data System (ADS)

Loisel, Thomas P.; Boujemaa, Rajaa; Pantaloni, Dominique; Carlier, Marie-France

1999-10-01

Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, α-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

Azaphilones inhibit tau aggregation and dissolve tau aggregates in vitro.

PubMed

Paranjape, Smita R; Riley, Andrew P; Somoza, Amber D; Oakley, C Elizabeth; Wang, Clay C C; Prisinzano, Thomas E; Oakley, Berl R; Gamblin, T Chris

2015-05-20

The aggregation of the microtubule-associated protein tau is a seminal event in many neurodegenerative diseases, including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activities. We have previously screened Aspergillus nidulans secondary metabolites for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol. One aggregation inhibitor identified was asperbenzaldehyde, an intermediate in azaphilone biosynthesis. We therefore tested 11 azaphilone derivatives to determine their tau assembly inhibition properties in vitro. All compounds tested inhibited tau filament assembly to some extent, and four of the 11 compounds had the advantageous property of disassembling preformed tau aggregates in a dose-dependent fashion. The addition of these compounds to the tau aggregates reduced both the total length and number of tau polymers. The most potent compounds were tested in in vitro reactions to determine whether they interfere with tau's normal function of stabilizing microtubules (MTs). We found that they did not completely inhibit MT assembly in the presence of tau. These derivatives are very promising lead compounds for tau aggregation inhibitors and, more excitingly, for compounds that can disassemble pre-existing tau filaments. They also represent a new class of anti-tau aggregation compounds with a novel structural scaffold.

Structure and mechanism of the phage T4 recombination mediator protein UvsY

DOE PAGES

Gajewski, Stefan; Waddell, Michael Brett; Vaithiyalingam, Sivaraja; ...

2016-03-07

The UvsY recombination mediator protein is critical for efficient homologous recombination in bacteriophage T4 and is the functional analog of the eukaryotic Rad52 protein. During T4 homologous recombination, the UvsX recombinase has to compete with the prebound gp32 single-stranded binding protein for DNA-binding sites and UvsY stimulates this filament nucleation event. We report here the crystal structure of UvsY in four similar open-barrel heptameric assemblies and provide structural and biophysical insights into its function. The UvsY heptamer was confirmed in solution by centrifugation and light scattering, and thermodynamic analyses revealed that the UvsY–ssDNA interaction occurs within the assembly via twomore » distinct binding modes. Using surface plasmon resonance, we also examined the binding of UvsY to both ssDNA and the ssDNA–gp32 complex. These analyses confirmed that ssDNA can bind UvsY and gp32 independently and also as a ternary complex. They also showed that residues located on the rim of the heptamer are required for optimal binding to ssDNA, thus identifying the putative ssDNA-binding surface. We propose a model in which UvsY promotes a helical ssDNA conformation that disfavors the binding of gp32 and initiates the assembly of the ssDNA–UvsX filament.« less

ROTATING MOTIONS AND MODELING OF THE ERUPTING SOLAR POLAR-CROWN PROMINENCE ON 2010 DECEMBER 6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Yingna; Van Ballegooijen, Adriaan, E-mail: ynsu@head.cfa.harvard.edu

2013-02-10

A large polar-crown prominence composed of different segments spanning nearly the entire solar disk erupted on 2010 December 6. Prior to the eruption, the filament in the active region part split into two layers: a lower layer and an elevated layer. The eruption occurs in several episodes. Around 14:12 UT, the lower layer of the active region filament breaks apart: One part ejects toward the west, while the other part ejects toward the east, which leads to the explosive eruption of the eastern quiescent filament. During the early rise phase, part of the quiescent filament sheet displays strong rolling motionmore » (observed by STEREO-B) in the clockwise direction (viewed from east to west) around the filament axis. This rolling motion appears to start from the border of the active region, then propagates toward the east. The Atmospheric Imaging Assembly (AIA) observes another type of rotating motion: In some other parts of the erupting quiescent prominence, the vertical threads turn horizontal, then turn upside down. The elevated active region filament does not erupt until 18:00 UT, when the erupting quiescent filament has already reached a very large height. We develop two simplified three-dimensional models that qualitatively reproduce the observed rolling and rotating motions. The prominence in the models is assumed to consist of a collection of discrete blobs that are tied to particular field lines of a helical flux rope. The observed rolling motion is reproduced by continuous twist injection into the flux rope in Model 1 from the active region side. Asymmetric reconnection induced by the asymmetric distribution of the magnetic fields on the two sides of the filament may cause the observed rolling motion. The rotating motion of the prominence threads observed by AIA is consistent with the removal of the field line dips in Model 2 from the top down during the eruption.« less

Anti-parallel EUV Flows Observed along Active Region Filament Threads with Hi-C

NASA Astrophysics Data System (ADS)

Alexander, Caroline E.; Walsh, Robert W.; Régnier, Stéphane; Cirtain, Jonathan; Winebarger, Amy R.; Golub, Leon; Kobayashi, Ken; Platt, Simon; Mitchell, Nick; Korreck, Kelly; DePontieu, Bart; DeForest, Craig; Weber, Mark; Title, Alan; Kuzin, Sergey

2013-09-01

Plasma flows within prominences/filaments have been observed for many years and hold valuable clues concerning the mass and energy balance within these structures. Previous observations of these flows primarily come from Hα and cool extreme-ultraviolet (EUV) lines (e.g., 304 Å) where estimates of the size of the prominence threads has been limited by the resolution of the available instrumentation. Evidence of "counter-steaming" flows has previously been inferred from these cool plasma observations, but now, for the first time, these flows have been directly imaged along fundamental filament threads within the million degree corona (at 193 Å). In this work, we present observations of an AR filament observed with the High-resolution Coronal Imager (Hi-C) that exhibits anti-parallel flows along adjacent filament threads. Complementary data from the Solar Dynamics Observatory (SDO)/Atmospheric Imaging Assembly (AIA) and Helioseismic and Magnetic Imager are presented. The ultra-high spatial and temporal resolution of Hi-C allow the anti-parallel flow velocities to be measured (70-80 km s-1) and gives an indication of the resolvable thickness of the individual strands (0.''8 ± 0.''1). The temperature of the plasma flows was estimated to be log T (K) = 5.45 ± 0.10 using Emission Measure loci analysis. We find that SDO/AIA cannot clearly observe these anti-parallel flows or measure their velocity or thread width due to its larger pixel size. We suggest that anti-parallel/counter-streaming flows are likely commonplace within all filaments and are currently not observed in EUV due to current instrument spatial resolution.

Topology effects on nonaffine behavior of semiflexible fiber networks

NASA Astrophysics Data System (ADS)

Hatami-Marbini, H.; Shriyan, V.

2017-12-01

Filamentous semiflexible networks define the mechanical and physical properties of many materials such as cytoskeleton. In the absence of a distinct unit cell, the Mikado fiber network model is commonly used algorithm for representing the microstructure of these networks in numerical models. Nevertheless, certain types of filamentous structures such as collagenous tissues, at early stages of their development, are assembled by growth of individual fibers from random nucleation sites. In this work, we develop a computational model to investigate the mechanical response of such networks by characterizing their nonaffine behavior. We show that the deformation of these networks is nonaffine at all length scales. Furthermore, similar to Mikado networks, the degree of nonaffinity in these structures decreases with increasing the probing length scale, the network fiber density, and/or the bending stiffness of constituting filaments. Nevertheless, despite the lower coordination number of these networks, their deformation field is more affine than that of the Mikado networks with the same fiber density and fiber mechanical properties.

Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change.

PubMed

Hetrick, Byron; Han, Min Suk; Helgeson, Luke A; Nolen, Brad J

2013-05-23

Actin-related protein 2/3 (Arp2/3) complex is a seven-subunit assembly that nucleates branched actin filaments. Small molecule inhibitors CK-666 and CK-869 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here, we use biochemical and structural methods to determine the mechanism of each inhibitor. Our data indicate that CK-666 stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short pitch) conformation, while CK-869 binds to a serendipitous pocket on Arp3 and allosterically destabilizes the short pitch Arp3-Arp2 interface. These results provide key insights into the relationship between conformation and activity in Arp2/3 complex and will be critical for interpreting the influence of the inhibitors on actin filament networks in vivo. Copyright © 2013 Elsevier Ltd. All rights reserved.

Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia

PubMed Central

Goldman, Robert D.; Louvard, Daniel

2010-01-01

Invasive cancer cells are believed to breach the basement membrane (BM) using specialized protrusions called invadopodia. We found that the crossing of a native BM is a three-stage process: invadopodia indeed form and perforate the BM, elongate into mature invadopodia, and then guide the cell toward the stromal compartment. We studied the remodeling of cytoskeleton networks during invadopodia formation and elongation using ultrastructural analysis, spatial distribution of molecular markers, and RNA interference silencing of protein expression. We show that formation of invadopodia requires only the actin cytoskeleton and filopodia- and lamellipodia-associated proteins. In contrast, elongation of invadopodia is mostly dependent on filopodial actin machinery. Moreover, intact microtubules and vimentin intermediate filament networks are required for further growth. We propose that invadopodia form by assembly of dendritic/diagonal and bundled actin networks and then mature by elongation of actin bundles, followed by the entry of microtubules and vimentin filaments. These findings provide a link between the epithelial to mesenchymal transition and BM transmigration. PMID:20421424

Nano-assembly of nanodiamonds by conjugation to actin filaments.

PubMed

Bradac, Carlo; Say, Jana M; Rastogi, Ishan D; Cordina, Nicole M; Volz, Thomas; Brown, Louise J

2016-03-01

Fluorescent nanodiamonds (NDs) are remarkable objects. They possess unique mechanical and optical properties combined with high surface areas and controllable surface reactivity. They are non-toxic and hence suited for use in biological environments. NDs are also readily available and commercially inexpensive. Here, the exceptional capability of controlling and tailoring their surface chemistry is demonstrated. Small, bright diamond nanocrystals (size ˜30 nm) are conjugated to protein filaments of actin (length ˜3-7 µm). The conjugation to actin filaments is extremely selective and highly target-specific. These unique features, together with the relative simplicity of the conjugation-targeting method, make functionalised nanodiamonds a powerful and versatile platform in biomedicine and quantum nanotechnologies. Applications ranging from using NDs as superior biological markers to, potentially, developing novel bottom-up approaches for the fabrication of hybrid quantum devices that would bridge across the bio/solid-state interface are presented and discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

THE KELVIN-HELMHOLTZ INSTABILITY AT CORONAL MASS EJECTION BOUNDARIES IN THE SOLAR CORONA: OBSERVATIONS AND 2.5D MHD SIMULATIONS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moestl, U. V.; Temmer, M.; Veronig, A. M., E-mail: ute.moestl@uni-graz.at

2013-03-20

The Atmospheric Imaging Assembly on board the Solar Dynamics Observatory observed a coronal mass ejection with an embedded filament on 2011 February 24, revealing quasi-periodic vortex-like structures at the northern side of the filament boundary with a wavelength of approximately 14.4 Mm and a propagation speed of about 310 {+-} 20 km s{sup -1}. These structures could result from the Kelvin-Helmholtz instability occurring on the boundary. We perform 2.5D numerical simulations of the Kelvin-Helmholtz instability and compare the simulated characteristic properties of the instability with the observations, where we obtain qualitative as well as quantitative accordance. We study the absencemore » of Kelvin-Helmholtz vortex-like structures on the southern side of the filament boundary and find that a magnetic field component parallel to the boundary with a strength of about 20% of the total magnetic field has stabilizing effects resulting in an asymmetric development of the instability.« less

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy.

PubMed

Loquet, Antoine; Tolchard, James; Berbon, Melanie; Martinez, Denis; Habenstein, Birgit

2017-09-17

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13 C/ 15 N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Astro-E2 Magnesium Diboride High Current Leads

NASA Technical Reports Server (NTRS)

Panek, J. S.; Tuttle, J. G.; Riall, S.; Mustafi, S.; Gray, A.; Edmonds, R.; Marrero, V.

2003-01-01

The recent discovery of superconducting properties in MgB_2 and rapid development of small diameter steel-clad wires has opened up the possibility of enhancing the design of the baseline Astro-E2 high current lead assembly. Replacing YBCO filaments with MgB_2 wires and modifying the heat sink location can give much higher margins against quench from temperature oscillations of the 4 K heat sink, although wih some overall thermal penalty. The design and performance of a new lead assembly during flight qualification is discussed, with emphasis on thermal, structural, and electrical test results.

De novo genome assembly of Geosmithia morbida, the causal agent of thousand cankers disease

Treesearch

Taruna A. Schuelke; Anthony Westbrook; Kirk Broders; Keith Woeste; Matthew D. MacManes

2016-01-01

Geosmithia morbida is a filamentous ascomycete that causes thousand cankers disease in the eastern black walnut tree. This pathogen is commonly found in the western U.S.; however, recently the disease was also detected in several eastern states where the black walnut lumber industry is concentrated. G. morbida is one of two...

ER-PM Contacts Define Actomyosin Kinetics for Proper Contractile Ring Assembly.

PubMed

Zhang, Dan; Bidone, Tamara C; Vavylonis, Dimitrios

2016-03-07

The cortical endoplasmic reticulum (ER), an elaborate network of tubules and cisternae [1], establishes contact sites with the plasma membrane (PM) through tethering machinery involving a set of conserved integral ER proteins [2]. The physiological consequences of forming ER-PM contacts are not fully understood. Here, we reveal a kinetic restriction role of ER-PM contacts over ring compaction process for proper actomyosin ring assembly in Schizosaccharomyces pombe. We show that fission yeast cells deficient in ER-PM contacts exhibit aberrant equatorial clustering of actin cables during ring assembly and are particularly susceptible to compromised actin filament crosslinking activity. Using quantitative image analyses and computer simulation, we demonstrate that ER-PM contacts function to modulate the distribution of ring components and to constrain their compaction kinetics. We propose that ER-PM contacts have evolved as important physical modulators to ensure robust ring assembly. Copyright © 2016 Elsevier Ltd. All rights reserved.

An analysis of FtsZ assembly using small angle X-ray scattering and electron microscopy.

PubMed

Kuchibhatla, Anuradha; Abdul Rasheed, A S; Narayanan, Janaky; Bellare, Jayesh; Panda, Dulal

2009-04-09

Small angle X-ray scattering (SAXS) was used for the first time to study the self-assembly of the bacterial cell division protein, FtsZ, with three different additives: calcium chloride, monosodium glutamate and DEAE-dextran hydrochloride in solution. The SAXS data were analyzed assuming a model form factor and also by a model-independent analysis using the pair distance distribution function. Transmission electron microscopy (TEM) was used for direct observation of the FtsZ filaments. By sectioning and negative staining with glow discharged grids, very high bundling as well as low bundling polymers were observed under different assembly conditions. FtsZ polymers formed different structures in the presence of different additives and these additives were found to increase the bundling of FtsZ protofilaments by different mechanisms. The combined use of SAXS and TEM provided us a significant insight of the assembly of FtsZ and microstructures of the assembled FtsZ polymers.

Structure and activity of full-length formin mDia1

PubMed Central

Maiti, Sankar; Michelot, Alphee; Gould, Christopher; Blanchoin, Laurent; Sokolova, Olga; Goode, Bruce L.

2012-01-01

Formins are a conserved family of actin assembly-promoting factors with essential and diverse biological roles. Most of our biochemical understanding of formin effects on actin dynamics is derived from studies using formin fragments. In addition, all structural information on formins has been limited to fragments. This has left open key questions about the structure, activity and regulation of intact formin proteins. Here, we isolated full-length mouse mDia1 (mDia1-FL) and found that it forms tightly autoinhibited dimers that can only be partially activated by RhoA. We solved the structure of autoinhibited mDia1-FL using electron microscopy and single particle analysis. Docking of crystal structures into the 3D reconstruction revealed that the fork-shaped N-terminal DID-CC region hangs over the ring-shaped FH2 domain, suggesting that autoinhibition results from steric obstruction of actin binding. Deletion of the C-terminal DAD domain extended mDia1 structure and activated it for actin assembly. Using TIRF microscopy, we observed that RhoA-activated mDia1-FL persistently accelerated filament elongation in the presence of profilin similar to mDia1 FH1-FH2 fragment. These observations validate the known activities of FH1-FH2 fragments as reflecting those of the intact molecule. Our results further suggest that mDia1-FL does not readily snap back into the autoinhibited conformation and dissociate from growing filament ends, and thus additional factors may be required to displace formins and restrict filament length. PMID:22605659

Amino Acid Substitutions of Coiled-Coil Protein Tpr Abrogate Anchorage to the Nuclear Pore Complex but Not Parallel, In-Register Homodimerization

PubMed Central

Hase, Manuela E.; Kuznetsov, Nikolai V.; Cordes, Volker C.

2001-01-01

Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil–forming proteins. Proposed functions for Tpr have included roles as a homo- or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have studied recombinant Tpr segments by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We show that polypeptides of the N-terminal domain homodimerize in vitro and represent α-helical molecules of extended rod-like shape. With the use of a yeast two-hybrid approach, arrangement of the coiled-coil is found to be in parallel and in register. To clarify whether Tpr can self-assemble further into homopolymeric filaments, the full-length protein and deletion mutants were overexpressed in human cells and then analyzed by confocal immunofluorescence microscopy, cell fractionation, and immuno-electron microscopy. Surplus Tpr, which does not bind to the NPC, remains in a soluble state of ∼7.5 S and occasionally forms aggregates of entangled molecules but neither self-assembles into extended linear filaments nor stably binds to other intranuclear structures. Binding to the NPC is shown to depend on the integrity of individual HRs; amino acid substitutions within these HRs abrogate NPC binding and render the protein soluble but do not abolish Tpr's general ability to homodimerize. Possible contributions of Tpr to the structural organization of the nuclear periphery in somatic cells are discussed. PMID:11514627

Effects of polymerization and nucleotide identity on the conformational dynamics of the bacterial actin homolog MreB

PubMed Central

Colavin, Alexandre; Hsin, Jen; Huang, Kerwyn Casey

2014-01-01

The assembly of protein filaments drives many cellular processes, from nucleoid segregation, growth, and division in single cells to muscle contraction in animals. In eukaryotes, shape and motility are regulated through cycles of polymerization and depolymerization of actin cytoskeletal networks. In bacteria, the actin homolog MreB forms filaments that coordinate the cell-wall synthesis machinery to regulate rod-shaped growth and contribute to cellular stiffness through unknown mechanisms. Like actin, MreB is an ATPase and requires ATP to polymerize, and polymerization promotes nucleotide hydrolysis. However, it is unclear whether other similarities exist between MreB and actin because the two proteins share low sequence identity and have distinct cellular roles. Here, we use all-atom molecular dynamics simulations to reveal surprising parallels between MreB and actin structural dynamics. We observe that MreB exhibits actin-like polymerization-dependent structural changes, wherein polymerization induces flattening of MreB subunits, which restructures the nucleotide-binding pocket to favor hydrolysis. MreB filaments exhibited nucleotide-dependent intersubunit bending, with hydrolyzed polymers favoring a straighter conformation. We use steered simulations to demonstrate a coupling between intersubunit bending and the degree of flattening of each subunit, suggesting cooperative bending along a filament. Taken together, our results provide molecular-scale insight into the diversity of structural states of MreB and the relationships among polymerization, hydrolysis, and filament properties, which may be applicable to other members of the broad actin family. PMID:24550504

Effects of polymerization and nucleotide identity on the conformational dynamics of the bacterial actin homolog MreB.

