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  1. Boolean gates on actin filaments

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    Siccardi, Stefano, E-mail: ssiccardi@2ssas.it [The Unconventional Computing Centre, University of the West of England, Bristol (United Kingdom); Tuszynski, Jack A., E-mail: jackt@ualberta.ca [Department of Oncology, University of Alberta, Edmonton, Alberta (Canada); Adamatzky, Andrew, E-mail: andrew.adamatzky@uwe.ac.uk [The Unconventional Computing Centre, University of the West of England, Bristol (United Kingdom)

    2016-01-08

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications. - Highlights: • We simulate interaction between voltage pulses using on actin filaments. • We use a coupled nonlinear transmission line model. • We design Boolean logical gates via interactions between the voltage pulses. • We construct one-bit half-adder with interacting voltage pulses.

  2. Actin filaments as tension sensors.

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    Galkin, Vitold E; Orlova, Albina; Egelman, Edward H

    2012-02-07

    The field of mechanobiology has witnessed an explosive growth over the past several years as interest has greatly increased in understanding how mechanical forces are transduced by cells and how cells migrate, adhere and generate traction. Actin, a highly abundant and anomalously conserved protein, plays a large role in forming the dynamic cytoskeleton that is so essential for cell form, motility and mechanosensitivity. While the actin filament (F-actin) has been viewed as dynamic in terms of polymerization and depolymerization, new results suggest that F-actin itself may function as a highly dynamic tension sensor. This property may help explain the unusual conservation of actin's sequence, as well as shed further light on actin's essential role in structures from sarcomeres to stress fibers. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Architecture dependence of actin filament network disassembly.

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    Gressin, Laurène; Guillotin, Audrey; Guérin, Christophe; Blanchoin, Laurent; Michelot, Alphée

    2015-06-01

    Turnover of actin networks in cells requires the fast disassembly of aging actin structures. While ADF/cofilin and Aip1 have been identified as central players, how their activities are modulated by the architecture of the networks remains unknown. Using our ability to reconstitute a diverse array of cellular actin organizations, we found that ADF/cofilin binding and ADF/cofilin-mediated disassembly both depend on actin geometrical organization. ADF/cofilin decorates strongly and stabilizes actin cables, whereas its weaker interaction to Arp2/3 complex networks is correlated with their dismantling and their reorganization into stable architectures. Cooperation of ADF/cofilin with Aip1 is necessary to trigger the full disassembly of all actin filament networks. Additional experiments performed at the single-molecule level indicate that this cooperation is optimal above a threshold of 23 molecules of ADF/cofilin bound as clusters along an actin filament. Our results indicate that although ADF/cofilin is able to dismantle selectively branched networks through severing and debranching, stochastic disassembly of actin filaments by ADF/cofilin and Aip1 represents an efficient alternative pathway for the full disassembly of all actin networks. Our data support a model in which the binding of ADF/cofilin is required to trigger a structural change of the actin filaments, as a prerequisite for their disassembly by Aip1. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Filamentous actin detected in rat spermatozoa.

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    Vogl, A W; Genereux, K; Pfeiffer, D C

    1993-01-01

    In this paper we report the positive staining of epididymal spermatozoa and testicular cells (late spermatids and spermatozoa) with fluorescent phallotoxins. Staining is most obvious with rhodamine phalloidin, but is also detectible with NBD-phallacidin. Specific fluorescence is emitted as a linear tract along the dorsal curvature of the head and as an inverted V-shaped structure in what appears to be the anterior aspect of the post-acrosomal region. We conclude that filamentous actin occurs in the heads of rat spermatozoa. Moreover, we speculate that this filamentous actin is concentrated in two regions of the perinuclear theca; in the subacrosomal space along the dorsal curvature of the nucleus, and in the post-acrosomal region in an area termed the ventral spur.

  5. Electron tomography reveals unbranched networks of actin filaments in lamellipodia.

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    Urban, Edit; Jacob, Sonja; Nemethova, Maria; Resch, Guenter P; Small, J Victor

    2010-05-01

    Eukaryotic cells can initiate movement using the forces exerted by polymerizing actin filaments to extend lamellipodial and filopodial protrusions. In the current model, actin filaments in lamellipodia are organized in a branched, dendritic network. We applied electron tomography to vitreously frozen 'live' cells, fixed cells and cytoskeletons, embedded in vitreous ice or in deep-negative stain. In lamellipodia from four cell types, including rapidly migrating fish keratocytes, we found that actin filaments are almost exclusively unbranched. The vast majority of apparent filament junctions proved to be overlapping filaments, rather than branched end-to-side junctions. Analysis of the tomograms revealed that actin filaments terminate at the membrane interface within a zone several hundred nanometres wide at the lamellipodium front, and yielded the first direct measurements of filament densities. Actin filament pairs were also identified as lamellipodium components and bundle precursors. These data provide a new structural basis for understanding actin-driven protrusion during cell migration.

  6. Tropomyosin prevents depolymerization of actin filaments from the pointed end.

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    Broschat, K O

    1990-12-05

    Regulation of the pointed, or slow-growing, end of actin filaments is essential to the regulation of filament length. The purpose of this study is to investigate the role of skeletal muscle tropomyosin (TM) in regulating pointed end assembly and disassembly in vitro. The effects of TM upon assembly and disassembly of actin monomers from the pointed filament end were measured using pyrenyl-actin fluorescence assays in which the barbed ends were capped by villin. Tropomyosin did not affect pointed end elongation; however, filament disassembly from the pointed end stopped in the presence of TM under conditions where control filaments disassembled within minutes. The degree of protection against depolymerization was dependent upon free TM concentration and upon filament length. When filaments were diluted to a subcritical actin concentration in TM, up to 95% of the filamentous actin remained after 24 h and did not depolymerize further. Longer actin filaments (150 monomers average length) were more effectively protected from depolymerization than short filaments (50 monomers average length). Although filaments stopped depolymerizing in the presence of TM, they were not capped as shown by elongation assays. This study demonstrates that a protein, such as TM, which binds to the side of the actin filament can prevent dissociation of monomers from the end without capping the end to elongation. In skeletal muscle, tropomyosin could prevent thin filament disassembly from the pointed end and constitute a mechanism for regulating filament length.

  7. Bundling Actin Filaments From Membranes: Some Novel Players

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    Clément eThomas

    2012-08-01

    Full Text Available Progress in live-cell imaging of the cytoskeleton has significantly extended our knowledge about the organization and dynamics of actin filaments near the plasma membrane of plant cells. Noticeably, two populations of filamentous structures can be distinguished. On the one hand, fine actin filaments which exhibit an extremely dynamic behavior basically characterized by fast polymerization and prolific severing events, a process referred to as actin stochastic dynamics. On the other hand, thick actin bundles which are composed of several filaments and which are comparatively more stable although they constantly remodel as well. There is evidence that the actin cytoskeleton plays critical roles in trafficking and signaling at both the cell cortex and organelle periphery but the exact contribution of actin bundles remains unclear. A common view is that actin bundles provide the long-distance tracks used by myosin motors to deliver their cargo to growing regions and accordingly play a particularly important role in cell polarization. However, several studies support that actin bundles are more than simple passive highways and display multiple and dynamic roles in the regulation of many processes, such as cell elongation, polar auxin transport, stomatal and chloroplast movement, and defense against pathogens. The list of identified plant actin-bundling proteins is ever expanding, supporting that plant cells shape structurally and functionally different actin bundles. Here I review the most recently characterized actin-bundling proteins, with a particular focus on those potentially relevant to membrane trafficking and/or signaling.

  8. Localization of actin filaments in internodal cells of characean algae. A scanning and transmission electron microscope study

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    1976-01-01

    New methods of visualizing subcortical actin filament bundles, or fibrils, in Characean internodes confirm that they are associated with chloroplasts at the surface facing the streaming endoplasm, and reveal that they are continuous over long distances. With the scanning electron microscope, an average of four to six fibrils are seen bridging a file of chloroplasts. The same configuration appears in negatively stained preparations of large blocks of chloroplast files connected by actin fibrils. Few branches of the subcortical fibrils are evident. These findings are discussed with respect to the mechanism of cytoplasmic streaming in Characeae. PMID:1245548

  9. Arabidopsis actin-depolymerizing factor7 severs actin filaments and regulates actin cable turnover to promote normal pollen tube growth.

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    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-09-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana actin-depolymerizing factor7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7-enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes.

  10. Histones bundle F-actin filaments and affect actin structure.

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    Blotnick, Edna; Sol, Asaf; Muhlrad, Andras

    2017-01-01

    Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

  11. Histones bundle F-actin filaments and affect actin structure.

    Directory of Open Access Journals (Sweden)

    Edna Blotnick

    Full Text Available Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

  12. Actin binding domain of filamin distinguishes posterior from anterior actin filaments in migrating Dictyostelium cells.

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    Shibata, Keitaro; Nagasaki, Akira; Adachi, Hiroyuki; Uyeda, Taro Q P

    2016-01-01

    Actin filaments in different parts of a cell interact with specific actin binding proteins (ABPs) and perform different functions in a spatially regulated manner. However, the mechanisms of those spatially-defined interactions have not been fully elucidated. If the structures of actin filaments differ in different parts of a cell, as suggested by previous in vitro structural studies, ABPs may distinguish these structural differences and interact with specific actin filaments in the cell. To test this hypothesis, we followed the translocation of the actin binding domain of filamin (ABDFLN) fused with photoswitchable fluorescent protein (mKikGR) in polarized Dictyostelium cells. When ABDFLN-mKikGR was photoswitched in the middle of a polarized cell, photoswitched ABDFLN-mKikGR rapidly translocated to the rear of the cell, even though actin filaments were abundant in the front. The speed of translocation (>3 μm/s) was much faster than that of the retrograde flow of cortical actin filaments. Rapid translocation of ABDFLN-mKikGR to the rear occurred normally in cells lacking GAPA, the only protein, other than actin, known to bind ABDFLN. We suggest that ABDFLN recognizes a certain feature of actin filaments in the rear of the cell and selectively binds to them, contributing to the posterior localization of filamin.

  13. Actin filament attachments for sustained motility in vitro are maintained by filament bundling.

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    Xiaohua Hu

    Full Text Available We reconstructed cellular motility in vitro from individual proteins to investigate how actin filaments are organized at the leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP function together to propel thin glass nanofibers or beads coated with N-WASP WCA domains. Thin nanofibers produced wide comet tails that showed more structural variation in actin filament organization than did bead substrates. During sustained motility, physiological concentrations of Mg(2+ generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg(2+ abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, or filament capping. Analysis of similar motility of microspheres showed that loss of filament bundling did not affect actin shell formation or symmetry breaking but eliminated sustained attachments between the comet tail and the particle surface. Addition of Mg(2+, Lys-Lys(2+, or fascin restored both comet tail attachment and sustained particle motility in low Mg(2+ buffers. TIRF microscopic analysis of filaments captured by WCA-coated beads in the absence of Arp2/3, profilin, and CP showed that filament bundling by polycation or fascin addition increased barbed end capture by WCA domains. We propose a model in which CP directs barbed ends toward the leading edge and polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.

  14. Mechanical Heterogeneity Favors Fragmentation of Strained Actin Filaments

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    De La Cruz, Enrique M.; Martiel, Jean-Louis; Blanchoin, Laurent

    2015-01-01

    We present a general model of actin filament deformation and fragmentation in response to compressive forces. The elastic free energy density along filaments is determined by their shape and mechanical properties, which were modeled in terms of bending, twisting, and twist-bend coupling elasticities. The elastic energy stored in filament deformation (i.e., strain) tilts the fragmentation-annealing reaction free-energy profile to favor fragmentation. The energy gradient introduces a local shear force that accelerates filament intersubunit bond rupture. The severing protein, cofilin, renders filaments more compliant in bending and twisting. As a result, filaments that are partially decorated with cofilin are mechanically heterogeneous (i.e., nonuniform) and display asymmetric shape deformations and energy profiles distinct from mechanically homogenous (i.e., uniform), bare actin, or saturated cofilactin filaments. The local buckling strain depends on the relative size of the compliant segment as well as the bending and twisting rigidities of flanking regions. Filaments with a single bare/cofilin-decorated boundary localize energy and force adjacent to the boundary, within the compliant cofilactin segment. Filaments with small cofilin clusters were predicted to fragment within the compliant cofilactin rather than at boundaries. Neglecting contributions from twist-bend coupling elasticity underestimates the energy density and gradients along filaments, and thus the net effects of filament strain to fragmentation. Spatial confinement causes compliant cofilactin segments and filaments to adopt higher deformation modes and store more elastic energy, thereby promoting fragmentation. The theory and simulations presented here establish a quantitative relationship between actin filament fragmentation thermodynamics and elasticity, and reveal how local discontinuities in filament mechanical properties introduced by regulatory proteins can modulate both the severing efficiency

  15. Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

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    Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C.; Navaza, Jorge; Dominguez, Roberto (IBS); (BBRI); (UPENN-MED)

    2013-11-20

    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 helix 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.

  16. Multiple-particle tracking measurements of heterogeneities in solutions of actin filaments and actin bundles.

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    Apgar, J; Tseng, Y; Fedorov, E; Herwig, M B; Almo, S C; Wirtz, D

    2000-08-01

    One of the central functions of actin cytoskeleton is to provide the mechanical support required for the establishment and maintenance of cell morphology. The mechanical properties of actin filament assemblies are a consequence of both the available polymer concentration and the actin regulatory proteins that direct the formation of higher order structures. By monitoring the displacement of well-dispersed microspheres via fluorescence microscopy, we probe the degree of spatial heterogeneity of F-actin gels and networks in vitro. We compare the distribution of the time-dependent mean-square displacement (MSD) of polystyrene microspheres imbedded in low- and high-concentration F-actin solutions, in the presence and absence of the F-actin-bundling protein fascin. The MSD distribution of a 2. 6-microM F-actin solution is symmetric and its standard deviation is similar to that of a homogeneous solution of glycerol of similar zero-shear viscosity. However, increasing actin concentration renders the MSD distribution wide and asymmetric, an effect enhanced by fascin. Quantitative changes in the shape of the MSD distribution correlate qualitatively with the presence of large heterogeneities in F-actin solutions produced by increased filament concentration and the presence of actin bundles, as detected by confocal microscopy. Multiple-particle tracking offers a new, quantitative method to characterize the organization of biopolymers in solution.

  17. Electrostatics control actin filament nucleation and elongation kinetics.

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    Crevenna, Alvaro H; Naredi-Rainer, Nikolaus; Schönichen, André; Dzubiella, Joachim; Barber, Diane L; Lamb, Don C; Wedlich-Söldner, Roland

    2013-04-26

    The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment.

  18. Electrostatics Control Actin Filament Nucleation and Elongation Kinetics*

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    Crevenna, Alvaro H.; Naredi-Rainer, Nikolaus; Schönichen, André; Dzubiella, Joachim; Barber, Diane L.; Lamb, Don C.; Wedlich-Söldner, Roland

    2013-01-01

    The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment. PMID:23486468

  19. Decidable and undecidable arithmetic functions in actin filament networks

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    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.

  20. Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

    NARCIS (Netherlands)

    Persson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A. Michael; Mansson, Alf; Kocer, Armagan

    2013-01-01

    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies.

  1. Swinholide A is a microfilament disrupting marine toxin that stabilizes actin dimers and severs actin filaments.

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    Bubb, M R; Spector, I; Bershadsky, A D; Korn, E D

    1995-02-24

    Swinholide A, isolated from the marien sponge Theonella swinhoei, is a 44-carbon ring dimeric dilactone macrolide with a 2-fold axis of symmetry. Recent studies have elucidated its unusual structure and shown that it has potent cytotoxic activity. We now report that swinholide A disrupts the actin cytoskeleton of cells grown in culture, sequesters actin dimers in vitro in both polymerizing and non-polymerizing buffers with a binding stoichiometry of one swinholide A molecule per actin dimer, and rapidly severs F-actin in vitro with high cooperativity. These unique properties are sufficient to explain the cytotoxicity of swinholide A. They also suggest that swinholide A might be a model for studies of the mechanism of action of F-actin severing proteins and be therapeutically useful in conditions where filamentous actin contributes to pathologically high viscosities.

  2. Microfluidics-Assisted TIRF Imaging to Study Single Actin Filament Dynamics.

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    Shekhar, Shashank

    2017-12-11

    Dynamic assembly of actin filaments is essential for many cellular processes. The rates of assembly and disassembly of actin filaments are intricately controlled by regulatory proteins that interact with the ends and the sides of filaments and with actin monomers. TIRF-based single-filament imaging techniques have proven instrumental in uncovering mechanisms of actin regulation. In this unit, novel single-filament approaches using microfluidics-assisted TIRF imaging are described. These methods can be used to grow anchored actin filaments aligned in a flow, thus making the analysis much easier as compared to open flow cell approaches. The microfluidic nature of the system also enables rapid change of biochemical conditions and allows simultaneous imaging of a large number of actin filaments. Support protocols for preparing microfluidic chambers and purifying spectrin-actin seeds used for nucleating anchored filaments are also described. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  3. INF2-mediated severing through actin filament encirclement and disruption.

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    Gurel, Pinar S; Ge, Peng; Grintsevich, Elena E; Shu, Rui; Blanchoin, Laurent; Zhou, Z Hong; Reisler, Emil; Higgs, Henry N

    2014-01-20

    INF2 is a formin protein with the unique ability to accelerate both actin polymerization and depolymerization, the latter requiring filament severing. Mutations in INF2 lead to the kidney disease focal segmental glomerulosclerosis (FSGS) and the neurological disorder Charcot-Marie Tooth disease (CMTD). Here, we compare the severing mechanism of INF2 with that of the well-studied severing protein cofilin. INF2, like cofilin, binds stoichiometrically to filament sides and severs in a manner that requires phosphate release from the filament. In contrast to cofilin, however, INF2 binds ADP and ADP-Pi filaments equally well. Furthermore, two-color total internal reflection fluorescence (TIRF) microscopy reveals that a low number of INF2 molecules, as few as a single INF2 dimer, are capable of severing, while measurable cofilin-mediated severing requires more extensive binding. Hence, INF2 is a more potent severing protein than cofilin. While a construct containing the FH1 and FH2 domains alone has some severing activity, addition of the C-terminal region increases severing potency by 40-fold, and we show that the WH2-resembling DAD motif is responsible for this increase. Helical 3D reconstruction from electron micrographs at 20 Å resolution provides a structure of filament-bound INF2, showing that the FH2 domain encircles the filament. We propose a severing model in which FH2 binding and phosphate release causes local filament deformation, allowing the DAD to bind adjacent actin protomers, further disrupting filament structure. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Direct imaging of reptation for semiflexible actin filaments.

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    Käs, J; Strey, H; Sackmann, E

    1994-03-17

    According to the reptation model of polymer diffusion, a polymer chain exhibits snake-like motion through the entangled mesh of surrounding molecules, in which the undulations of the chain are restricted to a tube-like region. The reptation model can account for many of the dynamic properties of entangled polymer solutions and melts, and has received support from observations of block copolymer diffusion across an interface; but reptative motion has not previously been imaged directly. Here we report such a direct observation of reptation, obtained by video microscopy of fluorescently labelled single, semiflexible filaments of actin in a solution of unlabelled actin filaments. From the restricted thermal undulations of these filaments we can measure the diameter of the confining tube, and we also observe the characteristic thermally excited sliding of the filament out of the end of the tube. We find that the chain self-diffusion coefficient decreases approximately linearly as the filament length increases, in agreement with the reptation model.

  5. Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites.

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    Skillman, Kristen M; Diraviyam, Karthikeyan; Khan, Asis; Tang, Keliang; Sept, David; Sibley, L David

    2011-10-01

    Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

  6. Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites.

    Directory of Open Access Journals (Sweden)

    Kristen M Skillman

    2011-10-01

    Full Text Available Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI and Plasmodium (PfACTI and PfACTII actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

  7. Stress generation and filament turnover during actin ring constriction.

    Directory of Open Access Journals (Sweden)

    Alexander Zumdieck

    Full Text Available We present a physical analysis of the dynamics and mechanics of contractile actin rings. In particular, we analyze the dynamics of ring contraction during cytokinesis in the Caenorhabditis elegans embryo. We present a general analysis of force balances and material exchange and estimate the relevant parameter values. We show that on a microscopic level contractile stresses can result from both the action of motor proteins, which cross-link filaments, and from the polymerization and depolymerization of filaments in the presence of end-tracking cross-linkers.

  8. αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors.

    Science.gov (United States)

    Hansen, Scott D; Kwiatkowski, Adam V; Ouyang, Chung-Yueh; Liu, Hongjun; Pokutta, Sabine; Watkins, Simon C; Volkmann, Niels; Hanein, Dorit; Weis, William I; Mullins, R Dyche; Nelson, W James

    2013-12-01

    The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell-cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell-cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.

  9. Force-velocity measurements of a few growing actin filaments.

    Directory of Open Access Journals (Sweden)

    Coraline Brangbour

    2011-04-01

    Full Text Available The polymerization of actin in filaments generates forces that play a pivotal role in many cellular processes. We introduce a novel technique to determine the force-velocity relation when a few independent anchored filaments grow between magnetic colloidal particles. When a magnetic field is applied, the colloidal particles assemble into chains under controlled loading or spacing. As the filaments elongate, the beads separate, allowing the force-velocity curve to be precisely measured. In the widely accepted Brownian ratchet model, the transduced force is associated with the slowing down of the on-rate polymerization. Unexpectedly, in our experiments, filaments are shown to grow at the same rate as when they are free in solution. However, as they elongate, filaments are more confined in the interspace between beads. Higher repulsive forces result from this higher confinement, which is associated with a lower entropy. In this mechanism, the production of force is not controlled by the polymerization rate, but is a consequence of the restriction of filaments' orientational fluctuations at their attachment point.

  10. Actin filaments growing against a barrier with fluctuating shape.

    Science.gov (United States)

    Sadhu, Raj Kumar; Chatterjee, Sakuntala

    2016-06-01

    We study force generation by a set of parallel actin filaments growing against a nonrigid obstacle, in the presence of an external load. The filaments polymerize by either moving the whole obstacle, with a large energy cost, or by causing local distortion in its shape which costs much less energy. The nonrigid obstacle also has local thermal fluctuations due to which its shape can change with time and we describe this using fluctuations in the height profile of a one-dimensional interface with Kardar-Parisi-Zhang dynamics. We find the shape fluctuations of the barrier strongly affect the force generation mechanism. The qualitative nature of the force-velocity curve is crucially determined by the relative time scale of filament and barrier dynamics. The height profile of the barrier also shows interesting variation with the external load. Our analytical calculations within mean-field theory show reasonable agreement with our simulation results.

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

    Science.gov (United States)

    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.

  12. Tracer diffusion through F-actin: effect of filament length and cross-linking

    OpenAIRE

    Jones, J.D.; Luby-Phelps, K.

    1996-01-01

    We have determined diffusion coefficients for small (50- to 70-nm diameter) fluorescein-thiocarbamoyl-labeled Ficoll tracers through F-actin as a function of filament length and cross-linking. fx45 was used to regulate filament length and avidin/biotinylated actin or ABP-280 was used to prepare cross-linked actin gels. We found that tracer diffusion was generally independent of filament length in agreement with theoretical predictions for diffusion through solutions of rods. However, in some ...

  13. Prokaryotic DNA segregation by an actin-like filament

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Löwe, Jan

    2002-01-01

    The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like filaments...... was ATP dependent, and depolymerization of ParM filaments required nucleotide hydrolysis. Our in vivo and in vitro results indicate that ParM polymerization generates the force required for directional movement of plasmids to opposite cell poles and that the ParR-parC complex functions as a nucleation...... point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus....

  14. Structural organization of actin in the sea urchin egg cortex: microvillar elongation in the absence of actin filament bundle formation.

    Science.gov (United States)

    Begg, D A; Rebhun, L I; Hyatt, H

    1982-04-01

    We have investigated the relationship between the formation of actin filament bundles and the elongation of microvilli (MV) after fertilization in sea urchin eggs. In a previous study (1979, J Cell Biol. 83:241-248) we demonstrated that increased pH induced the formation of actin filaments in isolated sea urchin egg cortices with the concomitant elongation of MV. On the basis of these results we suggested that increased cytoplasmic pH after fertilization causes a reorganization of cortical actin, which in turn provides the force for MV elongation. To test this hypothesis, we compared the morphology of microvilli in eggs activated with and without the release of fertilization acid. Activation of eggs in normal sea water with the calcium ionophore A23187 causes the release of fertilization acid and the elongation of MV containing core bundles of actin filaments. Eggs activated with A23187 in NA(+)-free water do not undergo normal fertilization acid release but develop elongated, flaccid MV. These MV contain an irregular network of actin filaments rather than the parallel bundles of filaments found in normal MV. The addition of 40 mM NaCl to these eggs results in the release of H(+) and the concomitant conversion of flaccid MV to erect MV containing typical core bundles of actin filaments. Identical results are obtained when 10 mM NH(4)Cl is substituted for NaCl. The induction of cytoplasmic alkalinization in unactivated eggs with NH(4)Cl does not cause either MV elongation or the formation of actin filament bundles . These results suggest that: (a) the elongation of MV is stimulated by a rise in intracellular free Ca(++) concentration; (b) actin filament bundle formation is triggered by an increase in cytoplasmic pH; and (c) the formation of actin filament bundles is not necessary for MV elongation but is required to provide rigid support for MV.

  15. Unidirectional growth of heavy meromyosin clusters along actin filaments revealed by real-time fluorescence microscopy.

    Science.gov (United States)

    Hirakawa, Rika; Nishikawa, Yusuke; Uyeda, Taro Q P; Tokuraku, Kiyotaka

    2017-12-01

    Heavy meromyosin (HMM) forms clusters along actin filaments under low ATP concentrations. Here, we observed the growth of HMM clusters under low concentrations of ATP in real time using fluorescence microscopy. When actin filaments were loosely immobilized on positively charged lipid bilayers, clusters of HMM-GFP were readily formed. Time-lapse observation revealed that the clusters grew unidirectionally. When we used a mixture of actin filaments and copolymers of actin and acto-S1dC, a chimeric protein of actin and the myosin motor domain, HMM-GFP preferentially formed clusters along the copolymers. We thus suggest that binding of myosin motors carrying ADP and Pi induces unidirectional conformational changes in actin filaments and allosterically recruits more myosin binding. In contrast, when actin filaments and copolymers were anchored to glass substrate via stable biotin-avidin linkage, higher concentrations of HMM-GFP were required to form clusters than on the lipid bilayer. Moreover, actin filaments and copolymers were not discriminated regarding preferential cluster formation. This is presumably because the myosin-induced cooperative conformational changes in actin filaments involve changes in the helical twist. Consistent with this, cofilin clusters, which supertwist the helix, were readily formed along loosely immobilized actin filaments, but not along those anchored via biotin-avidin linkage. © 2017 Wiley Periodicals, Inc.

  16. Turnover of branched actin filament networks by stochastic fragmentation with ADF/cofilin.

    Science.gov (United States)

    Reymann, Anne-Cécile; Suarez, Cristian; Guérin, Christophe; Martiel, Jean-Louis; Staiger, Christopher J; Blanchoin, Laurent; Boujemaa-Paterski, Rajaa

    2011-07-15

    Cell motility depends on the rapid assembly, aging, severing, and disassembly of actin filaments in spatially distinct zones. How a set of actin regulatory proteins that sustains actin-based force generation during motility work together in space and time remains poorly understood. We present our study of the distribution and dynamics of Arp2/3 complex, capping protein (CP), and actin-depolymerizing factor (ADF)/cofilin in actin "comet tails," using a minimal reconstituted system with nucleation-promoting factor (NPF)-coated beads. The Arp2/3 complex concentrates at nucleation sites near the beads as well as in the first actin shell. CP colocalizes with actin and is homogeneously distributed throughout the comet tail; it serves to constrain the spatial distribution of ATP/ADP-P(i) filament zones to areas near the bead. The association of ADF/cofilin with the actin network is therefore governed by kinetics of actin assembly, actin nucleotide state, and CP binding. A kinetic simulation accurately validates these observations. Following its binding to the actin networks, ADF/cofilin is able to break up the dense actin filament array of a comet tail. Stochastic severing by ADF/cofilin loosens the tight entanglement of actin filaments inside the comet tail and facilitates turnover through the macroscopic release of large portions of the aged actin network.

  17. Cysteine-rich protein 1 (CRP1 regulates actin filament bundling

    Directory of Open Access Journals (Sweden)

    Fraley Tamara S

    2005-12-01

    Full Text Available Abstract Background Cysteine-rich protein 1 (CRP1 is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein α-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified. Results CRP1 expression increased actin bundling in rat embryonic fibroblasts. Although CRP1 did not affect the bundling activity of α-actinin, CRP1 was found to stabilize the interaction of α-actinin with actin bundles and to directly bundle actin microfilaments. Using confocal and photobleaching fluorescence resonance energy transfer (FRET microscopy, we demonstrate that there are two populations of CRP1 localized along actin stress fibers, one associated through interaction with α-actinin and one that appears to bind the actin filaments directly. Consistent with a role in regulating actin filament cross-linking, CRP1 also localized to the membrane ruffles of spreading and PDGF treated fibroblasts. Conclusion CRP1 regulates actin filament bundling by directly cross-linking actin filaments and stabilizing the interaction of α-actinin with actin filament bundles.

  18. Structural organization of actin in the sea urchin egg cortex: microvillar elongation in the absence of actin filament bundle formation

    OpenAIRE

    Begg, DA; Rebhun, LI; Hyatt, H

    1982-01-01

    We have investigated the relationship between the formation of actin filament bundles and the elongation of microvilli (MV) after fertilization in sea urchin eggs. In a previous study (1979, J Cell Biol. 83:241-248) we demonstrated that increased pH induced the formation of actin filaments in isolated sea urchin egg cortices with the concomitant elongation of MV. On the basis of these results we suggested that increased cytoplasmic pH after fertilization causes a reorganization of cortical ac...

  19. Actin filaments as the fast pathways for calcium ions involved in ...

    Indian Academy of Sciences (India)

    We investigated the polyelectrolyte properties of actin filaments which are in interaction with myosin motors, basic participants in mechano-electrical transduction in the stereocilia of the inner ear. Here, we elaborated a model in which actin filaments play the role of guides or pathways for localized flow of calcium ions.

  20. Actin filaments connected with the microtubules of lipotubuloids, cytoplasmic domains rich in lipid bodies and microtubules.

    Science.gov (United States)

    Kwiatkowska, M; Popłońska, K; Stepiński, D

    2005-12-01

    Lipotubuloids, i.e., cytoplasmic domains containing an agglomeration of lipid bodies surrounded by half-unit membrane, entwined and held together by a system of microtubules, have been found in the ovary epidermis of Ornithogalum umbellatum. Ultrastructural studies demonstrated thin filaments in lipotubuloids that are probably actin filaments arranged parallel to microtubules. It is suggested that interaction of actin filaments with the microtubules determines the driving force for the rotary motion characteristic of lipotubuloids, as this movement is sensitive to cytochalasin B.

  1. Toxoplasma gondii profilin acts primarily to sequester G-actin while formins efficiently nucleate actin filament formation in vitro.

    Science.gov (United States)

    Skillman, Kristen M; Daher, Wassim; Ma, Christopher I; Soldati-Favre, Dominique; Sibley, L David

    2012-03-27

    Apicomplexan parasites employ gliding motility that depends on the polymerization of parasite actin filaments for host cell entry. Despite this requirement, parasite actin remains almost entirely unpolymerized at steady state; formation of filaments required for motility relies on a small repertoire of actin-binding proteins. Previous studies have shown that apicomplexan formins and profilin exhibit canonical functions on heterologous actins from higher eukaryotes; however, their biochemical properties on parasite actins are unknown. We therefore analyzed the impact of T. gondii profilin (TgPRF) and FH1-FH2 domains of two formin isoforms in T. gondii (TgFRM1 and TgFRM2) on the polymerization of T. gondii actin (TgACTI). Our findings based on in vitro assays demonstrate that TgFRM1-FH1-FH2 and TgFRM2-FH1-FH2 dramatically enhanced TgACTI polymerization in the absence of profilin, making them the sole protein factors known to initiate polymerization of this normally unstable actin. In addition, T. gondii formin domains were shown to both initiate polymerization and induce bundling of TgACTI filaments; however, they did not rely on TgPRF for these activities. In contrast, TgPRF sequestered TgACTI monomers, thus inhibiting polymerization even in the presence of formins. Collectively, these findings provide insight into the unusual control mechanisms of actin dynamics within the parasite.

  2. ADF/Cofilin Accelerates Actin Dynamics by Severing Filaments and Promoting Their Depolymerization at Both Ends.

    Science.gov (United States)

    Wioland, Hugo; Guichard, Berengere; Senju, Yosuke; Myram, Sarah; Lappalainen, Pekka; Jégou, Antoine; Romet-Lemonne, Guillaume

    2017-07-10

    Actin-depolymerizing factor (ADF)/cofilins contribute to cytoskeletal dynamics by promoting rapid actin filament disassembly. In the classical view, ADF/cofilin sever filaments, and capping proteins block filament barbed ends whereas pointed ends depolymerize, at a rate that is still debated. Here, by monitoring the activity of the three mammalian ADF/cofilin isoforms on individual skeletal muscle and cytoplasmic actin filaments, we directly quantify the reactions underpinning filament severing and depolymerization from both ends. We find that, in the absence of monomeric actin, soluble ADF/cofilin can associate with bare filament barbed ends to accelerate their depolymerization. Compared to bare filaments, ADF/cofilin-saturated filaments depolymerize faster from their pointed ends and slower from their barbed ends, resulting in similar depolymerization rates at both ends. This effect is isoform specific because depolymerization is faster for ADF- than for cofilin-saturated filaments. We also show that, unexpectedly, ADF/cofilin-saturated filaments qualitatively differ from bare filaments: their barbed ends are very difficult to cap or elongate, and consequently undergo depolymerization even in the presence of capping protein and actin monomers. Such depolymerizing ADF/cofilin-decorated barbed ends are produced during 17% of severing events. They are also the dominant fate of filament barbed ends in the presence of capping protein, because capping allows growing ADF/cofilin domains to reach the barbed ends, thereby promoting their uncapping and subsequent depolymerization. Our experiments thus reveal how ADF/cofilin, together with capping protein, control the dynamics of actin filament barbed and pointed ends. Strikingly, our results propose that significant barbed-end depolymerization may take place in cells. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  3. Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks.

    Science.gov (United States)

    Christensen, Jenna R; Hocky, Glen M; Homa, Kaitlin E; Morganthaler, Alisha N; Hitchcock-DeGregori, Sarah E; Voth, Gregory A; Kovar, David R

    2017-03-10

    The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks.

  4. Quantitative Analysis of Filament Branch Orientation in Listeria Actin Comet Tails.

    Science.gov (United States)

    Jasnin, Marion; Crevenna, Alvaro H

    2016-02-23

    Several bacterial and viral pathogens hijack the host actin cytoskeleton machinery to facilitate spread and infection. In particular, Listeria uses Arp2/3-mediated actin filament nucleation at the bacterial surface to generate a branched network that will help propel the bacteria. However, the mechanism of force generation remains elusive due to the lack of high-resolution three-dimensional structural data on the spatial organization of the actin mother and daughter (i.e., branch) filaments within this network. Here, we have explored the three-dimensional structure of Listeria actin tails in Xenopus laevis egg extracts using cryo-electron tomography. We found that the architecture of Listeria actin tails is shared between those formed in cells and in cell extracts. Both contained nanoscopic bundles along the plane of the substrate, where the bacterium lies, and upright filaments (also called Z filaments), both oriented tangentially to the bacterial cell wall. Here, we were able to identify actin filament intersections, which likely correspond to branches, within the tails. A quantitative analysis of putative Arp2/3-mediated branches in the actin network showed that mother filaments lie on the plane of the substrate, whereas daughter filaments have random deviations out of this plane. Moreover, the analysis revealed that branches are randomly oriented with respect to the bacterial surface. Therefore, the actin filament network does not push directly toward the surface but rather accumulates, building up stress around the Listeria surface. Our results favor a mechanism of force generation for Listeria movement where the stress is released into propulsive motion. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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 (mechanism from that of INF2. PMID:26124273

  6. Visualization of Actin Assembly and Filament Turnover by In Vitro Multicolor TIRF Microscopy.

    Science.gov (United States)

    Winterhoff, Moritz; Brühmann, Stefan; Franke, Christof; Breitsprecher, Dennis; Faix, Jan

    2016-01-01

    In response to chemotactic signals, motile cells develop a single protruding front to persistently migrate in direction of the chemotactic gradient. The highly dynamic reorganization of the actin cytoskeleton is an essential part during this process and requires the precise interplay of various actin filament assembly factors and actin-binding proteins (ABPs). Although many ABPs have been implicated in cell migration, as yet only a few of them have been well characterized concerning their specific functions during actin network assembly and disassembly. In this chapter, we describe a versatile method that allows the direct visualization of the assembly of single actin filaments and higher structures in real time by in vitro total internal reflection fluorescence microscopy (TIRF-M) using purified and fluorescently labeled actin and ABPs.

  7. Actin filaments as the fast pathways for calcium ions involved in ...

    Indian Academy of Sciences (India)

    2015-08-14

    Aug 14, 2015 ... Academic Press). Karkanevatos A 2001 Ultrastructural localization of cytoskeletal proteins in guinea-pig cochlear hair cells. M. Phil Thesis,. Keele University, UK. Lin EC and Cantiello HF 1993 A novel method to study the electrodynamic behavior of actin filaments. Evidence for cable-like properties of actin.

  8. In vitro reconstitution of dynamic microtubules interacting with actin filament networks

    NARCIS (Netherlands)

    Preciado Lopez, M.; Huber, F.; Grigoriev, Ilya; Steinmetz, M.O.; Akhmanova, Anna; Dogterom, M.; Koenderink, G.H.

    2014-01-01

    Interactions between microtubules and actin filaments (F-actin) are essential for eukaryotic cell migration, polarization, growth, and division. Although the importance of these interactions has been long recognized, the inherent complexity of the cell interior hampers a detailed mechanistic study

  9. Plant 115-kDa actin-filament bundling protein, P-115-ABP, is a homologue of plant villin and is widely distributed in cells.

    Science.gov (United States)

    Yokota, Etsuo; Vidali, Luis; Tominaga, Motoki; Tahara, Hiroshi; Orii, Hidefumi; Morizane, Yosuke; Hepler, Peter K; Shimmen, Teruo

    2003-10-01

    In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin

  10. Dynamics of actin cables in polarized growth of the filamentous fungus Aspergillus nidulans

    Directory of Open Access Journals (Sweden)

    Anna eBergs

    2016-05-01

    Full Text Available Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although specific marker proteins to visualize actin cables have been developed in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here we visualized actin cables using tropomyosin (TpmA and Lifeact fused to fluorescent proteins in Aspergillus nidulans and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules.

  11. A mathematical model of actin filament turnover for fitting FRAP data.

    Science.gov (United States)

    Halavatyi, Aliaksandr A; Nazarov, Petr V; Al Tanoury, Ziad; Apanasovich, Vladimir V; Yatskou, Mikalai; Friederich, Evelyne

    2010-03-01

    A novel mathematical model of the actin dynamics in living cells under steady-state conditions has been developed for fluorescence recovery after photobleaching (FRAP) experiments. As opposed to other FRAP fitting models, which use the average lifetime of actins in filaments and the actin turnover rate as fitting parameters, our model operates with unbiased actin association/dissociation rate constants and accounts for the filament length. The mathematical formalism is based on a system of stochastic differential equations. The derived equations were validated on synthetic theoretical data generated by a stochastic simulation algorithm adapted for the simulation of FRAP experiments. Consistent with experimental findings, the results of this work showed that (1) fluorescence recovery is a function of the average filament length, (2) the F-actin turnover and the FRAP are accelerated in the presence of actin nucleating proteins, (3) the FRAP curves may exhibit both a linear and non-linear behaviour depending on the parameters of actin polymerisation, and (4) our model resulted in more accurate parameter estimations of actin dynamics as compared with other FRAP fitting models. Additionally, we provide a computational tool that integrates the model and that can be used for interpretation of FRAP data on actin cytoskeleton.

  12. Self-assembly of actin monomers into long filaments: Brownian Dynamics simulations

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2009-01-01

    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 freemonomers and the relatively slow....../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. Theresults also show that the waiting time is governed by...

  13. F-actin-like filaments formed by plasmid segregation protein ParM

    DEFF Research Database (Denmark)

    van den Ent, Fusinita; Møller-Jensen, Jakob; Amos, Linda A.

    2002-01-01

    It was the general belief that DNA partitioning in prokaryotes is independent of a cytoskeletal structure, which in eukaryotic cells is indispensable for DNA segregation. Recently, however, immunofluorescence microscopy revealed highly dynamic, filamentous structures along the longitudinal axis...... of Escherichia coli formed by ParM, a plasmid-encoded protein required for accurate segregation of low-copy-number plasmid R1. We show here that ParM polymerizes into double helical protofilaments with a longitudinal repeat similar to filamentous actin (F-actin) and MreB filaments that maintain the cell shape...

  14. Antibodies covalently immobilized on actin filaments for fast myosin driven analyte transport.

    Directory of Open Access Journals (Sweden)

    Saroj Kumar

    Full Text Available Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm(-1. The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments.

  15. Differentiation-dependent rearrangements of actin filaments and microtubules hinder apical endocytosis in urothelial cells.

    Science.gov (United States)

    Tratnjek, Larisa; Romih, Rok; Kreft, Mateja Erdani

    2017-08-01

    During differentiation, superficial urothelial cells (UCs) of the urinary bladder form the apical surface, which is almost entirely covered by urothelial plaques containing densely packed uroplakin particles. These urothelial plaques are the main structural components of the blood-urine permeability barrier in the urinary bladder. We have shown previously that endocytosis from the apical plasma membrane decreases during urothelial cell differentiation. Here, we investigated the role of actin filament and microtubule rearrangements in apical endocytosis of differentiating UCs cells using hyperplastic and normoplastic porcine urothelial models. Partially differentiated normal porcine UCs contained actin filaments in the subapical cytoplasm, while microtubules had a net-like appearance. In highly differentiated UCs, actin filaments mostly disappeared from the subapical cytoplasm and microtubules remained as a thin layer close to the apical plasma membrane. Inhibition of actin filament formation with cytochalasin-D in partially differentiated UCs caused a decrease in apical endocytosis. Depolymerisation of microtubules with nocodazole did not prevent endocytosis of the endocytotic marker WGA into the subapical cytoplasm; however, it abolished WGA transport to endolysosomal compartments in the central cytoplasm. Cytochalasin-D or nocodazole treatment did not significantly change apical endocytosis in highly differentiated UCs. In conclusion, we showed that the physiological differentiation-dependent or chemically induced redistribution and reorganization of actin filaments and microtubules impair apical endocytosis in UCs. Importantly, reduced apical endocytosis due to cytoskeletal rearrangements in highly differentiated UCs, together with the formation of rigid urothelial plaques, reinforces the barrier function of the urothelium.

  16. Gestalt-binding of tropomyosin on actin during thin filament activation.

    Science.gov (United States)

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

    2013-08-01

    Our thesis is that thin filament function can only be fully understood and muscle regulation then elucidated if atomic structures of the thin filament are available to reveal the positions of tropomyosin on actin in all physiological states. After all, it is tropomyosin influenced by troponin that regulates myosin-crossbridge cycling on actin and therefore controls contraction in all muscles. In addition, we maintain that a complete appreciation of thin filament activation also requires that the mechanical properties of tropomyosin itself are recognized and then related to the effect of myosin-association on actin. Taking the Gestalt-binding of tropomyosin into account, coupled with our electron microscopy structures and computational chemistry, we propose a comprehensive mechanism for tropomyosin regulatory movement over the actin filament surface that explains the cooperative muscle activation process. In fact, well-known point mutations of critical amino acids on the actin-tropomyosin binding interface disrupt Gestalt-binding and are associated with a number of inherited myopathies. Moreover, dysregulation of tropomyosin may also be a factor that interferes with the gatekeeping operation of non-muscle tropomyosin in the controlling interactions of a wide variety of cellular actin-binding proteins. The clinical relevance of Gestalt-binding is discussed in articles by the Marston and the Gunning groups in this special journal issue devoted to the impact of tropomyosin on biological systems.

  17. Coupling of two non-processive myosin 5c dimers enables processive stepping along actin filaments.

    Science.gov (United States)

    Gunther, Laura K; Furuta, Ken'ya; Bao, Jianjun; Urbanowski, Monica K; Kojima, Hiroaki; White, Howard D; Sakamoto, Takeshi

    2014-05-09

    Myosin 5c (Myo5c) is a low duty ratio, non-processive motor unable to move continuously along actin filaments though it is believed to participate in secretory vesicle trafficking in vertebrate cells. Here, we measured the ATPase kinetics of Myo5c dimers and tested the possibility that the coupling of two Myo5c molecules enables processive movement. Steady-state ATPase activity and ADP dissociation kinetics demonstrated that a dimer of Myo5c-HMM (double-headed heavy meromyosin 5c) has a 6-fold lower Km for actin filaments than Myo5c-S1 (single-headed myosin 5c subfragment-1), indicating that the two heads of Myo5c-HMM increase F-actin-binding affinity. Nanometer-precision tracking analyses showed that two Myo5c-HMM dimers linked with each other via a DNA scaffold and moved processively along actin filaments. Moreover, the distance between the Myo5c molecules on the DNA scaffold is an important factor for the processive movement. Individual Myo5c molecules in two-dimer complexes move stochastically in 30-36 nm steps. These results demonstrate that two dimers of Myo5c molecules on a DNA scaffold increased the probability of rebinding to F-actin and enabled processive steps along actin filaments, which could be used for collective cargo transport in cells.

  18. Leukocytes Breach Endothelial Barriers by Insertion of Nuclear Lobes and Disassembly of Endothelial Actin Filaments

    Directory of Open Access Journals (Sweden)

    Sagi Barzilai

    2017-01-01

    Full Text Available The endothelial cytoskeleton is a barrier for leukocyte transendothelial migration (TEM. Mononuclear and polymorphonuclear leukocytes generate gaps of similar micron-scale size when squeezing through inflamed endothelial barriers in vitro and in vivo. To elucidate how leukocytes squeeze through these barriers, we co-tracked the endothelial actin filaments and leukocyte nuclei in real time. Nuclear squeezing involved either preexistent or de novo-generated lobes inserted into the leukocyte lamellipodia. Leukocyte nuclei reversibly bent the endothelial actin stress fibers. Surprisingly, formation of both paracellular gaps and transcellular pores by squeezing leukocytes did not require Rho kinase or myosin II-mediated endothelial contractility. Electron-microscopic analysis suggested that nuclear squeezing displaced without condensing the endothelial actin filaments. Blocking endothelial actin turnover abolished leukocyte nuclear squeezing, whereas increasing actin filament density did not. We propose that leukocyte nuclei must disassemble the thin endothelial actin filaments interlaced between endothelial stress fibers in order to complete TEM.

  19. Probing the flexibility of tropomyosin and its binding to filamentous actin using molecular dynamics simulations.

    Science.gov (United States)

    Zheng, Wenjun; Barua, Bipasha; Hitchcock-DeGregori, Sarah E

    2013-10-15

    Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  20. Crenactin forms actin-like double helical filaments regulated by arcadin-2

    Science.gov (United States)

    Izoré, Thierry; Kureisaite-Ciziene, Danguole; McLaughlin, Stephen H; Löwe, Jan

    2016-01-01

    The similarity of eukaryotic actin to crenactin, a filament-forming protein from the crenarchaeon Pyrobaculum calidifontis supports the theory of a common origin of Crenarchaea and Eukaryotes. Monomeric structures of crenactin and actin are similar, although their filament architectures were suggested to be different. Here we report that crenactin forms bona fide double helical filaments that show exceptional similarity to eukaryotic F-actin. With cryo-electron microscopy and helical reconstruction we solved the structure of the crenactin filament to 3.8 Å resolution. When forming double filaments, the 'hydrophobic plug' loop in crenactin rearranges. Arcadin-2, also encoded by the arcade gene cluster, binds tightly with its C-terminus to the hydrophobic groove of crenactin. Binding is reminiscent of eukaryotic actin modulators such as cofilin and thymosin β4 and arcadin-2 is a depolymeriser of crenactin filaments. Our work further supports the theory of shared ancestry of Eukaryotes and Crenarchaea. DOI: http://dx.doi.org/10.7554/eLife.21600.001 PMID:27852434

  1. Quantitative analysis of cortical actin filaments during polar body formation in starfish oocytes.

    Science.gov (United States)

    Hamaguchi, Yukihisa; Numata, Taketoshi; K Satoh, Setsuko

    2007-01-01

    Polar body formation is an extremely unequal cell division. In order to understand the mechanism of polar body formation, morphological changes at the animal pole were investigated in living oocytes of the starfish, Asterina pectinifera, and the amounts of cortical actin filaments were quantitatively estimated after staining the maturing oocytes with fluorescently-labeled phallotoxins using a computer and image-processing software. Formation of a bulge, which is presumed to become a polar body, and the anaphase separation of chromosomes occurred simultaneously. When the bulge became large, one group of chromatids moved into the bulge. The dividing furrow then formed and finally a polar body formed. Just at the time of bulge formation, the intensity of the fluorescence produced by the actin filaments at the top of the animal pole began to decrease, and subsequently the intensity at the top fell to half of the original value. On the other hand, the fluorescence intensity at the base of the bulge increased gradually. This actin accumulation at the base created a dividing furrow around the top of the animal pole as the bulge grew. Even when the polar body formation was inhibited mechanically, a similar pattern of actin deficiency and accumulation in the cortex near the animal pole was observed. This indicates that such regulation of filamentous actin can take place without bulging. Therefore, polar body formation is initiated by the bulging of the cortex weakened by actin deficiency and followed by contraction of the base of the bulge reinforced by actin accumulation.

  2. Direct actin binding to A- and B-type lamin tails and actin filament bundling by the lamin A tail.

    Science.gov (United States)

    Simon, Dan N; Zastrow, Michael S; Wilson, Katherine L

    2010-01-01

    Nuclear intermediate filament networks formed by A- and B-type lamins are major components of the nucleoskeleton. Lamins have growing links to human physiology and disease including Emery-Dreifuss muscular dystrophy (EDMD), lipodystrophy, cardiomyopathy, neuropathy, cerebellar disorders and segmental accelerated 'aging' syndromes. How lamins interact with other nucleoskeletal components, and even the identities of these other components, are open questions. Previous studies suggested lamins might bind actin. We report that the recombinant C-terminal tail domain of human A- and B-type lamins binds directly to purified actin in high-speed pelleting assays. This interaction maps to a conserved Actin Binding site (AB-1) comprising lamin A residues 461-536 in the Ig-fold domain, which are 54% identical in lamin B1. Two EDMD-causing missense mutations (R527P and L530P) in lamin A that are predicted to disrupt the Ig-fold, each reduced F-actin binding by ∼66%, whereas the surface-exposed lipodystrophy-causing R482Q mutation had no significant effect. The lamin A tail was unique among lamins in having a second actin-binding site (AB-2). This second site was mapped to lamin A tail residues 564-608, based on actin-binding results for the lamin C tail and internal deletions in the lamin A tail that cause Hutchinson-Gilford Progeria Syndrome (Δ35, Δ50) or restrictive dermopathy (Δ90). Supporting the presence of two actin-binding sites, recombinant precursor (unmodified) and mature lamin A tails (not C or B1 tails) each bundled F-actin in vitro: furthermore F-actin bundling was reduced 25-40% by the R527P, L530P, Δ35 and Δ50 mutations, and was abolished by Δ90. Unexpectedly, the mature lamin A tail bound F-actin significantly more efficiently than did the prelamin A tail; this suggested unmodified residues 647-664, unique to prelamin A, might auto-inhibit binding to actin (and potentially other partners). These biochemical results suggest direct mechanisms by which

  3. Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation.

    Science.gov (United States)

    Yamagishi, Yuka; Abe, Hiroshi

    2015-12-01

    We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation. © 2015 Yamagishi and Abe. 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).

  4. Assembly and turnover of short actin filaments by the formin INF2 and profilin.

    Science.gov (United States)

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

    2015-09-11

    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 (mechanism from that of INF2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Spatial localisation of actin filaments across developmental stages of the malaria parasite.

    Directory of Open Access Journals (Sweden)

    Fiona Angrisano

    Full Text Available Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu.

  6. TIRF microscopy analysis of human Cof1, Cof2, and ADF effects on actin filament severing and turnover.

    Science.gov (United States)

    Chin, Samantha M; Jansen, Silvia; Goode, Bruce L

    2016-04-24

    Dynamic remodeling and turnover of cellular actin networks requires actin filament severing by actin-depolymerizing factor (ADF)/Cofilin proteins. Mammals express three different ADF/Cofilins (Cof1, Cof2, and ADF), and genetic studies suggest that in vivo they perform both overlapping and unique functions. To gain mechanistic insights into their different roles, we directly compared their G-actin and F-actin binding affinities, and quantified the actin filament severing activities of human Cof1, Cof2, and ADF using in vitro total internal reflection fluorescence microscopy. All three ADF/Cofilins had similar affinities for G-actin and F-actin. However, Cof2 and ADF severed filaments much more efficiently than Cof1 at both lower and higher concentrations and using either muscle or platelet actin. Furthermore, Cof2 and ADF were more effective than Cof1 in promoting "enhanced disassembly" when combined with actin disassembly co-factors Coronin-1B and actin-interacting protein 1 (AIP1), and these differences were observed on both preformed and actively growing filaments. To probe the mechanism underlying these differences, we used multi-wavelength total internal reflection fluorescence microscopy to directly observe Cy3-Cof1 and Cy3-Cof2 interacting with actin filaments in real time during severing. Cof1 and Cof2 each bound to filaments with similar kinetics, yet Cof2 induced severing much more rapidly than Cof1, decreasing the time interval between initial binding on a filament and severing at the same location. These differences in ADF/Cofilin activities and mechanisms may be used in cells to tune filament turnover rates, which can vary widely for different actin structures. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. UNC-87, a calponin-related protein in C. elegans, antagonizes ADF/cofilin-mediated actin filament dynamics.

    Science.gov (United States)

    Yamashiro, Sawako; Gimona, Mario; Ono, Shoichiro

    2007-09-01

    Stabilization of actin filaments is critical for supporting actomyosin-based contractility and for maintaining stable cellular structures. Tropomyosin is a well-characterized ubiquitous actin stabilizer that inhibits ADF/cofilin-dependent actin depolymerization. Here, we show that UNC-87, a calponin-related Caenorhabditis elegans protein with seven calponin-like repeats, competes with ADF/cofilin for binding to actin filaments and inhibits ADF/cofilin-dependent filament severing and depolymerization in vitro. Mutations in the unc-87 gene suppress the disorganized actin phenotype in an ADF/cofilin mutant in the C. elegans body wall muscle, supporting their antagonistic roles in regulating actin stability in vivo. UNC-87 and tropomyosin exhibit synergistic effects in stabilizing actin filaments against ADF/cofilin, and direct comparison reveals that UNC-87 effectively stabilizes actin filaments at much lower concentrations than tropomyosin. However, the in vivo functions of UNC-87 and tropomyosin appear different, suggesting their distinct roles in the regulation of actomyosin assembly and cellular contractility. Our results demonstrate that actin binding via calponin-like repeats competes with ADF/cofilin-driven cytoskeletal turnover, and is critical for providing the spatiotemporal regulation of actin filament stability.

  8. Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

    Science.gov (United States)

    Kumar, Nitin; Gardel, Margaret

    Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

  9. Particle-Based Modeling of Living Actin Filaments in an Optical Trap

    Directory of Open Access Journals (Sweden)

    Thomas A. Hunt

    2016-09-01

    Full Text Available We report a coarse-grained molecular dynamics simulation study of a bundle of parallel actin filaments under supercritical conditions pressing against a loaded mobile wall using a particle-based approach where each particle represents an actin unit. The filaments are grafted to a fixed wall at one end and are reactive at the other end, where they can perform single monomer (depolymerization steps and push on a mobile obstacle. We simulate a reactive grand canonical ensemble in a box of fixed transverse area A, with a fixed number of grafted filaments N f , at temperature T and monomer chemical potential μ 1 . For a single filament case ( N f = 1 and for a bundle of N f = 8 filaments, we analyze the structural and dynamical properties at equilibrium where the external load compensates the average force exerted by the bundle. The dynamics of the bundle-moving-wall unit are characteristic of an over-damped Brownian oscillator in agreement with recent in vitro experiments by an optical trap setup. We analyze the influence of the pressing wall on the kinetic rates of (depolymerization events for the filaments. Both static and dynamic results compare reasonably well with recent theoretical treatments of the same system. Thus, we consider the proposed model as a good tool to investigate the properties of a bundle of living filaments.

  10. Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network

    National Research Council Canada - National Science Library

    Weiwei Luo; Zi Zhao Lieu; Ed Manser; Alexander D Bershadsky; Michael P Sheetz

    2016-01-01

    .... DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly...

  11. The Role of Formin Tails in Actin Nucleation, Processive Elongation, and Filament Bundling*

    Science.gov (United States)

    Vizcarra, Christina L.; Bor, Batbileg; Quinlan, Margot E.

    2014-01-01

    Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements. PMID:25246531

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

    Science.gov (United States)

    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

  13. Actin in dividing cells: contractile ring filaments bind heavy meromyosin.

    Science.gov (United States)

    Schroeder, T E

    1973-06-01

    Many microfilaments and microtubules are well preserved after glycerol-extraction of HeLa cells at room temperature (22 degrees ). Incubation in heavy meromyosin from rabbit skeletal muscle results in conspicuous and characteristic "decoration" of microfilaments of the contractile ring. Decoration is completely prevented by 10 mM ATP or 2 mM pyrophosphate, and fails to occur if heavy meromyosin is either omitted or replaced by egg albumin, a nonspecific protein. Decorated microfilaments have a substructure consisting of polarized, repeating arrowheads 27-35 nm apart. The specificity of these results strongly suggests that microfilaments of the contractile ring in HeLa cells are closely related to muscle actin. Very thin undecorated strands among the microfilaments of the contractile ring possibly represent a myosin component. These findings are discussed in terms of: the actomyosin-like properties of the contractile ring as a mechanochemical organelle that causes cell cleavage; the probable universal occurrence of actin-like protein in all dividing animal cells; and the contractile ring's combined sensitivity to cytochalasin B and its affinity for heavy meromyosin, a combination unique among microfilamentous organelles.

  14. Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

    Science.gov (United States)

    Bharat, Tanmay A M; Murshudov, Garib N; Sachse, Carsten; Löwe, Jan

    2015-07-02

    Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

  15. ATP-dependent regulation of actin monomer-filament equilibrium by cyclase-associated protein and ADF/cofilin.

    Science.gov (United States)

    Nomura, Kazumi; Ono, Shoichiro

    2013-07-15

    CAP (cyclase-associated protein) is a conserved regulator of actin filament dynamics. In the nematode Caenorhabditis elegans, CAS-1 is an isoform of CAP that is expressed in striated muscle and regulates sarcomeric actin assembly. In the present study, we report that CAS-2, a second CAP isoform in C. elegans, attenuates the actin-monomer-sequestering effect of ADF (actin depolymerizing factor)/cofilin to increase the steady-state levels of actin filaments in an ATP-dependent manner. CAS-2 binds to actin monomers without a strong preference for either ATP- or ADP-actin. CAS-2 strongly enhances the exchange of actin-bound nucleotides even in the presence of UNC-60A, a C. elegans ADF/cofilin that inhibits nucleotide exchange. UNC-60A induces the depolymerization of actin filaments and sequesters actin monomers, whereas CAS-2 reverses the monomer-sequestering effect of UNC-60A in the presence of ATP, but not in the presence of only ADP or the absence of ATP or ADP. A 1:100 molar ratio of CAS-2 to UNC-60A is sufficient to increase actin filaments. CAS-2 has two independent actin-binding sites in its N- and C-terminal halves, and the C-terminal half is necessary and sufficient for the observed activities of the full-length CAS-2. These results suggest that CAS-2 (CAP) and UNC-60A (ADF/cofilin) are important in the ATP-dependent regulation of the actin monomer-filament equilibrium.

  16. Two-Photon Correlation Spectroscopy in Single Dendritic Spines Reveals Fast Actin Filament Reorganization during Activity-Dependent Growth.