PubMed

Colavin, Alexandre; Hsin, Jen; Huang, Kerwyn Casey

2014-03-04

The assembly of protein filaments drives many cellular processes, from nucleoid segregation, growth, and division in single cells to muscle contraction in animals. In eukaryotes, shape and motility are regulated through cycles of polymerization and depolymerization of actin cytoskeletal networks. In bacteria, the actin homolog MreB forms filaments that coordinate the cell-wall synthesis machinery to regulate rod-shaped growth and contribute to cellular stiffness through unknown mechanisms. Like actin, MreB is an ATPase and requires ATP to polymerize, and polymerization promotes nucleotide hydrolysis. However, it is unclear whether other similarities exist between MreB and actin because the two proteins share low sequence identity and have distinct cellular roles. Here, we use all-atom molecular dynamics simulations to reveal surprising parallels between MreB and actin structural dynamics. We observe that MreB exhibits actin-like polymerization-dependent structural changes, wherein polymerization induces flattening of MreB subunits, which restructures the nucleotide-binding pocket to favor hydrolysis. MreB filaments exhibited nucleotide-dependent intersubunit bending, with hydrolyzed polymers favoring a straighter conformation. We use steered simulations to demonstrate a coupling between intersubunit bending and the degree of flattening of each subunit, suggesting cooperative bending along a filament. Taken together, our results provide molecular-scale insight into the diversity of structural states of MreB and the relationships among polymerization, hydrolysis, and filament properties, which may be applicable to other members of the broad actin family.

The detection of the imprint of filaments on cosmic microwave background lensing

NASA Astrophysics Data System (ADS)

He, Siyu; Alam, Shadab; Ferraro, Simone; Chen, Yen-Chi; Ho, Shirley

2018-05-01

Galaxy redshift surveys, such as the 2-Degree-Field Survey (2dF)1, Sloan Digital Sky Survey (SDSS)2, 6-Degree-Field Survey (6dF)3, Galaxy And Mass Assembly survey (GAMA)4 and VIMOS Public Extragalactic Redshift Survey (VIPERS)5, have shown that the spatial distribution of matter forms a rich web, known as the cosmic web6. Most galaxy survey analyses measure the amplitude of galaxy clustering as a function of scale, ignoring information beyond a small number of summary statistics. Because the matter density field becomes highly non-Gaussian as structure evolves under gravity, we expect other statistical descriptions of the field to provide us with additional information. One way to study the non-Gaussianity is to study filaments, which evolve non-linearly from the initial density fluctuations produced in the primordial Universe. In our study, we report the detection of lensing of the cosmic microwave background (CMB) by filaments, and we apply a null test to confirm our detection. Furthermore, we propose a phenomenological model to interpret the detected signal, and we measure how filaments trace the matter distribution on large scales through filament bias, which we measure to be around 1.5. Our study provides new scope to understand the environmental dependence of galaxy formation. In the future, the joint analysis of lensing and Sunyaev-Zel'dovich observations might reveal the properties of `missing baryons', the vast majority of the gas that resides in the intergalactic medium, which has so far evaded most observations.

Interaction of the Coronavirus Infectious Bronchitis Virus Membrane Protein with β-Actin and Its Implication in Virion Assembly and Budding

PubMed Central

Wang, Jibin; Fang, Shouguo; Xiao, Han; Chen, Bo; Tam, James P.; Liu, Ding Xiang

2009-01-01

Coronavirus M protein is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation. The M protein is integrated into the viral envelope with three transmembrane domains flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. In this study, we showed co-purification of the M protein from coronavirus infectious bronchitis virus (IBV) with actin. To understand the cellular factors that may be involved in virion assembly, budding and maturation processes, IBV M was used as the bait in a yeast two-hybrid screen, resulting in the identification of β-actin as a potentially interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescence microscopy in mammalian cells, and mutation of amino acids A159 and K160 on the M protein abolished the interaction. Introduction of the A159-K160 mutation into an infectious IBV clone system blocks the infectivity of the clone, although viral RNA replication and subgenomic mRNA transcription were actively detected. Disruption of actin filaments with cell-permeable agent cytochalasin D at early stages of the infection cycle led to the detection of viral protein synthesis in infected cells but not release of virus particles to the cultured media. However, the same treatment at late stages of the infection cycle did not affect the release of virus particles to the media, suggesting that disruption of the actin filaments might block virion assembly and budding, but not release of the virus particles. This study reveals an essential function of actin in the replication cycle of coronavirus. PMID:19287488

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage.

PubMed

Ploss, Martin; Kuhn, Andreas

2011-09-26

Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

PubMed Central

2011-01-01

Background Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. Results The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Conclusions Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies. PMID:21943062

Self-assembly of hexahistidine-tagged tobacco etch virus capsid protein into microfilaments that induce IgG2-specific response against a soluble porcine reproductive and respiratory syndrome virus chimeric protein.

PubMed

Manuel-Cabrera, Carlos Alberto; Vallejo-Cardona, Alba Adriana; Padilla-Camberos, Eduardo; Hernández-Gutiérrez, Rodolfo; Herrera-Rodríguez, Sara Elisa; Gutiérrez-Ortega, Abel

2016-11-29

Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-μm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.

Live-cell imaging of G-actin dynamics using sequential FDAP

PubMed Central

Kiuchi, Tai; Nagai, Tomoaki; Ohashi, Kazumasa; Watanabe, Naoki; Mizuno, Kensaku

2011-01-01

Various microscopic techniques have been developed to understand the mechanisms that spatiotemporally control actin filament dynamics in live cells. Kinetic data on the processes of actin assembly and disassembly on F-actin have been accumulated. However, the kinetics of cytoplasmic G-actin, a key determinant for actin polymerization, has remained unclear because of a lack of appropriate methods to measure the G-actin concentration quantitatively. We have developed two new microscopic techniques based on the fluorescence decay after photoactivation (FDAP) time-lapse imaging of photoswitchable Dronpa-labeled actin. These techniques, sequential FDAP (s-FDAP) and multipoint FDAP, were used to measure the time-dependent changes in and spatial distribution of the G-actin concentration in live cells. Use of s-FDAP provided data on changes in the G-actin concentration with high temporal resolution; these data were useful for the model analysis of actin assembly processes in live cells. The s-FDAP analysis also provided evidence that the cytoplasmic G-actin concentration substantially decreases after cell stimulation and that the extent of stimulus-induced actin assembly and cell size extension are linearly correlated with the G-actin concentration before cell stimulation. The advantages of using s-FDAP and multipoint FDAP to measure spatiotemporal G-actin dynamics and the roles of G-actin concentration and ADF/cofilin in stimulus-induced actin assembly and lamellipodium extension in live cells are discussed. PMID:22754616

Natural products from filamentous fungi and production by heterologous expression.

PubMed

Alberti, Fabrizio; Foster, Gary D; Bailey, Andy M

2017-01-01

Filamentous fungi represent an incredibly rich and rather overlooked reservoir of natural products, which often show potent bioactivity and find applications in different fields. Increasing the naturally low yields of bioactive metabolites within their host producers can be problematic, and yield improvement is further hampered by such fungi often being genetic intractable or having demanding culturing conditions. Additionally, total synthesis does not always represent a cost-effective approach for producing bioactive fungal-inspired metabolites, especially when pursuing assembly of compounds with complex chemistry. This review aims at providing insights into heterologous production of secondary metabolites from filamentous fungi, which has been established as a potent system for the biosynthesis of bioactive compounds. Numerous advantages are associated with this technique, such as the availability of tools that allow enhanced production yields and directing biosynthesis towards analogues of the naturally occurring metabolite. Furthermore, a choice of hosts is available for heterologous expression, going from model unicellular organisms to well-characterised filamentous fungi, which has also been shown to allow the study of biosynthesis of complex secondary metabolites. Looking to the future, fungi are likely to continue to play a substantial role as sources of new pharmaceuticals and agrochemicals-either as producers of novel natural products or indeed as platforms to generate new compounds through synthetic biology.

ANTI-PARALLEL EUV FLOWS OBSERVED ALONG ACTIVE REGION FILAMENT THREADS WITH HI-C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alexander, Caroline E.; Walsh, Robert W.; Régnier, Stéphane

Plasma flows within prominences/filaments have been observed for many years and hold valuable clues concerning the mass and energy balance within these structures. Previous observations of these flows primarily come from Hα and cool extreme-ultraviolet (EUV) lines (e.g., 304 Å) where estimates of the size of the prominence threads has been limited by the resolution of the available instrumentation. Evidence of 'counter-steaming' flows has previously been inferred from these cool plasma observations, but now, for the first time, these flows have been directly imaged along fundamental filament threads within the million degree corona (at 193 Å). In this work, wemore » present observations of an AR filament observed with the High-resolution Coronal Imager (Hi-C) that exhibits anti-parallel flows along adjacent filament threads. Complementary data from the Solar Dynamics Observatory (SDO)/Atmospheric Imaging Assembly (AIA) and Helioseismic and Magnetic Imager are presented. The ultra-high spatial and temporal resolution of Hi-C allow the anti-parallel flow velocities to be measured (70-80 km s{sup –1}) and gives an indication of the resolvable thickness of the individual strands (0.''8 ± 0.''1). The temperature of the plasma flows was estimated to be log T (K) = 5.45 ± 0.10 using Emission Measure loci analysis. We find that SDO/AIA cannot clearly observe these anti-parallel flows or measure their velocity or thread width due to its larger pixel size. We suggest that anti-parallel/counter-streaming flows are likely commonplace within all filaments and are currently not observed in EUV due to current instrument spatial resolution.« less

Kinetics of spontaneous filament nucleation via oligomers: Insights from theory and simulation

NASA Astrophysics Data System (ADS)

Šarić, Andela; Michaels, Thomas C. T.; Zaccone, Alessio; Knowles, Tuomas P. J.; Frenkel, Daan

2016-12-01

Nucleation processes are at the heart of a large number of phenomena, from cloud formation to protein crystallization. A recently emerging area where nucleation is highly relevant is the initiation of filamentous protein self-assembly, a process that has broad implications in many research areas ranging from medicine to nanotechnology. As such, spontaneous nucleation of protein fibrils has received much attention in recent years with many theoretical and experimental studies focussing on the underlying physical principles. In this paper we make a step forward in this direction and explore the early time behaviour of filamentous protein growth in the context of nucleation theory. We first provide an overview of the thermodynamics and kinetics of spontaneous nucleation of protein filaments in the presence of one relevant degree of freedom, namely the cluster size. In this case, we review how key kinetic observables, such as the reaction order of spontaneous nucleation, are directly related to the physical size of the critical nucleus. We then focus on the increasingly prominent case of filament nucleation that includes a conformational conversion of the nucleating building-block as an additional slow step in the nucleation process. Using computer simulations, we study the concentration dependence of the nucleation rate. We find that, under these circumstances, the reaction order of spontaneous nucleation with respect to the free monomer does no longer relate to the overall physical size of the nucleating aggregate but rather to the portion of the aggregate that actively participates in the conformational conversion. Our results thus provide a novel interpretation of the common kinetic descriptors of protein filament formation, including the reaction order of the nucleation step or the scaling exponent of lag times, and put into perspective current theoretical descriptions of protein aggregation.

Kinetics of spontaneous filament nucleation via oligomers: Insights from theory and simulation.

PubMed

Šarić, Anđela; Michaels, Thomas C T; Zaccone, Alessio; Knowles, Tuomas P J; Frenkel, Daan

2016-12-07

Nucleation processes are at the heart of a large number of phenomena, from cloud formation to protein crystallization. A recently emerging area where nucleation is highly relevant is the initiation of filamentous protein self-assembly, a process that has broad implications in many research areas ranging from medicine to nanotechnology. As such, spontaneous nucleation of protein fibrils has received much attention in recent years with many theoretical and experimental studies focussing on the underlying physical principles. In this paper we make a step forward in this direction and explore the early time behaviour of filamentous protein growth in the context of nucleation theory. We first provide an overview of the thermodynamics and kinetics of spontaneous nucleation of protein filaments in the presence of one relevant degree of freedom, namely the cluster size. In this case, we review how key kinetic observables, such as the reaction order of spontaneous nucleation, are directly related to the physical size of the critical nucleus. We then focus on the increasingly prominent case of filament nucleation that includes a conformational conversion of the nucleating building-block as an additional slow step in the nucleation process. Using computer simulations, we study the concentration dependence of the nucleation rate. We find that, under these circumstances, the reaction order of spontaneous nucleation with respect to the free monomer does no longer relate to the overall physical size of the nucleating aggregate but rather to the portion of the aggregate that actively participates in the conformational conversion. Our results thus provide a novel interpretation of the common kinetic descriptors of protein filament formation, including the reaction order of the nucleation step or the scaling exponent of lag times, and put into perspective current theoretical descriptions of protein aggregation.

The step-wise pathway of septin hetero-octamer assembly in budding yeast.

PubMed

Weems, Andrew; McMurray, Michael

2017-05-25

Septin proteins bind guanine nucleotides and form rod-shaped hetero-oligomers. Cells choose from a variety of available septins to assemble distinct hetero-oligomers, but the underlying mechanism was unknown. Using a new in vivo assay, we find that a stepwise assembly pathway produces the two species of budding yeast septin hetero-octamers: Cdc11/Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11/Shs1. Rapid GTP hydrolysis by monomeric Cdc10 drives assembly of the core Cdc10 homodimer. The extended Cdc3 N terminus autoinhibits Cdc3 association with Cdc10 homodimers until prior Cdc3-Cdc12 interaction. Slow hydrolysis by monomeric Cdc12 and specific affinity of Cdc11 for transient Cdc12•GTP drive assembly of distinct trimers, Cdc11-Cdc12-Cdc3 or Shs1-Cdc12-Cdc3. Decreasing the cytosolic GTP:GDP ratio increases the incorporation of Shs1 vs Cdc11, which alters the curvature of filamentous septin rings. Our findings explain how GTP hydrolysis controls septin assembly, and uncover mechanisms by which cells construct defined septin complexes.

Draft Genome Sequence of Tolypothrix boutellei Strain VB521301

PubMed Central

Chandrababunaidu, Mathu Malar; Singh, Deeksha; Sen, Diya; Bhan, Sushma; Das, Subhadeep; Gupta, Akash

2015-01-01

We report here the draft genome sequence of the filamentous nitrogen-fixing cyanobacterium Tolypothrix boutellei strain VB521301. The organism is lipid rich and hydrophobic and produces polyunsaturated fatty acids which can be harnessed for industrial purpose. The draft genome sequence assembled into 11,572,263 bp with 70 scaffolds and 7,777 protein coding genes. PMID:25700407

Embedded sensor having an identifiable orientation

DOEpatents

Bennett, Thomas E.; Nelson, Drew V.

2002-01-01

An apparatus and method is described wherein a sensor, such as a mechanical strain sensor, embedded in a fiber core, is "flagged" to identify a preferred orientation of the sensor. The identifying "flag" is a composite material, comprising a plurality of non-woven filaments distributed in a resin matrix, forming a small planar tab. The fiber is first subjected to a stimulus to identify the orientation providing the desired signal response, and then sandwiched between first and second layers of the composite material. The fiber, and therefore, the sensor orientation is thereby captured and fixed in place. The process for achieving the oriented fiber includes, after identifying the fiber orientation, carefully laying the oriented fiber onto the first layer of composite, moderately heating the assembled layer for a short period in order to bring the composite resin to a "tacky" state, heating the second composite layer as the first, and assembling the two layers together such that they merge to form a single consolidated block. The consolidated block achieving a roughly uniform distribution of composite filaments near the embedded fiber such that excess resin is prevented from "pooling" around the periphery of the fiber.

A growing family: the expanding universe of the bacterial cytoskeleton

PubMed Central

Ingerson-Mahar, Michael; Gitai, Zemer

2014-01-01

Cytoskeletal proteins are important mediators of cellular organization in both eukaryotes and bacteria. In the past, cytoskeletal studies have largely focused on three major cytoskeletal families, namely the eukaryotic actin, tubulin, and intermediate filament (IF) proteins and their bacterial homologs MreB, FtsZ, and crescentin. However, mounting evidence suggests that these proteins represent only the tip of the iceberg, as the cellular cytoskeletal network is far more complex. In bacteria, each of MreB, FtsZ, and crescentin represents only one member of large families of diverse homologs. There are also newly identified bacterial cytoskeletal proteins with no eukaryotic homologs, such as WACA proteins and bactofilins. Furthermore, there are universally conserved proteins, such as the metabolic enzyme CtpS, that assemble into filamentous structures that can be repurposed for structural cytoskeletal functions. Recent studies have also identified an increasing number of eukaryotic cytoskeletal proteins that are unrelated to actin, tubulin, and IFs, such that expanding our understanding of cytoskeletal proteins is advancing the understanding of the cell biology of all organisms. Here, we summarize the recent explosion in the identification of new members of the bacterial cytoskeleton and describe a hypothesis for the evolution of the cytoskeleton from self-assembling enzymes. PMID:22092065

Eukaryotic and Prokaryotic Cytoskeletons: Structure and Mechanics

NASA Astrophysics Data System (ADS)

Gopinathan, Ajay

2013-03-01

The eukaryotic cytoskeleton is an assembly of filamentous proteins and a host of associated proteins that collectively serve functional needs ranging from spatial organization and transport to the production and transmission of forces. These systems can exhibit a wide variety of non-equilibrium, self-assembled phases depending on context and function. While much recent progress has been made in understanding the self-organization, rheology and nonlinear mechanical properties of such active systems, in this talk, we will concentrate on some emerging aspects of cytoskeletal physics that are promising. One such aspect is the influence of cytoskeletal network topology and its dynamics on both active and passive intracellular transport. Another aspect we will highlight is the interplay between chirality of filaments, their elasticity and their interactions with the membrane that can lead to novel conformational states with functional implications. Finally we will consider homologs of cytoskeletal proteins in bacteria, which are involved in templating cell growth, segregating genetic material and force production, which we will discuss with particular reference to contractile forces during cell division. These prokaryotic structures function in remarkably similar yet fascinatingly different ways from their eukaryotic counterparts and can enrich our understanding of cytoskeletal functioning as a whole.

An ultra-fast EOD-based force-clamp detects rapid biomechanical transitions

NASA Astrophysics Data System (ADS)

Woody, Michael S.; Capitanio, Marco; Ostap, E. Michael; Goldman, Yale E.

2017-08-01

We assembled an ultra-fast infrared optical trapping system to detect mechanical events that occur less than a millisecond after a ligand binds to its filamentous substrate, such as myosin undergoing its 5 - 10 nm working stroke after actin binding. The instrument is based on the concept of Capitanio et al.1, in which a polymer bead-actin-bead dumbbell is held in two force-clamped optical traps. A force applied by the traps causes the filament to move at a constant velocity as hydrodynamic drag balances the applied load. When the ligand binds, the filament motion stops within 100 μs as the total force from the optical traps is transferred to the attachment. Subsequent translations signal active motions, such as the magnitude and timing of the motor's working stroke. In our instrument, the beads defining the dumbbell are held in independent force clamps utilizing a field-programmable gate array (FPGA) to update the trap beam positions at 250 kHz. We found that in our setup, acousto-optical deflectors (AODs) steering the beams were unsuitable for this purpose due to a slightly non-linear response in the beam intensity and deflection angle vs. the AOD ultra-sound wavelength, likely caused by low-amplitude standing acoustic waves in the deflectors. These aberrations caused instability in the force feedback loops leading to artefactual 20 nm jumps in position. This type of AOD non-linearity has been reported to be absent in electro-optical deflectors (EODs)2. We demonstrate that replacement of the AODs with EODs improves the performance of our instrument. Combining the superior beam-steering capability of the EODs, force acquisition via back-plane interferometry, and the dual high-speed FPGA-based feedback loops, we smoothly and precisely apply constant loads to study the dynamics of interactions between biological molecules such as actin and myosin.

Fast Two-Dimensional Bubble Analysis of Biopolymer Filamentous Networks Pore Size from Confocal Microscopy Thin Data Stacks

PubMed Central

Molteni, Matteo; Magatti, Davide; Cardinali, Barbara; Rocco, Mattia; Ferri, Fabio

2013-01-01

The average pore size ξ0 of filamentous networks assembled from biological macromolecules is one of the most important physical parameters affecting their biological functions. Modern optical methods, such as confocal microscopy, can noninvasively image such networks, but extracting a quantitative estimate of ξ0 is a nontrivial task. We present here a fast and simple method based on a two-dimensional bubble approach, which works by analyzing one by one the (thresholded) images of a series of three-dimensional thin data stacks. No skeletonization or reconstruction of the full geometry of the entire network is required. The method was validated by using many isotropic in silico generated networks of different structures, morphologies, and concentrations. For each type of network, the method provides accurate estimates (a few percent) of the average and the standard deviation of the three-dimensional distribution of the pore sizes, defined as the diameters of the largest spheres that can be fit into the pore zones of the entire gel volume. When applied to the analysis of real confocal microscopy images taken on fibrin gels, the method provides an estimate of ξ0 consistent with results from elastic light scattering data. PMID:23473499

Adhesion by pathogenic corynebacteria.

PubMed

Rogers, Elizabeth A; Das, Asis; Ton-That, Hung

2011-01-01

Pathogenic members of the genus Corynebacterium cause a wide range of serious infections in humans including diphtheria. Adhesion to host cells is a crucial step during infection. In Corynebacterium diphtheriae, adhesion is mediated primarily by filamentous structures called pili or fimbriae that are covalently attached to the bacterial cell wall. C. diphtheriae produces three distinct pilus structures, SpaA-, SpaD- and SpaH-type pili. Similar to other types, the prototype SpaA pilus consists of SpaA forming the pilus shaft and two minor pilins SpaB and SpaC located at the base and at the tip, respectively. The minor pilins SpaB/SpaC are critical for bacterial binding to human pharyngeal cells, and thus represent the major adhesins of corynebacteria. Like pili of many other gram-positive microbes, the assembly of corynebacterial pili occurs by a two-step mechanism, whereby pilins are covalently polymerized by a transpeptidase enzyme named pilin-specific sortase and the generated pilus polymer is subsequently anchored to the cell wall peptidoglycan via the base pilin by the housekeeping sortase or a non-polymerizing sortase. This chapter reviews the current knowledge of corynebacterial adhesion, with a specific focus on pilus structures, their assembly, and the mechanism of adhesion mediated by pili.

Diagnosis of femtosecond plasma filament by channeling microwaves along the filament

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alshershby, Mostafa; Ren, Yu; Qin, Jiang

2013-05-20

We introduce a simple, fast, and non-intrusive experimental method to obtain the basic parameters of femtosecond laser-generated plasma filament. The method is based on the channeling of microwaves along both a plasma filament and a well-defined conducting wire. By comparing the detected microwaves that propagate along the plasma filament and a copper wire with known conductivity and spatial dimension, the basic parameters of the plasma filament can be easily obtained. As a result of the possibility of channeling microwave radiation along the plasma filament, we were then able to obtain the plasma density distribution along the filament length.

Selective self-assembly of adenine-silver nanoparticles forms rings resembling the size of cells

NASA Astrophysics Data System (ADS)

Choi, Sungmoon; Park, Soonyoung; Yang, Seon-Ah; Jeong, Yujin; Yu, Junhua

2015-12-01

Self-assembly has played critical roles in the construction of functional nanomaterials. However, the structure of the macroscale multicomponent materials built by the self-assembly of nanoscale building blocks is hard to predict due to multiple intermolecular interactions of great complexity. Evaporation of solvents is usually an important approach to induce kinetically stable assemblies of building blocks with a large-scale specific arrangement. During such a deweting process, we tried to monitor the possible interactions between silver nanoparticles and nucleobases at a larger scale by epifluorescence microscopy, thanks to the doping of silver nanoparticles with luminescent silver nanodots. ssDNA oligomer-stabilized silver nanoparticles and adenine self-assemble to form ring-like compartments similar to the size of modern cells. However, the silver ions only dismantle the self-assembly of adenine. The rings are thermodynamically stable as the drying process only enrich the nanoparticles-nucleobase mixture to a concentration that activates the self-assembly. The permeable membrane-like edge of the ring is composed of adenine filaments glued together by silver nanoparticles. Interestingly, chemicals are partially confined and accumulated inside the ring, suggesting that this might be used as a microreactor to speed up chemical reactions during a dewetting process.

Functional Supramolecular Polymers*

PubMed Central

Aida, T.; Meijer, E.W.; Stupp, S.I.

2012-01-01

Supramolecular polymers can be random and entangled coils with the mechanical properties of plastics and elastomers, but with great capacity for processability, recycling, and self-healing due to their reversible monomer-to-polymer transitions. At the other extreme, supramolecular polymers can be formed by self-assembly among designed subunits to yield shape-persistent and highly ordered filaments. The use of strong and directional interactions among molecular subunits can achieve not only rich dynamic behavior but also high degrees of internal order that are not known in ordinary polymers. They can resemble, for example, the ordered and dynamic one-dimensional supramolecular assemblies of the cell cytoskeleton, and possess useful biological and electronic functions. PMID:22344437

Probing actin polymerization by intermolecular cross-linking.

PubMed

Millonig, R; Salvo, H; Aebi, U

1988-03-01

We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant beta-actin (Leavitt, J., G. Bushar, T. Kakunaga, H. Hamada, T. Hirakawa, D. Goldman, and C. Merril. 1982. Cell. 28:259-268) is arrested under polymerizing conditions.

A dynamic formin-dependent deep F-actin network in axons

PubMed Central

Ganguly, Archan; Tang, Yong; Wang, Lina; Ladt, Kelsey; Loi, Jonathan; Dargent, Bénédicte; Leterrier, Christophe

2015-01-01

Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles. PMID:26216902

Bacterial actin MreB forms antiparallel double filaments

PubMed Central

van den Ent, Fusinita; Izoré, Thierry; Bharat, Tanmay AM; Johnson, Christopher M; Löwe, Jan

2014-01-01

Filaments of all actin-like proteins known to date are assembled from pairs of protofilaments that are arranged in a parallel fashion, generating polarity. In this study, we show that the prokaryotic actin homologue MreB forms pairs of protofilaments that adopt an antiparallel arrangement in vitro and in vivo. We provide an atomic view of antiparallel protofilaments of Caulobacter MreB as apparent from crystal structures. We show that a protofilament doublet is essential for MreB's function in cell shape maintenance and demonstrate by in vivo site-specific cross-linking the antiparallel orientation of MreB protofilaments in E. coli. 3D cryo-EM shows that pairs of protofilaments of Caulobacter MreB tightly bind to membranes. Crystal structures of different nucleotide and polymerisation states of Caulobacter MreB reveal conserved conformational changes accompanying antiparallel filament formation. Finally, the antimicrobial agents A22/MP265 are shown to bind close to the bound nucleotide of MreB, presumably preventing nucleotide hydrolysis and destabilising double protofilaments. DOI: http://dx.doi.org/10.7554/eLife.02634.001 PMID:24843005

Bacterial actin MreB forms antiparallel double filaments.

PubMed

van den Ent, Fusinita; Izoré, Thierry; Bharat, Tanmay Am; Johnson, Christopher M; Löwe, Jan

2014-05-02

Filaments of all actin-like proteins known to date are assembled from pairs of protofilaments that are arranged in a parallel fashion, generating polarity. In this study, we show that the prokaryotic actin homologue MreB forms pairs of protofilaments that adopt an antiparallel arrangement in vitro and in vivo. We provide an atomic view of antiparallel protofilaments of Caulobacter MreB as apparent from crystal structures. We show that a protofilament doublet is essential for MreB's function in cell shape maintenance and demonstrate by in vivo site-specific cross-linking the antiparallel orientation of MreB protofilaments in E. coli. 3D cryo-EM shows that pairs of protofilaments of Caulobacter MreB tightly bind to membranes. Crystal structures of different nucleotide and polymerisation states of Caulobacter MreB reveal conserved conformational changes accompanying antiparallel filament formation. Finally, the antimicrobial agents A22/MP265 are shown to bind close to the bound nucleotide of MreB, presumably preventing nucleotide hydrolysis and destabilising double protofilaments.DOI: http://dx.doi.org/10.7554/eLife.02634.001. Copyright © 2014, van den Ent et al.

Filamentous, mixed micelles of triblock copolymers enhance tumor localization of indocyanine green in a murine xenograft model

PubMed Central

Kim, Tae Hee; Mount, Christopher W; Dulken, Benjamin W; Ramos, Jenelyn; Fu, Caroline J; Khant, Htet A; Chiu, Wah; Gombotz, Wayne R; Pun, Suzie H

2012-01-01

Polymeric micelles formed by the self-assembly of amphiphilic block copolymers can be used to encapsulate hydrophobic drugs for tumor-delivery applications. Filamentous carriers with high aspect ratios offer potential advantages over spherical carriers, including prolonged circulation times. In this work, mixed micelles comprised of poly (ethylene oxide)-poly-[(R)-3-hydroxybutyrate]-poly (ethylene oxide) (PEO-PHB-PEO) and Pluronic F-127 (PF-127) were used to encapsulate a near-infrared fluorophore. The micelle formulations were assessed for tumor accumulation after tail vein injection to xenograft tumor-bearing mice by non-invasive optical imaging. The mixed micelle formulation that facilitated the highest tumor accumulation was shown by cryo-electron microscopy to be filamentous in structure compared to spherical structures of pure PF-127 micelles. In addition, increased dye loading efficiency and dye stability was attained in this mixed micelle formulation compared to pure PEO-PHB-PEO micelles. Therefore, the optimized PEO-PHB-PEO/PF-127 mixed micelle formulation offers advantages for cancer delivery over micelles formed from the individual copolymer components. PMID:22118658

Signatures of a macroscopic switching transition for a dynamic microtubule

NASA Astrophysics Data System (ADS)

Aparna, J. S.; Padinhateeri, Ranjith; Das, Dibyendu

2017-04-01

Characterising complex kinetics of non-equilibrium self-assembly of bio-filaments is of general interest. Dynamic instability in microtubules, consisting of successive catastrophes and rescues, is observed to occur as a result of the non-equilibrium conversion of GTP-tubulin to GDP-tubulin. We study this phenomenon using a model for microtubule kinetics with GTP/GDP state-dependent polymerisation, depolymerisation and hydrolysis of subunits. Our results reveal a sharp switch-like transition in the mean velocity of the filaments, from a growth phase to a shrinkage phase, with an associated co-existence of the two phases. This transition is reminiscent of the discontinuous phase transition across the liquid-gas boundary. We probe the extent of discontinuity in the transition quantitatively using characteristic signatures such as bimodality in velocity distribution, variance and Binder cumulant, and also hysteresis behaviour of the system. We further investigate ageing behaviour in catastrophes of the filament, and find that the multi-step nature of catastrophes is intensified in the vicinity of the switching transition. This assumes importance in the context of Microtubule Associated Proteins which have the potential of altering kinetic parameter values.

Mechanism of synergistic activation of Arp2/3 complex by cortactin and N-WASP

PubMed Central

Helgeson, Luke A; Nolen, Brad J

2013-01-01

Nucleation promoting factors (NPFs) initiate branched actin network assembly by activating Arp2/3 complex, a branched actin filament nucleator. Cellular actin networks contain multiple NPFs, but how they coordinately regulate Arp2/3 complex is unclear. Cortactin is an NPF that activates Arp2/3 complex weakly on its own, but with WASP/N-WASP, another class of NPFs, potently activates. We dissect the mechanism of synergy and propose a model in which cortactin displaces N-WASP from nascent branches as a prerequisite for nucleation. Single-molecule imaging revealed that unlike WASP/N-WASP, cortactin remains bound to junctions during nucleation, and specifically targets junctions with a ∼160-fold increased on rate over filament sides. N-WASP must be dimerized for potent synergy, and targeted mutations indicate release of dimeric N-WASP from nascent branches limits nucleation. Mathematical modeling shows cortactin-mediated displacement but not N-WASP recycling or filament recruitment models can explain synergy. Our results provide a molecular basis for coordinate Arp2/3 complex regulation. DOI: http://dx.doi.org/10.7554/eLife.00884.001 PMID:24015358

Exciton antennas and concentrators from core-shell and corrugated carbon nanotube filaments of homogeneous composition.

PubMed

Han, Jae-Hee; Paulus, Geraldine L C; Maruyama, Ryuichiro; Heller, Daniel A; Kim, Woo-Jae; Barone, Paul W; Lee, Chang Young; Choi, Jong Hyun; Ham, Moon-Ho; Song, Changsik; Fantini, C; Strano, Michael S

2010-10-01

There has been renewed interest in solar concentrators and optical antennas for improvements in photovoltaic energy harvesting and new optoelectronic devices. In this work, we dielectrophoretically assemble single-walled carbon nanotubes (SWNTs) of homogeneous composition into aligned filaments that can exchange excitation energy, concentrating it to the centre of core-shell structures with radial gradients in the optical bandgap. We find an unusually sharp, reversible decay in photoemission that occurs as such filaments are cycled from ambient temperature to only 357 K, attributed to the strongly temperature-dependent second-order Auger process. Core-shell structures consisting of annular shells of mostly (6,5) SWNTs (E(g)=1.21 eV) and cores with bandgaps smaller than those of the shell (E(g)=1.17 eV (7,5)-0.98 eV (8,7)) demonstrate the concentration concept: broadband absorption in the ultraviolet-near-infrared wavelength regime provides quasi-singular photoemission at the (8,7) SWNTs. This approach demonstrates the potential of specifically designed collections of nanotubes to manipulate and concentrate excitons in unique ways.

Polymeric Nanoelectrodes for Investigating Cellular Adhesion

NASA Astrophysics Data System (ADS)

Thapa, Prem; Paneru, Govind; Flanders, Bret

2011-03-01

Polyethylene dioxythiophene nano-filaments were grown on lithographic electrode arrays by the recently developed directed electrochemical nanowire assembly technique. These filaments are firmly attached to the electrode but are not attached to the glass substrate. Hence, they behave like cantilevered rods (with one free end). Individual cells of the slime mold Dictystolium discoideum initiate contact by extending pseudopods to the nanoelectrodes when cultured on the electrode arrays. Scanning electron micrographs of the interfaces show the contact area to be of the order of 0.1 μ m 2 . Confocal images reveal the focal adhesions in the cell-electrode contact region. Deflection of the nanoelectrode by an individual cell can be used to measure the force exerted by the cell. Recent results on this innovative force sensing approach will be discussed. NSF.

Enabling a Community to Dissect an Organism: Overview of the Neurospora Functional Genomics Project

PubMed Central

Dunlap, Jay C.; Borkovich, Katherine A.; Henn, Matthew R.; Turner, Gloria E.; Sachs, Matthew S.; Glass, N. Louise; McCluskey, Kevin; Plamann, Michael; Galagan, James E.; Birren, Bruce W.; Weiss, Richard L.; Townsend, Jeffrey P.; Loros, Jennifer J.; Nelson, Mary Anne; Lambreghts, Randy; Colot, Hildur V.; Park, Gyungsoon; Collopy, Patrick; Ringelberg, Carol; Crew, Christopher; Litvinkova, Liubov; DeCaprio, Dave; Hood, Heather M.; Curilla, Susan; Shi, Mi; Crawford, Matthew; Koerhsen, Michael; Montgomery, Phil; Larson, Lisa; Pearson, Matthew; Kasuga, Takao; Tian, Chaoguang; Baştürkmen, Meray; Altamirano, Lorena; Xu, Junhuan

2013-01-01

A consortium of investigators is engaged in a functional genomics project centered on the filamentous fungus Neurospora, with an eye to opening up the functional genomic analysis of all the filamentous fungi. The overall goal of the four interdependent projects in this effort is to acccomplish functional genomics, annotation, and expression analyses of Neurospora crassa, a filamentous fungus that is an established model for the assemblage of over 250,000 species of nonyeast fungi. Building from the completely sequenced 43-Mb Neurospora genome, Project 1 is pursuing the systematic disruption of genes through targeted gene replacements, phenotypic analysis of mutant strains, and their distribution to the scientific community at large. Project 2, through a primary focus in Annotation and Bioinformatics, has developed a platform for electronically capturing community feedback and data about the existing annotation, while building and maintaining a database to capture and display information about phenotypes. Oligonucleotide-based microarrays created in Project 3 are being used to collect baseline expression data for the nearly 11,000 distinguishable transcripts in Neurospora under various conditions of growth and development, and eventually to begin to analyze the global effects of loss of novel genes in strains created by Project 1. cDNA libraries generated in Project 4 document the overall complexity of expressed sequences in Neurospora, including alternative splicing alternative promoters and antisense transcripts. In addition, these studies have driven the assembly of an SNP map presently populated by nearly 300 markers that will greatly accelerate the positional cloning of genes. PMID:17352902

Flux Cancelation: The Key to Solar Eruptions

NASA Technical Reports Server (NTRS)

Panesar, Navdeep K.; Sterling, Alphonse; Moore, Ronald; Chakrapani, Prithi; Innes, Davina; Schmit, Don; Tiwari, Sanjiv

2017-01-01

Solar coronal jets are magnetically channeled eruptions that occur in all types of solar environments (e.g. active regions, quiet-Sun regions and coronal holes). Recent studies show that coronal jets are driven by the eruption of small-scare filaments (minifilaments). Once the eruption is underway magnetic reconnection evidently makes the jet spire and the bright emission in the jet base. However, the triggering mechanism of these eruptions and the formation mechanism of the pre-jet minifilaments are still open questions. In this talk, mainly using SDO/AIA (Solar Dynamics Observatory / Atmospheric Imaging Assembly) and SDO/HIM (Solar Dynamics Observatory / Helioseismic and Magnetic Imager) data, first I will address the question: what triggers the jet-driving minifilament eruptions in different solar environments (coronal holes, quiet regions, active regions)? Then I will talk about the magnetic field evolution that produces the pre-jet minifilaments. By examining pre-jet evolutionary changes in line-of-sight HMI magnetograms while examining concurrent EUV (Extreme Ultra-Violet) images of coronal and transition-region emission, we find clear evidence that flux cancelation is the main process that builds pre-jet minifilaments, and is also the main process that triggers the eruptions. I will also present results from our ongoing work indicating that jet-driving minifilament eruptions are analogous to larger-scare filament eruptions that make flares and CMEs (Coronal Mass Ejections). We find that persistent flux cancellation at the neutral line of large-scale filaments often triggers their eruptions. From our observations we infer that flux cancelation is the fundamental process from the buildup and triggering of solar eruptions of all sizes.