    Directory of Open Access Journals (Sweden)

    Jian-Hua Chen

    Full Text Available Two-photon fluorescence correlation spectroscopy (2P-FCS within single dendritic spines of living hippocampal pyramidal neurons was used to resolve various subpopulations of mobile F-actin during activity-dependent structural changes such as potentiation induced spine head growth. Two major classes of mobile F-actin were discovered: very dynamic and about a hundred times less dynamic F-actin. Spine head enlargement upon application of Tetraethylammonium (TEA, a protocol previously used for the chemical induction of long-term potentiation (cLTP strictly correlated to changes in the dynamics and filament numbers in the different actin filament fractions. Our observations suggest that spine enlargement is governed by a mechanism in which longer filaments are first cut into smaller filaments that cooperate with the second, increasingly dynamic shorter actin filament population to quickly reorganize and expand the actin cytoskeleton within the spine head. This process would allow a fast and efficient spine head enlargement using a major fraction of the actin filament population that was already present before spine head growth.

  17. Actin filament turnover drives leading edge growth during myelin sheath formation in the central nervous system.

    Science.gov (United States)

    Nawaz, Schanila; Sánchez, Paula; Schmitt, Sebastian; Snaidero, Nicolas; Mitkovski, Mišo; Velte, Caroline; Brückner, Bastian R; Alexopoulos, Ioannis; Czopka, Tim; Jung, Sang Y; Rhee, Jeong S; Janshoff, Andreas; Witke, Walter; Schaap, Iwan A T; Lyons, David A; Simons, Mikael

    2015-07-27

    During CNS development, oligodendrocytes wrap their plasma membrane around axons to generate multilamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic, and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/cofilin1, which mediates high F-actin turnover rates, as an essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Critical forces for actin filament buckling and force transmission influence transport in actomyosin networks

    Science.gov (United States)

    Stam, Samantha; Gardel, Margaret

    Viscoelastic networks of biopolymers coordinate the motion of intracellular objects during transport. These networks have nonlinear mechanical properties due to events such as filament buckling or breaking of cross-links. The influence of such nonlinear properties on the time and length scales of transport is not understood. Here, we use in vitro networks of actin and the motor protein myosin II to clarify how intracellular forces regulate active diffusion. We observe two transitions in the mean-squared displacement of cross-linked actin with increasing motor concentration. The first is a sharp transition from initially subdiffusive to diffusive-like motion that requires filament buckling but does not cause net contraction of the network. Further increase of the motor density produces a second transition to network rupture and ballistic actin transport. This corresponds with an increase in the correlation of motion and thus may be caused when forces propagate far enough for global motion. We conclude that filament buckling and overall network contraction require different amounts of force and produce distinct transport properties. These nonlinear transitions may act as mechanical switches that can be turned on to produce observed motion within cells.

  19. α-Catenin Is a Molecular Switch that Binds E-Cadherin-β-Catenin and Regulates Actin-Filament Assembly

    Science.gov (United States)

    Drees, Frauke; Pokutta, Sabine; Yamada, Soichiro; Nelson, W. James; Weis, William I.

    2012-01-01

    SUMMARY Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that α-catenin links the adhesion protein E-cadherin through β-catenin to the actin cytoskeleton, in the accompanying paper we report that α-catenin does not bind simultaneously to both E-cadherin-β-catenin and actin filaments. Here we demonstrate that α-catenin exists as a monomer or a homodimer with different binding properties. Monomeric α-catenin binds more strongly to E-cadherin-β-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that α-catenin is an allosteric protein. Significantly, α-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for α-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion. PMID:16325583

  20. Alpha-catenin is a molecular switch that binds E-cadherin-beta-catenin and regulates actin-filament assembly.

    Science.gov (United States)

    Drees, Frauke; Pokutta, Sabine; Yamada, Soichiro; Nelson, W James; Weis, William I

    2005-12-02

    Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that alpha-catenin links the adhesion protein E-cadherin through beta-catenin to the actin cytoskeleton, in the accompanying paper we report that alpha-catenin does not bind simultaneously to both E-cadherin-beta-catenin and actin filaments. Here we demonstrate that alpha-catenin exists as a monomer or a homodimer with different binding properties. Monomeric alpha-catenin binds more strongly to E-cadherin-beta-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that alpha-catenin is an allosteric protein. Significantly, alpha-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for alpha-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion.

  1. Microrheometry of semiflexible actin networks through enforced single-filament reptation: frictional coupling and heterogeneities in entangled networks.

    Science.gov (United States)

    Dichtl, M A; Sackmann, E

    2002-05-14

    Magnetic tweezers are applied to study the enforced motion of single actin filaments in entangled actin networks to gain insight into friction-mediated entanglement in semiflexible macromolecular networks. Magnetic beads are coupled to one chain end of test filaments, which are pulled by 5 to 20 pN force pulses through entangled solutions of nonlabeled actin, the test filaments thus acting as linear force probes of the network. The transient filament motion is analyzed by microfluorescence, and the deflection-versus-time curves of the beads are evaluated in terms of a mechanical equivalent circuit to determine viscoelastic parameters, which are then interpreted in terms of viscoelastic moduli of the network. We demonstrate that the frictional coefficient characterizing the hydrodynamic coupling of the filaments to the surrounding network is much higher than predicted by the tube model, suggesting that friction-mediated interfilament coupling plays an important role in the entanglement of non-cross-linked actin networks. Furthermore, the local tube width along the filament contour (measured in terms of the root-mean-square displacement characterizing the lateral Brownian motion of the test filament) reveals strong fluctuations that can lead to transient local pinching of filaments.

  2. Automatic Actin Filament Quantification of Osteoblasts and Their Morphometric Analysis on Microtextured Silicon-Titanium Arrays

    Directory of Open Access Journals (Sweden)

    Claudia Matschegewski

    2012-06-01

    Full Text Available Microtexturing of implant surfaces is of major relevance in the endeavor to improve biorelevant implant designs. In order to elucidate the role of biomaterial’s topography on cell physiology, obtaining quantitative correlations between cellular behavior and distinct microarchitectural properties is in great demand. Until now, the microscopically observed reorganization of the cytoskeleton on structured biomaterials has been difficult to convert into data. We used geometrically microtextured silicon-titanium arrays as a model system. Samples were prepared by deep reactive-ion etching of silicon wafers, resulting in rectangular grooves (width and height: 2 µm and cubic pillars (pillar dimensions: 2 × 2 × 5 and 5 × 5 × 5 µm; finally sputter-coated with 100 nm titanium. We focused on the morphometric analysis of MG-63 osteoblasts, including a quantification of the actin cytoskeleton. By means of our novel software FilaQuant, especially developed for automatic actin filament recognition, we were first able to quantify the alterations of the actin network dependent on the microtexture of a material surface. The cells’ actin fibers were significantly reduced in length on the pillared surfaces versus the grooved array (4–5 fold and completely reorganized on the micropillars, but without altering the orientation of cells. Our morpho-functional approach opens new possibilities for the data correlation of cell-material interactions.

  3. Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Stall, Richard; Ramos, Joseph; Kent Fulcher, F.; Patel, Yashomati M., E-mail: ympatel@uncg.edu

    2014-03-10

    Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated that MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin.

  4. Characterization of Actin Filament Dynamics during Mitosis in Wheat Protoplasts under UV-B Radiation

    Science.gov (United States)

    Chen, Huize; Han, Rong

    2016-01-01

    Enhanced ultraviolet-B (UV-B) radiation is caused by the thinning ozone and affects photosynthesis and crop yield. Recently, UV-B radiation has been considered as an environmental signal that regulates plant growth. Elucidating the downstream effectors in UV-B-triggered pathways is of particular interest. Previous studies have shown that actin filaments (AFs) play many roles during cell physiological processes. However, the underlying response of AFs to UV-B radiation remains unclear. In this study, wheat protoplasts were isolated from 7-d-old leaves. The dynamics of AFs during mitosis were observed under different treatments. The protoplasts were treated with UV-B radiation, cytochalasin B (CB) and jasplakinolide (JAS). Ph-FITC labelling results revealed typical actin filament structures in the control group; AFs were rearranged under UV-B radiation. AFs polymerized into bundles during interphase, the preprophase band (PPB) structure was destroyed during prophase, and the AFs gathered into plaques during metaphase in response to UV-B radiation. During anaphase and telophase, the distribution of AFs was dispersed. Pharmacologic experiments revealed that CB induced apoptosis and JAS induced nuclear division without cytokinesis in wheat protoplasts. These results indicated that AFs respond to UV-B radiation during mitosis, supplying evidence of UV-B signal transduction in plants. PMID:26823006

  5. Nodularin Exposure Induces SOD1 Phosphorylation and Disrupts SOD1 Co-localization with Actin Filaments

    Directory of Open Access Journals (Sweden)

    Kari E. Fladmark

    2012-12-01

    Full Text Available Apoptotic cell death is induced in primary hepatocytes by the Ser/Thr protein phosphatase inhibiting cyanobacterial toxin nodularin after only minutes of exposure. Nodularin-induced apoptosis involves a rapid development of reactive oxygen species (ROS, which can be delayed by the Ca2+/calmodulin protein kinase II inhibitor KN93. This apoptosis model provides us with a unique population of highly synchronized dying cells, making it possible to identify low abundant phosphoproteins participating in apoptosis signaling. Here, we show that nodularin induces phosphorylation and possibly also cysteine oxidation of the antioxidant Cu,Zn superoxide dismutase (SOD1, without altering enzymatic SOD1 activity. The observed post-translational modifications of SOD1 could be regulated by Ca2+/calmodulin protein kinase II. In untreated hepatocytes, a high concentration of SOD1 was found in the sub-membranous area, co-localized with the cortical actin cytoskeleton. In the early phase of nodularin exposure, SOD1 was found in high concentration in evenly distributed apoptotic buds. Nodularin induced a rapid reorganization of the actin cytoskeleton and, at the time of polarized budding, SOD1 and actin filaments no longer co-localized.

  6. Individual actin filaments in a microfluidic flow reveal the mechanism of ATP hydrolysis and give insight into the properties of profilin.

    Directory of Open Access Journals (Sweden)

    Antoine Jégou

    2011-09-01

    Full Text Available The hydrolysis of ATP associated with actin and profilin-actin polymerization is pivotal in cell motility. It is at the origin of treadmilling of actin filaments and controls their dynamics and mechanical properties, as well as their interactions with regulatory proteins. The slow release of inorganic phosphate (Pi that follows rapid cleavage of ATP gamma phosphate is linked to an increase in the rate of filament disassembly. The mechanism of Pi release in actin filaments has remained elusive for over 20 years. Here, we developed a microfluidic setup to accurately monitor the depolymerization of individual filaments and determine their local ADP-Pi content. We demonstrate that Pi release in the filament is not a vectorial but a random process with a half-time of 102 seconds, irrespective of whether the filament is assembled from actin or profilin-actin. Pi release from the depolymerizing barbed end is faster (half-time of 0.39 seconds and further accelerated by profilin. Profilin accelerates the depolymerization of both ADP- and ADP-Pi-F-actin. Altogether, our data show that during elongation from profilin-actin, the dissociation of profilin from the growing barbed end is not coupled to Pi release or to ATP cleavage on the terminal subunit. These results emphasize the potential of microfluidics in elucidating actin regulation at the scale of individual filaments.

  7. Organization and polarity of actin filament networks in cells: implications for the mechanism of myosin-based cell motility.

    Science.gov (United States)

    Cramer, L P

    1999-01-01

    Force arising from myosin activity drives a number of different types of motility in eukaryotic cells. Outside of muscle tissue, the precise mechanism of myosin-based cell motility is for the most part theoretical. A large part of the problem is that, aside from cell surface features such as lamellipodia and microvilli, relatively little is known about the structural organization of potential actin substrates for myosin in non-muscle motile cells. Several groups [Cramer, Siebert and Mitchison (1997) J. Cell Biol. 136, 1287-1305; Guild, Connelly, Shaw and Tilney (1997) J. Cell Biol. 138, 783-797; Svitkina, Verkhovsky, McQuade and Borisy (1997) J. Cell Biol. 139, 397-415] have begun to address this issue by determining actin organization throughout entire non-muscle motile cells. These studies reveal that a single motile cell comprises up to four distinct structural groups of actin organization, distinguished by differences in actin filament polarity: alternating, uniform, mixed or graded. The relative abundance and spatial location in cells of a particular actin organization varies with cell type. The existence in non-muscle motile cells of alternating-polarity actin filament bundles, the organization of muscle sarcomeres, provides direct structural evidence that some forms of motility in non-muscle cells are based on sarcomeric contraction, a recurring theory in the literature since the early days of muscle research. In this scenario, as in muscle sarcomeres, myosin generates isometric force, which is ideally suited to driving symmetrical types of motility, e.g. healing of circular wounds in coherent groups of cells. In contrast, uniform-polarity actin filament bundles and oriented meshworks in cells allow oriented movement of myosin, potentially over relatively long distances. In this simple 'transport-based' scenario, the direction in which myosin generates force is inherently polarized, and is well placed for driving asymmetrical or polarized types of motility

  8. Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

    NARCIS (Netherlands)

    Szechynska-Hebda, M.; Wedzony, M.; Dubas, E.; Kieft, H.; Lammeren, van A.A.M.

    2006-01-01

    Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation,

  9. Rabbit alveolar macrophages contain a Ca2+-sensitive, 41,000-dalton protein which reversibly blocks the "barbed" ends of actin filaments but does not sever them.

    Science.gov (United States)

    Southwick, F S; DiNubile, M J

    1986-10-25

    A 41,000-dalton Ca2+-sensitive actin-modulating protein has been purified from rabbit alveolar macrophages using ion exchange and gel filtration chromatography. On sodium dodecyl-polyacrylamide gel electrophoresis, this macrophage protein migrates more rapidly than actin and fails to cross-react with polyclonal anti-actin antibody. It has a Stokes radius of 3.0 nm and an isoelectric point of 6.6. In the presence of micromolar Ca2+ this 41,000-Da protein: reduces the viscosity of polymerized actin, nucleates actin filament assembly, causes a nearly instantaneous increase in fluorescence intensity of subcritical concentrations of pyrenyl-actin (estimated KD of the pyrene actin-macrophage protein complex, 5 X 10(-8) M), increases the critical concentration of actin by 0.65 microM (molar ratios of protein/actin, 1/100-1/10), blocks actin monomer depolymerization from the "barbed" filament ends, and does not sever preformed actin filaments. The ability of this protein to block filament ends is rapidly and completely inhibited by lowering free calcium ion concentration below the micromolar range.

  10. Visualization of prosomes (MCP-proteasomes), intermediate filament and actin networks by "instantaneous fixation" preserving the cytoskeleton.

    Science.gov (United States)

    Arcangeletti, C; Sütterlin, R; Aebi, U; De Conto, F; Missorini, S; Chezzi, C; Scherrer, K

    1997-06-01

    A new "instantaneous" fixation/extraction procedure, yielding good preservation of intermediate filaments (IFs) and actin filaments when applied at 37 degrees C, has been explored to reexamine the relationships of the prosomes to the cytoskeleton. Prosomes are protein complexes of variable subunit composition, including occasionally a small RNA, which were originally observed as trans-acting factors in untranslated mRNPs. Constituting also the proteolytic core of the 26S proteasomes, they are also called "multicatalytic proteinase (MCP) complexes" or "20S-Proteasomes." In Triton X-100-extracted epithelial, fibroblastic, and muscle cells, prosome particles were found associated primarily with the IFs (Olink-Coux et al., 1994). Application of "instantaneous fixation" has now led to the new observation that a major fraction of prosome particles, composed of specific sets of subunits, is distributed in variable proportions between the IFs and the microfilament/ stress fiber system in PtK1 epithelial cells and human fibroblasts. Electron microscopy using gold-labeled antibodies confirms this dual localization on classical whole mounts and on cells exposed to instantaneous fixation. In contrast to the resistance of the prosome-IF association, a variable fraction of the prosome particles is released from the actin cytoskeleton by Triton X-100 when applied prior to fixation. Moreover, in vitro copolymerization of prosomes with G-actin made it possible to observe "ladder-like" filamentous structures in the electron microscope, in which the prosome particles, like the "rungs of a ladder," laterally crosslink two or more actin filaments in a regular pattern. These results demonstrate that prosomes are bound in the cell not only to IFs but also to the actin cytoskeleton and, furthermore, not only within large M(r) complexes (possibly mRNPs and/or 26S proteasomes), but also directly, as individual prosome particles.

  11. A function for filamentous alpha-smooth muscle actin: Retardation of motility in human breast fibroblasts

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Petersen, Ole William

    1996-01-01

    Actins are known to comprise six mammalian isoforms of which beta- and gamma-nonmuscle actins are present in all cells, whereas alpha-smooth muscle (alpha-sm) actin is normally restricted to cells of the smooth muscle lineages. alpha-Sm actin has been found also to be expressed transiently in cer...

  12. Enhanced Depolymerization of Actin Filaments by ADF/Cofilin and Monomer Funneling by Capping Protein Cooperate to Accelerate Barbed-End Growth.

    Science.gov (United States)

    Shekhar, Shashank; Carlier, Marie-France

    2017-07-10

    A living cell's ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers, which feed polarized filament growth [1, 2]. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like actin depolymerizing factor (ADF)/cofilin and filament cappers like capping protein (CP) are essential agonists of motility [3-8], but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades [9-12]. Whereas filament fragmentation by ADF/cofilin has long been demonstrated by total internal reflection fluorescence (TIRF) [13, 14], filament depolymerization was only inferred from bulk solution assays [15]. Using microfluidics-assisted TIRF microscopy, we provide the first direct visual evidence of ADF's simultaneous severing and rapid depolymerization of individual filaments. Using a conceptually novel assay to directly visualize ADF's effect on a population of pre-assembled filaments, we demonstrate how ADF's enhanced pointed-end depolymerization causes an increase in polymerizable actin monomers, thus promoting faster barbed-end growth. We further reveal that ADF-enhanced depolymerization synergizes with CP's long-predicted "monomer funneling" [16] and leads to skyrocketing of filament growth rates, close to estimated lamellipodial rates. The "funneling model" hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few non-capped filaments transiently grow much faster [15], an effect proposed to be very important for motility. We provide the first direct microscopic evidence of monomer funneling at the scale of individual filaments. These results significantly enhance our understanding of the turnover of cellular actin networks. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  13. Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

    Science.gov (United States)

    Pal Sharma, C; Goldmann, Wolfgang H

    2004-01-01

    Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

  14. Mechanism of filament nucleation and branch stability revealed by the structure of the Arp2/3 complex at actin branch junctions.

    Directory of Open Access Journals (Sweden)

    Coumaran Egile

    2005-11-01

    Full Text Available Actin branch junctions are conserved cytoskeletal elements critical for the generation of protrusive force during actin polymerization-driven cellular motility. Assembly of actin branch junctions requires the Arp2/3 complex, upon activation, to initiate a new actin (daughter filament branch from the side of an existing (mother filament, leading to the formation of a dendritic actin network with the fast growing (barbed ends facing the direction of movement. Using genetic labeling and electron microscopy, we have determined the structural organization of actin branch junctions assembled in vitro with 1-nm precision. We show here that the activators of the Arp2/3 complex, except cortactin, dissociate after branch formation. The Arp2/3 complex associates with the mother filament through a comprehensive network of interactions, with the long axis of the complex aligned nearly perpendicular to the mother filament. The actin-related proteins, Arp2 and Arp3, are positioned with their barbed ends facing the direction of daughter filament growth. This subunit map brings direct structural insights into the mechanism of assembly and mechanical stability of actin branch junctions.

  15. The AMP-Related Kinase (AMPK) Induces Ca2+-Independent Dilation of Resistance Arteries by Interfering With Actin Filament Formation.

    Science.gov (United States)

    Schubert, Kai Michael; Qiu, Jiehua; Blodow, Stephanie; Wiedenmann, Margarethe; Lubomirov, Lubomir T; Pfitzer, Gabriele; Pohl, Ulrich; Schneider, Holger

    2017-07-07

    Decreasing Ca2+ sensitivity of vascular smooth muscle (VSM) allows for vasodilation without lowering of cytosolic Ca2+. This may be particularly important in states requiring maintained dilation, such as hypoxia. AMP-related kinase (AMPK) is an important cellular energy sensor in VSM. Regulation of Ca2+ sensitivity usually is attributed to myosin light chain phosphatase activity, but findings in non-VSM identified changes in the actin cytoskeleton. The potential role of AMPK in this setting is widely unknown. To assess the influence of AMPK on the actin cytoskeleton in VSM of resistance arteries with regard to potential Ca2+ desensitization of VSM contractile apparatus. AMPK induced a slowly developing dilation at unchanged cytosolic Ca2+ levels in potassium chloride-constricted intact arteries isolated from mouse mesenteric tissue. This dilation was not associated with changes in phosphorylation of myosin light chain or of myosin light chain phosphatase regulatory subunit. Using ultracentrifugation and confocal microscopy, we found that AMPK induced depolymerization of F-actin (filamentous actin). Imaging of arteries from LifeAct mice showed F-actin rarefaction in the midcellular portion of VSM. Immunoblotting revealed that this was associated with activation of the actin severing factor cofilin. Coimmunoprecipitation experiments indicated that AMPK leads to the liberation of cofilin from 14-3-3 protein. AMPK induces actin depolymerization, which reduces vascular tone and the response to vasoconstrictors. Our findings demonstrate a new role of AMPK in the control of actin cytoskeletal dynamics, potentially allowing for long-term dilation of microvessels without substantial changes in cytosolic Ca2+. © 2017 American Heart Association, Inc.

  16. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

    Science.gov (United States)

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W; Gelfand, Vladimir I

    2014-07-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases. © FASEB.

  17. A tridimensional view of the organization of actin filaments in the central nervous system by use of fluorescent photooxidation.

    Science.gov (United States)

    Capani, Francisco; Saraceno, Ezequiel; Boti, Valeria Romina; Aon-Bertolino, Laura; Fernández, Juan Carlos; Gato, Fernándo; Kruse, Maria Sol; Krause, Maria Sol; Giraldez, Lisandro; Ellisman, Mark H; Coirini, Héctor

    2008-04-01

    Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions.

  18. Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells.

    Science.gov (United States)

    Yamanaka, Daisuke; Akama, Takeshi; Chida, Kazuhiro; Minami, Shiro; Ito, Koichi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830-840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250-1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and plays

  19. Phosphatidylinositol 3-kinase-associated protein (PI3KAP/XB130 crosslinks actin filaments through its actin binding and multimerization properties in vitro and enhances endocytosis in HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Daisuke Yamanaka

    2016-07-01

    Full Text Available Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP/XB130 has been reported to be localized to actin filaments (F-actin and required for cell migration in thyroid carcinoma cells. Here we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830-840 was required and sufficient for localization to F-actin in NIH3T3 cells and this region directly bound to F-actin in vitro. Moreover, actin crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native (BN-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250 kDa-1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in human embryonic kidney (HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin

  20. A peek into tropomyosin binding and unfolding on the actin filament.

    Directory of Open Access Journals (Sweden)

    Abhishek Singh

    Full Text Available BACKGROUND: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. PRINCIPAL FINDINGS: Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering, and chain dissociation (analyzed using circular dichroism. CONCLUSIONS: This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest

  1. Specific types of prosomes distribute differentially between intermediate and actin filaments in epithelial, fibroblastic and muscle cells.

    Science.gov (United States)

    Arcangeletti, C; De Conto, F; Sütterlin, R; Pinardi, F; Missorini, S; Géraud, G; Aebi, U; Chezzi, C; Scherrer, K

    2000-06-01

    First observed as components of non-translated mRNP complexes, prosomes harbour RNase and several proteinase activities; they are also the central constituent of the "Multicatalytic Proteinase (MCP) complexes" or "26S-proteasomes". In two recent publications (Arcangeletti et al., 1997b; De Conto et al., 1997) we have shown, by applying a new fixation technique, that these particles distribute differentially between the cytoskeletal networks of intermediate filament (IF) and actin types; previously they had been observed exclusively on the intermediate filaments. Here we further investigate the distribution of prosomes of several types, distinct by their subunit composition, between the IF of vimentin type and the actin network, as well as in the 3D space of the cell. It is shown that subtypes of prosomes occupy specific networks of the cytoskeleton, and that this pattern is specific for a given cell type. Confocal microscopy shows that prosome cytodistribution is not homogeneous in the 3D space: in the perinuclear area they colocalize most strongly with the IF, and more peripherally with the microfilament/stress fiber system; connections may exist between the two networks. Furthermore, new data indicate that the prosome-actin interaction may participate in the molecular structure of the stress fibers.

  2. AFAP-1L1-mediated actin filaments crosslinks hinder Trypanosoma cruzi cell invasion and intracellular multiplication.

    Science.gov (United States)

    de Araújo, Karine Canuto Loureiro; Teixeira, Thaise Lara; Machado, Fabrício Castro; da Silva, Aline Alves; Quintal, Amanda Pifano Neto; da Silva, Claudio Vieira

    2016-10-01

    Host actin cytoskeleton polymerization has been shown to play an important role during Trypanosoma cruzi internalization into mammalian cell. The structure and dynamics of the actin cytoskeleton in cells are regulated by a vast number of actin-binding proteins. Here we aimed to verify the impact of AFAP-1L1, during invasion and multiplication of T. cruzi. Knocking-down AFAP-1L1 increased parasite cell invasion and intracellular multiplication. Thus, we have shown that the integrity of the machinery formed by AFAP-1L1 in actin cytoskeleton polymerization is important to hinder parasite infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. EhCoactosin stabilizes actin filaments in the protist parasite Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Nitesh Kumar

    2014-09-01

    Full Text Available Entamoeba histolytica is a protist parasite that is the causative agent of amoebiasis, and is a highly motile organism. The motility is essential for its survival and pathogenesis, and a dynamic actin cytoskeleton is required for this process. EhCoactosin, an actin-binding protein of the ADF/cofilin family, participates in actin dynamics, and here we report our studies of this protein using both structural and functional approaches. The X-ray crystal structure of EhCoactosin resembles that of human coactosin-like protein, with major differences in the distribution of surface charges and the orientation of terminal regions. According to in vitro binding assays, full-length EhCoactosin binds both F- and G-actin. Instead of acting to depolymerize or severe F-actin, EhCoactosin directly stabilizes the polymer. When EhCoactosin was visualized in E. histolytica cells using either confocal imaging or total internal reflectance microscopy, it was found to colocalize with F-actin at phagocytic cups. Over-expression of this protein stabilized F-actin and inhibited the phagocytic process. EhCoactosin appears to be an unusual type of coactosin involved in E. histolytica actin dynamics.

  4. Using cell structures to develop functional nanomaterials and nanostructures--case studies of actin filaments and microtubules.

    Science.gov (United States)

    Wu, Kevin Chia-Wen; Yang, Chung-Yao; Cheng, Chao-Min

    2014-04-25

    This article is based on the continued development of biologically relevant elements (i.e., actin filaments and microtubules in living cells) as building blocks to create functional nanomaterials and nanostructures that can then be used to manufacture nature-inspired small-scale devices or systems. Here, we summarize current progress in the field and focus specifically on processes characterized by (1) robustness and ease of use, (2) inexpensiveness, and (3) potential expandability to mass production. This article, we believe, will provide scientists and engineers with a more comprehensive understanding of how to mine biological materials and natural design features to construct functional materials and devices.

  5. Actin filaments at the leading edge of cancer cells are characterized by a high mobile fraction and turnover regulation by profilin I.

    Directory of Open Access Journals (Sweden)

    Gisela Lorente

    Full Text Available Cellular motility is the basis for cancer cell invasion and metastasis. In the case of breast cancer, the most common type of cancer among women, metastasis represents the most devastating stage of the disease. The central role of cellular motility in cancer development emphasizes the importance of understanding the specific mechanisms involved in this process. In this context, tumor development and metastasis would be the consequence of a loss or defect of the mechanisms that control cytoskeletal remodeling. Profilin I belongs to a family of small actin binding proteins that are thought to assist in actin filament elongation at the leading edge of migrating cells. Traditionally, Profilin I has been considered to be an essential control element for actin polymerization and cell migration. Expression of Profilin I is down-regulated in breast and various other cancer cells. In MDA-MB-231 cells, a breast cancer cell line, further inhibition of Profilin I expression promotes hypermotility and metastatic spread, a finding that contrasts with the proposed role of Profilin in enhancing polymerization. In this report, we have taken advantage of the fluorescence recovery after photobleaching (FRAP of GFP-actin to quantify and compare actin dynamics at the leading edge level in both cancer and non-cancer cell models. Our results suggest that (i a high level of actin dynamics (i.e., a large mobile fraction of actin filaments and a fast turnover is a common characteristic of some cancer cells; (ii actin polymerization shows a high degree of independence from the presence of extracellular growth factors; and (iii our results also corroborate the role of Profilin I in regulating actin polymerization, as raising the intracellular levels of Profilin I decreased the mobile fraction ratio of actin filaments and slowed their polymerization rate; furthermore, increased Profilin levels also led to reduced individual cell velocity and directionality.

  6. Actin filament organization during endothelial wound healing in the rabbit cornea: comparison between transcorneal freeze and mechanical scrape injuries.

    Science.gov (United States)

    Ichijima, H; Petroll, W M; Barry, P A; Andrews, P M; Dai, M; Cavanagh, H D; Jester, J V

    1993-08-01

    To compare and contrast the in vivo mechanism of wound healing after mechanical scrape and transcorneal freeze (TCF) injury in a rabbit eye model by examining changes in the cytoskeletal organization of contractile, filamentous actin (f-actin) microfilaments as relates to differences in cell migration or translocation during endothelial repair. Endothelial wound healing after mechanical scrape and transcorneal freeze injury was studied in rabbit eyes using laser scanning confocal microscopy (LSCM). Central corneal mechanical scrape injury was made using an olive tip cannula, and TCF injury was made using a 3-mm diameter stainless steel probe cooled with liquid nitrogen. Cytoskeletal changes in f-actin stained with phalloidin-FITC were observed during wound healing using LSCM. At 6 hours after mechanical scrape, the leading edge of the migrating sheet showed a decrease in the intensity of phalloidin-FITC staining, suggesting a decrease in cortical f-actin. Migrating endothelial cells in vivo did not appear to develop stress fibers after mechanical scrape, which is consistent with an in vitro cell spreading mechanism of endothelial wound healing. By 24 hours, the denuded area was almost fully resurfaced by migrating endothelial cells. On the other hand, TCF injury produced fibroblastic changes in the endothelial cells with extension and elongation of spindle-shaped endothelial cells at the leading edge by 24 hours after injury. Fibroblastic endothelial cells developed prominent actin stress-fibers, which is consistent with an in vitro cell migration mechanism of endothelial wound healing. Three days after TCF, the wounded area was resurfaced with two cell types: rough, fibroblast-like cells forming a retrocorneal fibrous membrane having prominent f-actin bundles or stress fibers with few cell-cell junctions, and smooth, polygonal-shaped endothelial cells having tight cell junctions with a cortical distribution of f-actin. After 28 days the retrocorneal fibrous

  7. Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD

    Directory of Open Access Journals (Sweden)

    Yujian Chen

    2016-06-01

    Full Text Available Actin filament-associated protein-120kD (AFAP-120 is an alternatively spliced isoform of actin filament-associated protein-110kD (AFAP-110 and contains an additional neuronal insert (NINS fragment in addition to identical domains to the AFAP-110. Unlike AFAP-110 widely expressed in tissues, AFAP-120 is specifically expressed in the nervous system and plays a role in organizing dynamic actin structures during neuronal differentiation. However, anti-AFAP-120 antibody is still commercially unavailable, and this may hinder the function research for AFAP-120. In this study, we simultaneously used the ABCpred online server and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of human AFAP-120 protein, and found that a 16aa-peptide sequence was the consensus epitope predicted by both tools. This peptide was chemically synthesized and used as an immunogen to develop polyclonal antibody against AFAP-120 (anti-AFAP-120. The sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, immunoprecipitation, and immunofluorescence assays. Our results indicated that anti-AFAP-120 could react with over-expressed and endogenous human AFAP-120 protein under denatured condition, but not with human AFAP-110 protein. Moreover, native human AFAP-120 protein could also be recognized by the anti-AFAP-120 antibody. These results suggested that the prepared anit-AFAP-120 antibody would be a useful tool for studying the biochemical and biological functions of AFAP-120.

  8. Hydrogen peroxide formation and actin filament reorganization by Cdc42 are essential for ethanol-induced in vitro angiogenesis.

    Science.gov (United States)

    Qian, Yong; Luo, Jia; Leonard, Stephen S; Harris, Gabriel K; Millecchia, Lyndell; Flynn, Daniel C; Shi, Xianglin

    2003-05-02

    This report focuses on the identification of the molecular mechanisms of ethanol-induced in vitro angiogenesis. The manipulation of angiogenesis is an important therapeutic approach for the treatment of cancer, cardiovascular diseases, and chronic inflammation. Our results showed that ethanol stimulation altered the integrity of actin filaments and increased the formation of lamellipodia and filopodia in SVEC4-10 cells. Further experiments demonstrated that ethanol stimulation increased cell migration and invasion and induced in vitro angiogenesis in SVEC4-10 cells. Mechanistically, ethanol stimulation activated Cdc42 and produced H(2)O(2) a reactive oxygen species intermediate in SVEC4-10 cells. Measuring the time course of Cdc42 activation and H(2)O(2) production upon ethanol stimulation revealed that the Cdc42 activation and the increase of H(2)O(2) lasted more than 3 h, which indicates the mechanisms of the long duration effects of ethanol on the cells. Furthermore, either overexpression of a constitutive dominant negative Cdc42 or inhibition of H(2)O(2) production abrogated the effects of ethanol on SVEC4-10 cells, indicating that both the activation of Cdc42 and the production of H(2)O(2) are essential for the actions of ethanol. Interestingly, we also found that overexpression of a constitutive dominant positive Cdc42 itself was sufficient to produce H(2)O(2) and to induce in vitro angiogenesis. Taken together, our results suggest that ethanol stimulation can induce H(2)O(2) production through the activation of Cdc42, which results in reorganizing actin filaments and increasing cell motility and in vitro angiogenesis.

  9. The vital role of actin and the intermediate filaments in the ...

    African Journals Online (AJOL)

    The cytoskeleton is a system of structural protein primarily involved in the cellular architectural maintenance, intracellular transport, cell division and movement. The classical major components of the cytoskeleton are microtubules, intermediate filaments and microfilaments. Traditionally the microtubules are involved in ...

  10. Acceleration of the sliding movement of actin filaments with the use of a non-motile mutant myosin in in vitro motility assays driven by skeletal muscle heavy meromyosin.

    Directory of Open Access Journals (Sweden)

    Kohei Iwase

    Full Text Available We examined the movement of an actin filament sliding on a mixture of normal and genetically modified myosin molecules that were attached to a glass surface. For this purpose, we used a Dictyostelium G680V mutant myosin II whose release rates of Pi and ADP were highly suppressed relative to normal myosin, leading to a significantly extended life-time of the strongly bound state with actin and virtually no motility. When the mixing ratio of G680V mutant myosin II to skeletal muscle HMM (heavy myosin was 0.01%, the actin filaments moved intermittently. When they moved, their sliding velocities were about two-fold faster than the velocity of skeletal HMM alone. Furthermore, sliding movements were also faster when the actin filaments were allowed to slide on skeletal muscle HMM-coated glass surfaces in the motility buffer solution containing G680V HMM. In this case no intermittent movement was observed. When the actin filaments used were copolymerized with a fusion protein consisting of Dictyostelium actin and Dictyostelium G680V myosin II motor domain, similar faster sliding movements were observed on skeletal muscle HMM-coated surfaces. The filament sliding velocities were about two-fold greater than the velocities of normal actin filaments. We found that the velocity of actin filaments sliding on skeletal muscle myosin molecules increased in the presence of a non-motile G680V mutant myosin motor.

  11. Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin

    Energy Technology Data Exchange (ETDEWEB)

    Coffey, Greg P.; Rajapaksa, Ranjani; Liu, Raymond; Sharpe, Orr; Kuo, Chiung-Chi; Wald Krauss, Sharon; Sagi, Yael; Davis, R. Eric; Staudt, Louis M.; Sharman, Jeff P.; Robinson, William H.; Levy, Shoshana

    2009-06-09

    CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of Blymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. Ezrin colocalized with CD81 and F-actin upon stimulation and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This may be a mechanism explaining the pleiotropic effects induced in response to stimulating cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

  12. Entamoeba histolytica: comparison of the role of receptors and filamentous actin among various endocytic processes.

    Science.gov (United States)

    Welter, B H; Powell, R R; Laughlin, R C; McGugan, G C; Bonner, M; King, A; Temesvari, L A

    2006-06-01

    Entamoeba histolytica is the causative agent of amoebic dysentery. Uptake of iron is critical for E. histolytica growth and iron-bound human transferrin (holo-transferrin) has been shown to serve as an iron source in vitro. Although a transferrin-binding protein has been identified in E. histolytica, the mechanism by which this iron source is taken up by this pathogen is not well understood. To gain insight into this process, the uptake of fluorescent-dextran, -holo-transferrin, and human red blood cells (hRBCs) was compared. Both dextran and transferrin were taken up in an apparent receptor-independent fashion as compared to hRBCs, which were taken up in a receptor-mediated fashion. Interestingly, the uptake of FITC-dextran and FITC-holo-transferrin differentially relied on an intact actin cytoskeleton suggesting that their internalization routes may be regulated independently.

  13. Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

    Directory of Open Access Journals (Sweden)

    Kefeng Fang

    Full Text Available A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II through borate diester linkages. Phenylboronic acid (PBA can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.

  14. Distribution and dynamics of the cytoskeleton in graviresponding protonemata and rhizoids of characean algae: exclusion of microtubules and a convergence of actin filaments in the apex suggest an actin-mediated gravitropism.

    Science.gov (United States)

    Braun, M; Wasteneys, G O

    1998-05-01

    The organization of the microtubule (MT) and actin microfilament (MF) cytoskeleton of tip-growing rhizoids and protonemata of characean green algae was examined by confocal laser scanning microscopy. This analysis included microinjection of fluorescent tubulin and phallotoxins into living cells, as well as immunofluorescence labeling of fixed material and fluorescent phallotoxin labeling of unfixed material. Although the morphologically very similar positively gravitropic (downward growing) rhizoids and negatively gravitropic (upward growing) protonemata show opposite gravitropic responses, no differences were detected in the extensive three-dimensional distribution of actin MFs and MTs in both cell types. Tubulin microinjection revealed that in contrast to internodal cells, fluorescent tubulin incorporated very slowly into the MT arrays of rhizoids, suggesting that MT dynamics are very different in tip-growing and diffusely expanding cells. Microtubules assembled from multiple sites at the plasma membrane in the basal zone, and a dense subapical array emerged from a diffuse nucleation centre on the basal side of the nuclear envelope. Immunofluorescence confirmed these distribution patterns but revealed more extensive MT arrays. In the basal zone, short branching clusters of MTs form two cortical hemicylinders. Subapical, axially oriented MTs are distributed in equal density throughout the peripheral and inner cytoplasm and are closely associated with subapical organelles. Microtubules, however, are completely absent from the apical zones of rhizoids and protonemata. Actin MFs were found in all zones of rhizoids and protonemata including the apex. Two files of axially oriented bundles of subcortical actin MFs and ring-like actin structures in the streaming endoplasm of rhizoids were detected in the basal zones by microinjection or rhodamine-phalloidin labeling. The subapical zone contains a dense array of mainly axially oriented actin MFs that co-distribute with

  15. Genetic deletion of ABP-120 alters the three-dimensional organization of actin filaments in Dictyostelium pseudopods

    OpenAIRE

    1995-01-01

    This study extends the observations on the defects in pseudopod formation of ABP-120+ and ABP-120- cells by a detailed morphological and biochemical analysis of the actin based cytoskeleton. Both ABP-120+ and ABP-120- cells polymerize the same amount of F-actin in response to stimulation with cAMP. However, unlike ABP-120+ cells, ABP-120- cells do not incorporate actin into the Triton X-100-insoluble cytoskeleton at 30-50 s, the time when ABP-120 is incorporated into the cytoskeleton and when...

  16. The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter

    Directory of Open Access Journals (Sweden)

    Sérgio Carvalho Leite

    2016-04-01

    Full Text Available The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings.

  17. Using the peptide BP100 as a cell-penetrating tool for the chemical engineering of actin filaments within living plant cells.

    Science.gov (United States)

    Eggenberger, Kai; Mink, Christian; Wadhwani, Parvesh; Ulrich, Anne S; Nick, Peter

    2011-01-03

    The delivery of externally applied macromolecules or nanoparticles into living cells still represents a critically limiting step before the full capabilities of chemical engineering can be explored. Molecular transporters such as cell-penetrating peptides, peptoids, and other mimetics can be used to carry cargo across the cellular membrane, but it is still difficult to find suitable sequences that operate efficiently for any particular type of cell. Here we report that BP100 (KKLFKKILKYL-amide), originally designed as an antimicrobial peptide against plant pathogens, can be employed as a fast and efficient cell-penetrating agent to transport fluorescent test cargoes into the cytosol of walled plant cells. The uptake of BP100 proceeds slightly more slowly than the endocytosis of fluorescent dextranes, but BP100 accumulates more efficiently and to much higher levels (by an order of magnitude). The entry of BP100 can be efficiently blocked by latrunculin B; this suggests that actin filaments are essential to the uptake mechanism. To test whether this novel transporter can also be used to deliver functional cargoes, we designed a fusion construct of BP100 with the actin-binding Lifeact peptide (MGVADLIKKFESISKEE). We demonstrated that the short BP100 could transport the attached 17-residue sequence quickly and efficiently into tobacco cells. The Lifeact construct retained its functionality as it successfully labeled the actin bundles that tether the nucleus in the cell center.

  18. Prosome cytodistribution relative to desmin and actin filaments in dividing C2.7 myoblasts and during myotube formation in vitro.

    Science.gov (United States)

    De Conto, F; Missorini, S; Arcangeletti, C; Pinardi, F; Montarras, D; Pinset, C; Vassy, J; Géraud, G; Chezzi, C; Scherrer, K

    1997-05-25

    Prosomes constitute the multicatalytic proteinase (MCP) core of the 26S proteasomes, but were first observed as subcomplexes of untranslated mRNP; this suggests that they play a putative role in the control of protein biosynthesis in addition to their catabolic enzymatic function. In previous investigations it was shown that some prosomes colocalize with the intermediate filaments (IF) of the cytoskeleton, of the cytokeratin type in epithelial cells, and of the vimentin type in fibroblasts. Studies on adult rat muscle carried out with prosome-specific monoclonal antibodies (p-mAbs) have shown, surprisingly, that specific types of prosomes predominantly occupy a particular zone in between the M and the Z lines of the sarcomeric structure. The data presented here show that the subunit composition of prosomes changes when the dividing C2.7 myoblasts fuse into myotubes. We show furthermore that, in dividing C2.7 myoblasts, prosomes colocalize with the desmin network as well as with that of actin, in a distribution that changes with the subunit pattern of the prosomes investigated by individual p-mAbs. Surprisingly, when myogenic fusion is induced, specific types of prosomes move first to the nuclei; later on, they reappear in the cytoplasm. There, superimposing initially onto the reorganizing desmin filaments that run from one pole of the prefusion myoblast to the other, prosomes gradually colocalize with the actin fibers in the fusing myotubes, finally forming a "pearl on a string" pattern. These results are discussed in relation to parallel observations of prosome distribution between the actin and IF networks not only in epithelial cells but also in fusing muscle satellite cells, which made it possible to monitor the complete buildup of the sarcomeric structure.

  19. Purification of recombinant human and Drosophila septin hexamers for TIRF assays of actin-septin filament assembly.

    Science.gov (United States)

    Mavrakis, M; Tsai, F-C; Koenderink, G H

    2016-01-01

    Septins are guanine nucleotide-binding proteins that are conserved from fungi to humans. Septins assemble into heterooligomeric complexes and higher-order structures with key roles in various cellular functions including cell migration and division. The mechanisms by which septins assemble and interact with other cytoskeletal elements like actin remain elusive. A powerful approach to address this question is by cell-free reconstitution of purified cytoskeletal proteins combined with fluorescence microscopy. Here, we describe procedures for the purification of recombinant Drosophila and human septin hexamers from Escherichia coli and reconstitution of actin-septin coassembly. These procedures can be used to compare assembly of Drosophila and human septins and their coassembly with the actin cytoskeleton by total internal reflection fluorescence microscopy. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Filamentous fungal-specific septin AspE is phosphorylated in vivo and interacts with actin, tubulin and other septins in the human pathogen Aspergillus fumigatus

    Energy Technology Data Exchange (ETDEWEB)

    Juvvadi, Praveen Rao; Belina, Detti [Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, NC (United States); Soderblom, Erik J.; Moseley, M. Arthur [Duke Proteomics Core Facility, Institute for Genome Sciences and Policy, Duke University, Durham, NC (United States); Steinbach, William J., E-mail: bill.steinbach@duke.edu [Division of Pediatric Infectious Diseases, Department of Pediatrics, Duke University Medical Center, Durham, NC (United States); Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC (United States)

    2013-02-15

    Highlights: ► In vivo interactions of the novel septin AspE were identified by GFP-Trap® affinity purification. ► Septins AspA, AspB, AspC and AspD interacted with AspE in vivo. ► Actin and tubulin interacted with AspE in vivo. ► AspE is phosphorylated at six serine residues in vivo. -- Abstract: We previously analyzed the differential localization patterns of five septins (AspA–E), including a filamentous fungal-specific septin, AspE, in the human pathogen Aspergillus fumigatus. Here we utilized the A. fumigatus strain expressing an AspE–EGFP fusion protein and show that this novel septin with a tubular localization pattern in hyphae is phosphorylated in vivo and interacts with the other septins, AspA, AspB, AspC and AspD. The other major proteins interacting with AspE included the cytoskeletal proteins, actin and tubulin, which may be involved in the organization and transport of the septins. This is the first report analyzing the phosphorylation of AspE and localizing the sites of phosphorylation, and opens opportunities for further analysis on the role of post-translational modifications in the assembly and organization of A. fumigatus septins. This study also describes the previously unknown interaction of AspE with the actin-microtubule network. Furthermore, the novel GFP-Trap® affinity purification method used here complements widely-used GFP localization studies in fungal systems.

  1. Myosins, Actin and Autophagy

    National Research Council Canada - National Science Library

    Kruppa, Antonina J; Kendrick‐Jones, John; Buss, Folma

    2016-01-01

    .... In this review, we will discuss the importance of actin filament dynamics for autophagy progression and highlight the distinct requirement for three classes of myosins during different stages of the autophagy pathway...

  2. Extracting the ridge set as a graph for actin filament length estimation from confocal laser scanning microscopic images

    Science.gov (United States)

    Birkholz, Harald

    2012-04-01

    The progress in image acquisition techniques provides life sciences with an abundance of data. Image analysis facilitates the assessment. The actin cytoskeleton plays a crucial role in understanding the behavior of osteoblastic cells on biomaterials. In the flat basal part of the cells, it can be visualized by confocal laser scanning microscopy. In the microscopic images, the stained cytoskeleton appears as a dense network of bright ridges which is so far only qualitatively assessed. For its quantification, there is a need for ridge detection techniques that provide a geometrical description of this graph feature. The state of the art methods do not cope with the systematical degradation by noise, unspecific luminance, and uneven dye uptake. This work presents the key part of a ridge-tracking technique, which makes more efficient use of context information, and evaluate it by its length measurement accuracy. Two random models illustrate the performance against ground truth. Representative microscopic images confirm the applicability.

  3. Monophasic Pulsed 200-μA Current Promotes Galvanotaxis With Polarization of Actin Filament and Integrin α2β1 in Human Dermal Fibroblasts.

    Science.gov (United States)

    Uemura, Mikiko; Maeshige, Noriaki; Koga, Yuka; Ishikawa-Aoyama, Michiko; Miyoshi, Makoto; Sugimoto, Masaharu; Terashi, Hiroto; Usami, Makoto

    2016-01-01

    The monophasic pulsed microcurrent is used to promote wound healing, and galvanotaxis regulation has been reported as one of the active mechanisms in the promotion of tissue repair with monophasic pulsed microcurrent. However, the optimum monophasic pulsed microcurrent parameters and intracellular changes caused by the monophasic pulsed microcurrent have not been elucidated in human dermal fibroblasts. The purpose of this study was to investigate the optimum intensity for promoting galvanotaxis and the effects of electrical stimulation on integrin α2β1 and actin filaments in human dermal fibroblasts. Human dermal fibroblasts were treated with the monophasic pulsed microcurrent of 0, 100, 200, or 300 μA for 8 hours, and cell migration and cell viability were measured 24 hours after starting monophasic pulsed microcurrent stimulation. Polarization of integrin α2β1 and lamellipodia formation were detected by immunofluorescent staining 10 minutes after starting monophasic pulsed microcurrent stimulation. The migration toward the cathode was significantly higher in the cells treated with the 200-μA monophasic pulsed microcurrent than in the controls (P microcurrent did not alter the migration ratio. The electrostimulus of 200 μA also promoted integrin α2β1 polarization and lamellipodia formation at the cathode edge (P microcurrent intensity to promote migration toward the cathode, and this intensity could regulate polarization of migration-related intracellular factors in human dermal fibroblasts.

  4. Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM).

    Science.gov (United States)

    Wang, Yilin; Kanchanawong, Pakorn

    2016-12-01

    Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and > 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale. Here, we describe the application of a three-dimensional super resolution microscopy method based on single-molecule localization microscopy and multiphase interferometry, called interferometric PhotoActivated Localization Microscopy (iPALM). This method provides nearly isotropic resolution on the order of 20 nm in all three dimensions. Protocols for visualizing the filamentous actin cytoskeleton, including specimen preparation and operation of the iPALM instrument, are described here. These protocols are also readily adaptable and instructive for the study of other ultrastructural features in cells.

  5. Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

    Science.gov (United States)

    Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1997-01-01

    Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

  6. Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia

    DEFF Research Database (Denmark)

    Rasmussen, Izabela; Pedersen, Line Hjortshøj; Byg, Luise

    2010-01-01

    Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin...

  7. Actin nucleation: spire - actin nucleator in a class of its own.

    Science.gov (United States)

    Baum, Buzz; Kunda, Patricia

    2005-04-26

    The rate limiting step for actin filament polymerisation is nucleation, and two types of nucleator have been described: the Arp2/3 complex and the formins. A recent study has now identified in Spire a third class of actin nucleator. The four short WH2 repeats within Spire bind four consecutive actin monomers to form a novel single strand nucleus for 'barbed end' actin filament elongation.

  8. Neuropsychology of subcortical dementias.

    Science.gov (United States)

    Savage, C R

    1997-12-01

    Subcortical dementias are a heterogeneous group of disorders that share primary pathology in subcortical structure and a characteristic pattern of neuropsychological impairment. This article describes the neurobiological and cognitive features of three prototypical subcortical dementias, Parkinson's disease, Huntington's disease, and progressive supranuclear palsy, concentrating of traits shared by disorders. Clinical features are also discussed, especially those which differentiate subcortical dementias from cortical dementias, such as Alzheimer's disease. The cortical-subcortical nomenclature has been criticized over the years, but it continues to provide an effective means of classifying dementia profiles in clinically and theoretically useful ways.

  9. Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex.

    Science.gov (United States)

    Serra-Peinado, Carla; Sicart, Adrià; Llopis, Juan; Egea, Gustavo

    2016-04-01

    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Comparative Biomechanics of Thick Filaments and Thin Filaments with Functional Consequences for Muscle Contraction

    Directory of Open Access Journals (Sweden)

    Mark S. Miller

    2010-01-01

    Full Text Available The scaffold of striated muscle is predominantly comprised of myosin and actin polymers known as thick filaments and thin filaments, respectively. The roles these filaments play in muscle contraction are well known, but the extent to which variations in filament mechanical properties influence muscle function is not fully understood. Here we review information on the material properties of thick filaments, thin filaments, and their primary constituents; we also discuss ways in which mechanical properties of filaments impact muscle performance.

  11. Structural Differences Explain Diverse Functions of Plasmodium Actins

    Science.gov (United States)

    Vahokoski, Juha; Martinez, Silvia Muñico; Ignatev, Alexander; Lepper, Simone; Frischknecht, Friedrich; Sidén-Kiamos, Inga; Sachse, Carsten; Kursula, Inari

    2014-01-01

    Actins are highly conserved proteins and key players in central processes in all eukaryotic cells. The two actins of the malaria parasite are among the most divergent eukaryotic actins and also differ from each other more than isoforms in any other species. Microfilaments have not been directly observed in Plasmodium and are presumed to be short and highly dynamic. We show that actin I cannot complement actin II in male gametogenesis, suggesting critical structural differences. Cryo-EM reveals that Plasmodium actin I has a unique filament structure, whereas actin II filaments resemble canonical F-actin. Both Plasmodium actins hydrolyze ATP more efficiently than α-actin, and unlike any other actin, both parasite actins rapidly form short oligomers induced by ADP. Crystal structures of both isoforms pinpoint several structural changes in the monomers causing the unique polymerization properties. Inserting the canonical D-loop to Plasmodium actin I leads to the formation of long filaments in vitro. In vivo, this chimera restores gametogenesis in parasites lacking actin II, suggesting that stable filaments are required for exflagellation. Together, these data underline the divergence of eukaryotic actins and demonstrate how structural differences in the monomers translate into filaments with different properties, implying that even eukaryotic actins have faced different evolutionary pressures and followed different paths for developing their polymerization properties. PMID:24743229

  12. Modification of actins by phallotoxins.

    Science.gov (United States)

    Wieland, T

    1977-06-01

    Phallotoxins bind to filamentous actin (F-actin) from liver or rabbit muscle with Kdiss approximately 10(-8) M. By this combination the structure of F-actin is stabilized to such an extent that it will be resistant to the depolymerizing action of 0.6 M KI and of deoxyribonuclease I, to denaturation by heat (70 degrees C), and to local ruptures caused by ultrasonication or by cytochalasin B. The structural features of phallotoxins essential for affinity to F-actin are specific.

  13. Actin turnover is required for myosin-dependent mitochondrial movements in Arabidopsis root hairs.

    Directory of Open Access Journals (Sweden)

    Maozhong Zheng

    Full Text Available BACKGROUND: Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements. METHODOLOGY/PRINCIPAL FINDINGS: We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria. CONCLUSIONS/SIGNIFICANCE: Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.

  14. In inflammatory reactive astrocytes co-cultured with brain endothelial cells nicotine-evoked Ca(2+) transients are attenuated due to interleukin-1beta release and rearrangement of actin filaments.

    Science.gov (United States)

    Delbro, D; Westerlund, A; Björklund, U; Hansson, E

    2009-03-17

    The aim of this study was to investigate whether nicotine acetylcholine receptors (nAChRs) are expressed in a more pronounced way in astrocytes co-cultured with microvascular endothelial cells from adult rat brain, compared with monocultured astrocytes, as a sign of a more developed signal transduction system. Also investigated was whether nicotine plays a role in the control of neuroinflammatory reactivity in astrocytes. Ca(2+) imaging experiments were performed using cells loaded with the Ca(2+) indicator Fura-2/AM. Co-cultured astrocytes responded to lower concentrations of nicotine than did monocultured astrocytes, indicating that they are more sensitive to nicotine. Co-cultured astrocytes also expressed a higher selectivity for alpha7nAChR and alpha4/beta2 subunits and evoked higher Ca(2+) transients compared with monocultured astrocytes. The Ca(2+) transients referred to are activators of Ca(2+)-induced Ca(2+) release from intracellular stores, both IP(3) and ryanodine, triggered by influx through receptor channels. The nicotine-induced Ca(2+) transients were attenuated after incubation with the inflammatory mediator lipopolysaccharide (LPS), but were not attenuated after incubation with the pain-transmitting peptides substance P and calcitonin-gene-related peptide, nor with the infection and inflammation stress mediator, leptin. Furthermore, LPS-induced release of interleukin-1beta (IL-1beta) measured by enzyme-linked immunosorbent assay (ELISA) was more pronounced in co-cultured versus monocultured astrocytes. Incubation with both LPS and IL-1beta further attenuated nicotine-induced Ca(2+) response. We also found that LPS and IL-1beta induced rearrangement of the F-actin filaments, as measured with an Alexa488-conjugated phalloidin probe. The rearrangements consisted of increases in ring formations and a more dispersed appearance of the filaments. These results indicate that there is a connection between a dysfunction of nicotine Ca(2+) signaling in

  15. Dendritic Actin Cytoskeleton: Structure, Functions, and Regulations

    Directory of Open Access Journals (Sweden)

    Anja Konietzny

    2017-05-01

    Full Text Available Actin is a versatile and ubiquitous cytoskeletal protein that plays a major role in both the establishment and the maintenance of neuronal polarity. For a long time, the most prominent roles that were attributed to actin in neurons were the movement of growth cones, polarized cargo sorting at the axon initial segment, and the dynamic plasticity of dendritic spines, since those compartments contain large accumulations of actin filaments (F-actin that can be readily visualized using electron- and fluorescence microscopy. With the development of super-resolution microscopy in the past few years, previously unknown structures of the actin cytoskeleton have been uncovered: a periodic lattice consisting of actin and spectrin seems to pervade not only the whole axon, but also dendrites and even the necks of dendritic spines. Apart from that striking feature, patches of F-actin and deep actin filament bundles have been described along the lengths of neurites. So far, research has been focused on the specific roles of actin in the axon, while it is becoming more and more apparent that in the dendrite, actin is not only confined to dendritic spines, but serves many additional and important functions. In this review, we focus on recent developments regarding the role of actin in dendrite morphology, the regulation of actin dynamics by internal and external factors, and the role of F-actin in dendritic protein trafficking.

  16. A Continuum Model of Actin Waves in Dictyostelium discoideum

    Science.gov (United States)

    Khamviwath, Varunyu; Hu, Jifeng; Othmer, Hans G.

    2013-01-01

    Actin waves are complex dynamical patterns of the dendritic network of filamentous actin in eukaryotes. We developed a model of actin waves in PTEN-deficient Dictyostelium discoideum by deriving an approximation of the dynamics of discrete actin filaments and combining it with a signaling pathway that controls filament branching. This signaling pathway, together with the actin network, contains a positive feedback loop that drives the actin waves. Our model predicts the structure, composition, and dynamics of waves that are consistent with existing experimental evidence, as well as the biochemical dependence on various protein partners. Simulation suggests that actin waves are initiated when local actin network activity, caused by an independent process, exceeds a certain threshold. Moreover, diffusion of proteins that form a positive feedback loop with the actin network alone is sufficient for propagation of actin waves at the observed speed of . Decay of the wave back can be caused by scarcity of network components, and the shape of actin waves is highly dependent on the filament disassembly rate. The model allows retraction of actin waves and captures formation of new wave fronts in broken waves. Our results demonstrate that a delicate balance between a positive feedback, filament disassembly, and local availability of network components is essential for the complex dynamics of actin waves. PMID:23741312

  17. Direct determination of actin polarity in the cell.

    Science.gov (United States)

    Narita, Akihiro; Mueller, Jan; Urban, Edit; Vinzenz, Marlene; Small, J Victor; Maéda, Yuichiro

    2012-06-22

    Actin filaments are polar structures that exhibit a fast growing plus end and a slow growing minus end. According to their organization in cells, in parallel or antiparallel arrays, they can serve, respectively, in protrusions or in contractions. The determination of actin filament polarity in subcellular compartments is therefore required to establish their local function. Myosin binding has previously been the sole method of polarity determination. Here, we report the first direct determination of actin filament polarity in the cell without myosin binding. Negatively stained cytoskeletons of lamellipodia were analyzed by adapting electron tomography and a single particle analysis for filamentous complexes. The results of the stained cytoskeletons confirmed that all actin filament ends facing the cell membrane were the barbed ends. In general, this approach should be applicable to the analysis of actin polarity in tomograms of the actin cytoskeleton. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Actin cortex architecture regulates cell surface tension.

    Science.gov (United States)

    Chugh, Priyamvada; Clark, Andrew G; Smith, Matthew B; Cassani, Davide A D; Dierkes, Kai; Ragab, Anan; Roux, Philippe P; Charras, Guillaume; Salbreux, Guillaume; Paluch, Ewa K

    2017-06-01

    Animal cell shape is largely determined by the cortex, a thin actin network underlying the plasma membrane in which myosin-driven stresses generate contractile tension. Tension gradients result in local contractions and drive cell deformations. Previous cortical tension regulation studies have focused on myosin motors. Here, we show that cortical actin network architecture is equally important. First, we observe that actin cortex thickness and tension are inversely correlated during cell-cycle progression. We then show that the actin filament length regulators CFL1, CAPZB and DIAPH1 regulate mitotic cortex thickness and find that both increasing and decreasing thickness decreases tension in mitosis. This suggests that the mitotic cortex is poised close to a tension maximum. Finally, using a computational model, we identify a physical mechanism by which maximum tension is achieved at intermediate actin filament lengths. Our results indicate that actin network architecture, alongside myosin activity, is key to cell surface tension regulation.