Microparticle based morphology engineering of filamentous microorganisms for industrial bio-production.

PubMed

Walisko, Robert; Krull, Rainer; Schrader, Jens; Wittmann, Christoph

2012-11-01

Filamentous microorganisms are important work horses in industrial biotechnology and supply enzymes, antibiotics, pharmaceuticals, bulk and fine chemicals. Here we highlight recent findings on the use of microparticles in the cultivation of filamentous bacteria and fungi, with the aim of enabling a more precise control of their morphology towards better production performance. First examples reveal a broad application range of microparticle based processes, since multiple filamentous organisms are controllable in their growth characteristics and respond by enhanced product formation.

Draft Genome Sequence of Tolypothrix boutellei Strain VB521301.

PubMed

Chandrababunaidu, Mathu Malar; Singh, Deeksha; Sen, Diya; Bhan, Sushma; Das, Subhadeep; Gupta, Akash; Adhikary, Siba Prasad; Tripathy, Sucheta

2015-02-19

We report here the draft genome sequence of the filamentous nitrogen-fixing cyanobacterium Tolypothrix boutellei strain VB521301. The organism is lipid rich and hydrophobic and produces polyunsaturated fatty acids which can be harnessed for industrial purpose. The draft genome sequence assembled into 11,572,263 bp with 70 scaffolds and 7,777 protein coding genes. Copyright © 2015 Chandrababunaidu et al.

A new design of indirectly heated cathode based strip type electron gun.

PubMed

Maiti, Namita; Lijeesh, K; Barve, U D; Quadri, Nishad; Tembhare, G U; Mukherjee, S; Thakur, K B; Das, A K

2013-08-01

A new design of indirectly heated solid cathode based electron gun (200 kW, 45 kV, 270° bent strip type electron gun) has been presented. The design issue addressed is the uniformity of temperature on the solid cathode using (a) a multi-segmented filament with variable height as the primary heat source and (b) trapezoidal shaped single long filament as the primary heat source. The proposed design in this paper is based on computer simulation and validated by extensive experimentations. The design emphasis is on maintaining uniform temperature on the solid cathode. The designed multi-segment filament and the single long filament provide a temperature uniformity on the solid cathode of about 250 K and 110 K, respectively. The better temperature uniformity inspite of the thermal expansion, in case of a single long filament tightly clamped at two ends, has been possible due to shaping of the single filament with a number of constituent sections such that the thermal expansion of different sections forming the actual filament takes care of not only the mechanical stability but also does not affect the emitting surface of the filament. Experiments show that the modified design achieves a one to one correspondence of the solid cathode length and the electron beam length emitted from the solid cathode.

Large-amplitude Longitudinal Oscillations Triggered by the Merging of Two Solar Filaments: Observations and Magnetic Field Analysis

NASA Astrophysics Data System (ADS)

Luna, M.; Su, Y.; Schmieder, B.; Chandra, R.; Kucera, T. A.

2017-12-01

We follow the eruption of two related intermediate filaments observed in Hα (from GONG) and EUV (from Solar Dynamics Observatory SDO/Atmospheric Imaging assembly AIA) and the resulting large-amplitude longitudinal oscillations of the plasma in the filament channels. The events occurred in and around the decayed active region AR12486 on 2016 January 26. Our detailed study of the oscillation reveals that the periods of the oscillations are about one hour. In Hα, the period decreases with time and exhibits strong damping. The analysis of 171 Å images shows that the oscillation has two phases: an initial long-period phase and a subsequent oscillation with a shorter period. In this wavelength, the damping appears weaker than in Hα. The velocity is the largest ever detected in a prominence oscillation, approximately 100 {km} {{{s}}}-1. Using SDO/HMI magnetograms, we reconstruct the magnetic field of the filaments, modeled as flux ropes by using a flux-rope insertion method. Applying seismological techniques, we determine that the radii of curvature of the field lines in which cool plasma is condensed are in the range 75-120 Mm, in agreement with the reconstructed field. In addition, we infer a field strength of ≥7 to 30 Gauss, depending on the electron density assumed, that is also in agreement with the values from the reconstruction (8-20 Gauss). The poloidal flux is zero and the axis flux is on the order of 1020 to 1021 Mx, confirming the high shear existing even in a non-active filament.

Effects of Plectin Depletion on Keratin Network Dynamics and Organization

PubMed Central

Moch, Marcin; Windoffer, Reinhard; Schwarz, Nicole; Pohl, Raphaela; Omenzetter, Andreas; Schnakenberg, Uwe; Herb, Fabian; Chaisaowong, Kraisorn; Merhof, Dorit; Ramms, Lena; Fabris, Gloria; Hoffmann, Bernd; Merkel, Rudolf; Leube, Rudolf E.

2016-01-01

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells. PMID:27007410

Aspergillus nidulans ArfB Plays a Role in Endocytosis and Polarized Growth ▿ †

PubMed Central

Lee, Soo Chan; Schmidtke, Sabrina N.; Dangott, Lawrence J.; Shaw, Brian D.

2008-01-01

Filamentous fungi undergo polarized growth throughout most of their life cycles. The Spitzenkörper is an apical organelle composed primarily of vesicles that is unique to filamentous fungi and is likely to act as a vesicle supply center for tip growth. Vesicle assembly and trafficking are therefore important for hyphal growth. ADP ribosylation factors (Arfs), a group of small GTPase proteins, play an important role in nucleating vesicle assembly. Little is known about the role of Arfs in filamentous hyphal growth. We found that Aspergillus nidulans is predicted to encode six Arf family proteins. Analysis of protein sequence alignments suggests that A. nidulans ArfB shares similarity with ARF6 of Homo sapiens and Arf3p of Saccharomyces cerevisiae. An arfB null allele (arfB disrupted by a transposon [arfB::Tn]) was characterized by extended isotropic growth of germinating conidia followed by cell lysis or multiple, random germ tube emergence, consistent with a failure to establish polarity. The mutant germ tubes and hyphae that do form initially meander abnormally off of the axis of polarity and frequently exhibit dichotomous branching at cell apices, consistent with a defect in polarity maintenance. FM4-64 staining of the arfB::Tn strain revealed that another phenotypic characteristic seen for arfB::Tn is a reduction and delay in endocytosis. ArfB is myristoylated at its N terminus. Green fluorescent protein-tagged ArfB (ArfB::GFP) localizes to the plasma membrane and endomembranes and mutation (ArfBG2A::GFP) of the N-terminal myristoylation motif disperses the protein to the cytoplasm rather than to the membranes. These results demonstrate that ArfB functions in endocytosis to play important roles in polarity establishment during isotropic growth and polarity maintenance during hyphal extension. PMID:18539885

Development of Self-Assembled Nanoscale Templates via Microphase Separation Induced by Polymer Brushes

NASA Astrophysics Data System (ADS)

Chu, Elza

Phase separation in soft matter has been the crucial element in generating hybrid materials, such as polymer blends and mixed polymer brushes. This dissertation discusses two methods of developing self-assembled nanoscale templates via microphase separation induced by polymer brush synthesis. This work introduces a novel soft substrate approach with renewable grafting sites where polyacrylamide is "grafted through" chitosan soft substrates. The mechanism of grafting leads to ordered arrays of filament-like nanostructures spanning the chitosan-air interface. Additionally, the chemical composition of the filaments allows for post-chemical modification to change the physical properties of the filaments, and subsequently tailor surfaces for specific application. Unlike traditional materials, multi-functional or "smart" materials, such as binary polymer brushes (BPB) are capable of spontaneously changing the spatial distribution of functional groups and morphology at the surface upon external stimuli. Although promising in principle, the limited range of available complementary polymers with common non-selective solvents confines the diversity of usable materials and restricts any further advancement in the field. This dissertation also covers the fabrication and characterization of responsive nanoscale polystyrene templates or "mosaic" brushes that are capable of changing interfacial composition upon exposure to varying solvent qualities. Using a "mosaic" brush template is a unique approach that allows the fabrication of strongly immiscible polymer BPB without the need for a common solvent. The synthesis of such BPB is exemplified by two strongly immiscible polymers, i.e. polystyrene (polar) and polyacrylamide (non-polar), where polyacrylamide brush is "graft through" a Si-substrate modified with the polystyrene collapsed "mosaic" brush. The surface exhibits solvent-triggered responses, as well as application potential for anti-biofouling.

Self-assembly of amphiphilic molecules in organic liquids

NASA Astrophysics Data System (ADS)

Tung, Shih-Huang

2007-12-01

Amphiphilic molecules are well-known for their ability to self-assemble in water to form structures such as micelles and vesicles. In comparison, much less is known about amphiphilic self-assembly in nonpolar organic liquids. Such "reverse" self assembly can produce many of the counterparts to structures found in water. In this dissertation, we focus on the formation and dynamics of such reverse structures. We seek to obtain fundamental insight into the driving forces for reverse self-assembly processes. Three specific types of reverse structures are studied: (a) reverse wormlike micelles, i.e., long, flexible micellar chains; (b) reverse vesicles, i.e., hollow containers enclosed by reverse bilayers; and (c) organogel networks. While our focus is on the fundamentals, we note that reverse structures can be useful in a variety of applications ranging from drug delivery, controlled release, hosts for enzymatic reactions, and templates for nanomaterials synthesis. In the first part of this study, we describe a new route for forming reverse wormlike micelles in nonpolar organic liquids. This route involves the addition of trace amounts of a bile salt to solutions of the phospholipid, lecithin. We show that bile salts, due to their unique "facially amphiphilic" structure, can promote the aggregation of lecithin molecules into these reverse micellar chains. The resulting samples are viscoelastic and show interesting rheological properties. Unusual trends are seen in the temperature dependence of their rheology, which indicates the importance of hydrogen-bonding interactions in the formation of these micelles. Another remarkable feature of their rheology is the presence of strain-stiffening, where the material becomes stiffer at high deformations. Strain-stiffening has been seen before for elastic gels of biopolymers; here, we demonstrate the same properties for viscoelastic micellar solutions. The second reverse aggregate we deal with is the reverse vesicle. We present a new route for forming stable unilamellar reverse vesicles, and this involves mixing short- and long-chain lipids (lecithins) with a trace of sodium chloride. The ratio of the short to long-chain lipid controls the type and size of self-assembled structure formed, and as this ratio is increased, a transition from reverse micelles to vesicles occurs. The structural changes can be explained in terms of molecular geometry, with the sodium chloride acting as a "glue" in binding lipid headgroups together through electrostatic interactions. The final part of this dissertation focuses on organogels. The two-tailed anionic surfactant, AOT, is well-known to form spherical reverse micelles in organic solvents. We have found that trace amounts (e.g., less than 1 mM) of the dihydroxy bile salt, sodium deoxycholate (SDC) can transform these dilute micellar solutions into self-supporting, transparent organogels. The structure and rheology of these organogels is reminiscent of the self-assembled networks formed by proteins such as actin in water. The organogels are based on networks of long, rigid, cylindrical filaments, with SDC molecules stacked together in the filament core.

A common 'aggregation-prone' interface possibly participates in the self-assembly of human zona pellucida proteins.

PubMed

Louros, Nikolaos N; Chrysina, Evangelia D; Baltatzis, Georgios E; Patsouris, Efstratios S; Hamodrakas, Stavros J; Iconomidou, Vassiliki A

2016-03-01

Human zona pellucida (ZP) is composed of four glycoproteins, namely ZP1, ZP2, ZP3 and ZP4. ZP proteins form heterodimers, which are incorporated into filaments through a common bipartite polymerizing component, designated as the ZP domain. The latter is composed of two individually folded subdomains, named ZP-N and ZP-C. Here, we have synthesized six 'aggregation-prone' peptides, corresponding to a common interface of human ZP2, ZP3 and ZP4. Experimental results utilizing electron microscopy, X-ray diffraction, ATR FT-IR spectroscopy and polarizing microscopy indicate that these peptides self-assemble forming fibrils with distinct amyloid-like features. Finally, by performing detailed modeling and docking, we attempt to shed some light in the self-assembly mechanism of human ZP proteins. © 2016 Federation of European Biochemical Societies.

First genomic insights into members of a candidate bacterial phylum responsible for wastewater bulking

PubMed Central

Ohashi, Akiko; Parks, Donovan H.; Yamauchi, Toshihiro; Tyson, Gene W.

2015-01-01

Filamentous cells belonging to the candidate bacterial phylum KSB3 were previously identified as the causative agent of fatal filament overgrowth (bulking) in a high-rate industrial anaerobic wastewater treatment bioreactor. Here, we obtained near complete genomes from two KSB3 populations in the bioreactor, including the dominant bulking filament, using differential coverage binning of metagenomic data. Fluorescence in situ hybridization with 16S rRNA-targeted probes specific for the two populations confirmed that both are filamentous organisms. Genome-based metabolic reconstruction and microscopic observation of the KSB3 filaments in the presence of sugar gradients indicate that both filament types are Gram-negative, strictly anaerobic fermenters capable of non-flagellar based gliding motility, and have a strikingly large number of sensory and response regulator genes. We propose that the KSB3 filaments are highly sensitive to their surroundings and that cellular processes, including those causing bulking, are controlled by external stimuli. The obtained genomes lay the foundation for a more detailed understanding of environmental cues used by KSB3 filaments, which may lead to more robust treatment options to prevent bulking. PMID:25650158

Visualization of bacterial flagella dynamics in a viscous shear flow

NASA Astrophysics Data System (ADS)

Ali, Jamel; Kim, Minjun

2016-11-01

We report on the dynamics of tethered bacterial flagella in an applied viscous shear flow and analyze their behavior using image processing. Flagellin proteins were repolymerized into flagellar filaments functionalized with biotin at their proximal end, and allowed to self-assemble within a micro channel coated with streptavidin. It was observed that all attached flagellar filaments aligned with the steady shear flow of various polymeric solutions. Furthermore it was observed that many of the filaments were stretched, and at elevated flow rates began to undergo polymorphic transformations, which were initiated at one end of the flagellum. When undergoing a change to a different helical form the flagellum was observed to transform to an oppositely handed helix, as to counteract the viscous torque imparted by the shear flow. It was also observed that some flagellar filaments did not undergo polymorphic transformations, but rotated about their helical axis. The rate of this rotation appears to be a function of the applied flow rate. These results expand on previous experimental work and aid in the development of a novel platform that harnesses the autonomic response of a 'forest' of bacterial flagella for engineering applications. This work was funded by NSF Grant CMMI-1000255, KEIT MOTIE Grant No. 10052980, and with Government support under and awarded by DoD, Air Force Office of Scientific Research, National Defense Science and Engineering Graduate (NDSEG) Fellowship, 32 CFR 168a.

Active turnover regulates pattern formation and stress transmission in disordered acto-myosin networks

NASA Astrophysics Data System (ADS)

McCall, Patrick; Stam, Samantha; Kovar, David; Gardel, Margaret

The shape and mechanics of animal cells are controlled by a dynamic, thin network of semiflexible actin filaments and myosin-II motor proteins called the actomyosin cortex. Motor-generated stresses in the cortex drive changes in cell shape during cell division and morphogenesis, while dynamic turnover of actin filaments dissipates stress. The relative effects that force generation, force dissipation, and disassembly and reassembly of material have on motion in these networks are unknown. We find that cross-linked actin networks in vitro contract under myosin-generated stresses, resulting in partial filament disassembly, the formation of asters, and clustering of myosin motors. We observe a rapid restoration of uniform polymer density in the presence of the assembly factors which catalyze network turnover through elongation of severed actin filaments. When severing is accelerated further by the addition of a severing protein, network contraction and motor clustering are dramatically suppressed. We test the relative effects of material regeneration and force transmission using image analysis, and conclude that the dominant mechanism for this effect is relatively short-lived stresses that do not propagate over considerable distance or push network deformation into the nonlinear contractile regime we have previously characterized. Our results present a framework to understand cytoskeletal active matter that are influenced by a complex interplay between stress generation, network reorganization, and polymer turnover.

A structured interdomain linker directs self-polymerization of human uromodulin

PubMed Central

Bokhove, Marcel; Nishimura, Kaoru; Brunati, Martina; Han, Ling; de Sanctis, Daniele; Rampoldi, Luca

2016-01-01

Uromodulin (UMOD)/Tamm–Horsfall protein, the most abundant human urinary protein, plays a key role in chronic kidney diseases and is a promising therapeutic target for hypertension. Via its bipartite zona pellucida module (ZP-N/ZP-C), UMOD forms extracellular filaments that regulate kidney electrolyte balance and innate immunity, as well as protect against renal stones. Moreover, salt-dependent aggregation of UMOD filaments in the urine generates a soluble molecular net that captures uropathogenic bacteria and facilitates their clearance. Despite the functional importance of its homopolymers, no structural information is available on UMOD and how it self-assembles into filaments. Here, we report the crystal structures of polymerization regions of human UMOD and mouse ZP2, an essential sperm receptor protein that is structurally related to UMOD but forms heteropolymers. The structure of UMOD reveals that an extensive hydrophobic interface mediates ZP-N domain homodimerization. This arrangement is required for filament formation and is directed by an ordered ZP-N/ZP-C linker that is not observed in ZP2 but is conserved in the sequence of deafness/Crohn’s disease-associated homopolymeric glycoproteins α-tectorin (TECTA) and glycoprotein 2 (GP2). Our data provide an example of how interdomain linker plasticity can modulate the function of structurally similar multidomain proteins. Moreover, the architecture of UMOD rationalizes numerous pathogenic mutations in both UMOD and TECTA genes. PMID:26811476

A novel modeling to predict the critical current behavior of Nb3Sn PIT strand under transverse load based on a scaling law and Finite Element Analysis

NASA Astrophysics Data System (ADS)

Wang, Tiening; Chiesa, Luisa; Takayasu, Makoto; Bordini, Bernardo

2014-09-01

Superconducting Nb3Sn Powder-In-Tube (PIT) strands could be used for the superconducting magnets of the next generation Large Hadron Collider. The strands are cabled into the typical flat Rutherford cable configuration. During the assembly of a magnet and its operation the strands experience not only longitudinal but also transverse load due to the pre-compression applied during the assembly and the Lorentz load felt when the magnets are energized. To properly design the magnets and guarantee their safe operation, mechanical load effects on the strand superconducting properties are studied extensively; particularly, many scaling laws based on tensile load experiments have been established to predict the critical current dependence on strain. However, the dependence of the superconducting properties on transverse load has not been extensively studied so far. One of the reasons is that transverse loading experiments are difficult to conduct due to the small diameter of the strand (about 1 mm) and the data currently available do not follow a common measurement standard making the comparison between different data sets difficult. Recently at the University of Geneva, a new device has been developed to characterize the critical current of Nb3Sn strands under transverse loads. In this work we present a new 2D Finite Element Analysis (FEA) to predict the electro-mechanical response of a PIT strand that was tested at the University of Geneva when transverse load is applied. The FEA provides the strain map for the superconducting filaments when the load is applied. Those strain maps are then used to evaluate the critical current behavior of a PIT strand using a recently developed scaling law that correlates the superconducting properties of a wire with the strain invariants due to the load applied on the superconductor. The benefits and limitations of this method are discussed based on the comparison between the critical current simulation results obtained with the filament strain map and the experimental results available for PIT strands.

Altering lamina assembly reveals lamina-dependent and -independent functions for A-type lamins.

PubMed

Zwerger, Monika; Roschitzki-Voser, Heidi; Zbinden, Reto; Denais, Celine; Herrmann, Harald; Lammerding, Jan; Grütter, Markus G; Medalia, Ohad

2015-10-01

Lamins are intermediate filament proteins that form a fibrous meshwork, called the nuclear lamina, between the inner nuclear membrane and peripheral heterochromatin of metazoan cells. The assembly and incorporation of lamin A/C into the lamina, as well as their various functions, are still not well understood. Here, we employed designed ankyrin repeat proteins (DARPins) as new experimental tools for lamin research. We screened for DARPins that specifically bound to lamin A/C, and interfered with lamin assembly in vitro and with incorporation of lamin A/C into the native lamina in living cells. The selected DARPins inhibited lamin assembly and delocalized A-type lamins to the nucleoplasm without modifying lamin expression levels or the amino acid sequence. Using these lamin binders, we demonstrate the importance of proper integration of lamin A/C into the lamina for nuclear mechanical properties and nuclear envelope integrity. Finally, our study provides evidence for cell-type-specific differences in lamin functions. © 2015. Published by The Company of Biologists Ltd.

Altering lamina assembly reveals lamina-dependent and -independent functions for A-type lamins

PubMed Central

Zwerger, Monika; Roschitzki-Voser, Heidi; Zbinden, Reto; Denais, Celine; Herrmann, Harald; Lammerding, Jan; Grütter, Markus G.; Medalia, Ohad

2015-01-01

ABSTRACT Lamins are intermediate filament proteins that form a fibrous meshwork, called the nuclear lamina, between the inner nuclear membrane and peripheral heterochromatin of metazoan cells. The assembly and incorporation of lamin A/C into the lamina, as well as their various functions, are still not well understood. Here, we employed designed ankyrin repeat proteins (DARPins) as new experimental tools for lamin research. We screened for DARPins that specifically bound to lamin A/C, and interfered with lamin assembly in vitro and with incorporation of lamin A/C into the native lamina in living cells. The selected DARPins inhibited lamin assembly and delocalized A-type lamins to the nucleoplasm without modifying lamin expression levels or the amino acid sequence. Using these lamin binders, we demonstrate the importance of proper integration of lamin A/C into the lamina for nuclear mechanical properties and nuclear envelope integrity. Finally, our study provides evidence for cell-type-specific differences in lamin functions. PMID:26275827

Immobile myosin-II plays a scaffolding role during cytokinesis in budding yeast

PubMed Central

Wloka, Carsten; Vallen, Elizabeth A.; Thé, Lydia; Fang, Xiaodong; Oh, Younghoon

2013-01-01

Core components of cytokinesis are conserved from yeast to human, but how these components are assembled into a robust machine that drives cytokinesis remains poorly understood. In this paper, we show by fluorescence recovery after photobleaching analysis that Myo1, the sole myosin-II in budding yeast, was mobile at the division site before anaphase and became immobilized shortly before cytokinesis. This immobility was independent of actin filaments or the motor domain of Myo1 but required a small region in the Myo1 tail that is thought to be involved in higher-order assembly. As expected, proteins involved in actin ring assembly (tropomyosin and formin) and membrane trafficking (myosin-V and exocyst) were dynamic during cytokinesis. Strikingly, proteins involved in septum formation (the chitin synthase Chs2) and/or its coordination with the actomyosin ring (essential light chain, IQGAP, F-BAR, etc.) displayed Myo1-dependent immobility during cytokinesis, suggesting that Myo1 plays a scaffolding role in the assembly of a cytokinesis machine. PMID:23358243

Force feedback controls motor activity and mechanical properties of self-assembling branched actin networks

PubMed Central

Bieling, Peter; Li, Tai-De; Weichsel, Julian; McGorty, Ryan; Jreij, Pamela; Huang, Bo; Fletcher, Daniel A.; Mullins, R. Dyche

2016-01-01

Branched actin networks–created by the Arp2/3 complex, capping protein, and a nucleation promoting factor– generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency. Microscopically, increased density reflects increased filament number and altered geometry, but no change in average length. Macroscopically, increased density enhances network stiffness and resistance to mechanical failure beyond those of isotropic actin networks. These effects endow branched actin networks with memory of their mechanical history that shapes their material properties and motor activity. This work reveals intrinsic force feedback mechanisms by which mechanical resistance makes self-assembling actin networks stiffer, stronger, and more powerful. PMID:26771487

Mapping Flexibility and the Assembly Switch of Cell Division Protein FtsZ by Computational and Mutational Approaches*♦

PubMed Central

Martín-Galiano, Antonio J.; Buey, Rubén M.; Cabezas, Marta; Andreu, José M.

2010-01-01

The molecular switch for nucleotide-regulated assembly and disassembly of the main prokaryotic cell division protein FtsZ is unknown despite the numerous crystal structures that are available. We have characterized the functional motions in FtsZ with a computational consensus of essential dynamics, structural comparisons, sequence conservation, and networks of co-evolving residues. Employing this information, we have constructed 17 mutants, which alter the FtsZ functional cycle at different stages, to modify FtsZ flexibility. The mutant phenotypes ranged from benign to total inactivation and included increased GTPase, reduced assembly, and stabilized assembly. Six mutations clustering at the long cleft between the C-terminal β-sheet and core helix H7 deviated FtsZ assembly into curved filaments with inhibited GTPase, which still polymerize cooperatively. These mutations may perturb the predicted closure of the C-terminal domain onto H7 required for switching between curved and straight association modes and for GTPase activation. By mapping the FtsZ assembly switch, this work also gives insight into FtsZ druggability because the curved mutations delineate the putative binding site of the promising antibacterial FtsZ inhibitor PC190723. PMID:20472561

Mechanisms Underlying the Active Self-Assembly of Microtubule Rings and Spools.

PubMed

VanDelinder, Virginia; Brener, Stephanie; Bachand, George D

2016-03-14

Active self-assembly offers a powerful route for the creation of dynamic multiscale structures that are presently inaccessible with standard microfabrication techniques. One such system uses the translation of microtubule filaments by surface-tethered kinesin to actively assemble nanocomposites with bundle, ring, and spool morphologies. Attempts to observe mechanisms involved in this active assembly system have been hampered by experimental difficulties with performing observation during buffer exchange and photodamage from fluorescent excitation. In the present work, we used a custom microfluidic device to remove these limitations and directly study ring/spool formation, including the earliest events (nucleation) that drive subsequent nanocomposite assembly. Three distinct formation events were observed: pinning, collisions, and induced curvature. Of these three, collisions accounted for the majority of event leading to ring/spool formation, while the rate of pinning was shown to be dependent on the amount of photodamage in the system. We further showed that formation mechanism directly affects the diameter and rotation direction of the resultant rings and spools. Overall, the fundamental understanding described in this work provides a foundation by which the properties of motor-driven, actively assembled nanocomposites may be tailored toward specific applications.

Mapping flexibility and the assembly switch of cell division protein FtsZ by computational and mutational approaches.

PubMed

Martín-Galiano, Antonio J; Buey, Rubén M; Cabezas, Marta; Andreu, José M

2010-07-16

The molecular switch for nucleotide-regulated assembly and disassembly of the main prokaryotic cell division protein FtsZ is unknown despite the numerous crystal structures that are available. We have characterized the functional motions in FtsZ with a computational consensus of essential dynamics, structural comparisons, sequence conservation, and networks of co-evolving residues. Employing this information, we have constructed 17 mutants, which alter the FtsZ functional cycle at different stages, to modify FtsZ flexibility. The mutant phenotypes ranged from benign to total inactivation and included increased GTPase, reduced assembly, and stabilized assembly. Six mutations clustering at the long cleft between the C-terminal beta-sheet and core helix H7 deviated FtsZ assembly into curved filaments with inhibited GTPase, which still polymerize cooperatively. These mutations may perturb the predicted closure of the C-terminal domain onto H7 required for switching between curved and straight association modes and for GTPase activation. By mapping the FtsZ assembly switch, this work also gives insight into FtsZ druggability because the curved mutations delineate the putative binding site of the promising antibacterial FtsZ inhibitor PC190723.

Mechanisms underlying the active self-assembly of microtubule rings and spools

DOE PAGES

VanDelinder, Virginia; Brener, Stephanie; Bachand, George D.

2016-02-04

Here, active self-assembly offers a powerful route for the creation of dynamic multiscale structures that are presently inaccessible with standard microfabrication techniques. One such system uses the translation of microtubule filaments by surface-tethered kinesin to actively assemble nanocomposites with bundle, ring, and spool morphologies. Attempts to observe mechanisms involved in this active assembly system have been hampered by experimental difficulties with performing observation during buffer exchange and photodamage from fluorescent excitation. In the present work, we used a custom microfluidic device to remove these limitations and directly study ring/spool formation, including the earliest events (nucleation) that drive subsequent nanocomposite assembly.more » Three distinct formation events were observed: pinning, collisions, and induced curvature. Of these three, collisions accounted for the majority of event leading to ring/spool formation, while the rate of pinning was shown to be dependent on the amount of photodamage in the system. We further showed that formation mechanism directly affects the diameter and rotation direction of the resultant rings and spools. Overall, the fundamental understanding described in this work provides a foundation by which the properties of motor-driven, actively assembled nanocomposites may be tailored toward specific applications.« less

Structure and function of the interacting domains of Spire and Fmn-family formins.

PubMed

Vizcarra, Christina L; Kreutz, Barry; Rodal, Avital A; Toms, Angela V; Lu, Jun; Zheng, Wei; Quinlan, Margot E; Eck, Michael J

2011-07-19

Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.

Simulation of the M13 life cycle I: Assembly of a genetically-structured deterministic chemical kinetic simulation.

PubMed

Smeal, Steven W; Schmitt, Margaret A; Pereira, Ronnie Rodrigues; Prasad, Ashok; Fisk, John D

2017-01-01

To expand the quantitative, systems level understanding and foster the expansion of the biotechnological applications of the filamentous bacteriophage M13, we have unified the accumulated quantitative information on M13 biology into a genetically-structured, experimentally-based computational simulation of the entire phage life cycle. The deterministic chemical kinetic simulation explicitly includes the molecular details of DNA replication, mRNA transcription, protein translation and particle assembly, as well as the competing protein-protein and protein-nucleic acid interactions that control the timing and extent of phage production. The simulation reproduces the holistic behavior of M13, closely matching experimentally reported values of the intracellular levels of phage species and the timing of events in the M13 life cycle. The computational model provides a quantitative description of phage biology, highlights gaps in the present understanding of M13, and offers a framework for exploring alternative mechanisms of regulation in the context of the complete M13 life cycle. Copyright © 2016 Elsevier Inc. All rights reserved.

Beyond taxol: microtubule-based treatment of disease and injury of the nervous system

PubMed Central

Ahmad, Fridoon J.

2013-01-01

Contemporary research has revealed a great deal of information on the behaviours of microtubules that underlie critical events in the lives of neurons. Microtubules in the neuron undergo dynamic assembly and disassembly, bundling and splaying, severing, and rapid transport as well as integration with other cytoskeletal elements such as actin filaments. These various behaviours are regulated by signalling pathways that affect microtubule-related proteins such as molecular motor proteins and microtubule severing enzymes, as well as a variety of proteins that promote the assembly, stabilization and bundling of microtubules. In recent years, translational neuroscientists have earmarked microtubules as a promising target for therapy of injury and disease of the nervous system. Proof-of-principle has come mainly from studies using taxol and related drugs to pharmacologically stabilize microtubules in animal models of nerve injury and disease. However, concerns persist that the negative consequences of abnormal microtubule stabilization may outweigh the positive effects. Other potential approaches include microtubule-active drugs with somewhat different properties, but also expanding the therapeutic toolkit to include intervention at the level of microtubule regulatory proteins. PMID:23811322

CHIRALITY AND MAGNETIC CONFIGURATIONS OF SOLAR FILAMENTS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ouyang, Y.; Zhou, Y. H.; Chen, P. F.

It has been revealed that the magnetic topology in the solar atmosphere displays hemispheric preference, i.e., helicity is mainly negative/positive in the northern/southern hemispheres, respectively. However, the strength of the hemispheric rule and its cyclic variation are controversial. In this paper, we apply a new method based on the filament drainage to 571 erupting filaments from 2010 May to 2015 December in order to determine the filament chirality and its hemispheric preference. It is found that 91.6% of our sample of erupting filaments follows the hemispheric rule of helicity sign. It is also found that the strength of the hemisphericmore » preference of the quiescent filaments decreases slightly from ∼97% in the rising phase to ∼85% in the declining phase of solar cycle 24, whereas the strength of the intermediate filaments keeps a high value around 96 ± 4% at all times. Only the active-region filaments show significant variations. Their strength of the hemispheric rule rises from ∼63% to ∼95% in the rising phase, and keeps a high value of 82% ± 5% during the declining phase. Furthermore, during a half-year period around the solar maximum, their hemispheric preference totally vanishes. Additionally, we also diagnose the magnetic configurations of the filaments based on our indirect method and find that in our sample of erupting events, 89% are inverse-polarity filaments with a flux rope magnetic configuration, whereas 11% are normal-polarity filaments with a sheared arcade configuration.« less

Chirality and Magnetic Configurations of Solar Filaments

NASA Astrophysics Data System (ADS)

Ouyang, Y.; Zhou, Y. H.; Chen, P. F.; Fang, C.

2017-01-01

It has been revealed that the magnetic topology in the solar atmosphere displays hemispheric preference, I.e., helicity is mainly negative/positive in the northern/southern hemispheres, respectively. However, the strength of the hemispheric rule and its cyclic variation are controversial. In this paper, we apply a new method based on the filament drainage to 571 erupting filaments from 2010 May to 2015 December in order to determine the filament chirality and its hemispheric preference. It is found that 91.6% of our sample of erupting filaments follows the hemispheric rule of helicity sign. It is also found that the strength of the hemispheric preference of the quiescent filaments decreases slightly from ˜97% in the rising phase to ˜85% in the declining phase of solar cycle 24, whereas the strength of the intermediate filaments keeps a high value around 96 ± 4% at all times. Only the active-region filaments show significant variations. Their strength of the hemispheric rule rises from ˜63% to ˜95% in the rising phase, and keeps a high value of 82% ± 5% during the declining phase. Furthermore, during a half-year period around the solar maximum, their hemispheric preference totally vanishes. Additionally, we also diagnose the magnetic configurations of the filaments based on our indirect method and find that in our sample of erupting events, 89% are inverse-polarity filaments with a flux rope magnetic configuration, whereas 11% are normal-polarity filaments with a sheared arcade configuration.

Additive Manufacturing of Syntactic Foams: Part 1: Development, Properties, and Recycling Potential of Filaments

NASA Astrophysics Data System (ADS)

Singh, Ashish Kumar; Patil, Balu; Hoffmann, Niklas; Saltonstall, Brooks; Doddamani, Mrityunjay; Gupta, Nikhil

2018-03-01

This work focuses on developing filaments of high-density polyethylene (HDPE) and their hollow particle-filled syntactic foams for commercial three-dimensional (3D) printers based on fused filament fabrication technology. Hollow fly-ash cenospheres were blended by 40 wt.% in a HDPE matrix to produce syntactic foam (HDPE40) filaments. Further, the recycling potential was studied by pelletizing the filaments again to extrude twice (2×) and three times (3×). The filaments were tensile tested at 10-4 s-1, 10-3 s-1, and 10-2 s-1 strain rates. HDPE40 filaments show an increasing trend in modulus and strength with the strain rate. Higher density and modulus were noticed for 2× filaments compared to 1× filaments because of the crushing of some cenospheres in the extrusion cycle. However, 2× and 3× filament densities are nearly the same, showing potential for recycling them. The filaments show better properties than the same materials processed by conventional injection molding. Micro-CT scans show a uniform dispersion of cenospheres in all filaments.

[Energy saving achieved by limited filamentous bulking under low dissolved oxygen: derivation, originality and theoretical basis].

PubMed

Peng, Yong-zhen; Guo, Jian-hua; Wang, Shu-ying; Chen, Ying

2008-12-01

How to prevent and control filamentous bulking sludge has being a research focus and attracted much attention. To date despite the extensive research that has been done on bulking sludge, filamentous bulking still occurs world-wide and a comprehensive solution does not seem to be available. Particularly, there are few studies about making use of the characteristics of filamentous bacteria and achieving energy saving by filamentous bulking. Limited filamentous bulking, a novel method for energy saving while equal or better treatment performance by allowing slight and controlled filamentous bulking sludge at low dissolved oxygen (DO), was proposed based on full-scale field observations. The practical operation showed that limited filamentous bulking resulted from a decline in DO concentration. COD, SS and TP removal could be enhanced and energy saving could be achieved by limited filamentous bulking at low DO. The derivation of limited filamentous bulking is introduced firstly, and then the theoretical fundamentals of the technique of limited filamentous bulking are presented and analyzed, including the occurrence of limited filamentous bulking caused by low DO, the enhanced effect of filamentous bacteria on pollutants removal and the energy saving mechanism by using limited filamentous bulking. Besides, the paper provides some new perspectives about the application and research direction of limited filamentous bulking in future.