  19. LL-37 induces polymerization and bundling of actin and affects actin structure.

    Directory of Open Access Journals (Sweden)

    Asaf Sol

    Full Text Available Actin exists as a monomer (G-actin which can be polymerized to filaments F-actin that under the influence of actin-binding proteins and polycations bundle and contribute to the formation of the cytoskeleton. Bundled actin from lysed cells increases the viscosity of sputum in lungs of cystic fibrosis patients. The human host defense peptide LL-37 was previously shown to induce actin bundling and was thus hypothesized to contribute to the pathogenicity of this disease. In this work, interactions between actin and the cationic LL-37 were studied by optical, proteolytic and surface plasmon resonance methods and compared to those obtained with scrambled LL-37 and with the cationic protein lysozyme. We show that LL-37 binds strongly to CaATP-G-actin while scrambled LL-37 does not. While LL-37, at superstoichiometric LL-37/actin concentrations polymerizes MgATP-G-actin, at lower non-polymerizing concentrations LL-37 inhibits actin polymerization by MgCl(2 or NaCl. LL-37 bundles Mg-F-actin filaments both at low and physiological ionic strength when in equimolar or higher concentrations than those of actin. The LL-37 induced bundles are significantly less sensitive to increase in ionic strength than those induced by scrambled LL-37 and lysozyme. LL-37 in concentrations lower than those needed for actin polymerization or bundling, accelerates cleavage of both monomer and polymer actin by subtilisin. Our results indicate that the LL-37-actin interaction is partially electrostatic and partially hydrophobic and that a specific actin binding sequence in the peptide is responsible for the hydrophobic interaction. LL-37-induced bundles, which may contribute to the accumulation of sputum in cystic fibrosis, are dissociated very efficiently by DNase-1 and also by cofilin.

  20. FMNL2 drives actin-based protrusion and migration downstream of Cdc42

    DEFF Research Database (Denmark)

    Block, Jennifer; Breitsprecher, Dennis; Kühn, Sonja

    2012-01-01

    Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation...

  1. Mesoscopic model of actin-based propulsion.

    Directory of Open Access Journals (Sweden)

    Jie Zhu

    Full Text Available Two theoretical models dominate current understanding of actin-based propulsion: microscopic polymerization ratchet model predicts that growing and writhing actin filaments generate forces and movements, while macroscopic elastic propulsion model suggests that deformation and stress of growing actin gel are responsible for the propulsion. We examine both experimentally and computationally the 2D movement of ellipsoidal beads propelled by actin tails and show that neither of the two models can explain the observed bistability of the orientation of the beads. To explain the data, we develop a 2D hybrid mesoscopic model by reconciling these two models such that individual actin filaments undergoing nucleation, elongation, attachment, detachment and capping are embedded into the boundary of a node-spring viscoelastic network representing the macroscopic actin gel. Stochastic simulations of this 'in silico' actin network show that the combined effects of the macroscopic elastic deformation and microscopic ratchets can explain the observed bistable orientation of the actin-propelled ellipsoidal beads. To test the theory further, we analyze observed distribution of the curvatures of the trajectories and show that the hybrid model's predictions fit the data. Finally, we demonstrate that the model can explain both concave-up and concave-down force-velocity relations for growing actin networks depending on the characteristic time scale and network recoil. To summarize, we propose that both microscopic polymerization ratchets and macroscopic stresses of the deformable actin network are responsible for the force and movement generation.

  2. Non-Straub type actin from molluscan catch muscle

    Energy Technology Data Exchange (ETDEWEB)

    Shelud' ko, Nikolay S., E-mail: sheludko@stl.ru; Girich, Ulyana V.; Lazarev, Stanislav S.; Vyatchin, Ilya G.

    2016-05-27

    We have developed a method of obtaining natural actin from smooth muscles of the bivalves on the example of the Crenomytilus grayanus catch muscle. The muscles were previously rigorized to prevent a loss of thin filaments during homogenization and washings. Thin filaments were isolated with a low ionic strength solution in the presence of ATP and sodium pyrophosphate. Surface proteins of thin filaments-tropomyosin, troponin, calponin and some minor actin-binding proteins-were dissociated from actin filaments by increasing the ionic strength to 0.6 M KCL. Natural fibrillar actin obtained in that way depolymerizes easily in low ionic strength solutions commonly used for the extraction of Straub-type actin from acetone powder. Purification of natural actin was carried out by the polymerization–depolymerization cycle. The content of inactivated actin remaining in the supernatant is much less than at a similar purification of Straub-type actin. A comparative investigation was performed between the natural mussel actin and the Straub-type rabbit skeletal actin in terms of the key properties of actin: polymerization, activation of Mg-ATPase activity of myosin, and the electron-microscopic structure of actin polymers. -- Highlights: •We developed method of repolymerizable invertebrate smooth muscle actin obtaining. •Our method does not involve use of denaturating agents, which could modify proteins. •Viscosity and polymerization rate of actin, gained that way, is similar to Straub one. •Electron microscopy showed that repolymerized mussel actin is similar to Straub one. •Repolymerized mussel actin has greater ATPase activating capacity, than Straub actin.

  3. Purification of Actin from Fission Yeast Schizosaccharomyces pombe and Characterization of Functional Differences from Muscle Actin*

    Science.gov (United States)

    Ti, Shih-Chieh; Pollard, Thomas D.

    2011-01-01

    Fission yeast Schizosaccharomyces pombe is an important genetic model organism for studying the mechanisms of endocytosis and cytokinesis. However, most work on the biochemical properties of fission yeast actin-binding proteins has been done with skeletal muscle actin for matters of convenience. When simulations of mathematical models of the mechanism of endocytosis were compared with events in live cells, some of the reactions appeared to be much faster than observed in biochemical experiments with muscle actin. Here, we used gelsolin affinity chromatography to purify actin from fission yeast. S. pombe actin shares many properties with skeletal muscle actin but has higher intrinsic nucleotide exchange rate, faster trimer nucleus formation, faster phosphate dissociation rate from polymerized actin, and faster nucleation of actin filaments with Arp2/3 complex. These properties close the gap between the biochemistry and predictions made by mathematical models of endocytosis in S. pombe cells. PMID:21148484

  4. Actin filament-associated protein 1 (AFAP-1) is a key mediator in inflammatory signaling-induced rapid attenuation of intrinsic P-gp function in human brain capillary endothelial cells.

    Science.gov (United States)

    Hoshi, Yutaro; Uchida, Yasuo; Tachikawa, Masanori; Ohtsuki, Sumio; Terasaki, Tetsuya

    2017-04-01

    The purpose of this study was to identify regulatory molecule(s) involved in the inflammatory signaling-induced decrease in P-glycoprotein (P-gp) efflux function at the blood-brain barrier (BBB) that may occur in brain diseases. We confirmed that in vivo P-gp efflux activity at the BBB was decreased without any change in P-gp protein expression level in a mouse model of acute inflammation induced by 3 mg/kg lipopolysaccharide. In a human BBB model cell line (human brain capillary endothelial cells; hCMEC/D3), 1-h treatment with 10 ng/mL tumor necrosis factor-α (TNF-α; an inflammatory mediator) rapidly reduced P-gp efflux activity, but had no effect on P-gp protein expression level. To clarify the non-transcriptional mechanism that causes the decrease in intrinsic efflux activity of P-gp in acute inflammation, we applied comprehensive quantitative phosphoproteomics to compare hCMEC/D3 cells treated with TNF-α and vehicle (control). Actin filament-associated protein-1 (AFAP-1), MAPK1, and transcription factor AP-1 (AP-1) were significantly phosphorylated in TNF-α-treated cells, and were selected as candidate proteins. In validation experiments, knockdown of AFAP-1 expression blocked the reduction in P-gp efflux activity by TNF-α treatment, whereas inhibition of MAPK function or knockdown of AP-1 expression did not. Quantitative targeted absolute proteomics revealed that the reduction in P-gp activity by TNF-α did not require any change in P-gp protein expression levels in the plasma membrane. Our results demonstrate that AFAP-1 is a key mediator in the inflammatory signaling-induced, translocation-independent rapid attenuation of P-gp efflux activity in human brain capillary endothelial cells. © 2017 International Society for Neurochemistry.

  5. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.

    Science.gov (United States)

    Spracklen, Andrew J; Fagan, Tiffany N; Lovander, Kaylee E; Tootle, Tina L

    2014-09-15

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools--Utrophin, Lifeact, and F-tractin--for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool

  6. Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis

    NARCIS (Netherlands)

    Sampathkumar, A.; Lindeboom, J.J.; Debolt, S.; Gutierrez, R.; Ehrhardt, D.W.; Ketelaar, T.; Persson, S.

    2011-01-01

    In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic

  7. Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

    Science.gov (United States)

    Srivastava, Jyoti; Barber, Diane

    2008-01-01

    The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape. Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling. A number of proteins can bind to the actin cytoskeleton. The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin. The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin. Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays. PMID:19066573

  8. Single Actin Bundle Rheology.

    Science.gov (United States)

    Strehle, Dan; Mollenkopf, Paul; Glaser, Martin; Golde, Tom; Schuldt, Carsten; Käs, Josef A; Schnauß, Jörg

    2017-10-24

    Bundled actin structures play an essential role in the mechanical response of the actin cytoskeleton in eukaryotic cells. Although responsible for crucial cellular processes, they are rarely investigated in comparison to single filaments and isotropic networks. Presenting a highly anisotropic structure, the determination of the mechanical properties of individual bundles was previously achieved through passive approaches observing bending deformations induced by thermal fluctuations. We present a new method to determine the bending stiffness of individual bundles, by measuring the decay of an actively induced oscillation. This approach allows us to systematically test anisotropic, bundled structures. Our experiments revealed that thin, depletion force-induced bundles behave as semiflexible polymers and obey the theoretical predictions determined by the wormlike chain model. Thickening an individual bundle by merging it with other bundles enabled us to study effects that are solely based on the number of involved filaments. These thicker bundles showed a frequency-dependent bending stiffness, a behavior that is inconsistent with the predictions of the wormlike chain model. We attribute this effect to internal processes and give a possible explanation with regard to the wormlike bundle theory.

  9. Single Actin Bundle Rheology

    Directory of Open Access Journals (Sweden)

    Dan Strehle

    2017-10-01

    Full Text Available Bundled actin structures play an essential role in the mechanical response of the actin cytoskeleton in eukaryotic cells. Although responsible for crucial cellular processes, they are rarely investigated in comparison to single filaments and isotropic networks. Presenting a highly anisotropic structure, the determination of the mechanical properties of individual bundles was previously achieved through passive approaches observing bending deformations induced by thermal fluctuations. We present a new method to determine the bending stiffness of individual bundles, by measuring the decay of an actively induced oscillation. This approach allows us to systematically test anisotropic, bundled structures. Our experiments revealed that thin, depletion force-induced bundles behave as semiflexible polymers and obey the theoretical predictions determined by the wormlike chain model. Thickening an individual bundle by merging it with other bundles enabled us to study effects that are solely based on the number of involved filaments. These thicker bundles showed a frequency-dependent bending stiffness, a behavior that is inconsistent with the predictions of the wormlike chain model. We attribute this effect to internal processes and give a possible explanation with regard to the wormlike bundle theory.

  10. Dynamics of an F-actin aggresome generated by the actin-stabilizing toxin jasplakinolide.

    Science.gov (United States)

    Lázaro-Diéguez, Francisco; Aguado, Carmen; Mato, Eugenia; Sánchez-Ruíz, Yován; Esteban, Inmaculada; Alberch, Jordi; Knecht, Erwin; Egea, Gustavo

    2008-05-01

    In this study, we report the formation of several cytoplasmic inclusion bodies composed of filamentous actin (F-actin) and generated by experimental treatments using depolymerizing or stabilizing actin toxins in neuronal and non-neuronal mammalian cell lines. The actin-stabilizing toxin jasplakinolide (Jpk) induced, in a microtubule-dependent manner, a single, large F-actin aggregate, which contained beta- and gamma-actin, ADF/cofilin, cortactin, and the actin nucleator Arp2/3. This aggregate was tightly associated with the Golgi complex and mitochondria, and was surrounded by vimentin intermediate filaments, microtubules and MAP4. Therefore, the Jpk-induced single, large F-actin aggregate fits the established criteria for being considered an aggresome. Lysosomes and/or autophagic vacuoles, proteasomes and microtubules were found to directly participate in the dissolution of this F-actin aggresome. Finally, the model reported here is simple, highly reproducible and reversible, and it provides an opportunity to test pharmacological agents that interfere with the formation, maintenance and/or disappearance of F-actin-enriched pathological inclusion bodies.

  11. Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation

    DEFF Research Database (Denmark)

    Fritzsche, Marco; Fernandes, Ricardo A.; Chang, Veronica T.

    2017-01-01

    T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced...... optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse...... lengths of two differently sized filamentous actin populations, wherein forminmediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact...

  12. An invertebrate smooth muscle with striated muscle myosin filaments

    Science.gov (United States)

    Sulbarán, Guidenn; Alamo, Lorenzo; Pinto, Antonio; Márquez, Gustavo; Méndez, Franklin; Padrón, Raúl; Craig, Roger

    2015-01-01

    Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and actin filaments are in register, whereas in smooth muscles, the actin filaments are attached to dense bodies and the myosin and actin filaments are out of register. The structure of the filaments in smooth muscles is also different from that in striated muscles. Here we have studied the structure of myosin filaments from the smooth muscles of the human parasite Schistosoma mansoni. We find, surprisingly, that they are indistinguishable from those in an arthropod striated muscle. This structural similarity is supported by sequence comparison between the schistosome myosin II heavy chain and known striated muscle myosins. In contrast, the actin filaments of schistosomes are similar to those of smooth muscles, lacking troponin-dependent regulation. We conclude that schistosome muscles are hybrids, containing striated muscle-like myosin filaments and smooth muscle-like actin filaments in a smooth muscle architecture. This surprising finding has broad significance for understanding how muscles are built and how they evolved, and challenges the paradigm that smooth and striated muscles always have distinctly different components. PMID:26443857

  13. The unusual dynamics of parasite actin result from isodesmic polymerization.

    Science.gov (United States)

    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, 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. Polymerization kinetics of actin in T. gondii 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 actin filaments in T. gondii 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.

  14. Deconstructing the cadherin-catenin-actin complex.

    Science.gov (United States)

    Yamada, Soichiro; Pokutta, Sabine; Drees, Frauke; Weis, William I; Nelson, W James

    2005-12-02

    Spatial and functional organization of cells in tissues is determined by cell-cell adhesion, thought to be initiated through trans-interactions between extracellular domains of the cadherin family of adhesion proteins, and strengthened by linkage to the actin cytoskeleton. Prevailing dogma is that cadherins are linked to the actin cytoskeleton through beta-catenin and alpha-catenin, although the quaternary complex has never been demonstrated. We test this hypothesis and find that alpha-catenin does not interact with actin filaments and the E-cadherin-beta-catenin complex simultaneously, even in the presence of the actin binding proteins vinculin and alpha-actinin, either in solution or on isolated cadherin-containing membranes. Direct analysis in polarized cells shows that mobilities of E-cadherin, beta-catenin, and alpha-catenin are similar, regardless of the dynamic state of actin assembly, whereas actin and several actin binding proteins have higher mobilities. These results suggest that the linkage between the cadherin-catenin complex and actin filaments is more dynamic than previously appreciated.

  15. Mechanics model for actin-based motility.

    Science.gov (United States)

    Lin, Yuan

    2009-02-01

    We present here a mechanics model for the force generation by actin polymerization. The possible adhesions between the actin filaments and the load surface, as well as the nucleation and capping of filament tips, are included in this model on top of the well-known elastic Brownian ratchet formulation. A closed form solution is provided from which the force-velocity relationship, summarizing the mechanics of polymerization, can be drawn. Model predictions on the velocity of moving beads driven by actin polymerization are consistent with experiment observations. This model also seems capable of explaining the enhanced actin-based motility of Listeria monocytogenes and beads by the presence of Vasodilator-stimulated phosphoprotein, as observed in recent experiments.

  16. Morphological changes in liposomes caused by polymerization of encapsulated actin and spontaneous formation of actin bundles.

    OpenAIRE

    Miyata, H.; Hotani, H.

    1992-01-01

    Spherical giant liposomes that had encapsulated skeletal-muscle G-actin were made by swelling a dried lipid mixture of dimyristoyl phosphatidylcholine/cardiolipin, 1:1 (wt/wt), in a solution of G-actin/CaCl2 at 0 degree C. Polymerization of the encapsulated G-actin into actin filaments was achieved by raising the temperature to 30 degrees C. We observed the subsequent shape changes of the liposomes by dark-field and differential interference-contrast light microscopy. After approximately 40 m...

  17. Probing the phalloidin binding site of actin.

    Science.gov (United States)

    Faulstich, H; Zobeley, S; Heintz, D; Drewes, G

    1993-03-08

    Phallotoxins form tight complexes with filamentous actin and stabilize the polymer against shearing stress. In the present study a phalloidin derivative containing a thiol-capturing moiety was prepared and reacted with single thiol groups of monomeric muscle actin. Sites of attachment in the protein were Cys-374 next to the C-terminus and Cys-10, close to the N-terminus; the latter was recently shown to be uncovered during a slow but reversible conformational transition occurring in ADP-G-actin. Phalloidin bound to Cys-374 stabilizes filaments against shearing stress almost as effectively as free phalloidin, indicating that the phalloidin binding site cannot be far from the C-terminus of actin. Stabilization was also achieved when the phalloidin reagent was added to F-actin, however, the subsequent formation of a covalent linkage with Cys-374 was not observed, most likely due to a restricted mobility of the reactants. In contrast to the efficient stabilization of filaments by phalloidin linked to Cys-374 a destabilizing effect was observed when phalloidin was attached to Cys-10. It appears that phalloidin located close to the N-terminus is unable to bind to the normal binding site in its own filament. Pronounced gelification of this actin derivative suggests that the toxin is able to mediate crosslinking with neighbouring filaments. From these results we conclude that the phalloidin binding site of actin is distant from the N-terminus, but close to the C-terminus. Furthermore, the data provide evidence that binding of phalloidin reduces the mobility of the C-terminus.

  18. Force-induced dynamical properties of multiple cytoskeletal filaments are distinct from that of single filaments

    CERN Document Server

    Das, Dipjyoti; Padinhateeri, Ranjith

    2014-01-01

    How cytoskeletal filaments collectively undergo growth and shrinkage is an intriguing question. Collective properties of multiple bio-filaments (actin or microtubules) undergoing hydrolysis, have not been studied extensively earlier, within simple theoretical frameworks. In this paper, we show that collective properties of multiple filaments under force are very distinct from the properties of a single filament under similar conditions -- these distinctions manifest as follows: (i) the collapse time during collective catastrophe for a multifilament system is much larger than that of a single filament with the same average length, (ii) force-dependence of the cap-size distribution of multiple filaments are quantitatively different from that of single filament, (iii) the diffusion constant associated with the system length fluctuations is distinct for multiple filaments, (iv) switching dynamics of multiple filaments between capped and uncapped states and the fluctuations therein are also distinct. We build a un...

  19. Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis

    Science.gov (United States)

    Spracklen, Andrew J.; Kelpsch, Daniel J.; Chen, Xiang; Spracklen, Cassandra N.; Tootle, Tina L.

    2014-01-01

    Prostaglandins (PGs)—lipid signals produced downstream of cyclooxygenase (COX) enzymes—regulate actin dynamics in cell culture and platelets, but their roles during development are largely unknown. Here we define a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton—temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). In addition, loss of Pxt results in extensive early actin remodeling, including actin filaments and aggregates, within the posterior nurse cells of S9 follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts-RC) and Villin (Quail), an actin bundler, localize to all early actin structures, whereas Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to a model in which PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, whereas at S10B, it promotes Ena-dependent actin remodeling. PMID:24284900

  20. In vivo dynamics of the cortical actin network revealed by fast-scanning atomic force microscopy.

    Science.gov (United States)

    Zhang, Yanshu; Yoshida, Aiko; Sakai, Nobuaki; Uekusa, Yoshitsugu; Kumeta, Masahiro; Yoshimura, Shige H

    2017-08-01

    Together with lamellipodia and stress fibers, a dynamic network of actin filaments in the cell cortex plays a major role in the maintenance of cell morphology and motility. In contrast to lamellipodia, which have been well studied in various motile cells, the dynamics of actin filaments in the cell cortex have not yet been clarified due to a lack of proper imaging techniques. Here, we utilized high-speed atomic force microscopy for live-cell imaging and analyzed cortical actin dynamics in living cells. We successfully measured the polymerization rate and the frequency of filament synthesis in living COS-7 cells, and examined the associated effects of various inhibitors and actin-binding proteins. Actin filaments are synthesized beneath the plasma membrane and eventually descend into the cytoplasm. The inhibitors, cytochalasin B inhibited the polymerization, while jasplakinolide, inhibited the turnover of actin filaments as well as descension of the newly synthesized filaments, suggesting that actin polymerization near the membrane drives turnover of the cortical actin meshwork. We also determined how actin turnover is maintained and regulated by the free G-actin pool and G-actin binding proteins such as profilin and thymosin β4, and found that only a small amount of free G-actin was present in the cortex. Finally, we analyzed several different cell types, and found that the mesh size and the orientation of actin filaments were highly divergent, indicating the involvement of various actin-binding proteins in the maintenance and regulation of cortical actin architecture in each cell type. © The Author 2017. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Actin dynamics and the elasticity of cytoskeletal networks

    Directory of Open Access Journals (Sweden)

    2009-09-01

    Full Text Available The structural integrity of a cell depends on its cytoskeleton, which includes an actin network. This network is transient and depends upon the continual polymerization and depolymerization of actin. The degradation of an actin network, and a corresponding reduction in cell stiffness, can indicate the presence of disease. Numerical simulations will be invaluable for understanding the physics of these systems and the correlation between actin dynamics and elasticity. Here we develop a model that is capable of generating actin network structures. In particular, we develop a model of actin dynamics which considers the polymerization, depolymerization, nucleation, severing, and capping of actin filaments. The structures obtained are then fed directly into a mechanical model. This allows us to qualitatively assess the effects of changing various parameters associated with actin dynamics on the elasticity of the material.

  2. Organization of the actin cytoskeleton of resting and activated platelets in suspension.

    Science.gov (United States)

    Escolar, G; Krumwiede, M; White, J G

    1986-04-01

    The present study has employed lysine, phalloidin, and a low concentration of osmic acid to protect the actin cytoskeleton of resting and thrombin-activated platelets during detergent extraction and fixation in suspension. Thin sections of resting platelets revealed a fine amorphous layer containing a few short actin filaments mimicking discoid shape and a randomly dispersed network of actin polymers in the cytoplasm. Following thrombin activation, the cytoskeleton consisted of a peripheral layer of submembrane actin filaments following the contour of shape change, a variable number of filaments in peripheral cytoplasm and extending into pseudopods, and a concentric mass of actin filaments with constricted microtubule rings in cell centers. Prior treatment with cytochalasin B (CB) appeared to reduce the number of actin filaments in resting platelets. Thrombin activation of CB-treated cells resulted in separation of actin filaments, which became concentrated in cell centers, from microtubule coils remaining at the cell periphery. The appearance of detergent-extracted cytoskeletons of platelet actin protected during fixation in suspension by lysine and phalloidin was remarkably similar to that of resting or CB-treated platelets before and after thrombin activation when viewed in conventionally prepared thin sections without exposure to detergent during fixation. The advantage of the new procedure is revelation of the actin filament organization in suspended platelets, which is obscured by the usual glutaraldehyde-osmic acid fixation technique.

  3. Measuring F-actin properties in dendritic spines

    Directory of Open Access Journals (Sweden)

    Mikko eKoskinen

    2014-08-01

    Full Text Available During the last decade, numerous studies have demonstrated that the actin cytoskeleton plays a pivotal role in the control of dendritic spine shape. Synaptic stimulation rapidly changes the actin dynamics and many actin regulators have been shown to play roles in neuron functionality. Accordingly, defects in the regulation of the actin cytoskeleton in neurons have been implicated in memory disorders. Due to the small size of spines, it is difficult to detect changes in the actin structures in dendritic spines by conventional light microscopy imaging. Instead, to know how tightly actin filaments are bundled together, and how fast the filaments turnover, we need to use advanced microscopy techniques, such as fluorescence recovery after photobleaching (FRAP, photoactivatable green fluorescent protein (PAGFP fluorescence decay and fluorescence anisotropy. Fluorescence anisotropy, which measures the Förster resonance energy transfer (FRET between two GFP fluorophores, has been proposed as a method to measure the level of actin polymerization. Here, we propose a novel idea that fluorescence anisotropy could be more suitable to study the level of actin filament bundling instead of actin polymerization. We validate the method in U2OS cell line where the actin structures can be clearly distinguished and apply the method to analyze how actin filament organization in dendritic spines changes during neuronal maturation. In addition to fluorescence anisotropy validation, we take a critical look at the properties and limitations of FRAP and PAGFP fluorescence decay methods and offer our proposals for the analysis methods for these approaches. These three methods complement each other, each providing additional information about actin dynamics and organization in dendritic spines.

  4. PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

    Science.gov (United States)

    Hong, Nan Hyung; Qi, Aidong

    2015-01-01

    Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover. PMID:26323691

  5. Transport of single cells using an actin bundle-myosin bionanomotor transport system

    Energy Technology Data Exchange (ETDEWEB)

    Takatsuki, Hideyo; Rice, Kevin M; Kohama, Kazuhiro; Blough, Eric R [Department of Biological Sciences, Marshall University, Huntington, WV (United States); Tanaka, Hideyuki [Department of Molecular and Cellular Pharmacology, Gunma University Graduate School of Medicine, Maebashi (Japan); Kolli, Madhukar B; Nalabotu, Siva K [Center for Diagnostic Nanosystems, Marshall University, Huntington, WV (United States); Famouri, Parviz, E-mail: blough@marshall.edu [Lane Department of Computer Science and Electrical Engineering, West Virginia University, Morgantown, WV (United States)

    2011-06-17

    The potential of using actin bundles for the transport of liposomes and single cells across myosin-coated surfaces is investigated. Compared to that observed with filamentous actin, the liposome transport using actin bundles was more linear in nature and able to occur over longer distances. Bundles, but not filamentous actin, were capable of moving single cells. Cargo unloading from bundles was achieved by incubation with Triton X-100. These data suggest that actin bundling may improve the ability of the myosin motor system for nanotransport applications.

  6. Actinic keratosis

    DEFF Research Database (Denmark)

    Erlendsson, Andrés M; Egekvist, Henrik; Lorentzen, Henrik F

    2015-01-01

    cryotherapy (57.7%) and photodynamic therapy (PDT) with methyl aminolevulinate (17.1%) and imiquimod (11.2%). The likelihood of receiving cryotherapy was higher for men (odds ratio [OR] 1.65, 95% confidence interval [CI] 1.10-2.47) and increased with age (2.2% per year, 0.4-4.0%). PDT represented the most...... treatment overall, except in instances of severe actinic damage, in which PDT is the first-choice treatment. © 2016 International Society of Dermatology....

  7. FMNL3 FH2-actin structure gives insight into formin-mediated actin nucleation and elongation.

    Science.gov (United States)

    Thompson, Morgan E; Heimsath, Ernest G; Gauvin, Timothy J; Higgs, Henry N; Kull, F Jon

    2013-01-01

    Formins are actin-assembly factors that act in a variety of actin-based processes. The conserved formin homology 2 (FH2) domain promotes filament nucleation and influences elongation through interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4-Å structure of a mouse FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2 actin-binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding, whereas those in the lasso-post interface are important for the stepping mechanism. Biochemical and cellular experiments test the importance of individual residues for function. This structure provides details for FH2-mediated filament elongation by processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus.

  8. Active multistage coarsening of actin networks driven by myosin motors

    Science.gov (United States)

    Silva, Marina Soares e; Depken, Martin; Stuhrmann, Björn; Korsten, Marijn; MacKintosh, Fred C.; Koenderink, Gijsje H.

    2011-01-01

    In cells, many vital processes involve myosin-driven motility that actively remodels the actin cytoskeleton and changes cell shape. Here we study how the collective action of myosin motors organizes actin filaments into contractile structures in a simplified model system devoid of biochemical regulation. We show that this self-organization occurs through an active multistage coarsening process. First, motors form dense foci by moving along the actin network structure followed by coalescence. Then the foci accumulate actin filaments in a shell around them. These actomyosin condensates eventually cluster due to motor-driven coalescence. We propose that the physical origin of this multistage aggregation is the highly asymmetric load response of actin filaments: they can support large tensions but buckle easily under piconewton compressive loads. Because the motor-generated forces well exceed this threshold, buckling is induced on the connected actin network that resists motor-driven filament sliding. We show how this buckling can give rise to the accumulation of actin shells around myosin foci and subsequent coalescence of foci into superaggregates. This new physical mechanism provides an explanation for the formation and contractile dynamics of disordered condensed actomyosin states observed in vivo. PMID:21593409

  9. Symmetrical retrograde actin flow in the actin fusion structure is involved in osteoclast fusion

    Directory of Open Access Journals (Sweden)

    Jiro Takito

    2017-07-01

    Full Text Available The aim of this study was to elucidate the role of the zipper-like structure (ZLS, a podosome-related structure that transiently appears at the cell contact zone, in osteoclast fusion. Live-cell imaging of osteoclasts derived from RAW264.7 cells transfected with EGFP-actin revealed consistent symmetrical retrograde actin flow in the ZLS, but not in the podosome cluster, the podosome ring or the podosome belt. Confocal imaging showed that the distributions of F-actin, vinculin, paxillin and zyxin in the ZLS were different from those in the podosome belt. Thick actin filament bundles running outside the ZLS appeared to recruit non-muscle myosin IIA. The F-actin-rich domain of the ZLS contained actin-related protein 2/3 complex (Arp2/3. Inhibition of Arp2/3 activity disorganized the ZLS, disrupted actin flow, deteriorated cell-cell adhesion and inhibited osteoclast hypermultinucleation. In contrast, ML-7, an inhibitor of myosin light chain kinase, had little effect on the structure of ZLS and promoted osteoclast hypermultinucleation. These results reveal a link between actin flow in the ZLS and osteoclast fusion. Osteoclast fusion was promoted by branched actin elongation and negatively regulated by actomyosin contraction.