Kinetic factors determining conducting filament formation in solid polymer electrolyte based planar devices

NASA Astrophysics Data System (ADS)

Krishnan, Karthik; Aono, Masakazu; Tsuruoka, Tohru

2016-07-01

Resistive switching characteristics and conducting filament formation dynamics in solid polymer electrolyte (SPE) based planar-type atomic switches, with opposing active Ag and inert Pt electrodes, have been investigated by optimizing the device configuration and experimental parameters such as the gap distance between the electrodes, the salt inclusion in the polymer matrix, and the compliance current applied in current-voltage measurements. The high ionic conductivities of SPE enabled us to make scanning electron microscopy observations of the filament formation processes in the sub-micrometer to micrometer ranges. It was found that switching behaviour and filament growth morphology depend strongly on several kinetic factors, such as the redox reaction rate at the electrode-polymer interfaces, ion mobility in the polymer matrix, electric field strength, and the reduction sites for precipitation. Different filament formations, resulting from unidirectional and dendritic growth behaviours, can be controlled by tuning specified parameters, which in turn improves the stability and performance of SPE-based devices.Resistive switching characteristics and conducting filament formation dynamics in solid polymer electrolyte (SPE) based planar-type atomic switches, with opposing active Ag and inert Pt electrodes, have been investigated by optimizing the device configuration and experimental parameters such as the gap distance between the electrodes, the salt inclusion in the polymer matrix, and the compliance current applied in current-voltage measurements. The high ionic conductivities of SPE enabled us to make scanning electron microscopy observations of the filament formation processes in the sub-micrometer to micrometer ranges. It was found that switching behaviour and filament growth morphology depend strongly on several kinetic factors, such as the redox reaction rate at the electrode-polymer interfaces, ion mobility in the polymer matrix, electric field strength, and the reduction sites for precipitation. Different filament formations, resulting from unidirectional and dendritic growth behaviours, can be controlled by tuning specified parameters, which in turn improves the stability and performance of SPE-based devices. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr00569a

Dynamics of myosin II organization into contractile networks and fibers at the medial cell cortex

NASA Astrophysics Data System (ADS)

Nie, Wei

The cellular morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of non-muscle myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin (which disrupts actomyosin stress fibers). Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared to studies by other groups. This analysis suggested the following processes: myosin minifilament assembly and disassembly; aligning and contraction; myosin filament stabilization upon increasing contractile tension. Numerical simulations that include those processes capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. We discuss methods to monitor myosin reorganization using non-linear imaging methods.

A growing family: the expanding universe of the bacterial cytoskeleton.

PubMed

Ingerson-Mahar, Michael; Gitai, Zemer

2012-01-01

Cytoskeletal proteins are important mediators of cellular organization in both eukaryotes and bacteria. In the past, cytoskeletal studies have largely focused on three major cytoskeletal families, namely the eukaryotic actin, tubulin, and intermediate filament (IF) proteins and their bacterial homologs MreB, FtsZ, and crescentin. However, mounting evidence suggests that these proteins represent only the tip of the iceberg, as the cellular cytoskeletal network is far more complex. In bacteria, each of MreB, FtsZ, and crescentin represents only one member of large families of diverse homologs. There are also newly identified bacterial cytoskeletal proteins with no eukaryotic homologs, such as WACA proteins and bactofilins. Furthermore, there are universally conserved proteins, such as the metabolic enzyme CtpS, that assemble into filamentous structures that can be repurposed for structural cytoskeletal functions. Recent studies have also identified an increasing number of eukaryotic cytoskeletal proteins that are unrelated to actin, tubulin, and IFs, such that expanding our understanding of cytoskeletal proteins is advancing the understanding of the cell biology of all organisms. Here, we summarize the recent explosion in the identification of new members of the bacterial cytoskeleton and describe a hypothesis for the evolution of the cytoskeleton from self-assembling enzymes. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Selective self-assembly of adenine-silver nanoparticles forms rings resembling the size of cells

PubMed Central

Choi, Sungmoon; Park, Soonyoung; Yang, Seon-Ah; Jeong, Yujin; Yu, Junhua

2015-01-01

Self-assembly has played critical roles in the construction of functional nanomaterials. However, the structure of the macroscale multicomponent materials built by the self-assembly of nanoscale building blocks is hard to predict due to multiple intermolecular interactions of great complexity. Evaporation of solvents is usually an important approach to induce kinetically stable assemblies of building blocks with a large-scale specific arrangement. During such a deweting process, we tried to monitor the possible interactions between silver nanoparticles and nucleobases at a larger scale by epifluorescence microscopy, thanks to the doping of silver nanoparticles with luminescent silver nanodots. ssDNA oligomer-stabilized silver nanoparticles and adenine self-assemble to form ring-like compartments similar to the size of modern cells. However, the silver ions only dismantle the self-assembly of adenine. The rings are thermodynamically stable as the drying process only enrich the nanoparticles-nucleobase mixture to a concentration that activates the self-assembly. The permeable membrane-like edge of the ring is composed of adenine filaments glued together by silver nanoparticles. Interestingly, chemicals are partially confined and accumulated inside the ring, suggesting that this might be used as a microreactor to speed up chemical reactions during a dewetting process. PMID:26643504

3D printing of high drug loaded dosage forms using thermoplastic polyurethanes.

PubMed

Verstraete, G; Samaro, A; Grymonpré, W; Vanhoorne, V; Van Snick, B; Boone, M N; Hellemans, T; Van Hoorebeke, L; Remon, J P; Vervaet, C

2018-01-30

It was the aim of this study to develop high drug loaded (>30%, w/w), thermoplastic polyurethane (TPU)-based dosage forms via fused deposition modelling (FDM). Model drugs with different particle size and aqueous solubility were pre-processed in combination with diverse TPU grades via hot melt extrusion (HME) into filaments with a diameter of 1.75 ± 0.05 mm. Subsequently, TPU-based filaments which featured acceptable quality attributes (i.e. consistent filament diameter, smooth surface morphology and good mechanical properties) were printed into tablets. The sustained release potential of the 3D printed dosage forms was tested in vitro. Moreover, the impact of printing parameters on the in vitro drug release was investigated. TPU-based filaments could be loaded with 60% (w/w) fine drug powder without observing severe shark skinning or inconsistent filament diameter. During 3D printing experiments, HME filaments based on hard TPU grades were successfully converted into personalized dosage forms containing a high concentration of crystalline drug (up to 60%, w/w). In vitro release kinetics were mainly affected by the matrix composition and tablet infill degree. Therefore, this study clearly demonstrated that TPU-based FDM feedstock material offers a lot of formulation freedom for the development of personalized dosage forms. Copyright © 2017 Elsevier B.V. All rights reserved.

Bio-Source of di-n-butyl phthalate production by filamentous fungi

NASA Astrophysics Data System (ADS)

Tian, Congkui; Ni, Jinren; Chang, Fang; Liu, Sitong; Xu, Nan; Sun, Weiling; Xie, Yuan; Guo, Yongzhao; Ma, Yanrong; Yang, Zhenxing; Dang, Chenyuan; Huang, Yuefei; Tian, Zhexian; Wang, Yiping

2016-02-01

Although DBP (di-n-butyl phthalate) is commonly encountered as an artificially-synthesized plasticizer with potential to impair fertility, we confirm that it can also be biosynthesized as microbial secondary metabolites from naturally occurring filamentous fungi strains cultured either in an artificial medium or natural water. Using the excreted crude enzyme from the fungi for catalyzing a variety of substrates, we found that the fungal generation of DBP was largely through shikimic acid pathway, which was assembled by phthalic acid with butyl alcohol through esterification. The DBP production ability of the fungi was primarily influenced by fungal spore density and incubation temperature. This study indicates an important alternative natural waterborne source of DBP in addition to artificial synthesis, which implied fungal contribution must be highlighted for future source control and risk management of DBP.

Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function*

PubMed Central

Lowery, Jason; Kuczmarski, Edward R.; Herrmann, Harald; Goldman, Robert D.

2015-01-01

Intermediate filaments (IFs) are composed of one or more members of a large family of cytoskeletal proteins, whose expression is cell- and tissue type-specific. Their importance in regulating the physiological properties of cells is becoming widely recognized in functions ranging from cell motility to signal transduction. IF proteins assemble into nanoscale biopolymers with unique strain-hardening properties that are related to their roles in regulating the mechanical integrity of cells. Furthermore, mutations in the genes encoding IF proteins cause a wide range of human diseases. Due to the number of different types of IF proteins, we have limited this short review to cover structure and function topics mainly related to the simpler homopolymeric IF networks composed of vimentin, and specifically for diseases, the related muscle-specific desmin IF networks. PMID:25957409

Weak-lensing detection of intracluster filaments with ground-based data

NASA Astrophysics Data System (ADS)

Maturi, Matteo; Merten, Julian

2013-11-01

According to the current standard model of cosmology, matter in the Universe arranges itself along a network of filamentary structure. These filaments connect the main nodes of this so-called "cosmic web", which are clusters of galaxies. Although its large-scale distribution is clearly characterized by numerical simulations, constraining the dark-matter content of the cosmic web in reality turns out to be difficult. The natural method of choice is gravitational lensing. However, the direct detection and mapping of the elusive filament signal is challenging and in this work we present two methods that are specifically tailored to achieve this task. A linear matched filter aims at detecting the smooth mass-component of filaments and is optimized to perform a shear decomposition that follows the anisotropic component of the lensing signal. Filaments clearly inherit this property due to their morphology. At the same time, the contamination arising from the central massive cluster is controlled in a natural way. The filament 1σ detection is of about κ ~ 0.01 - 0.005 according to the filter's template width and length, enabling the detection of structures beyond reach with other approaches. The second, complementary method seeks to detect the clumpy component of filaments. The detection is determined by the number density of subclump identifications in an area enclosing the potential filament, as was found within the observed field with the filter approach. We tested both methods against mocked observations based on realistic N-body simulations of filamentary structure and proved the feasibility of detecting filaments with ground-based data.

The unusual dynamics of parasite actin result from isodesmic polymerization

PubMed Central

Skillman, Kristen M.; Ma, Christopher I.; Fremont, Daved H.; Diraviyam, Karthikeyan; Cooper, John A.; Sept, David; Sibley, L. David

2013-01-01

Previous reports have indicated that parasite actins are short and inherently unstable, despite being required for motility. Here, we re-examine the polymerization properties of actin in Toxoplasma gondii (TgACTI), unexpectedly finding that it exhibits isodesmic polymerization in contrast to the conventional nucleation-elongation process of all previously studied actins from both eukaryotes and bacteria. TgACTI polymerization kinetics lacks both a lag phase and critical concentration, normally characteristic of actins. Unique among actins, the kinetics of assembly can be fit with a single set of rate constants for all subunit interactions, without need for separate nucleation and elongation rates. This isodesmic model accurately predicts the assembly, disassembly, and the size distribution of TgACTI filaments in vitro, providing a mechanistic explanation for actin dynamics in vivo. Our findings expand the repertoire of mechanisms by which actin polymerization is governed and offer clues about the evolution of self-assembling, stabilized protein polymers. PMID:23921463

Galactic cold cores. VII. Filament formation and evolution: Methods and observational constraints

NASA Astrophysics Data System (ADS)

Rivera-Ingraham, A.; Ristorcelli, I.; Juvela, M.; Montillaud, J.; Men'shchikov, A.; Malinen, J.; Pelkonen, V.-M.; Marston, A.; Martin, P. G.; Pagani, L.; Paladini, R.; Paradis, D.; Ysard, N.; Ward-Thompson, D.; Bernard, J.-P.; Marshall, D. J.; Montier, L.; Tóth, L. V.

2016-06-01

Context. The association of filaments with protostellar objects has made these structures a priority target in star formation studies. However, little is known about the link between filament properties and their local environment. Aims: The datasets from the Herschel Galactic Cold cores key programme allow for a statistical study of filaments with a wide range of intrinsic and environmental characteristics. Characterisation of this sample can therefore be used to identify key physical parameters and quantify the role of the environment in the formation of supercritical filaments. These results are necessary to constrain theoretical models of filament formation and evolution. Methods: Filaments were extracted from fields at distance D< 500 pc with the getfilaments algorithm and characterised according to their column density profiles and intrinsic properties. Each profile was fitted with a beam-convolved Plummer-like function, and the filament structure was quantified based on the relative contributions from the filament "core", represented by a Gaussian, and "wing" component, dominated by the power-law behaviour of the Plummer-like function. These filament parameters were examined for populations associated with different background levels. Results: Filaments increase their core (Mline,core) and wing (Mline,wing) contributions while increasing their total linear mass density (Mline,tot). Both components appear to be linked to the local environment, with filaments in higher backgrounds having systematically more massive Mline,core and Mline,wing. This dependence on the environment supports an accretion-based model of filament evolution in the local neighbourhood (D ≤ 500 pc). Structures located in the highest backgrounds develop the highest central AV, Mline,core, and Mline,wing as Mline,tot increases with time, favoured by the local availability of material and the enhanced gravitational potential. Our results indicate that filaments acquiring a significantly massive central region with Mline,core≳Mcrit/2 may become supercritical and form stars. This translates into a need for filaments to become at least moderately self-gravitating to undergo localised star formation or become star-forming filaments. Herschel is an ESA space observatory with science instruments provided by European-led Principal Investigator consortia and with important participation from NASA.

Non-equilibrium assembly of microtubules: from molecules to autonomous chemical robots.

PubMed

Hess, H; Ross, Jennifer L

2017-09-18

Biological systems have evolved to harness non-equilibrium processes from the molecular to the macro scale. It is currently a grand challenge of chemistry, materials science, and engineering to understand and mimic biological systems that have the ability to autonomously sense stimuli, process these inputs, and respond by performing mechanical work. New chemical systems are responding to the challenge and form the basis for future responsive, adaptive, and active materials. In this article, we describe a particular biochemical-biomechanical network based on the microtubule cytoskeletal filament - itself a non-equilibrium chemical system. We trace the non-equilibrium aspects of the system from molecules to networks and describe how the cell uses this system to perform active work in essential processes. Finally, we discuss how microtubule-based engineered systems can serve as testbeds for autonomous chemical robots composed of biological and synthetic components.

Electron microscopy of intermediate filaments: teaming up with atomic force and confocal laser scanning microscopy.

PubMed

Kreplak, Laurent; Richter, Karsten; Aebi, Ueli; Herrmann, Harald

2008-01-01

Intermediate filaments (IFs) were originally discovered and defined by electron microscopy in myoblasts. In the following it was demonstrated and confirmed that they constitute, in addition to microtubules and microfilaments, a third independent, general filament system in the cytoplasm of most metazoan cells. In contrast to the other two systems, IFs are present in cells in two principally distinct cytoskeletal forms: (i) extended and free-running filament arrays in the cytoplasm that are integrated into the cytoskeleton by associated proteins of the plakin type; and (ii) a membrane- and chromatin-bound thin 'lamina' of a more or less regular network of interconnected filaments made from nuclear IF proteins, the lamins, which differ in several important structural aspects from cytoplasmic IF proteins. In man, more than 65 genes code for distinct IF proteins that are expressed during embryogenesis in various routes of differentiation in a tightly controlled manner. IF proteins exhibit rather limited sequence identity implying that the different types of IFs have distinct biochemical properties. Hence, to characterize the structural properties of the various IFs, in vitro assembly regimes have been developed in combination with different visualization methods such as transmission electron microscopy of fixed and negatively stained samples as well as methods that do not use staining such as scanning transmission electron microscopy (STEM) and cryoelectron microscopy as well as atomic force microscopy. Moreover, with the generation of both IF-type specific antibodies and chimeras of fluorescent proteins and IF proteins, it has become possible to investigate the subcellular organization of IFs by correlative fluorescence and electron microscopic methods. The combination of these powerful methods should help to further develop our understanding of nuclear architecture, in particular how nuclear subcompartments are organized and in which way lamins are involved.

DIRECT OBSERVATIONS OF TETHER-CUTTING RECONNECTION DURING A MAJOR SOLAR EVENT FROM 2014 FEBRUARY 24 TO 25

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Huadong; Zhang, Jun; Yang, Shuhong

2014-12-20

Using multi-wavelength data from the Atmospheric Imaging Assembly on board the Solar Dynamics Observatory, we investigated two successive solar flares, a C5.1 confined flare and an X4.9 ejective flare with a halo coronal mass ejection, in NOAA active region 11990 from 2014 February 24 to 25. Before the confined flare onset, EUV brightening beneath the filament was detected. As the flare began, a twisted helical flux rope (FR) wrapping around the filament moved upward and then stopped, and in the meantime an obvious X-ray source below it was observed. Prior to the ejective X4.9 flare, some pre-existing loop structures inmore » the active region interacted with each other, which produced a brightening region beneath the filament. Meanwhile, a small flaring loop appeared below the interaction region and some new helical lines connecting the far ends of the loop structures were gradually formed and continually added into the former twisted FR. Then, due to the resulting imbalance between the magnetic pressure and tension, the new FR, together with the filament, erupted outward. Our observations coincide well with a tether-cutting model, suggesting that the two flares probably have the same triggering mechanism, i.e., tether-cutting reconnection. To our knowledge, this is the first direct observation of tether-cutting reconnection occurring between pre-existing loops in an active region. In the ejective flare case, the erupting filament exhibited an Ω-like kinked structure and underwent an exponential rise after a slow-rise phase, indicating that the kink instability might be also responsible for the eruption initiation.« less

OBSERVATION OF A QUASIPERIODIC PULSATION IN HARD X-RAY, RADIO, AND EXTREME-ULTRAVIOLET WAVELENGTHS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Pankaj; Cho, Kyung-Suk; Nakariakov, Valery M., E-mail: pankaj@kasi.re.kr

2016-05-01

We present a multiwavelength analysis of a quasiperiodic pulsation (QPP) observed in the hard X-ray (HXR), radio, and extreme-ultraviolet (EUV) channels during an M1.9 flare that occurred on 2011 September 23–24. The nonthermal HXR emission in 25–50 keV observed by RHESSI shows five distinct impulsive peaks of decaying amplitude with a period of about 3 minutes. A similar QPP was observed in the microwave emission recorded by the Nobeyama Radioheliograph and Polarimeter in the 2, 3.75, 9.4, and 17 GHz channels. Interestingly, the 3-minute QPP was also observed in the metric and decimetric radio frequencies (25–180, 245, 610 MHz) asmore » repetitive type III bursts. Multiwavelength observations from the Solar Dynamics Observatory /Atmospheric Image Assembly, Hinode /SOT, and Solar TErrestrial RElations Observatory /SECCHI suggest a fan-spine topology at the eruption site, associated with the formation of a quasi-circular ribbon during the flare. A small filament was observed below the fan loops before the flare onset. The filament rose slowly and interacted with the ambient field. This behavior was followed by an untwisting motion of the filament. Two different structures of the filament showed an approximately 3-minute periodic alternate rotation in the clockwise and counterclockwise directions. The 3-minute QPP was found to highly correlate with 3-minute oscillations in a nearby sunspot. We suggest that the periodic reconnection (modulated either by a sunspot slow-mode wave or by an untwisting filament) at a magnetic null point most likely causes the repetitive particle acceleration, generating the QPP observed in HXR, microwave, and type III radio bursts.« less

Allele-specific Effects of Human Deafness γ-Actin Mutations (DFNA20/26) on the Actin/Cofilin Interaction*

PubMed Central

Bryan, Keith E.; Rubenstein, Peter A.

2009-01-01

Auditory hair cell function requires proper assembly and regulation of the nonmuscle gamma isoactin-rich cytoskeleton, and six point mutations in this isoactin cause a type of delayed onset autosomal dominant nonsyndromic progressive hearing loss, DFNA20/26. The molecular basis underlying this actin-dependent hearing loss is unknown. To address this problem, the mutations have been introduced into yeast actin, and their effects on actin function were assessed in vivo and in vitro. Because we previously showed that polymerization was unaffected in five of the six mutants, we have focused on proteins that regulate actin, in particular cofilin, which severs F-actin and sequesters actin monomers. The mutations do not affect the interaction of cofilin with G-actin. However, T89I and V370A mutant F-actins are much more susceptible to cofilin disassembly than WT filaments in vitro. Conversely, P332A filaments demonstrate enhanced resistance. Wild type actin solutions containing T89I, K118M, or P332A mutant actins at mole fractions similar to those found in the hair cell respond in vitro toward cofilin in a manner proportional to the level of the mutant present. Finally, depression of cofilin action in vivo by elimination of the cofilin-activating protein, Aip1p, rescues the inability to grow on glycerol caused by K118M, T278I, P332A, and V370A. These results suggest that a filament instability caused by these mutations can be balanced by decreasing a system in vivo that promotes increased filament turnover. Such mutant-dependent filament destabilization could easily result in hair cell malfunction leading to the late-onset hearing loss observed in these patients. PMID:19419963

Axon Initial Segment Cytoskeleton: Architecture, Development, and Role in Neuron Polarity

PubMed Central

Svitkina, Tatyana M.

2016-01-01

The axon initial segment (AIS) is a specialized structure in neurons that resides in between axonal and somatodendritic domains. The localization of the AIS in neurons is ideal for its two major functions: it serves as the site of action potential firing and helps to maintain neuron polarity. It has become increasingly clear that the AIS cytoskeleton is fundamental to AIS functions. In this review, we discuss current understanding of the AIS cytoskeleton with particular interest in its unique architecture and role in maintenance of neuron polarity. The AIS cytoskeleton is divided into two parts, submembrane and cytoplasmic, based on localization, function, and molecular composition. Recent studies using electron and subdiffraction fluorescence microscopy indicate that submembrane cytoskeletal components (ankyrin G, βIV-spectrin, and actin filaments) form a sophisticated network in the AIS that is conceptually similar to the polygonal/triangular network of erythrocytes, with some important differences. Components of the AIS cytoplasmic cytoskeleton (microtubules, actin filaments, and neurofilaments) reside deeper within the AIS shaft and display structural features distinct from other neuronal domains. We discuss how the AIS submembrane and cytoplasmic cytoskeletons contribute to different aspects of AIS polarity function and highlight recent advances in understanding their AIS cytoskeletal assembly and stability. PMID:27493806

Multiwavelength Diagnostics of the Precursor and Main Phases of an M1.8 Flare on 2011 April 22

NASA Technical Reports Server (NTRS)

Awasthi, A. K.; Jain, R.; Gadhiya, P. D.; Aschwanden, M. J.; Uddin, W.; Srivastava, A. K.; Chandra, R.; Gopalswamy, N.; Nitta, N. V.; Yashiro, S.;

Comparison of Brownian-dynamics-based estimates of polymer tension with direct force measurements.

PubMed

Arsenault, Mark E; Purohit, Prashant K; Goldman, Yale E; Shuman, Henry; Bau, Haim H

2010-11-01

With the aid of brownian dynamics models, it is possible to estimate polymer tension by monitoring polymers' transverse thermal fluctuations. To assess the precision of the approach, brownian dynamics-based tension estimates were compared with the force applied to rhodamine-phalloidin labeled actin filaments bound to polymer beads and suspended between two optical traps. The transverse thermal fluctuations of each filament were monitored with a CCD camera, and the images were analyzed to obtain the filament's transverse displacement variance as a function of position along the filament, the filament's tension, and the camera's exposure time. A linear Brownian dynamics model was used to estimate the filament's tension. The estimated force was compared and agreed within 30% (when the tension <0.1 pN ) and 70% (when the tension <1 pN ) with the applied trap force. In addition, the paper presents concise asymptotic expressions for the mechanical compliance of a system consisting of a filament attached tangentially to bead handles (dumbbell system). The techniques described here can be used for noncontact estimates of polymers' and fibers' tension.

Dynamics and Breakup of a Contracting Viscous Filament

NASA Astrophysics Data System (ADS)

Wilkes, Edward; Notz, Patrick; Ambravaneswaran, Bala; Basaran, Osman

1999-11-01

Free viscous filaments are formed during the breakup of liquid drops and jets. Such filaments are typically precursors of satellite droplets that are often undesirable in applications such as ink-jet printing. In this paper, the contraction of an axisymmetric liquid filament due to action of surface tension is studied theoretically. The analysis is based on solving (a) the full Navier-Stokes system in two-dimensions (2-d) and (b) a one-dimensional (1-d) approximation of the exact equations based on slender-jet theory. The rigorous, 2-d calculations are carried out with finite element algorithms using either algebraic or elliptic mesh generation. As the filament contracts, bulbous regions form at its two ends. When the initial aspect ratio a/b and/or the Reynolds number Re are sufficiently low, the ends coalesce into an oscillating free drop. Filament breakup occurs when a/b and/or Re are sufficiently high. The 2-d algorithms reveal for the first time that liquid filaments of finite viscosity can overturn prior to interface rupture. The power of elliptic mesh generation over algebraic methods in analyzing such situations is highlighted.

Dynamics and mechanics of motor-filament systems

NASA Astrophysics Data System (ADS)

Kruse, K.; Jülicher, F.

2006-08-01

Motivated by the cytoskeleton of eukaryotic cells, we develop a general framework for describing the large-scale dynamics of an active filament network. In the cytoskeleton, active cross-links are formed by motor proteins that are able to induce relative motion between filaments. Starting from pair-wise interactions of filaments via such active processes, our framework is based on momentum conservation and an analysis of the momentum flux. This allows us to calculate the stresses in the filament network generated by the action of motor proteins. We derive effective theories for the filament dynamics which can be related to continuum theories of active polar gels. As an example, we discuss the stability of homogenous isotropic filament distributions in two spatial dimensions.

The Multiple Roles of Cyk1p in the Assembly and Function of the Actomyosin Ring in Budding Yeast

PubMed Central

Shannon, Katie B.; Li, Rong

1999-01-01

The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud junction during anaphase and has been shown to be required for the completion of cytokinesis. In this study, video microscopy analysis of cells expressing green fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a dynamic component of the contractile ring during cytokinesis. Furthermore, in the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p in actomyosin ring assembly and dynamics, we have investigated the role of the structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino terminal portion containing the calponin homology domain binds to actin filaments and is required for the assembly of actin filaments to the ring. This result supports the hypothesis that Cyk1p/Iqg1p plays a direct role in F-actin recruitment. Deletion of the domain harboring the eight IQ motifs abolishes the localization of Cyk1p/Iqg1p to the bud neck, suggesting that Cyk1p/Iqg1p may be localized through interactions with a calmodulin-like protein. Interestingly, deletion of the COOH-terminal GTPase-activating protein-related domain does not affect Cyk1p/Iqg1p localization or actin recruitment to the ring but prevents actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p binds to calmodulin, Cmd1p, in a calcium-dependent manner, and to Tem1p, a small GTP-binding protein previously found to be required for the completion of anaphase. These results demonstrate the critical function of Cyk1p/Iqg1p in regulating various steps of actomyosin ring assembly and cytokinesis. PMID:9950677

Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding.

PubMed

Frantz, Christian; Barreiro, Gabriela; Dominguez, Laura; Chen, Xiaoming; Eddy, Robert; Condeelis, John; Kelly, Mark J S; Jacobson, Matthew P; Barber, Diane L

2008-12-01

Newly generated actin free barbed ends at the front of motile cells provide sites for actin filament assembly driving membrane protrusion. Growth factors induce a rapid biphasic increase in actin free barbed ends, and we found both phases absent in fibroblasts lacking H(+) efflux by the Na-H exchanger NHE1. The first phase is restored by expression of mutant cofilin-H133A but not unphosphorylated cofilin-S3A. Constant pH molecular dynamics simulations and nuclear magnetic resonance (NMR) reveal pH-sensitive structural changes in the cofilin C-terminal filamentous actin binding site dependent on His133. However, cofilin-H133A retains pH-sensitive changes in NMR spectra and severing activity in vitro, which suggests that it has a more complex behavior in cells. Cofilin activity is inhibited by phosphoinositide binding, and we found that phosphoinositide binding is pH-dependent for wild-type cofilin, with decreased binding at a higher pH. In contrast, phosphoinositide binding by cofilin-H133A is attenuated and pH insensitive. These data suggest a molecular mechanism whereby cofilin acts as a pH sensor to mediate a pH-dependent actin filament dynamics.

Magnetic Actuation of Self-Assembled DNA Hinges

NASA Astrophysics Data System (ADS)

Lauback, S.; Mattioli, K.; Armstrong, M.; Miller, C.; Pease, C.; Castro, C.; Sooryakumar, R.

DNA nanotechnology offers a broad range of applications spanning from the creation of nanoscale devices, motors and nanoparticle templates to the development of precise drug delivery systems. Central to advancing this technology is the ability to actuate or reconfigure structures in real time, which is currently achieved primarily by DNA strand displacement yielding slow actuation times (about 1-10min). Here we exploit superparamagnetic beads to magnetically actuate DNA structures which also provides a system to measure forces associated with molecular interactions. DNA nanodevices are folded using DNA origami, whereby a long single-stranded DNA is folded into a precise compact geometry using hundreds of short oligonucleotides. Our DNA nanodevice is a nanohinge from which rod shaped DNA nanostructures are polymerized into micron-scale filaments forming handles for actuation. By functionalizing one arm of the hinge and the filament ends, the hinge can be attached to a surface while still allowing an arm to rotate and the filaments can be labeled with magnetic beads enabling the hinge to be actuated almost instantaneously by external magnetic fields. These results lay the groundwork to establish real-time manipulation and direct force application of DNA constructs.

Investigating Deinococcus radiodurans RecA protein filament formation on dsDNA by a real-time single-molecule approach

PubMed Central

Hsu, Hsin-Fang; Ngo, Khanh V.; Chitteni-Pattu, Sindhu; Cox, Michael M.; Li, Hung-Wen

2011-01-01

With the aid of an efficient, precise, and almost error-free DNA repair system, Deinococcus radiodurans can survive hundreds of double strand breaks inflicted by high doses of irradiation or desiccation. The RecA of Deinococcus radiodurans (DrRecA) plays a central role both in the early phase of repair by an extended synthesis-dependent strand annealing process and in the later more general homologous recombination phase. Both roles likely require DrRecA filament formation on duplex DNA. We have developed single-molecule tethered particle motion (TPM) experiments to study the assembly dynamics of RecA proteins on individual duplex DNA molecules by observing changes in DNA tether length resulting from RecA binding. We demonstrate that DrRecA nucleation on dsDNA is much faster than Escherichia coli (Ec) RecA protein, but the extension is slower. This combination of attributes would tend to increase the number and decrease the length of DrRecA filaments relative to those of EcRecA, a feature that may reflect the requirement to repair hundreds of genomic double strand breaks concurrently in irradiated Deinococcus cells. PMID:21853996

Electrostatic interaction map reveals a new binding position for tropomyosin on F-actin.

PubMed

Rynkiewicz, Michael J; Schott, Veronika; Orzechowski, Marek; Lehman, William; Fischer, Stefan

2015-12-01

Azimuthal movement of tropomyosin around the F-actin thin filament is responsible for muscle activation and relaxation. Recently a model of αα-tropomyosin, derived from molecular-mechanics and electron microscopy of different contractile states, showed that tropomyosin is rather stiff and pre-bent to present one specific face to F-actin during azimuthal transitions. However, a new model based on cryo-EM of troponin- and myosin-free filaments proposes that the interacting-face of tropomyosin can differ significantly from that in the original model. Because resolution was insufficient to assign tropomyosin side-chains, the interacting-face could not be unambiguously determined. Here, we use structural analysis and energy landscapes to further examine the proposed models. The observed bend in seven crystal structures of tropomyosin is much closer in direction and extent to the original model than to the new model. Additionally, we computed the interaction map for repositioning tropomyosin over the F-actin surface, but now extended over a much larger surface than previously (using the original interacting-face). This map shows two energy minima-one corresponding to the "blocked-state" as in the original model, and the other related by a simple 24 Å translation of tropomyosin parallel to the F-actin axis. The tropomyosin-actin complex defined by the second minimum fits perfectly into the recent cryo-EM density, without requiring any change in the interacting-face. Together, these data suggest that movement of tropomyosin between regulatory states does not require interacting-face rotation. Further, they imply that thin filament assembly may involve an interplay between initially seeded tropomyosin molecules growing from distinct binding-site regions on actin.

Two distinct modes of RecA action are required for DNA polymerase V-catalyzed translesion synthesis.

PubMed

Pham, Phuong; Seitz, Erica M; Saveliev, Sergei; Shen, Xuan; Woodgate, Roger; Cox, Michael M; Goodman, Myron F

2002-08-20

SOS mutagenesis in Escherichia coli requires DNA polymerase V (pol V) and RecA protein to copy damaged DNA templates. Here we show that two distinct biochemical modes for RecA protein are necessary for pol V-catalyzed translesion synthesis. One RecA mode is characterized by a strong stimulation in nucleotide incorporation either directly opposite a lesion or at undamaged template sites, but by the absence of lesion bypass. A separate RecA mode is necessary for translesion synthesis. The RecA1730 mutant protein, which was identified on the basis of its inability to promote pol V (UmuD'(2)C)-dependent UV-mutagenesis, appears proficient for the first mode of RecA action but is deficient in the second mode. Data are presented suggesting that the two RecA modes are "nonfilamentous". That is, contrary to current models for SOS mutagenesis, formation of a RecA nucleoprotein filament may not be required for copying damaged DNA templates. Instead, SOS mutagenesis occurs when pol V interacts with two RecA molecules, first at a 3' primer end, upstream of a template lesion, where RecA mode 1 stimulates pol V activity, and subsequently at a site immediately downstream of the lesion, where RecA mode 2 cocatalyzes lesion bypass. We posit that in vivo assembly of a RecA nucleoprotein filament may be required principally to target pol V to a site of DNA damage and to stabilize the pol V-RecA interaction at the lesion. However, it is only a RecA molecule located at the 3' filament tip, proximal to a damaged template base, that is directly responsible for translesion synthesis.

Two distinct modes of RecA action are required for DNA polymerase V-catalyzed translesion synthesis

PubMed Central

Pham, Phuong; Seitz, Erica M.; Saveliev, Sergei; Shen, Xuan; Woodgate, Roger; Cox, Michael M.; Goodman, Myron F.

2002-01-01

SOS mutagenesis in Escherichia coli requires DNA polymerase V (pol V) and RecA protein to copy damaged DNA templates. Here we show that two distinct biochemical modes for RecA protein are necessary for pol V-catalyzed translesion synthesis. One RecA mode is characterized by a strong stimulation in nucleotide incorporation either directly opposite a lesion or at undamaged template sites, but by the absence of lesion bypass. A separate RecA mode is necessary for translesion synthesis. The RecA1730 mutant protein, which was identified on the basis of its inability to promote pol V (UmuD′2C)-dependent UV-mutagenesis, appears proficient for the first mode of RecA action but is deficient in the second mode. Data are presented suggesting that the two RecA modes are “nonfilamentous”. That is, contrary to current models for SOS mutagenesis, formation of a RecA nucleoprotein filament may not be required for copying damaged DNA templates. Instead, SOS mutagenesis occurs when pol V interacts with two RecA molecules, first at a 3′ primer end, upstream of a template lesion, where RecA mode 1 stimulates pol V activity, and subsequently at a site immediately downstream of the lesion, where RecA mode 2 cocatalyzes lesion bypass. We posit that in vivo assembly of a RecA nucleoprotein filament may be required principally to target pol V to a site of DNA damage and to stabilize the pol V-RecA interaction at the lesion. However, it is only a RecA molecule located at the 3′ filament tip, proximal to a damaged template base, that is directly responsible for translesion synthesis. PMID:12177433

Deformation and deceleration of coronal wave

NASA Astrophysics Data System (ADS)

Xue, Z. K.; Qu, Z. Q.; Yan, X. L.; Zhao, L.; Ma, L.

2013-08-01

Aims: We studied the kinematics and morphology of two coronal waves to better understand the nature and origin of coronal waves. Methods: Using multi-wavelength observations of the Atmospheric Imaging Assembly (AIA) on board the Solar Dynamics Observatory (SDO) and the Extreme Ultraviolet Imager (EUVI) on board the twin spacecraft Solar-TErrestrial RElations Observatory (STEREO), we present morphological and dynamic characteristics of consecutive coronal waves on 2011 March 24. We also show the coronal magnetic field based on the potential field source surface model. Results: This event contains several interesting aspects. The first coronal wave initially appeared after a surge-like eruption. Its front was changed and deformed significantly from a convex shape to a line-shaped appearance, and then to a concave configuration during its propagation to the northwest. The initial speeds ranged from 947 km s-1 to 560 km s-1. The first wave decelerated significantly after it passed through a filament channel. After the deceleration, the final propagation speeds of the wave were from 430 km s-1 to 312 km s-1. The second wave was found to appear after the first wave in the northwest side of the filament channel. Its wave front was more diffused and the speed was around 250 km s-1, much slower than that of the first wave. Conclusions: The deformation of the first coronal wave was caused by the different speeds along different paths. The sudden deceleration implies that the refraction of the first wave took place at the boundary of the filament channel. The event provides evidence that the first coronal wave may be a coronal MHD shock wave, and the second wave may be the apparent propagation of the brightenings caused by successive stretching of the magnetic field lines.

Filament turnover tunes both force generation and dissipation to control long-range flows in a model actomyosin cortex

PubMed Central

McCall, Patrick M.; Gardel, Margaret L.; Munro, Edwin M.