  10. The binding of fluorescent phallotoxins to actin in myofibrils.

    Science.gov (United States)

    Szczesna, D; Lehrer, S S

    1993-12-01

    Fluorescence microscope observation of myofibrils incubated with rhodamine-phalloidin and coumarine-phallacidin showed an initial appearance of fluorescence bands at the Z-lines and near the middle of the sarcomeres indicating preferential binding of dye to actin subunits located at both actin filament ends. After long incubation times (1-3 h) however, a final pattern is reached which consists of fluorescent Z-lines in the center of uniformly labelled actin bands, with greater fluorescence in the Z-lines than in the uniform region outside the Z-lines. Increasing the temperature or the ionic strength increased the rate of change to the final pattern. These data indicate: (1) that the ends of the actin filament are kinetically more accessible to phallotoxins; (2) at long times when equilibrium binding presumably occurs, the concentration of actin subunits in the Z-band is greater than in the rest of the sarcomere.

  11. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

    Directory of Open Access Journals (Sweden)

    Mirco Müller

    Full Text Available Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

  12. F-actin accumulates in the vulva of female Strongyloides venezuelensis.

    Science.gov (United States)

    Silva, C V; Gonçalves, A L R; Cruz, L; Cruz, M C; Ueta, M T; Costa-Cruz, J M

    2013-09-01

    Little is known about the actin cytoskeleton architecture in female Strongyloides venezuelensis and thus to investigate the distribution and concentration of actin, female worms were labelled with phalloidin-rhodamine and visualized under confocal microscopy. Our results demonstrate that filamentous actin accumulates in the vulva and the concentration of F-actin at this site suggests its important role, especially during oviposition, in the life cycle of S. venezuelensis.

  13. Actin, actin-related proteins and profilin in diatoms: a comparative genomic analysis.

    Science.gov (United States)

    Aumeier, Charlotte; Polinski, Ellen; Menzel, Diedrik

    2015-10-01

    Diatoms are heterokont unicellular algae with a widespread distribution throughout all aquatic habitats. Research on diatoms has advanced significantly over the last decade due to available genetic transformation methods and publicly available genome databases. Yet up to now, proteins involved in the regulation of the cytoskeleton in diatoms are largely unknown. Consequently, this work focuses on actin and actin-related proteins (ARPs) encoded in the diatom genomes of Thalassiosira pseudonana, Thalassiosira oceanica, Phaeodactylum tricornutum, Fragilariopsis cylindrus and Pseudo-nitzschia multiseries. Our comparative genomic study revealed that most diatoms possess only a single conventional actin and a small set of ARPs. Among these are the highly conserved cytoplasmic Arp1 protein and the nuclear Arp4 as well as Arp6. Diatom genomes contain genes coding for two structurally different homologues of Arp4 that might serve specific functions. All diatom species examined here lack ARP2 and ARP3 proteins, suggesting that diatoms are not capable of forming the Arp2/3 complex, which is essential in most eukaryotes for actin filament branching and plus-end dynamics. Interestingly, none of the sequenced representatives of the Bacillariophyta phylum code for profilin. Profilin is an essential actin-binding protein regulating the monomer actin pool and is involved in filament plus-end dynamics. This is the first report of organisms not containing profilin. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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

  15. A unique profilin-actin interface is important for malaria parasite motility.

    Directory of Open Access Journals (Sweden)

    Catherine A Moreau

    2017-05-01

    Full Text Available Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like β-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites.

  16. Magnetic manipulation of actin orientation, polymerization, and gliding on myosin using superparamagnetic iron oxide particles

    Energy Technology Data Exchange (ETDEWEB)

    Chen Yun; Guzik, Stephanie; Sumner, James P; Koretsky, Alan P [National Institutes of Health, Bethesda, MD (United States); Moreland, John, E-mail: koretskya@ninds.nih.gov [National Institute of Standards and Technology, Boulder, CO (United States)

    2011-02-11

    The actin cytoskeleton controls cell shape, motility, as well as intracellular molecular trafficking. The ability to remotely manipulate actin is therefore highly desirable as a tool to probe and manipulate biological processes at the molecular level. We demonstrate actin manipulation by labeling actin filaments with superparamagnetic iron oxide particles (IOPs) and applying a uniform magnetic field to affect actin orientation, polymerization and gliding on myosin. We show for the first time magnetic manipulation of magnetizable actin filaments at the molecular level while gliding on a bed of myosin molecules and during polymerization. A model for the magnetic alignment and guiding mechanism is proposed based on the torque from the induced molecular anisotropy due to interactions between neighboring IOPs distributed along magnetically labeled actin molecules.

  17. Plant villin, lily P-135-ABP, possesses G-actin binding activity and accelerates the polymerization and depolymerization of actin in a Ca2+-sensitive manner.

    Science.gov (United States)

    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+).

  18. Lifeact-mEGFP reveals a dynamic apical F-actin network in tip growing plant cells.

    Directory of Open Access Journals (Sweden)

    Luis Vidali

    2009-05-01

    Full Text Available Actin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments.In this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore.Lifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells.

  19. Mechanical Properties of Re-constituted Actin Networks at an Oil/Water Interface Determined by Microrheology

    NARCIS (Netherlands)

    Ershov, D.S.; Cohen Stuart, M.A.; Gucht, van der J.

    2012-01-01

    There have been various attempts to investigate the mechanical properties of the actin cortex in cells, but the factors that control them remain poorly understood. To make progress, we develop a reconstituted model of the actin cortex that mimics its structure. We attach actin filaments to lipids

  20. Actin structure-dependent stepping of myosin 5a and 10 during processive movement.

    Directory of Open Access Journals (Sweden)

    Jianjun Bao

    Full Text Available How myosin 10, an unconventional myosin, walks processively along actin is still controversial. Here, we used single molecule fluorescence techniques, TIRF and FIONA, to study the motility and the stepping mechanism of dimerized myosin 10 heavy-meromyosin-like fragment on both single actin filaments and two-dimensional F-actin rafts cross-linked by fascin or α-actinin. As a control, we also tracked and analyzed the stepping behavior of the well characterized processive motor myosin 5a. We have shown that myosin 10 moves processively along both single actin filaments and F-actin rafts with a step size of 31 nm. Moreover, myosin 10 moves more processively on fascin-F-actin rafts than on α-actinin-F-actin rafts, whereas myosin 5a shows no such selectivity. Finally, on fascin-F-actin rafts, myosin 10 has more frequent side steps to adjacent actin filaments than myosin 5a in the F-actin rafts. Together, these results reveal further single molecule features of myosin 10 on various actin structures, which may help to understand its cellular functions.

  1. Regulation of cell structure and function by actin-binding proteins: villin's perspective.

    Science.gov (United States)

    Khurana, Seema; George, Sudeep P

    2008-06-18

    Villin is a tissue-specific actin modifying protein that is associated with actin filaments in the microvilli and terminal web of epithelial cells. It belongs to a large family of actin-binding proteins which includes actin-capping, -nucleating and/or -severing proteins such as gelsolin, severin, fragmin, adseverin/scinderin and actin crosslinking proteins such as dematin and supervillin. Studies done in epithelial cell lines and villin knock-out mice have demonstrated the function of villin in regulating actin dynamics, cell morphology, epithelial-to-mesenchymal transition, cell migration and cell survival. In addition, the ligand-binding properties of villin (F-actin, G-actin, calcium, phospholipids and phospholipase C-gamma1) are mechanistically important for the crosstalk between signaling pathways and actin reorganization in epithelial cells.

  2. Allyl Isothiocyanate Inhibits Actin-Dependent Intracellular Transport in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Bjørnar Sporsheim

    2015-12-01

    Full Text Available Volatile allyl isothiocyanate (AITC derives from the biodegradation of the glucosinolate sinigrin and has been associated with growth inhibition in several plants, including the model plant Arabidopsis thaliana. However, the underlying cellular mechanisms of this feature remain scarcely investigated in plants. In this study, we present evidence of an AITC-induced inhibition of actin-dependent intracellular transport in A. thaliana. A transgenic line of A. thaliana expressing yellow fluorescent protein (YFP-tagged actin filaments was used to show attenuation of actin filament movement by AITC. This appeared gradually in a time- and dose-dependent manner and resulted in actin filaments appearing close to static. Further, we employed four transgenic lines with YFP-fusion proteins labeling the Golgi apparatus, endoplasmic reticulum (ER, vacuoles and peroxisomes to demonstrate an AITC-induced inhibition of actin-dependent intracellular transport of or, in these structures, consistent with the decline in actin filament movement. Furthermore, the morphologies of actin filaments, ER and vacuoles appeared aberrant following AITC-exposure. However, AITC-treated seedlings of all transgenic lines tested displayed morphologies and intracellular movements similar to that of the corresponding untreated and control-treated plants, following overnight incubation in an AITC-absent environment, indicating that AITC-induced decline in actin-related movements is a reversible process. These findings provide novel insights into the cellular events in plant cells following exposure to AITC, which may further expose clues to the physiological significance of the glucosinolate-myrosinase system.

  3. Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay.

    Science.gov (United States)

    Beausang, John F; Schroeder, Harry W; Nelson, Philip C; Goldman, Yale E

    2008-12-15

    The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay to measure the rotation of an actin filament about its axis ("twirling") as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeled actin monomers, using polarized total internal reflection microscopy. To determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement fourfold. We found that whole myosin II and myosin V both twirl actin with a relatively long (approximately 1 microm), left-handed pitch that is insensitive to myosin concentration, filament length, and filament velocity.

  4. Actin-ADF/cofilin rod formation in Caenorhabditis elegans muscle requires a putative F-actin binding site of ADF/cofilin at the C-terminus.

    Science.gov (United States)

    Ono, Kanako; Ono, Shoichiro

    2009-07-01

    Under a number of stress or pathological conditions, actin and actin depolymerizing factor (ADF)/cofilin form rod-like structures that contain abnormal bundles of actin filaments that are heavily decorated with ADF/cofilin. However, the mechanism of actin rod formation and the physiological role of actin rods are not clearly understood. Here, we report that overexpression of green fluorescent protein-fused UNC-60B, a muscle-specific ADF/cofilin isoform, in Caenorhabditis elegans body wall muscle induces formation of rod-like structures. The rods contained GFP-UNC-60B, actin-interacting protein 1 (AIP1), and actin, but not other major actin-associated proteins, thus resembling actin-ADF/cofilin rods found in other organisms. However, depletion or overexpression of AIP1 did not affect formation of the actin-GFP-UNC-60B rods, suggesting that AIP1 does not play a significant role in the rod assembly. Truncation of the C-terminal tail, a putative F-actin binding site, of UNC-60B abolished induction of the rod formation, strongly suggesting that stable association of UNC-60B with F-actin, which is mediated by its C-terminus, is required for inducing actin-ADF/cofilin rods. This study suggests that C. elegans can be a new model to study functions of actin-ADF/cofilin rods. (c) 2009 Wiley-Liss, Inc.

  5. The role of actin in root hair morphogenesis : studies with lipochito-oligosaccharide as a growth stimulator and cytochalasin as an actin perturbing drug

    NARCIS (Netherlands)

    Miller, D.D.; Ruijter, de N.C.A.; Bisseling, T.; Emons, A.M.C.

    1999-01-01

    Root hairs develop from bulges on root epidermal cells and elongate by tip growth, in which Golgi vesicles are targeted, released and inserted into the plasma membrane on one side of the cell. We studied the role of actin in vesicle delivery and retention by comparing the actin filament

  6. Unique properties of eukaryote-type actin and profilin horizontally transferred to cyanobacteria.

    Directory of Open Access Journals (Sweden)

    Arthur Guljamow

    Full Text Available A eukaryote-type actin and its binding protein profilin encoded on a genomic island in the cyanobacterium Microcystis aeruginosa PCC 7806 co-localize to form a hollow, spherical enclosure occupying a considerable intracellular space as shown by in vivo fluorescence microscopy. Biochemical and biophysical characterization reveals key differences between these proteins and their eukaryotic homologs. Small-angle X-ray scattering shows that the actin assembles into elongated, filamentous polymers which can be visualized microscopically with fluorescent phalloidin. Whereas rabbit actin forms thin cylindrical filaments about 100 µm in length, cyanobacterial actin polymers resemble a ribbon, arrest polymerization at 5-10 µm and tend to form irregular multi-strand assemblies. While eukaryotic profilin is a specific actin monomer binding protein, cyanobacterial profilin shows the unprecedented property of decorating actin filaments. Electron micrographs show that cyanobacterial profilin stimulates actin filament bundling and stabilizes their lateral alignment into heteropolymeric sheets from which the observed hollow enclosure may be formed. We hypothesize that adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes has driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity.

  7. Cell elasticity is regulated by the tropomyosin isoform composition of the actin cytoskeleton.

    Directory of Open Access Journals (Sweden)

    Iman Jalilian

    Full Text Available The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm, in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

  8. Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breaking

    Science.gov (United States)

    Carvalho, Kevin; Lemière, Joël; Faqir, Fahima; Manzi, John; Blanchoin, Laurent; Plastino, Julie; Betz, Timo; Sykes, Cécile

    2013-01-01

    Cells use complex biochemical pathways to drive shape changes for polarization and movement. One of these pathways is the self-assembly of actin filaments and myosin motors that together produce the forces and tensions that drive cell shape changes. Whereas the role of actin and myosin motors in cell polarization is clear, the exact mechanism of how the cortex, a thin shell of actin that is underneath the plasma membrane, can drive cell shape changes is still an open question. Here, we address this issue using biomimetic systems: the actin cortex is reconstituted on liposome membranes, in an ‘outside geometry’. The actin shell is either grown from an activator of actin polymerization immobilized at the membrane by a biotin–streptavidin link, or built by simple adsorption of biotinylated actin filaments to the membrane, in the presence or absence of myosin motors. We show that tension in the actin network can be induced either by active actin polymerization on the membrane via the Arp2/3 complex or by myosin II filament pulling activity. Symmetry breaking and spontaneous polarization occur above a critical tension that opens up a crack in the actin shell. We show that this critical tension is reached by growing branched networks, nucleated by the Arp2/3 complex, in a concentration window of capping protein that limits actin filament growth and by a sufficient number of motors that pull on actin filaments. Our study provides the groundwork to understanding the physical mechanisms at work during polarization prior to cell shape modifications. PMID:24062578

  9. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Juan Du

    Full Text Available Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin or polymeric form (F-actin. Members of the actin-depolymerizing factor (ADF/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1 in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin.

  10. Virulent Burkholderia species mimic host actin polymerases to drive actin-based motility.

    Science.gov (United States)

    Benanti, Erin L; Nguyen, Catherine M; Welch, Matthew D

    2015-04-09

    Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, whereas their close relative B. thailandensis is non-pathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion, and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate, and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Structure of the FMNL3 FH2/actin complex provides insight into formin-mediated actin nucleation and elongation

    Science.gov (United States)

    Thompson, Morgan E.; Heimsath, Ernest G.; Gauvin, Timothy J.; Higgs, Henry N.; Kull, F. Jon

    2012-01-01

    Summary Formins are actin assembly factors that act in a variety of actin-based processes. The conserved formin homology 2 (FH2) domain promotes filament nucleation and influences elongation via interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4 Å structure of an FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2-actin binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding while those in the lasso/post interface are important for the stepping mechanism. Biochemical and cellular experiments test the importance of individual residues for function. This structure provides details for FH2 mediated filament elongation via processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus. PMID:23222643

  12. A novel three-filament model of force generation in eccentric contraction of skeletal muscles.

    Directory of Open Access Journals (Sweden)

    Gudrun Schappacher-Tilp

    Full Text Available We propose and examine a three filament model of skeletal muscle force generation, thereby extending classical cross-bridge models by involving titin-actin interaction upon active force production. In regions with optimal actin-myosin overlap, the model does not alter energy and force predictions of cross-bridge models for isometric contractions. However, in contrast to cross-bridge models, the three filament model accurately predicts history-dependent force generation in half sarcomeres for eccentric and concentric contractions, and predicts the activation-dependent forces for stretches beyond actin-myosin filament overlap.

  13. The Arabidopsis Wave Complex: Mechanisms Of Localized Actin Polymerization And Growth

    Energy Technology Data Exchange (ETDEWEB)

    Daniel Szymanski

    2012-10-23

    The objective of this project was to discover the protein complexes and control mechanisms that determine the location of actin filament roadways in plant cells. Our work provided the first molecular description of protein complexes that are converted from inactive complexes to active actin filament nucleators in the cell. These discoveries provided a conceptual framework to control to roadways in plant cells that determine the location and delivery of plant metabolites and storage molecules that are relevant to the bioenergy economy.

  14. Transformation of actin-encapsulating liposomes induced by cytochalasin D.

    OpenAIRE

    Miyata, H.; Kinosita, K

    1994-01-01

    Liposomes encapsulating actin filaments were prepared by swelling at 0 degrees C lipid film consisting of a mixture of dimyristoyl phosphatidylcholine and cardiolipin (equal amounts by weight) in 100 microM rabbit skeletal muscle actin and 0.5 mM CaCl2 followed by polymerization of actin at 30 degrees C. Liposomes initially assumed either disk or dumbbell shape, but when cytochalasin D was added to the medium surrounding the liposomes, they were found to become spindle shaped. Liposomes conta...

  15. Visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin-Dendra2 fusion proteins.

    Directory of Open Access Journals (Sweden)

    Athanassios Dovas

    Full Text Available Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin-Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red G-actin species to become incorporated into pre-existing (green actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.

  16. Rocket launcher mechanism of collaborative actin assembly defined by single-molecule imaging

    Science.gov (United States)

    Breitsprecher, Dennis; Jaiswal, Richa; Bombardier, Jeffrey P.; Gould, Christopher J.; Gelles, Jeff; Goode, Bruce L.

    2013-01-01

    Interacting sets of actin assembly factors work together in cells, but the underlying mechanisms have remained obscure. We used triple-color single molecule fluorescence microscopy to image the tumor-suppressor Adenomateous polyposis coli (APC) and the formin mDia1 during filament assembly. Complexes consisting of APC, mDia1, and actin monomers intiated actin filament formation, overcoming inhibition by capping protein and profilin. Upon filament polymerization, the complexes separated, with mDia1 moving processively on growing barbed ends while APC remained at the site of nucleation. Thus, the two assembly factors directly interact to initiate filament assembly, and then separate but retain independent associations with either end of the growing filament. PMID:22654058

  17. Initiation of DNA replication requires actin dynamics and formin activity.

    Science.gov (United States)

    Parisis, Nikolaos; Krasinska, Liliana; Harker, Bethany; Urbach, Serge; Rossignol, Michel; Camasses, Alain; Dewar, James; Morin, Nathalie; Fisher, Daniel

    2017-11-02

    Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin- and redox-dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)-cargo release from RanGTP-importin complexes. Nuclear formin activity is further required to promote loading of cyclin-dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms. © 2017 The Authors.

  18. Analysis of actin assembly by in vitro TIRF microscopy.

    Science.gov (United States)

    Breitsprecher, Dennis; Kiesewetter, Antje K; Linkner, Joern; Faix, Jan

    2009-01-01

    Since directed movement toward an extracellular chemoattractant requires rapid and continuous reorganization of the actin cytoskeleton to form complex structures such as a protruding lamellipodium, it is of great interest to analyze and understand the individual contribution of proteins specifically involved in this process. Over the last decade, enormous progress has been made toward understanding the versatile molecular mechanisms underlying actin-based cell motility and the regulation of site-specific F-actin assembly and disassembly. In spite of this wealth of knowledge and due to the constant discovery of novel regulatory factors, many questions remain to be answered. In this chapter, we describe a powerful method that allows to study the effects of actin-binding proteins on the assembly of single filaments by in vitro total internal reflection fluorescence (TIRF) microscopy using purified proteins and fluorescently labeled actin.

  19. Pattern formation in polymerising actin flocks: spirals, spots and waves without nonlinear chemistry

    CERN Document Server

    Goff, Thomas Le; Marenduzzo, Davide

    2016-01-01

    We propose a model solely based on actin treadmilling and polymerisation which describes many characteristic states of actin wave formation: spots, spirals and travelling waves. In our model, as in experiments on cell recovering motility following actin depolymerisation, we choose an isotropic low density initial condition; polymerisation of actin filaments then raises the density towards the Onsager threshold where they align. We show that this alignment, in turn, destabilizes the isotropic phase and generically induces transient actin spots or spirals as part of the dynamical pathway towards a polarized phase which can either be uniform or consist of a series of actin-wave trains (flocks). Our results uncover a universal route to actin wave formation in the absence of any system specific nonlinear biochemistry, and it may help understand the mechanism underlying the observation of actin spots and waves in vivo. They also suggest a minimal setup to design similar patterns in vitro.

  20. Formin-mediated actin polymerization at endothelial junctions is required for vessel lumen formation and stabilization.

    Science.gov (United States)

    Phng, Li-Kun; Gebala, Véronique; Bentley, Katie; Philippides, Andrew; Wacker, Andrin; Mathivet, Thomas; Sauteur, Loïc; Stanchi, Fabio; Belting, Heinz-Georg; Affolter, Markus; Gerhardt, Holger

    2015-01-12

    During blood vessel formation, endothelial cells (ECs) establish cell-cell junctions and rearrange to form multicellular tubes. Here, we show that during lumen formation, the actin nucleator and elongation factor, formin-like 3 (fmnl3), localizes to EC junctions, where filamentous actin (F-actin) cables assemble. Fluorescent actin reporters and fluorescence recovery after photobleaching experiments in zebrafish embryos identified a pool of dynamic F-actin with high turnover at EC junctions in vessels. Knockdown of fmnl3 expression, chemical inhibition of formin function, and expression of dominant-negative fmnl3 revealed that formin activity maintains a stable F-actin content at EC junctions by continual polymerization of F-actin cables. Reduced actin polymerization leads to destabilized endothelial junctions and consequently to failure in blood vessel lumenization and lumen instability. Our findings highlight the importance of formin activity in blood vessel morphogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Actin-Dynamics in Plant Cells: The Function of Actin-Perturbing Substances: Jasplakinolide, Chondramides, Phalloidin, Cytochalasins, and Latrunculins.

    Science.gov (United States)

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

    This chapter gives an overview of the most common F-actin-perturbing substances that are used to study actin dynamics in living plant cells in studies on morphogenesis, motility, organelle movement, or when apoptosis has to be induced. These substances can be divided into two major subclasses: F-actin-stabilizing and -polymerizing substances like jasplakinolide and chondramides and F-actin-severing compounds like chytochalasins and latrunculins. Jasplakinolide was originally isolated form a marine sponge, and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane-permeable F-actin-stabilizing and -polymerizing agent, which may even have anticancer activities. Cytochalasins, derived from fungi, show an F-actin-severing function and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from red sea sponges; however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide a stable complex with actin dimers is formed resulting also in severing of F-actin. For influencing F-actin dynamics in plant cells only membrane permeable drugs are useful in a broad range. We however introduce also the phallotoxins and synthetic derivatives, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes, but has also been described in siphonal giant algae. In the present chapter the focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; however methods by fluorescence applications including phalloidin and antibody staining as well as immunofluorescence-localization of the inhibitor drugs are given.

  2. Actin-Dynamics in Plant Cells: The Function of Actin Perturbing Substances Jasplakinolide, Chondramides, Phalloidin, Cytochalasins, and Latrunculins

    Science.gov (United States)

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

    This chapter will give an overview of the most common F-actin perturbing substances, that are used to study actin dynamics in living plant cells in studies on morphogenesis, motility, organelle movement or when apoptosis has to be induced. These substances can be divided into two major subclasses – F-actin stabilizing and polymerizing substances like jasplakinolide, chondramides and F-actin severing compounds like chytochalasins and latrunculins. Jasplakinolide was originally isolated form a marine sponge, and can now be synthesized and has become commercially available, which is responsible for its wide distribution as membrane permeable F-actin stabilizing and polymerizing agent, which may even have anti-cancer activities. Cytochalasins, derived from fungi show an F-actin severing function and many derivatives are commercially available (A, B, C, D, E, H, J), also making it a widely used compound for F-actin disruption. The same can be stated for latrunculins (A, B), derived from red sea sponges, however the mode of action is different by binding to G-actin and inhibiting incorporation into the filament. In the case of swinholide a stable complex with actin dimers is formed resulting also in severing of F-actin. For influencing F-actin dynamics in plant cells only membrane permeable drugs are useful in a broad range. We however introduce also the phallotoxins and synthetic derivatives, as they are widely used to visualize F-actin in fixed cells. A particular uptake mechanism has been shown for hepatocytes, but has also been described in siphonal giant algae. In the present chapter the focus is set on F-actin dynamics in plant cells where alterations in cytoplasmic streaming can be particularly well studied; however methods by fluorescence applications including phalloidin- and antibody staining as well as immunofluorescence-localization of the inhibitor drugs are given. PMID:26498789

  3. Microtubules Modulate F-actin Dynamics during Neuronal Polarization.

    Science.gov (United States)

    Zhao, Bing; Meka, Durga Praveen; Scharrenberg, Robin; König, Theresa; Schwanke, Birgit; Kobler, Oliver; Windhorst, Sabine; Kreutz, Michael R; Mikhaylova, Marina; Calderon de Anda, Froylan

    2017-08-29

    Neuronal polarization is reflected by different dynamics of microtubule and filamentous actin (F-actin). Axonal microtubules are more stable than those in the remaining neurites, while dynamics of F-actin in axonal growth cones clearly exceed those in their dendritic counterparts. However, whether a functional interplay exists between the microtubule network and F-actin dynamics in growing axons and whether this interplay is instrumental for breaking cellular symmetry is currently unknown. Here, we show that an increment on microtubule stability or number of microtubules is associated with increased F-actin dynamics. Moreover, we show that Drebrin E, an F-actin and microtubule plus-end binding protein, mediates this cross talk. Drebrin E segregates preferentially to growth cones with a higher F-actin treadmilling rate, where more microtubule plus-ends are found. Interruption of the interaction of Drebrin E with microtubules decreases F-actin dynamics and arrests neuronal polarization. Collectively the data show that microtubules modulate F-actin dynamics for initial axon extension during neuronal development.

  4. The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration

    Energy Technology Data Exchange (ETDEWEB)

    Blom, Magdalena; Reis, Katarina [Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm (Sweden); Heldin, Johan [Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala SE-751 22 Uppsala (Sweden); Kreuger, Johan [Department of Medical Cell Biology, Science for Life Laboratory, Uppsala University, SE-751 23 Uppsala (Sweden); Aspenström, Pontus, E-mail: pontus.aspenstrom@ki.se [Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm (Sweden)

    2017-03-15

    RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as cortical actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration. - Highlights: • Increased RhoD expression leads to loss of actin structures, e.g. stress fibers and gives rise to decreased actin dynamics. • RhoD knockdown induces various actin-containing structures such as edge ruffles, stress fibers and cortical actin, in a cell-type specific manner. • RhoD induces specific actin rearrangements depending on its subcellular localization. • RhoD knockdown has effects on cellular processes, such as directed cell migration and proliferation.

  5. The actin homologue MreB organizes the bacterial cell membrane

    NARCIS (Netherlands)

    Strahl, H.; Burmann, F.; Hamoen, L.W.

    2014-01-01

    The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate

  6. Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers

    NARCIS (Netherlands)

    Honing, van der H.S.; Ruijter, de N.C.A.; Emons, A.M.C.; Ketelaar, T.

    2010-01-01

    Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm.

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

    Science.gov (United States)

    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.

  8. Intermediate Filaments as Organizers of Cellular Space: How They Affect Mitochondrial Structure and Function

    Directory of Open Access Journals (Sweden)

    Nicole Schwarz

    2016-07-01

    Full Text Available Intermediate filaments together with actin filaments and microtubules form the cytoskeleton, which is a complex and highly dynamic 3D network. Intermediate filaments are the major mechanical stress protectors but also affect cell growth, differentiation, signal transduction, and migration. Using intermediate filament-mitochondrial crosstalk as a prominent example, this review emphasizes the importance of intermediate filaments as crucial organizers of cytoplasmic space to support these functions. We summarize observations in different mammalian cell types which demonstrate how intermediate filaments influence mitochondrial morphology, subcellular localization, and function through direct and indirect interactions and how perturbations of these interactions may lead to human diseases.

  9. Actin-binding protein (ABP-280) filamin gene (FLN) maps telomeric to the color vision locus (R/GCP) and centromeric to G6PD in Xq28

    Energy Technology Data Exchange (ETDEWEB)

    Gorlin, J.B. (Brigham and Women' s Hospital, Boston, MA (United States) Dana-Farber Cancer Institute, Boston, MA (United States)); Henske, E.; Hartwig, J.H.; Kwiatkowski, D.J. (Brigham and Women' s Hospital, Boston, MA (United States)); Warren, S.T.; Kunst, C.B. (Emory Univ. School of Medicine, Atlanta, GA (United States)); D' Urso, M.; Palmieri, G. (International Institute of Genetics and Biophysics, Naples, (Italy)); Bruns, G. (Children' s Hospital, Boston, MA (United States))

    1993-08-01

    Actin-binding protein-280 (ABP-280) is a dimeric actin filament-crosslinking protein that promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The authors have mapped the ABP-280 filamin gene (FLN) to Xq28 by Southern blot analysis of somatic cell hybrid lines, by fluorescence in situ hybridization, and through identification of portions of the FLN gene within cosmids and YACs mapped to Xq28. The FLN gene is found within a 200-kb region centromeric to the G6PD locus and telomeric to DSX52 and the color vision locus. 23 refs., 2 figs.

  10. TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics

    Science.gov (United States)

    Bailly, Aurelien; Zwiewka, Marta; Sovero, Valpuri; Ge, Pei; Aryal, Bibek; Hao, Pengchao; Linnert, Miriam; Burgardt, Noelia Inés; Lücke, Christian; Weiwad, Matthias; Michel, Max; Weiergräber, Oliver H.; Pollmann, Stephan; Azzarello, Elisa; Fukao, Yoichiro; Hoffmann, Céline; Wedlich-Söldner, Roland

    2016-01-01

    Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity. PMID:27053424

  11. Actin and Apical Constriction: Some (Re)-Assembly Required.

    Science.gov (United States)

    Spracklen, Andrew J; Peifer, Mark

    2015-12-21

    Linkage of the actomyosin cytoskeleton to cell-cell junctions drives cell shape change in development and homeostasis. In this issue of Developmental Cell, Jodoin et al. (2015) provide new insights into the underlying mechanisms, revealing that factors driving actin filament disassembly and thus dynamics also play key roles in apical constriction. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Control of the actin cytoskeleton in plant cell growth

    NARCIS (Netherlands)

    Hussey, P.J.; Ketelaar, M.J.; Deeks, M.J.

    2006-01-01

    Plant cells grow through increases in volume and cell wall surface area. The mature morphology of a plant cell is a product of the differential rates of expansion between neighboring zones of the cell wall during this process. Filamentous actin arrays are associated with plant cell growth, and the

  13. Covalent modification of G-actin by pyridoxal 5'-phosphate: polymerization properties and interaction with DNase I and myosin subfragment 1.

    Science.gov (United States)

    Combeau, C; Carlier, M F

    1992-01-14

    Pyridoxal 5'-phosphate (PLP), a lysine-specific reagent, has been used to modify G-actin. At pH 7.5, PLP reacted with 1.7-2 lysines on G-actin. Limited proteolytic digestion experiments indicated that, in agreement with previous works, essentially lysine-61 was modified in a 1:1 fashion by PLP, other lysines being much less reactive. A PLP-derivatized affinity label of ATP binding sites, AMPPLP, reacted with two additional lysines that do not appear to be located in the ATP site on G-actin. PLP-G-actin did not polymerize spontaneously up to 30 microM; however, it retained other essential native properties of G-actin. PLP-actin bound to the barbed ends of actin filaments with an equilibrium dissociation constant of 4 microM and prevented dilution-induced depolymerization like a capping protein. PLP-actin copolymerized with unmodified actin. The stability of F-actin copolymers decreased with the fraction of PLP-actin incorporated, consistent with a model within which the actin-PLP-actin interactions in the copolymer are 50-fold weaker, and PLP-actin-PLP-actin interactions are 200-fold weaker than regular actin-actin interactions. PLP-actin bound DNase I with an equilibrium association constant of 2 nM-1, i.e., 10-fold lower than that of unmodified actin. PLP modification did not affect the binding of G-actin to myosin subfragment 1. However, polymerization of PLP-actin by myosin subfragment 1 was not observed in low ionic strength buffers, whereas PLP-F-actin-S1 filaments, in which the stoichiometry PLP-actin:S1 is 1:1, were formed with an apparent critical concentration of 4.5 microM in the presence of 0.1 M KCl.

  14. Two Functionally Distinct Sources of Actin Monomers Supply the Leading Edge of Lamellipodia

    Directory of Open Access Journals (Sweden)

    Eric A. Vitriol

    2015-04-01

    Full Text Available Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin regulated by the ordered assembly from and disassembly into actin monomers (G-actin. Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer-binding protein thymosin β4 (Tβ4 for optimal leading-edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it does not interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions.

  15. Actin growth profile in clathrin-mediated endocytosis

    Science.gov (United States)

    Tweten, D. J.; Bayly, P. V.; Carlsson, A. E.

    2017-05-01

    Clathrin-mediated endocytosis in yeast is driven by a protein patch containing close to 100 different types of proteins. Among the proteins are 5000 -10 000 copies of polymerized actin, and successful endocytosis requires growth of the actin network. Since it is not known exactly how actin network growth drives endocytosis, we calculate the spatial distribution of actin growth required to generate the force that drives the process. First, we establish the force distribution that must be supplied by actin growth, by combining membrane-bending profiles obtained via electron microscopy with established theories of membrane mechanics. Next, we determine the profile of actin growth, using a continuum mechanics approach and an iterative procedure starting with an actin growth profile obtained from a linear analysis. The profile has fairly constant growth outside a central hole of radius 45-50 nm, but very little growth in this hole. This growth profile can reproduce the required forces if the actin shear modulus exceeds 80 kPa, and the growing filaments can exert very large polymerization forces. The growth profile prediction could be tested via electron-microscopy or super-resolution experiments in which the turgor pressure is suddenly turned off.

  16. A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin.

    Directory of Open Access Journals (Sweden)

    Yao Liu

    2017-01-01

    Full Text Available Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.

  17. Monoubiquitination Inhibits the Actin Bundling Activity of Fascin.

    Science.gov (United States)

    Lin, Shengchen; Lu, Shuang; Mulaj, Mentor; Fang, Bin; Keeley, Tyler; Wan, Lixin; Hao, Jihui; Muschol, Martin; Sun, Jianwei; Yang, Shengyu

    2016-12-30

    Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys247 and Lys250, two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC50, delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Importance of a Lys113-Glu195 intermonomer ionic bond in F-actin stabilization and regulation by yeast formins Bni1p and Bnr1p.

    Science.gov (United States)

    Wen, Kuo-Kuang; McKane, Melissa; Rubenstein, Peter A

    2013-06-28

    Proper actin cytoskeletal function requires actin's ability to generate a stable filament and requires that this reaction be regulated by actin-binding proteins via allosteric effects on the actin. A proposed ionic interaction in the actin filament interior between Lys(113) of one monomer and Glu(195) of a monomer in the apposing strand potentially fosters cross-strand stabilization and allosteric communication between the filament interior and exterior. We interrupted the potential interaction by creating either K113E or E195K actin. By combining the two, we also reversed the interaction with a K113E/E195K (E/K) mutant. In all cases, we isolated viable cells expressing only the mutant actin. Either single mutant cell displays significantly decreased growth in YPD medium. This deficit is rescued in the double mutant. All three mutants display abnormal phalloidin cytoskeletal staining. K113E actin exhibits a critical concentration of polymerization 4 times higher than WT actin, nucleates more poorly, and forms shorter filaments. Restoration of the ionic bond, E/K, eliminates most of these problems. E195K actin behaves much more like WT actin, indicating accommodation of the neighboring lysines. Both Bni1 and Bnr1 formin FH1-FH2 fragment accelerate polymerization of WT, E/K, and to a lesser extent E195K actin. Bni1p FH1-FH2 dramatically inhibits K113E actin polymerization, consistent with barbed end capping. However, Bnr1p FH1-FH2 restores K113E actin polymerization, forming single filaments. In summary, the proposed ionic interaction plays an important role in filament stabilization and in the propagation of allosteric changes affecting formin regulation in an isoform-specific fashion.

  19. Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Gomibuchi, Yuki [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Uyeda, Taro Q.P. [Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan); Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp [Graduate School of Science and Engineering, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan); Department of Judo Therapy, Faculty of Medical Technology, Teikyo University, Toyosatodai 1-1, Utsunomiya 320-8551 (Japan)

    2013-11-29

    Highlights: •The effect of mutation of Tyr143 that becomes more exposed on assembly was examined. •Mutation of tyrosine-143 of Dictyostelium actin changed actin polymerizability. •The bulkiness or aromatic nature of Tyr143 is important for the weak binding. •The weak interaction between myosin and actin strengthened by Tyr143Trp mutation. -- Abstract: Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å{sup 2} to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V{sub max}) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K{sub app}) than that of the wild-type actin, with the V{sub max} being almost unchanged. The K{sub app} and V{sub max} of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K{sub app} was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA

  20. Reversibility and Viscoelastic Properties of Micropillar Supported and Oriented Magnesium Bundled F-Actin.

    Directory of Open Access Journals (Sweden)

    Timo Maier

    Full Text Available Filamentous actin is one of the most important cytoskeletal elements. Not only is it responsible for the elastic properties of many cell types, but it also plays a vital role in cellular adhesion and motility. Understanding the bundling kinetics of actin filaments is important in the formation of various cytoskeletal structures, such as filopodia and stress fibers. Utilizing a unique pillar-structured microfluidic device, we investigated the time dependence of bundling kinetics of pillar supported free-standing actin filaments. Microparticles attached to the filaments allowed the measurement of thermal motion, and we found that bundling takes place at lower concentrations than previously found in 3-dimensional actin gels, i.e. actin filaments formed bundles in the presence of 5-12 mM of magnesium chloride in a time-dependent manner. The filaments also displayed long term stability for up to hours after removing the magnesium ions from the buffer, which suggests that there is an extensive hysteresis between cation induced crosslinking and decrosslinking.

  1. Fast synaptic subcortical control of hippocampal circuits.

    Science.gov (United States)

    Varga, Viktor; Losonczy, Attila; Zemelman, Boris V; Borhegyi, Zsolt; Nyiri, Gábor; Domonkos, Andor; Hangya, Balázs; Holderith, Noémi; Magee, Jeffrey C; Freund, Tamás F

    2009-10-16

    Cortical information processing is under state-dependent control of subcortical neuromodulatory systems. Although this modulatory effect is thought to be mediated mainly by slow nonsynaptic metabotropic receptors, other mechanisms, such as direct synaptic transmission, are possible. Yet, it is currently unknown if any such form of subcortical control exists. Here, we present direct evidence of a strong, spatiotemporally precise excitatory input from an ascending neuromodulatory center. Selective stimulation of serotonergic median raphe neurons produced a rapid activation of hippocampal interneurons. At the network level, this subcortical drive was manifested as a pattern of effective disynaptic GABAergic inhibition that spread throughout the circuit. This form of subcortical network regulation should be incorporated into current concepts of normal and pathological cortical function.

  2. Latrunculin B-induced plant dwarfism: Plant cell elongation is F-actin-dependent.

    Science.gov (United States)

    Baluska, F; Jasik, J; Edelmann, H G; Salajová, T; Volkmann, D

    2001-03-01

    Marine macrolides latrunculins are highly specific toxins which effectively depolymerize actin filaments (generally F-actin) in all eukaryotic cells. We show that latrunculin B is effective on diverse cell types in higher plants and describe the use of this drug in probing F-actin-dependent growth and in plant development-related processes. In contrast to other eukaryotic organisms, cell divisions occurs in plant cells devoid of all actin filaments. However, the alignment of the division planes is often distorted. In addition to cell division, postembryonic development and morphogenesis also continue in the absence of F-actin. These experimental data suggest that F-actin is of little importance in the morphogenesis of higher plants, and that plants can develop more or less normally without F-actin. In contrast, F-actin turns out to be essential for cell elongation. When latrunculin B was added during germination, morphologically normal Arabidopsis and rye seedlings developed but, as a result of the absence of cell elongation, these were stunted, resembling either genetic dwarfs or environmental bonsai plants. In conclusion, F-actin is essential for the plant cell elongation, while this F-actin-dependent cell elongation is not an essential feature of plant-specific developmental programs.

  3. Altered Cell Mechanics from the Inside: Dispersed Single Wall Carbon Nanotubes Integrate with and Restructure Actin

    Directory of Open Access Journals (Sweden)

    Mohammad F. Islam

    2012-05-01

    Full Text Available With a range of desirable mechanical and optical properties, single wall carbon nanotubes (SWCNTs are a promising material for nanobiotechnologies. SWCNTs also have potential as biomaterials for modulation of cellular structures. Previously, we showed that highly purified, dispersed SWCNTs grossly alter F-actin inside cells. F-actin plays critical roles in the maintenance of cell structure, force transduction, transport and cytokinesis. Thus, quantification of SWCNT-actin interactions ranging from molecular, sub-cellular and cellular levels with both structure and function is critical for developing SWCNT-based biotechnologies. Further, this interaction can be exploited, using SWCNTs as a unique actin-altering material. Here, we utilized molecular dynamics simulations to explore the interactions of SWCNTs with actin filaments. Fluorescence lifetime imaging microscopy confirmed that SWCNTs were located within ~5 nm of F-actin in cells but did not interact with G-actin. SWCNTs did not alter myosin II sub-cellular localization, and SWCNT treatment in cells led to significantly shorter actin filaments. Functionally, cells with internalized SWCNTs had greatly reduced cell traction force. Combined, these results demonstrate direct, specific SWCNT alteration of F-actin structures which can be exploited for SWCNT-based biotechnologies and utilized as a new method to probe fundamental actin-related cellular processes and biophysics.

  4. The actin cytoskeleton may control the polar distribution of an auxin transport protein

    Science.gov (United States)

    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  5. Filamentous Growth in Eremothecium Fungi

    DEFF Research Database (Denmark)

    Oskarsson, Therese

    The fungal kingdom encompasses a diverse group of organisms some of which have a great impact on human lives, either as domesticated benefactors or as human and crop pathogens. Using the filamentous fungus Ashbya gossypii and its close relative Eremothecium cymbalariae as model organisms, this th......The fungal kingdom encompasses a diverse group of organisms some of which have a great impact on human lives, either as domesticated benefactors or as human and crop pathogens. Using the filamentous fungus Ashbya gossypii and its close relative Eremothecium cymbalariae as model organisms......, this thesis deals with some of the aspects of hyphal growth, which is an important virulence factor for pathogenic fungi infecting both humans and plants. Hyphal establishment through continuous polar growth is a complex process, requiring the careful coordination of a large subset of proteins involved......-regulatory activity of AgGts1, the protein could have additional actin organizing properties. In the second and third part, this thesis addresses the use of A. gossypii and its relative E. cymbalariae as model organisms for filamentous growth. A series of assays analyzed the capability of Eremothecium genus fungi...

  6. Variable actin dynamics requirement for the exit of different cargo from the trans-Golgi network.

    Science.gov (United States)

    Lázaro-Diéguez, Francisco; Colonna, Cecilia; Cortegano, Miguel; Calvo, María; Martínez, Susana E; Egea, Gustavo

    2007-08-07

    Efficient post-Golgi trafficking depends on microtubules, but actin filaments and actin-associated proteins are also postulated. Here we examined, by inverse fluorescence recovery after photobleaching, the role of actin dynamics in the exit from the TGN of fluorescent-tagged apical or basolateral and raft or non-raft-associated cargoes. Either the actin-stabilizing jasplakinolide or the actin-depolymerising latrunculin B variably but significantly inhibited post-Golgi traffic of non-raft associated apical p75NTR and basolateral VSV-G cargoes. The TGN-exit of the apical-destined VSV-G mutant was impaired only by latrunculin B. Strikingly, the raft-associated GPI-anchor protein was not affected by either actin toxin. Results indicate that actin dynamics participates in the TGN egress of both apical- and basolateral-targeted proteins but is not needed for apical raft-associated cargo.

  7. Essential role of ADF/cofilin for assembly of contractile actin networks in the C. elegans somatic gonad.

    Science.gov (United States)

    Ono, Kanako; Yamashiro, Sawako; Ono, Shoichiro

    2008-08-15

    The somatic gonad of the nematode Caenorhabditis elegans contains a myoepithelial sheath, which surrounds oocytes and provides contractile forces during ovulation. Contractile apparatuses of the myoepithelial-sheath cells are non-striated and similar to those of smooth muscle. We report the identification of a specific isoform of actin depolymerizing factor (ADF)/cofilin as an essential factor for assembly of contractile actin networks in the gonadal myoepithelial sheath. Two ADF/cofilin isoforms, UNC-60A and UNC-60B, are expressed from the unc-60 gene by alternative splicing. RNA interference of UNC-60A caused disorganization of the actin networks in the myoepithelial sheath. UNC-60B, which is known to function in the body-wall muscle, was not necessary or sufficient for actin organization in the myoepithelial sheath. However, mutant forms of UNC-60B with reduced actin-filament-severing activity rescued the UNC-60A-depletion phenotype. UNC-60A has a much weaker filament-severing activity than UNC-60B, suggesting that an ADF/cofilin with weak severing activity is optimal for assembly of actin networks in the myoepithelial sheath. By contrast, strong actin-filament-severing activity of UNC-60B was required for assembly of striated myofibrils in the body-wall muscle. Our results suggest that an optimal level of actin-filament-severing activity of ADF/cofilin is required for assembly of actin networks in the somatic gonad.

  8. Self-organizing actin patterns shape membrane architecture but not cell mechanics

    Science.gov (United States)

    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.

  9. Prokaryotic cytoskeletons: protein filaments organizing small cells.

    Science.gov (United States)

    Wagstaff, James; Löwe, Jan

    2018-01-22

    Most, if not all, bacterial and archaeal cells contain at least one protein filament system. Although these filament systems in some cases form structures that are very similar to eukaryotic cytoskeletons, the term 'prokaryotic cytoskeletons' is used to refer to many different kinds of protein filaments. Cytoskeletons achieve their functions through polymerization of protein monomers and the resulting ability to access length scales larger than the size of the monomer. Prokaryotic cytoskeletons are involved in many fundamental aspects of prokaryotic cell biology and have important roles in cell shape determination, cell division and nonchromosomal DNA segregation. Some of the filament-forming proteins have been classified into a small number of conserved protein families, for example, the almost ubiquitous tubulin and actin superfamilies. To understand what makes filaments special and how the cytoskeletons they form enable cells to perform essential functions, the structure and function of cytoskeletal molecules and their filaments have been investigated in diverse bacteria and archaea. In this Review, we bring these data together to highlight the diverse ways that linear protein polymers can be used to organize other molecules and structures in bacteria and archaea.

  10. Cofilin Is a pH Sensor for Actin Free Barbed End Formation: Role of Phosphoinositide Binding

    National Research Council Canada - National Science Library

    Christian Frantz; Gabriela Barreiro; Laura Dominguez; Xiaoming Chen; Robert Eddy; John Condeelis; Mark J. S. Kelly; Matthew P. Jacobson; Diane L. Barber

    2008-01-01

    .... 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 His 133...

  11. Mechanical integration of actin and adhesion dynamics in cell migration.

    Science.gov (United States)

    Gardel, Margaret L; Schneider, Ian C; Aratyn-Schaus, Yvonne; Waterman, Clare M

    2010-01-01

    Directed cell migration is a physical process that requires dramatic changes in cell shape and adhesion to the extracellular matrix. For efficient movement, these processes must be spatiotemporally coordinated. To a large degree, the morphological changes and physical forces that occur during migration are generated by a dynamic filamentous actin (F-actin) cytoskeleton. Adhesion is regulated by dynamic assemblies of structural and signaling proteins that couple the F-actin cytoskeleton to the extracellular matrix. Here, we review current knowledge of the dynamic organization of the F-actin cytoskeleton in cell migration and the regulation of focal adhesion assembly and disassembly with an emphasis on how mechanical and biochemical signaling between these two systems regulate the coordination of physical processes in cell migration.

  12. Excitable actin dynamics in lamellipodial protrusion and retraction.

    Science.gov (United States)

    Ryan, Gillian L; Petroccia, Heather M; Watanabe, Naoki; Vavylonis, Dimitrios

    2012-04-04

    Many animal cells initiate crawling by protruding lamellipodia, consisting of a dense network of actin filaments, at their leading edge. We imaged XTC cells that exhibit flat lamellipodia on poly-L-lysine-coated coverslips. Using active contours, we tracked the leading edge and measured the total amount of F-actin by summing the pixel intensities within a 5-μm band. We observed protrusion and retraction with period 130-200 s and local wavelike features. Positive (negative) velocities correlated with minimum (maximum) integrated actin concentration. Approximately constant retrograde flow indicated that protrusions and retractions were driven by fluctuations of the actin polymerization rate. We present a model of these actin dynamics as an excitable system in which a diffusive, autocatalytic activator causes actin polymerization; F-actin accumulation in turn inhibits further activator accumulation. Simulations of the model reproduced the pattern of actin polymerization seen in experiments. To explore the model's assumption of an autocatalytic activation mechanism, we imaged cells expressing markers for both F-actin and the p21 subunit of the Arp2/3 complex. We found that integrated Arp2/3-complex concentrations spike several seconds before spikes of F-actin concentration. This suggests that the Arp2/3 complex participates in an activation mechanism that includes additional diffuse components. Response of cells to stimulation by fetal calf serum could be reproduced by the model, further supporting the proposed dynamical picture. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. Visualization of endothelial actin cytoskeleton in the mouse retina.

    Directory of Open Access Journals (Sweden)

    Alessia Fraccaroli

    Full Text Available Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs, orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood - largely because of the difficulty to visualize filamentous actin (F-actin structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs, enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation.

  14. MicroScale Thermophoresis (MST) for studying actin polymerization kinetics.

    Science.gov (United States)

    Topf, Andrea; Franz, Peter; Tsiavaliaris, Georgios

    2017-10-01

    Here, we present a MicroScale Thermophoresis (MST)-based assay for in vitro assessment of actin polymerization. By monitoring the thermophoretic behavior of ATTO488-labeled actin in a temperature gradient over time, we could follow polymerization in real time and resolve its three characteristic phases: nucleation, elongation, and steady-state equilibration. Titration experiments allowed us to evaluate the effects of actin-binding proteins (ABPs) on polymerization, including DNase I-induced inhibition and mDia2FH1FH2 (mDia2)-assisted acceleration of nucleation. The corresponding rates of actin filament elongation were quantitatively determined, yielding values in good agreement with those obtained using the pyrene-actin polymerization assay. Finally, we measured the effect of myosin on actin polymerization, circumventing the problems of fluorescence quenching and signal disturbance that occur with other techniques. MST is a simple and valuable research tool for investigating actin kinetics covering a wide range of molecular interactions, with low protein consumption.

  15. Part I---Evaluating Effects of Oligomer Formation on Cytochrome P450 2C9 Electron Transfer and Drug Metabolism, Part II---Utilizing Molecular Modeling Techniques to Study the Src-Interacting Proteins Actin Filament Associated Protein of 110 kDa (AFAP-110) and Cortactin

    Science.gov (United States)

    Jett, John Edward, Jr.

    The dissertation has been divided into two parts to accurately reflect the two distinct areas of interest pursued during my matriculation in the School of Pharmacy at West Virginia University. In Part I, I discuss research probing the nature of electron transfer in the Cytochrome P450 family of proteins, a group of proteins well-known for their role in drug metabolism. In Part II, I focus on in silico and in vitro work developed in concert to probe protein structure and protein-protein interactions involved in actin filament reorganization and cellular motility. Part I. Cytochrome P450s (P450s) are an important class of enzymes known to metabolize a variety of endogenous and xenobiotic compounds. P450s are most commonly found in liver and intestinal endothelial cells and are responsible for the metabolism of approximately 75% of pharmaceutical drugs on the market. CYP2C9---one of the six major P450 isoforms---is responsible for ˜20% of drug metabolism. Elucidation of the factors that affect in vitro drug metabolism is crucial to the accurate prediction of in vivo drug metabolism kinetics. Currently, the two major techniques for studying in vitro drug metabolism are solution-based. However, it is known that the results of solution-based studies can vary from in vivo drug metabolism. One reason suggested to account for this variation is the state of P450 oligomer formation in solution compared to the in vivo environment, where P450s are membrane-bound. To understand the details of how oligomer formation affects in vitro drug metabolism, it is imperative that techniques be developed which will allow for the unequivocal control of oligomer formation without altering other experimental parameters. Our long term goal of this research is to develop methods to more accurately predict in vivo drug metabolism from in vitro data. This section of the dissertation will discuss the development of a platform consisting of a doped silicon surface containing a large array of gold

  16. Nuclear actin and protein 4.1: Essential interactions during nuclear assembly in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon; Heald, Rebecca

    2003-06-11

    Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

  17. 3D Filament Network Segmentation with Multiple Active Contours

    Science.gov (United States)

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2014-03-01

    Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and microtubules. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we developed a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D TIRF Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy.

  18. Safety of eptifibatide for subcortical stroke progression.

    Science.gov (United States)

    Martin-Schild, Sheryl; Shaltoni, Hashem; Abraham, Anitha T; Barreto, Andrew D; Hallevi, Hen; Gonzales, Nicole R; Grotta, James C; Savitz, Sean I

    2009-01-01

    There is no proven treatment for stroke progression in patients with subcortical infarcts. Eptifibatide, a glycoprotein IIb/IIIa inhibitor, might halt stroke progression by improving flow in the microcirculation. We conducted a retrospective analysis of patients with subcortical stroke who experienced deterioration and were treated with eptifibatide (loading dose 180 microg/kg; infusion 2 m microg/kg/min) for 24-48 h. Oral antiplatelet agents were started 6 h before discontinuation of eptifibatide. Twenty-four patients with subcortical strokes were treated. The median admission NIHSS score was 5.0, which worsened to 8.5 (motor 5.0) before starting eptifibatide. The median NIHSS score 24 h after starting eptifibatide was 5.5. At 24 h, 42% had motor NIHSS scores less than or equal to pre-deterioration scores (50% for total NIHSS), and 50% had improved at least 1 motor point compared to pre-eptifibatide scores, which was sustained until hospital discharge. At discharge, the median total NIHSS score was 4.5. Ninety-two percent of patients were discharged home or to inpatient rehabilitation. Treatment was stopped early in 1 case due to a platelet drop Eptifibatide infusion may be safe in patients with subcortical ischemic strokes. Future studies are needed to test the safety and potential efficacy of this agent in subcortical stroke progression. Copyright (c) 2009 S. Karger AG, Basel.

  19. Actin-myosin network is required for proper assembly of influenza virus particles

    Energy Technology Data Exchange (ETDEWEB)

    Kumakura, Michiko; Kawaguchi, Atsushi, E-mail: ats-kawaguchi@md.tsukuba.ac.jp; Nagata, Kyosuke, E-mail: knagata@md.tsukuba.ac.jp

    2015-02-15

    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 aggregated 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.

  20. HopW1 from Pseudomonas syringae disrupts the actin cytoskeleton to promote virulence in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Yongsung Kang

    2014-06-01

    Full Text Available A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.

  1. Localization of actin in pollen tubes of Ornithogalum virens L.

    Directory of Open Access Journals (Sweden)

    Małgorzata Stępka

    2014-01-01

    Full Text Available The germinating pollen grain (in vivo on the stigma or in vitro in germination medium forms a pollen tube which transports the vegetative nucleus and generative cell/two sperm cells participating in the process of double fertilization. The growth of the tube and the transport of organelles and the cells occur due to two major motor systems existing in the pollen tubes of higher plants: the tubuline-dynein/kinesin and the actin-myosin system. In pollen tubes of Ornithogalum virens the actin filaments were labelled with TRITC-phalloidin (2 µg/ml in the PIPES buffer and the 10% sucrose, without the fixative and DMSO. Omission of the fixative and permeabilizing agent (DMSO allowed better preservation of the structure, and the "fluorescence" of actin was observed in living pollen tubes. Observations in CLSM (confocal laser scanning microscope showed that actin is distributed in the vicinity of the cell membrane. This could support the view that actin filaments and the plasmalemma form the pollen tube cortex along which the cytoplasmic movement of organelles, and cell transport occurs.

  2. TIRF assays for real-time observation of microtubules and actin coassembly: Deciphering tau effects on microtubule/actin interplay.

    Science.gov (United States)

    Prezel, Eléa; Stoppin-Mellet, Virginie; Elie, Auréliane; Zala, Ninon; Denarier, Eric; Serre, Laurence; Arnal, Isabelle

    2017-01-01

    Microtubule and actin cytoskeletons are key players in vital processes in cells. Although the importance of microtubule-actin interaction for cell development and function has been highlighted for years, the properties of these two cytoskeletons have been mostly studied separately. Thus we now need procedures to simultaneously assess actin and microtubule properties to decipher the basic mechanisms underlying microtubule-actin crosstalk. Here we describe an in vitro assay that allows the coassembly of both filaments and the real-time observation of their interaction by TIRF microscopy. We show how this assay can be used to demonstrate that tau, a neuronal microtubule-associated protein, is a bona fide actin-microtubule cross-linker. The procedure relies on the use of highly purified proteins and chemically passivated perfusion chambers. We present a step-by-step protocol to obtain actin and microtubule coassembly and discuss the major pitfalls. An ImageJ macro to quantify actin and microtubule interaction is also provided. © 2017 Elsevier Inc. All rights reserved.

  3. Actin Cytoskeleton and Golgi Involvement in Barley stripe mosaic virus Movement and Cell Wall Localization of Triple Gene Block Proteins

    NARCIS (Netherlands)

    Lim, H.S.; Lee, M.Y.; Moon, J.S.; Moon, J.K.; Yu, Y.M.; Cho, I.S.; Bae, H.; Boer, de S.M.; Ju, H.; Hammond, J.; Jackson, A.O.

    2013-01-01

    Barley stripe mosaic virus (BSMV) induces massive actin filament thickening at the infection front of infected Nicotiana benthamiana leaves. To determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the BSMV triple gene block (TGB) proteins were coexpressed in cells

  4. Actin and microtubule networks contribute differently to cell response for small and large strains

    Science.gov (United States)

    Kubitschke, H.; Schnauss, J.; Nnetu, K. D.; Warmt, E.; Stange, R.; Kaes, J.

    2017-09-01

    Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.

  5. Contributions of the lower dimer to supramolecular actin patterning revealed by TIRF microscopy.

    Science.gov (United States)

    Silván, Unai; Hyotyla, Janne; Mannherz, Hans-Georg; Ringler, Philippe; Müller, Shirley A; Aebi, Ueli; Maier, Timm; Schoenenberger, Cora-Ann

    2016-08-01

    Two distinct dimers are formed during the initial steps of actin polymerization. The first one, referred to as the 'lower dimer' (LD) was discovered many years ago by means of chemical crosslinking. Owing to its transient nature, a biological relevance had long been precluded when, using LD-specific antibodies, we detected LD-like contacts in actin assemblies that are associated with the endolysosomal compartment in a number of different cell lines. Moreover, immunofluorescence showed the presence of LD-related structures at the cell periphery of migrating fibroblasts, in the nucleus, and in association with the centrosome of interphase cells. Here, we explore contributions of the LD to the assembly of supramolecular actin structures in real time by total internal reflection fluorescence (TIRF) microscopy. Our data shows that while LD on its own cannot polymerize under filament forming conditions, it is able to incorporate into growing F-actin filaments. This incorporation of LD triggers the formation of X-shaped filament assemblies with barbed ends that are pointing in the same direction in the majority of cases. Similarly, an increased frequency of junction sites was observed when filaments were assembled in the presence of oxidized actin. This data suggests that a disulfide bridge between Cys374 residues might stabilize LD-contacts. Based on our findings, we propose two possible models for the molecular mechanism underlying the supramolecular actin patterning in LD-related structures. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Intermediate Filaments and Polarization in the Intestinal Epithelium

    Directory of Open Access Journals (Sweden)

    Richard A. Coch

    2016-07-01

    Full Text Available The cytoplasmic intermediate filament cytoskeleton provides a tissue-specific three-dimensional scaffolding with unique context-dependent organizational features. This is particularly apparent in the intestinal epithelium, in which the intermediate filament network is localized below the apical terminal web region and is anchored to the apical junction complex. This arrangement is conserved from the nematode Caenorhabditis elegans to humans. The review summarizes compositional, morphological and functional features of the polarized intermediate filament cytoskeleton in intestinal cells of nematodes and mammals. We emphasize the cross talk of intermediate filaments with the actin- and tubulin-based cytoskeleton. Possible links of the intermediate filament system to the distribution of apical membrane proteins and the cell polarity complex are highlighted. Finally, we discuss how these properties relate to the establishment and maintenance of polarity in the intestine.