2017-01-01

Actomyosin-based cortical flow is a fundamental engine for cellular morphogenesis. Cortical flows are generated by cross-linked networks of actin filaments and myosin motors, in which active stress produced by motor activity is opposed by passive resistance to network deformation. Continuous flow requires local remodeling through crosslink unbinding and and/or filament disassembly. But how local remodeling tunes stress production and dissipation, and how this in turn shapes long range flow, remains poorly understood. Here, we study a computational model for a cross-linked network with active motors based on minimal requirements for production and dissipation of contractile stress: Asymmetric filament compliance, spatial heterogeneity of motor activity, reversible cross-links and filament turnover. We characterize how the production and dissipation of network stress depend, individually, on cross-link dynamics and filament turnover, and how these dependencies combine to determine overall rates of cortical flow. Our analysis predicts that filament turnover is required to maintain active stress against external resistance and steady state flow in response to external stress. Steady state stress increases with filament lifetime up to a characteristic time τm, then decreases with lifetime above τm. Effective viscosity increases with filament lifetime up to a characteristic time τc, and then becomes independent of filament lifetime and sharply dependent on crosslink dynamics. These individual dependencies of active stress and effective viscosity define multiple regimes of steady state flow. In particular our model predicts that when filament lifetimes are shorter than both τc and τm, the dependencies of effective viscosity and steady state stress on filament turnover cancel one another, such that flow speed is insensitive to filament turnover, and shows a simple dependence on motor activity and crosslink dynamics. These results provide a framework for understanding how animal cells tune cortical flow through local control of network remodeling. PMID:29253848

Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules

PubMed Central

1988-01-01

We have correlated the motility of the leading edge of fibroblasts, monitored by phase-contrast cinematography, with the relative distributions of several cytoskeletal elements (vinculin, tubulin, and actin) as well as with the contact patterns determined by interference reflection microscopy. This analysis has revealed the involvement of both ruffles and microspikes, as well as microtubules in the initiation of focal contact formation. Nascent vinculin sites within the leading edge or at its base, taken as primordial cell-substrate contacts, were invariably colocalized with sites that showed a history of transient, prolonged, or cyclic ruffling activity. Extended microspike structures, often preceded the formation of ruffles. Immunofluorescent labeling indicated that some of these primordial contacts were in close apposition to the ends of microtubules that penetrated into the leading edge. By fluorescence and electron microscopy short bundles of actin filaments found at the base of the leading edge were identified as presumptive, primordial contacts. It is concluded that ruffles and microspikes, either independently or in combination, initiate and mark the sites for future contact. Plaque proteins then accumulate (within 10-30 s) at the contract site and, beneath ruffles, induce localized bundling of actin filaments. We propose that all primordial contacts support traction for leading edge protrusion but that only some persist long enough to nucleate stress fiber assembly. Microtubules are postulated as the elements that select, stabilize, and potentiate the formation of these latter, long-lived contacts. PMID:3126193

MotD of Sinorhizobium meliloti and Related α-Proteobacteria Is the Flagellar-Hook-Length Regulator and Therefore Reassigned as FliK

PubMed Central

Eggenhofer, Elke; Rachel, Reinhard; Haslbeck, Martin; Scharf, Birgit

2006-01-01

The flagella of the soil bacterium Sinorhizobium meliloti differ from the enterobacterial paradigm in the complex filament structure and modulation of the flagellar rotary speed. The mode of motility control in S. meliloti has a molecular corollary in two novel periplasmic motility proteins, MotC and MotE, that are present in addition to the ubiquitous MotA/MotB energizing proton channel. A fifth motility gene is located in the mot operon downstream of the motB and motC genes. Its gene product was originally designated MotD, a cytoplasmic motility protein having an unknown function. We report here reassignment of MotD as FliK, the regulator of flagellar hook length. The FliK gene is one of the few flagellar genes not annotated in the contiguous flagellar regulon of S. meliloti. Characteristic for its class, the 475-residue FliK protein contains a conserved, compactly folded Flg hook domain in its carboxy-terminal region. Deletion of fliK leads to formation of prolonged flagellar hooks (polyhooks) with missing filament structures. Extragenic suppressor mutations all mapped in the cytoplasmic region of the transmembrane export protein FlhB and restored assembly of a flagellar filament, and thus motility, in the presence of polyhooks. The structural properties of FliK are consistent with its function as a substrate specificity switch of the flagellar export apparatus for switching from rod/hook-type substrates to filament-type substrates. PMID:16513744

Resemblance of actin-binding protein/actin gels to covalently crosslinked networks

NASA Astrophysics Data System (ADS)

Janmey, Paul A.; Hvidt, Søren; Lamb, Jennifer; Stossel, Thomas P.

1990-05-01

THE maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex1,2. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel sol' transformations result from the rearrangement of cortical actin-rich networks3. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, α-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments4: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels Theologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.

Direct Evidence of an Eruptive, Filament-hosting Magnetic Flux Rope Leading to a Fast Solar Coronal Mass Ejection

NASA Astrophysics Data System (ADS)

Chen, Bin; Bastian, T. S.; Gary, D. E.

2014-10-01

Magnetic flux ropes (MFRs) are believed to be at the heart of solar coronal mass ejections (CMEs). A well-known example is the prominence cavity in the low corona that sometimes makes up a three-part white-light (WL) CME upon its eruption. Such a system, which is usually observed in quiet-Sun regions, has long been suggested to be the manifestation of an MFR with relatively cool filament material collecting near its bottom. However, observational evidence of eruptive, filament-hosting MFR systems has been elusive for those originating in active regions. By utilizing multi-passband extreme-ultraviolet (EUV) observations from Solar Dynamics Observatory/Atmospheric Imaging Assembly, we present direct evidence of an eruptive MFR in the low corona that exhibits a hot envelope and a cooler core; the latter is likely the upper part of a filament that undergoes a partial eruption, which is later observed in the upper corona as the coiled kernel of a fast, WL CME. This MFR-like structure exists more than 1 hr prior to its eruption, and displays successive stages of dynamical evolution, in which both ideal and non-ideal physical processes may be involved. The timing of the MFR kinematics is found to be well correlated with the energy release of the associated long-duration C1.9 flare. We suggest that the long-duration flare is the result of prolonged energy release associated with the vertical current sheet induced by the erupting MFR.

Mind the gap; seven reasons to close fragmented genome assemblies.

PubMed

Thomma, Bart P H J; Seidl, Michael F; Shi-Kunne, Xiaoqian; Cook, David E; Bolton, Melvin D; van Kan, Jan A L; Faino, Luigi

2016-05-01

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions. Copyright © 2015 Elsevier Inc. All rights reserved.

The JCMT Gould Belt Survey: properties of star-forming filaments in Orion A North

NASA Astrophysics Data System (ADS)

Salji, C. J.; Richer, J. S.; Buckle, J. V.; di Francesco, J.; Hatchell, J.; Hogerheijde, M.; Johnstone, D.; Kirk, H.; Ward-Thompson, D.; JCMT GBS Consortium

2015-05-01

We develop and apply a Hessian-based filament detection algorithm to submillimetre continuum observations of Orion A North. The resultant filament radial density profiles are fitted with beam-convolved line-of-sight Plummer-profiles using Markov chain Monte Carlo techniques. The posterior distribution of the radial decay parameter demonstrates that the majority of filaments exhibit p = 1.5-3, with a mode at p = 2.2, suggesting deviation from the Ostriker p = 4 isothermal, equilibrium, self-gravitating cylinder. The spatial distribution of young stellar objects relative to the high column density filaments is investigated, yielding a lower limit on the star-forming age of the integral-shaped filament ˜1.4 Myr. Additionally, inferred lifetimes of filaments are examined which suggest long-term filament accretion, varying rates of star formation, or both. Theoretical filament stability measures are determined with the aid of HARP C18O J = 3-2 observations and indicate that the majority of filaments are gravitationally subcritical, despite the presence of young protostars. The results from this investigation are consistent with the one-dimensional accretion flow filament model recently observed in numerical simulations.

Diffusion rate limitations in actin-based propulsion of hard and deformable particles.

PubMed

Dickinson, Richard B; Purich, Daniel L

2006-08-15

The mechanism by which actin polymerization propels intracellular vesicles and invasive microorganisms remains an open question. Several recent quantitative studies have examined propulsion of biomimetic particles such as polystyrene microspheres, phospholipid vesicles, and oil droplets. In addition to allowing quantitative measurement of parameters such as the dependence of particle speed on its size, these systems have also revealed characteristic behaviors such a saltatory motion of hard particles and oscillatory deformation of soft particles. Such measurements and observations provide tests for proposed mechanisms of actin-based motility. In the actoclampin filament end-tracking motor model, particle-surface-bound filament end-tracking proteins are involved in load-insensitive processive insertion of actin subunits onto elongating filament plus-ends that are persistently tethered to the surface. In contrast, the tethered-ratchet model assumes working filaments are untethered and the free-ended filaments grow as thermal ratchets in a load-sensitive manner. This article presents a model for the diffusion and consumption of actin monomers during actin-based particle propulsion to predict the monomer concentration field around motile particles. The results suggest that the various behaviors of biomimetic particles, including dynamic saltatory motion of hard particles and oscillatory vesicle deformations, can be quantitatively and self-consistently explained by load-insensitive, diffusion-limited elongation of (+)-end-tethered actin filaments, consistent with predictions of the actoclampin filament-end tracking mechanism.

Self-organizing actin patterns shape membrane architecture but not cell mechanics

NASA Astrophysics Data System (ADS)

Fritzsche, M.; Li, D.; Colin-York, H.; Chang, V. T.; Moeendarbary, E.; Felce, J. H.; Sezgin, E.; Charras, G.; Betzig, E.; Eggeling, C.

2017-02-01

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

Self-organizing actin patterns shape membrane architecture but not cell mechanics

PubMed Central

Fritzsche, M.; Li, D.; Colin-York, H.; Chang, V. T.; Moeendarbary, E.; Felce, J. H.; Sezgin, E.; Charras, G.; Betzig, E.; Eggeling, C.

2017-01-01

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties. PMID:28194011

RecA: Regulation and Mechanism of a Molecular Search Engine.

PubMed

Bell, Jason C; Kowalczykowski, Stephen C

2016-06-01

Homologous recombination maintains genomic integrity by repairing broken chromosomes. The broken chromosome is partially resected to produce single-stranded DNA (ssDNA) that is used to search for homologous double-stranded DNA (dsDNA). This homology driven 'search and rescue' is catalyzed by a class of DNA strand exchange proteins that are defined in relation to Escherichia coli RecA, which forms a filament on ssDNA. Here, we review the regulation of RecA filament assembly and the mechanism by which RecA quickly and efficiently searches for and identifies a unique homologous sequence among a vast excess of heterologous DNA. Given that RecA is the prototypic DNA strand exchange protein, its behavior affords insight into the actions of eukaryotic RAD51 orthologs and their regulators, BRCA2 and other tumor suppressors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Miniature, low-power X-ray tube using a microchannel electron generator electron source

NASA Technical Reports Server (NTRS)

Elam, Wm. Timothy (Inventor); Kelliher, Warren C. (Inventor); Hershyn, William (Inventor); DeLong, David P. (Inventor)

2011-01-01

Embodiments of the invention provide a novel, low-power X-ray tube and X-ray generating system. Embodiments of the invention use a multichannel electron generator as the electron source, thereby increasing reliability and decreasing power consumption of the X-ray tube. Unlike tubes using a conventional filament that must be heated by a current power source, embodiments of the invention require only a voltage power source, use very little current, and have no cooling requirements. The microchannel electron generator comprises one or more microchannel plates (MCPs), Each MCP comprises a honeycomb assembly of a plurality of annular components, which may be stacked to increase electron intensity. The multichannel electron generator used enables directional control of electron flow. In addition, the multichannel electron generator used is more robust than conventional filaments, making the resulting X-ray tube very shock and vibration resistant.

Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function.

PubMed

Lowery, Jason; Kuczmarski, Edward R; Herrmann, Harald; Goldman, Robert D

2015-07-10

Intermediate filaments (IFs) are composed of one or more members of a large family of cytoskeletal proteins, whose expression is cell- and tissue type-specific. Their importance in regulating the physiological properties of cells is becoming widely recognized in functions ranging from cell motility to signal transduction. IF proteins assemble into nanoscale biopolymers with unique strain-hardening properties that are related to their roles in regulating the mechanical integrity of cells. Furthermore, mutations in the genes encoding IF proteins cause a wide range of human diseases. Due to the number of different types of IF proteins, we have limited this short review to cover structure and function topics mainly related to the simpler homopolymeric IF networks composed of vimentin, and specifically for diseases, the related muscle-specific desmin IF networks. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Spontaneous actin dynamics in contractile rings

NASA Astrophysics Data System (ADS)

Kruse, Karsten; Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Riveline, Daniel

Networks of polymerizing actin filaments are known to be capable to self-organize into a variety of structures. For example, spontaneous actin polymerization waves have been observed in living cells in a number of circumstances, notably, in crawling neutrophils and slime molds. During later stages of cell division, they can also spontaneously form a contractile ring that will eventually cleave the cell into two daughter cells. We present a framework for describing networks of polymerizing actin filaments, where assembly is regulated by various proteins. It can also include the effects of molecular motors. We show that the molecular processes driven by these proteins can generate various structures that have been observed in contractile rings of fission yeast and mammalian cells. We discuss a possible functional role of each of these patterns. The work was supported by Agence Nationale de la Recherche, France, (ANR-10-LABX-0030-INRT) and by Deutsche Forschungsgemeinschaft through SFB1027.

Emergent complexity of the cytoskeleton: from single filaments to tissue

PubMed Central

Huber, F.; Schnauß, J.; Rönicke, S.; Rauch, P.; Müller, K.; Fütterer, C.; Käs, J.

2013-01-01

Despite their overwhelming complexity, living cells display a high degree of internal mechanical and functional organization which can largely be attributed to the intracellular biopolymer scaffold, the cytoskeleton. Being a very complex system far from thermodynamic equilibrium, the cytoskeleton's ability to organize is at the same time challenging and fascinating. The extensive amounts of frequently interacting cellular building blocks and their inherent multifunctionality permits highly adaptive behavior and obstructs a purely reductionist approach. Nevertheless (and despite the field's relative novelty), the physics approach has already proved to be extremely successful in revealing very fundamental concepts of cytoskeleton organization and behavior. This review aims at introducing the physics of the cytoskeleton ranging from single biopolymer filaments to multicellular organisms. Throughout this wide range of phenomena, the focus is set on the intertwined nature of the different physical scales (levels of complexity) that give rise to numerous emergent properties by means of self-organization or self-assembly. PMID:24748680

Enhancement of branching efficiency by the actin filament-binding activity of N-WASP/WAVE2.

PubMed

Suetsugu, S; Miki, H; Yamaguchi, H; Obinata, T; Takenawa, T

2001-12-01

The actin-related protein (Arp) 2/3 complex is an essential regulator of de novo actin filament formation. Arp2/3 nucleates the polymerization of actin and creates branched actin filaments when activated by Arp2/3-complex activating domain (VCA) of Wiskott-Aldrich syndrome proteins (WASP family proteins). We found that the branching of actin filaments on pre-existing ADP filaments mediated by the Arp2/3 complex is twice as efficient when Arp2/3 was activated by wild-type neural WASP (N-WASP) or WASP-family verprolin-homologous protein (WAVE) 2 than when activated by the VCA domain alone. By contrast, there was no difference between wild-type N-WASP or WAVE2 and VCA in the branching efficiency on de novo filaments, which are thought to consist mainly of ADP-phosphate filaments. This increased branching efficiency on ADP filaments is due to the basic region located in the center of N-WASP and WAVE2, which was found to associate with ADP actin filaments. Actin filaments and phosphatidylinositol bisphosphate (PIP2) associate with N-WASP at different sites. This association of N-WASP and WAVE2 with actin filaments enhanced recruitment of Arp2/3 to the pre-existing filaments, presumably leading to efficient nucleation and branch formation on pre-existing filaments. These data together suggest that the actin filament binding activity of N-WASP and WAVE2 in the basic region increases the number of barbed ends created on pre-existing filaments. Efficient branching on ADP filaments may be important for initiation of actin-based motility.

Structure of the Tropomyosin Overlap Complex from Chicken Smooth Muscle: Insight into the Diversity of N-Terminal Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frye, Jeremiah; Klenchin, Vadim A.; Rayment, Ivan

Tropomyosin is a stereotypical {alpha}-helical coiled coil that polymerizes to form a filamentous macromolecular assembly that lies on the surface of F-actin. The interaction between the C-terminal and N-terminal segments on adjacent molecules is known as the overlap region. We report here two X-ray structures of the chicken smooth muscle tropomyosin overlap complex. A novel approach was used to stabilize the C-terminal and N-terminal fragments. Globular domains from both the human DNA ligase binding protein XRCC4 and bacteriophage {phi}29 scaffolding protein Gp7 were fused to 37 and 28 C-terminal amino acid residues of tropomyosin, respectively, whereas the 29 N-terminal aminomore » acids of tropomyosin were fused to the C-terminal helix bundle of microtubule binding protein EB1. The structures of both the XRCC4 and Gp7 fusion proteins complexed with the N-terminal EB1 fusion contain a very similar helix bundle in the overlap region that encompasses {approx}15 residues. The C-terminal coiled coil opens to allow formation of the helix bundle, which is stabilized by hydrophobic interactions. These structures are similar to that observed in the NMR structure of the rat skeletal overlap complex [Greenfield, N. J., et al. (2006) J. Mol. Biol. 364, 80-96]. The interactions between the N- and C-terminal coiled coils of smooth muscle tropomyosin show significant curvature, which differs somewhat between the two structures and implies flexibility in the overlap complex, at least in solution. This is likely an important attribute that allows tropomyosin to assemble around the actin filaments. These structures provide a molecular explanation for the role of N-acetylation in the assembly of native tropomyosin.« less

The Connection Between Solar Coronal Cavities and Solar Filaments

NASA Astrophysics Data System (ADS)

Zawadzki, B.; Karna, N.; Prchlik, J.; Reeves, K.; Kempton, D.; Angryk, R.

2017-12-01

Filaments are structures in the solar corona made up of relatively cool, dense, partially ionized plasma. Coronal cavities, circular or elliptical regions of low plasma density, are observed above prominences on the solar limb when viewed in EUV and white light coronal images. Since most filament/cavity eruptions lead to a coronal mass ejection (CME), determining the likelihood of an eruption event will improve our ability to predict space weather. We examine SDO/AIA cavity metadata and HEK filament metadata to determine which cavities are associated with which filaments from 2012 to 2015. Our study involved 140 cavities and 368 filaments that appeared poleward of +-30 degrees. We categorized the cavities and filaments based on the stability of the structures, defined by whether or not the cavity and filament exist long enough to track fully across the solar disk. Using these categories we perform a statistical study on various filament qualities within the metadata. Our findings indicate that filaments with cavities are observed more often at high latitude in compared to filaments without cavities. Moreover, our study indicates that a statistically significant difference exists between the filament length and tilt distributions for certain categories. This work supported by the NSF-REU solar physics program at SAO, grant number AGS-1560313, and the NSF-DIBBS project, grant number ACI-1443061.