  7. Intermediate Filaments and Polarization in the Intestinal Epithelium.

    Science.gov (United States)

    Coch, Richard A; Leube, Rudolf E

    2016-07-15

    The cytoplasmic intermediate filament cytoskeleton provides a tissue-specific three-dimensional scaffolding with unique context-dependent organizational features. This is particularly apparent in the intestinal epithelium, in which the intermediate filament network is localized below the apical terminal web region and is anchored to the apical junction complex. This arrangement is conserved from the nematode Caenorhabditis elegans to humans. The review summarizes compositional, morphological and functional features of the polarized intermediate filament cytoskeleton in intestinal cells of nematodes and mammals. We emphasize the cross talk of intermediate filaments with the actin- and tubulin-based cytoskeleton. Possible links of the intermediate filament system to the distribution of apical membrane proteins and the cell polarity complex are highlighted. Finally, we discuss how these properties relate to the establishment and maintenance of polarity in the intestine.

  8. Filamentous Growth in Eremothecium Fungi

    DEFF Research Database (Denmark)

    Oskarsson, Therese

    , this thesis deals with some of the aspects of hyphal growth, which is an important virulence factor for pathogenic fungi infecting both humans and plants. Hyphal establishment through continuous polar growth is a complex process, requiring the careful coordination of a large subset of proteins involved......-regulatory activity of AgGts1, the protein could have additional actin organizing properties. In the second and third part, this thesis addresses the use of A. gossypii and its relative E. cymbalariae as model organisms for filamentous growth. A series of assays analyzed the capability of Eremothecium genus fungi...... of molecular tools for E. cymbalariae to enable a faster and more efficient approach for genetic comparisons between Eremothecium genus fungi....

  9. The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

    Science.gov (United States)

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

  10. Nonequilibrium dynamics of probe filaments in actin-myosin networks.

    Science.gov (United States)

    Gladrow, J; Broedersz, C P; Schmidt, C F

    2017-08-01

    Active dynamic processes of cells are largely driven by the cytoskeleton, a complex and adaptable semiflexible polymer network, motorized by mechanoenzymes. Small dimensions, confined geometries, and hierarchical structures make it challenging to probe dynamics and mechanical response of such networks. Embedded semiflexible probe polymers can serve as nonperturbing multiscale probes to detect force distributions in active polymer networks. We show here that motor-induced forces transmitted to the probe polymers are reflected in nonequilibrium bending dynamics, which we analyze in terms of spatial eigenmodes of an elastic beam under steady-state conditions. We demonstrate how these active forces induce correlations among the mode amplitudes, which furthermore break time-reversal symmetry. This leads to a breaking of detailed balance in this mode space. We derive analytical predictions for the magnitude of resulting probability currents in mode space in the white-noise limit of motor activity. We relate the structure of these currents to the spatial profile of motor-induced forces along the probe polymers and provide a general relation for observable currents on two-dimensional hyperplanes.

  11. Inhibitory effects of pectenotoxins from marine algae on the polymerization of various actin isoforms.

    Science.gov (United States)

    Butler, Suzanne C; Miles, Christopher O; Karim, Amna; Twiner, Michael J

    2012-04-01

    Pectenotoxins (PTXs) are marine toxins produced by dinoflagellates and which accumulate in shellfish. There are at least 14 different analogs of PTX with slight variations in structure leading to different chemical properties and consequently different toxicities. Since preliminary studies have shown that the parent compound PTX1 targets actin, we investigated the effects of two analogs, PTX2 and PTX2 seco acid, on the polymerization and depolymerization of skeletal muscle actin, smooth muscle actin, cardiac muscle actin, and non-muscle actin. Optimized actin assays using fluorescently labeled skeletal muscle actin and SDS-PAGE were jointly used to determine the relative amounts of filamentous and globular actin formed during polymerization and depolymerization experiments. Our findings suggest that PTX2 causes a dose-dependent decrease in both the rate and yield of skeletal muscle actin polymerization (IC50 values of 44 and 177 nM; respectively), with no significant effects on depolymerization. Moreover, the inhibitory effects of PTX2 are conserved towards other actin isoforms (i.e., smooth muscle, cardiac muscle, and non-muscle), as the inhibitory effects on actin polymerization were also observed with similar IC50 values (range: 19-94 nM). No inhibitory effects on polymerization were observed for PTX2 seco acid, suggesting an intact lactone ring is necessary for bioactivity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Developmental expression of the actin depolymerizing factor ADF in the mouse inner ear and spiral ganglia.

    Science.gov (United States)

    Herde, Michel K; Friauf, Eckhard; Rust, Marco B

    2010-05-15

    Hair cells, the inner ear's sensory cells, are characterized by tens to hundreds of actin-rich stereocilia that form the hair bundle apparatus necessary for mechanoelectrical transduction. Both the number and length of actin filaments are precisely regulated in stereocilia. Proper cochlear and vestibular function also depends on actin filaments in nonsensory supporting cells. The formation of actin filaments is a dynamic, treadmill-like process in which actin-binding proteins play crucial roles. However, little is known about the presence and function of actin binding molecules in the inner ear, which set up, and maintain, actin-rich structures and regulate actin turnover. Here we examined the expression and subcellular location of the actin filament depolymerizing factor (ADF) in the cochlea and vestibular organs. By means of immunocytochemistry and confocal microscopy, we analyzed whole-mount preparations and cross-sections in fetal and postnatal mice (E15-P26). We found a transient ADF expression in immature hair cells of the organ of Corti, the utricle, and the saccule. Interestingly, the stereocilia were not labeled. By P26, ADF expression was restricted to supporting cells. In addition, we localized ADF in presynaptic terminals of medio-olivocochlear projections after hearing onset. A small population of spiral ganglion neurons strongly expressed ADF. Based on their relative number, peripheral location within the ganglion, smaller soma size, and coexpression of neurofilament 200, we identified these cells as Type II spiral ganglion neurons. The developmentally regulated ADF expression suggests a temporally restricted function in the stereocilia and, thus, a hitherto undescribed role of ADF. (c) 2009 Wiley-Liss, Inc.

  13. Encoding Mechano-Memories in Actin Networks

    Science.gov (United States)

    Foucard, Louis; Majumdar, Sayantan; Levine, Alex; Gardel, Margaret

    The ability of cells to sense and adapt to external mechanical stimuli is vital to many of its biological functions. A critical question is therefore to understand how mechanosensory mechanisms arise in living matter, with implications in both cell biology and smart materials design. Experimental work has demonstrated that the mechanical properties of semiflexible actin networks in Eukaryotic cells can be modulated (either transiently or irreversibly) via the application of external forces. Previous work has also shown with a combination of numerical simulations and analytic calculations shows that the broken rotational symmetry of the filament orientational distribution in semiflexible networks leads to dramatic changes in the mechanical response. Here we demonstrate with a combination of numerical and analytic calculations that the observed long-lived mechano-memory in the actin networks arise from changes in the nematic order of the constituent filaments. These stress-induced changes in network topology relax slowly under zero stress and can be observed through changes in the nonlinear mechanics. Our results provide a strategy for designing a novel class of materials and demonstrate a new putative mechanism of mechanical sensing in eukaryotic cells.

  14. System-wide organization of actin cytoskeleton determines organelle transport in hypocotyl plant cells

    Science.gov (United States)

    Nowak, Jacqueline; Ivakov, Alexander; Somssich, Marc; Persson, Staffan; Nikoloski, Zoran

    2017-01-01

    The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms. PMID:28655850

  15. Isolation from thyroid cells or purified plasma membranes with associated actin microfilaments. Proteins bound to actin.

    Science.gov (United States)

    Regnouf, F; Delobbe, A; Gabrion, J; Mesnier, D; Pradel, L A

    1982-02-01

    Plasma membranes of thyroid cells were purified from hog thyroid glands following two procedures. Their homogeneity was tested by electron microscopy and by measurements of the activity of membrane-bound enzyme markers. According to the procedure used the membrane fractions obtained present some differences in their morphological features as well as in the repartition of the activities of the membrane-bound enzyme markers. However, whatever the composition of the membrane fraction examined (membrane vesicles, single membrane sheets with junctional complexes), decoration with heavy meromyosin clearly shows the presence of actin filaments attached to these fragments. Analysis of proteins by polyacrylamide gel electrophoresis indicates the presence of about twelve major components with actin. Treatment of membranes with Triton X-100 results in an insoluble core which contains all the actin and most of the major proteins. The selective extraction of these components by buffers differing in their ionic strength, pH, or the presence or absence of ATP X Mg has been used to characterize some of the proteins associated to actin; among them are filamin, myosin, alpha-actinin, tropomyosin.

  16. Nonmuscle myosin II exerts tension but does not translocate actin in vertebrate cytokinesis.

    Science.gov (United States)

    Ma, Xuefei; Kovács, Mihály; Conti, Mary Anne; Wang, Aibing; Zhang, Yingfan; Sellers, James R; Adelstein, Robert S

    2012-03-20

    During vertebrate cytokinesis it is thought that contractile ring constriction is driven by nonmuscle myosin II (NM II) translocation of antiparallel actin filaments. Here we report in situ, in vitro, and in vivo observations that challenge this hypothesis. Graded knockdown of NM II in cultured COS-7 cells reveals that the amount of NM II limits ring constriction. Restoration of the constriction rate with motor-impaired NM II mutants shows that the ability of NM II to translocate actin is not required for cytokinesis. Blebbistatin inhibition of cytokinesis indicates the importance of myosin strongly binding to actin and exerting tension during cytokinesis. This role is substantiated by transient kinetic experiments showing that the load-dependent mechanochemical properties of mutant NM II support efficient tension maintenance despite the inability to translocate actin. Under loaded conditions, mutant NM II exhibits a prolonged actin attachment in which a single mechanoenzymatic cycle spans most of the time of cytokinesis. This prolonged attachment promotes simultaneous binding of NM II heads to actin, thereby increasing tension and resisting expansion of the ring. The detachment of mutant NM II heads from actin is enhanced by assisting loads, which prevent mutant NM II from hampering furrow ingression during cytokinesis. In the 3D context of mouse hearts, mutant NM II-B R709C that cannot translocate actin filaments can rescue multinucleation in NM II-B ablated cardiomyocytes. We propose that the major roles of NM II in vertebrate cell cytokinesis are to bind and cross-link actin filaments and to exert tension on actin during contractile ring constriction.

  17. Dysphagia Post Subcortical and Supratentorial Stroke.

    Science.gov (United States)

    Wan, Ping; Chen, Xuhui; Zhu, Lequn; Xu, Shuangjin; Huang, Li; Li, Xiangcui; Ye, Qing; Ding, Ruiying

    2016-01-01

    Studies have recognized that the damage in the subcortical and supratentorial regions may affect voluntary and involuntary aspects of the swallowing function. The current study attempted to explore the dysphagia characteristics in patients with subcortical and supratentorial stroke. Twelve post first or second subcortical and supratentorial stroke patients were included in the study. The location of the stroke was ascertained by computed tomography and magnetic resonance imaging. The characteristics of swallowing disorder were assessed by video fluoroscopic swallowing assessment/fiberoptic endoscopic evaluation of swallowing. The following main parameters were analyzed: oral transit time, pharyngeal delay time, presence of cricopharyngeal muscle achalasia (CMA), distance of laryngeal elevation, the amounts of vallecular residue and pyriform sinus residue (PSR), and the extent of pharyngeal contraction. Eighty-three percent of the 12 patients were found suffering from pharyngeal dysphagia, with 50% having 50%-100% PSRs, 50% having pharyngeal delay, and 41.6% cases demonstrating CMA. Simple regression analysis showed PSRs were most strongly associated with CMA. Pharyngeal delay in the study can be caused by infarcts of basal ganglia/thalamus, infarcts of sensory tract, infarcts of swallowing motor pathways in the centrum semiovale, or a combination of the three. Subcortical and supratentorial stroke may result in pharyngeal dysphagia such as PSR and pharyngeal delay. PSR was mainly caused by CMA. Copyright © 2015 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  18. Hippocampal atrophy in subcortical vascular dementia

    NARCIS (Netherlands)

    van de Pol, L.A.; Gertz, H.J.; Scheltens, P.; Wolf, H

    2011-01-01

    Background and Purpose: New research criteria for subcortical vascular dementia (SVaD) have been suggested to define a more homogeneous subgroup of vascular dementia. Hippocampal (Hc) atrophy is a hallmark of Alzheimer's disease (AD), but it also occurs in other dementia disorders including vascular

  19. Subcortical Facilitation of Behavioral Responses to Threat.

    Science.gov (United States)

    Vida, Mark D; Behrmann, Marlene

    2017-10-12

    Behavioral responses to threat are critical to survival. Several cortical and subcortical brain regions respond selectively to threat. However, the relation of these neural responses and their underlying representations to behavior is unclear. We examined the contribution of lower-order subcortical representations to behavioral responses to threat in adult humans. In Experiments 1 and 2, participants viewed pairs of images presented to the same eye or to different eyes. We observed a monocular advantage, which indicates subcortical facilitation, for ancestral threats (snakes, spiders), but not for modern threats, positive images, or neutral images. In Experiment 3, we presented pairs of snakes or neutral images into the temporal or nasal hemifield. For snakes only, we observed a temporal hemifield advantage, which indicates facilitation by the retino-tectal subcortical pathway. These results advance the current understanding of processing of threat by adult humans by revealing the characteristics of behaviors driven by a lower-order neural mechanism that is specialized for the processing of ancestral threats. The results also contribute to ongoing debates concerning the biological generality of neural mechanisms for processing of complex, emotionally-relevant stimuli by providing evidence for conservation of lower-order neural mechanisms for processing of ancestral threats across both ontogeny and phylogeny.

  20. Actin Cytoskeleton and Golgi Involvement in Barley stripe mosaic virus Movement and Cell Wall Localization of Triple Gene Block Proteins

    Directory of Open Access Journals (Sweden)

    Hyoun-Sub Lim

    2013-03-01

    Full Text Available Barley stripe mosaic virus (BSMV induces massive actin filament thickening at the infection front of infected Nicotiana benthamiana leaves. To determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the BSMV triple gene block (TGB proteins were coexpressed in cells with the actin marker DsRed: Talin. TGB ectopic expression experiments revealed that TGB3 is a major elicitor of filament thickening, that TGB2 resulted in formation of intermediate DsRed:Talin filaments, and that TGB1 alone had no obvious effects on actin filament structure. Latrunculin B (LatB treatments retarded BSMV cell-to-cell movement, disrupted actin filament organization, and dramatically decreased the proportion of paired TGB3 foci appearing at the cell wall (CW. BSMV infection of transgenic plants tagged with GFP-KDEL exhibited membrane proliferation and vesicle formation that were especially evident around the nucleus. Similar membrane proliferation occurred in plants expressing TGB2 and/or TGB3, and DsRed: Talin fluorescence in these plants colocalized with the ER vesicles. TGB3 also associated with the Golgi apparatus and overlapped with cortical vesicles appearing at the cell periphery. Brefeldin A treatments disrupted Golgi and also altered vesicles at the CW, but failed to interfere with TGB CW localization. Our results indicate that actin cytoskeleton interactions are important in BSMV cell-to-cell movement and for CW localization of TGB3.

  1. Arabidopsis VILLIN1 and VILLIN3 have overlapping and distinct activities in actin bundle formation and turnover

    Energy Technology Data Exchange (ETDEWEB)

    Khurana, P; Henty, Jessica L; Huang, S; Staiger, A M; Blanchoin, L; Staiger, Chris J

    2010-09-28

    Actin filament bundles are higher-order cytoskeletal structures that are crucial for the maintenance of cellular architecture and cell expansion. They are generated from individual actin filaments by the actions of bundling proteins like fimbrins, LIMs, and villins. However, the molecular mechanisms of dynamic bundle formation and turnover are largely unknown. Villins belong to the villin/gelsolin/fragmin superfamily and comprise at least five isovariants in Arabidopsis thaliana. Different combinations of villin isovariants are coexpressed in various tissues and cells. It is not clear whether these isovariants function together and act redundantly or whether they have unique activities. VILLIN1 (VLN1) is a simple filament-bundling protein and is Ca{sup 2+} insensitive. Based on phylogenetic analyses and conservation of Ca{sup 2+} binding sites, we predict that VLN3 is a Ca{sup 2+}-regulated villin capable of severing actin filaments and contributing to bundle turnover. The bundling activity of both isovariants was observed directly with time-lapse imaging and total internal reflection fluorescence (TIRF) microscopy in vitro, and the mechanism mimics the “catch and zipper” action observed in vivo. Using time-lapse TIRF microscopy, we observed and quantified the severing of individual actin filaments by VLN3 at physiological calcium concentrations. Moreover, VLN3 can sever actin filament bundles in the presence of VLN1 when calcium is elevated to micromolar levels. Collectively, these results demonstrate that two villin isovariants have overlapping and distinct activities.

  2. Arabidopsis VILLIN1 and VILLIN3 Have Overlapping and Distinct Activities in Actin Bundle Formation and Turnover[W

    Science.gov (United States)

    Khurana, Parul; Henty, Jessica L.; Huang, Shanjin; Staiger, Andrew M.; Blanchoin, Laurent; Staiger, Christopher J.

    2010-01-01

    Actin filament bundles are higher-order cytoskeletal structures that are crucial for the maintenance of cellular architecture and cell expansion. They are generated from individual actin filaments by the actions of bundling proteins like fimbrins, LIMs, and villins. However, the molecular mechanisms of dynamic bundle formation and turnover are largely unknown. Villins belong to the villin/gelsolin/fragmin superfamily and comprise at least five isovariants in Arabidopsis thaliana. Different combinations of villin isovariants are coexpressed in various tissues and cells. It is not clear whether these isovariants function together and act redundantly or whether they have unique activities. VILLIN1 (VLN1) is a simple filament-bundling protein and is Ca2+ insensitive. Based on phylogenetic analyses and conservation of Ca2+ binding sites, we predict that VLN3 is a Ca2+-regulated villin capable of severing actin filaments and contributing to bundle turnover. The bundling activity of both isovariants was observed directly with time-lapse imaging and total internal reflection fluorescence (TIRF) microscopy in vitro, and the mechanism mimics the “catch and zipper” action observed in vivo. Using time-lapse TIRF microscopy, we observed and quantified the severing of individual actin filaments by VLN3 at physiological calcium concentrations. Moreover, VLN3 can sever actin filament bundles in the presence of VLN1 when calcium is elevated to micromolar levels. Collectively, these results demonstrate that two villin isovariants have overlapping and distinct activities. PMID:20807878

  3. Involvement of the conserved adaptor protein Alix in actin cytoskeleton assembly.

    Science.gov (United States)

    Pan, Shujuan; Wang, Ruoning; Zhou, Xi; He, Guangan; Koomen, John; Kobayashi, Ryuji; Sun, Le; Corvera, Joe; Gallick, Gary E; Kuang, Jian

    2006-11-10

    The conserved adaptor protein Alix, also called AIP1 or Hp95, promotes flattening and alignment of cultured mammalian fibroblasts; however, the mechanism by which Alix regulates fibroblast morphology is not understood. Here we demonstrate that Alix in WI38 cells, which require Alix expression for maintaining typical fibroblast morphology, associates with filamentous actin (F-actin) and F-actin-based structures lamellipodia and stress fibers. Reducing Alix expression by small interfering RNA (siRNA) decreases F-actin content and inhibits stress fiber assembly. In cell-free systems, Alix directly interacts with F-actin at both the N-terminal Bro1 domain and the C-terminal proline-rich domain. In Alix immunoprecipitates from WI38 cell lysates, actin is the most abundant partner protein of Alix. In addition, the N-terminal half of the middle region of Alix binds cortactin, an activator of the ARP2/3 complex-mediated initiation of actin polymerization. Alix is required for lamellipodial localization of cortactin. The C-terminal half of the middle region of Alix interacts with alpha-actinin, a key factor that bundles F-actin in stress fibers. Alix knockdown decreases the amount of alpha-actinin that associates with F-actin. These findings establish crucial involvement of Alix in actin cytoskeleton assembly.

  4. Caldesmon regulates actin dynamics to influence cranial neural crest migration in Xenopus

    OpenAIRE

    Nie, Shuyi; Kee, Yun; Bronner-Fraser, Marianne

    2011-01-01

    Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells t...

  5. Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions.

    Directory of Open Access Journals (Sweden)

    Shenping Wu

    2010-09-01

    Full Text Available Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very

  6. Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Shenping; Liu, Jun; Reedy, Mary C.; Tregear, Richard T.; Winkler, Hanspeter; Franzini-Armstrong, Clara; Sasaki, Hiroyuki; Lucaveche, Carmen; Goldman, Yale E.; Reedy, Michael K.; Taylor, Kenneth A. (UPENN); (Duke); (MRCLMB); (FSU); (Jikei-Med)

    2010-10-22

    Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the 'target zone', situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77{sup o}/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127{sup o} range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are

  7. ER-mitochondria contacts: Actin dynamics at the ER control mitochondrial fission via calcium release.

    Science.gov (United States)

    Steffen, Janos; Koehler, Carla M

    2018-01-02

    The formin-like protein INF2 is an important player in the polymerization of actin filaments. In this issue, Chakrabarti et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201709111) demonstrate that INF2 mediates actin polymerization at the endoplasmic reticulum (ER), resulting in increased ER-mitochondria contacts, calcium uptake by mitochondria, and mitochondrial division. © 2018 Steffen and Koehler.

  8. Solar Features - Prominences and Filaments - Filaments

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Filaments are formed in magnetic loops that hold relatively cool, dense gas suspended above the surface of the Sun (David Hathaway/NASA)

  9. Physical principles of filamentous protein self-assembly kinetics

    Science.gov (United States)

    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.

  10. Fascin 2b is a component of stereocilia that lengthens actin-based protrusions.

    Directory of Open Access Journals (Sweden)

    Shih-Wei Chou

    Full Text Available Stereocilia are actin-filled protrusions that permit mechanotransduction in the internal ear. To identify proteins that organize the cytoskeleton of stereocilia, we scrutinized the hair-cell transcriptome of zebrafish. One promising candidate encodes fascin 2b, a filamentous actin-bundling protein found in retinal photoreceptors. Immunolabeling of zebrafish hair cells and the use of transgenic zebrafish that expressed fascin 2b fused to green fluorescent protein demonstrated that fascin 2b localized to stereocilia specifically. When filamentous actin and recombinant fusion protein containing fascin 2b were combined in vitro to determine their dissociation constant, a K(d≈0.37 µM was observed. Electron microscopy showed that fascin 2b-actin filament complexes formed parallel actin bundles in vitro. We demonstrated that expression of fascin 2b or espin, another actin-bundling protein, in COS-7 cells induced the formation of long filopodia. Coexpression showed synergism between these proteins through the formation of extra-long protrusions. Using phosphomutant fascin 2b proteins, which mimicked either a phosphorylated or a nonphosphorylated state, in COS-7 cells and in transgenic hair cells, we showed that both formation of long filopodia and localization of fascin 2b to stereocilia were dependent on serine 38. Overexpression of wild-type fascin 2b in hair cells was correlated with increased stereociliary length relative to controls. These findings indicate that fascin 2b plays a key role in shaping stereocilia.

  11. Antiparallel dimer and actin assembly†

    OpenAIRE

    Grintsevich, Elena E.; Phillips, Martin; Pavlov, Dmitry; Phan, Mai; Reisler, Emil; Muhlrad, Andras

    2010-01-01

    Antiparallel dimer (APD) is a unique actin species, which can be detected in the early stages of actin polymerization. In this work, we introduce novel tools to examine the effects of the APD on actin polymerization. We document that bifunctional methanothiosulfonate (MTS) reagents are an attractive alternative to the routinely used p-phenylenemaleimide (pPDM) for APD detection, allowing for a fast and efficient cross-linking under conditions of actin polymerization at neutral pH. We report a...

  12. Qdot Labeled Actin Super Resolution Motility Assay Measures Low Duty Cycle Muscle Myosin Step-Size

    Science.gov (United States)

    Wang, Yihua; Ajtai, Katalin; Burghardt, Thomas P.

    2013-01-01

    Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescence labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdot’s were imaged in time with total internal reflection fluorescence microscopy then spatially localized to 1-3 nanometers using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full length β-cardiac myosin (MYH7). Average Qdot-actin velocity matches measurements with rhodamine-phalloidin labeled actin. The sHMM Qdot-actin velocity histogram contains low velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm, and, larger compliance than sHMM depending on MYH7 surface concentration. Low duty cycle skeletal and cardiac myosin present challenges for a single molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space then characterized using super-resolution. The approach provides quick, quantitative, and inexpensive step-size measurement for low duty cycle muscle myosin. PMID:23383646

  13. DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.

    Directory of Open Access Journals (Sweden)

    Imre Molnár

    2014-02-01

    Full Text Available During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin.

  14. GMF is an evolutionarily developed Adf/cofilin-super family protein involved in the Arp2/3 complex-mediated organization of the actin cytoskeleton.

    Science.gov (United States)

    Nakano, Kentaro; Kuwayama, Hidekazu; Kawasaki, Masato; Numata, Osamu; Takaine, Masak

    2010-06-01

    Actin-depolymerizing factor (ADF)/cofilin is widely expressed in eukaryotes and plays a central role in reorganizing the actin cytoskeleton by disassembling actin filaments. The ADF-homologous domain (ADF-H) is conserved in several other actin-modulating proteins such as twinfilin, Abp1/drebrin, and coactosin. Although these proteins interact with actin via ADF-H, their effects on actin are not identical to each other. Here, we report a novel ADF/cofilin-super family protein, Gmf1 (Glia maturation factor-like protein 1), from the fission yeast Schizosaccharomyces pombe. Gmf1 is a component of actin patches, which are located on the cell cortex and required for endocytosis, and may be involved in the control of the disassembly of actin patches since its overexpression diminishes them. We provide evidence that Gmf1 binds weakly if at all to actin, but it associates with actin-related protein (Arp) 2/3 complex and suppresses its functions such as the promotion of actin polymerization and branching filaments. Importantly, Arp2/3 complex-suppressing activity is conserved among GMF-family proteins from other organisms. Given the functional plasticity of ADF-H, GMF-family proteins possibly have changed their target from conventional actin to Arps through molecular evolution.

  15. Neuropsychological performance in patients with subcortical stroke

    Directory of Open Access Journals (Sweden)

    Silviane Pinheiro Campos de Andrade

    2012-05-01

    Full Text Available Vascular cognitive impairment (VCI is characterized by cognitive compromise predominantly of executive dysfunction. OBJECTIVES: To assess cognitive functions in VCI, focusing on executive functions, to observe functional losses in relation to activities of daily living (ADLs and to detect early symptoms prior to the onset of dementia. METHODS: We evaluated healthy subjects matched for gender, education and age to patients with diagnosis of subcortical vascular disease who had a stroke classified into three groups: 1 vascular lesions and no impairment; 2 vascular cognitive impairment with no dementia (VCIND; 3 vascular dementia (VaD. RESULTS AND DISCUSSION: The performance on neuropsychological tests differed among groups, worsening with increased impairment level. The probable VaD group demonstrated impaired performance in memory, processing speed and verbal production, while the VCIND group showed attention deficits. CONCLUSION: Impairment in executive functions and difficulties in ADLs allow us to differentiate levels of impairment in groups of subcortical vascular disease.

  16. Implications of Subcortical structures in Aphasia.

    Directory of Open Access Journals (Sweden)

    Saleh Alamri

    2015-04-01

    Taken together, the results indicate that aphasia is a common outcome after a lesion to subcortical structures. Findings show that 110 out of 394 aphasic patients with lesion in the basal ganglia exhibited comprehension deficits, while 31 participants out of 288 with thalamic aphasia. Likewise, 129 aphasics of affected basal ganglia out of 394 had impaired naming, whereas 12 participants had impaired naming out of 288 individuals with thalamic aphasia. See figure 1. Figure 1: The percentage of language impairment in two sets of aphasic patients (the thalamus and the basal ganglia. Despite contradictory results and even cases of double dissociation (for an example of absence of language deficits in the event of thalamic lesions see Cappa et al., 1986, our literature review confirms the major role of subcortical structures in language processing.

  17. Actin-based motility propelled by molecular motors

    Science.gov (United States)

    Upadyayula, Sai Pramod; Rangarajan, Murali

    2012-09-01

    Actin-based motility of Listeria monocytogenes propelled by filament end-tracking molecular motors has been simulated. Such systems may act as potential nanoscale actuators and shuttles useful in sorting and sensing biomolecules. Filaments are modeled as three-dimensional elastic springs distributed on one end of the capsule and persistently attached to the motile bacterial surface through an end-tracking motor complex. Filament distribution is random, and monomer concentration decreases linearly as a function of position on the bacterial surface. Filament growth rate increases with monomer concentration but decreases with the extent of compression. The growing filaments exert push-pull forces on the bacterial surface. In addition to forces, torques arise due to two factors—distribution of motors on the bacterial surface, and coupling of torsion upon growth due to the right-handed helicity of F-actin—causing the motile object to undergo simultaneous translation and rotation. The trajectory of the bacterium is simulated by performing a force and torque balance on the bacterium. All simulations use a fixed value of torsion. Simulations show strong alignment of the filaments and the long axis of the bacterium along the direction of motion. In the absence of torsion, the bacterial surface essentially moves along the direction of the long axis. When a small amount of the torsion is applied to the bacterial surface, the bacterium is seen to move in right-handed helical trajectories, consistent with experimental observations.

  18. Subcortical cerebral infarctions in sickle cell trait.

    OpenAIRE

    Reyes, M G

    1989-01-01

    At necropsy, two patients with sickle cell trait and progressive motor and visual deficits, lethargy and coma showed infarctions of the deep cerebral white matter and brain stem. The findings in these patients and another reported in the literature suggest that subcortical infarctions may be more common in sickle cell trait than has been recognised and should be suspected in any patient with sickle cell trait who presents with an unusual neurological illness.

  19. ADP-ribosylation of actins in fibroblasts and myofibroblasts by botulinum C2 toxin: Influence on microfilament morphology and migratory behavior

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Petersen, Ole William

    1996-01-01

    (myofibroblasts) as well. The significance of this molecular conversion has remained largely unknown. We have recently shown that a reduction in filamentous -sm actin by electroinjected specific antibodies or antisense oligodeoxynucleotides leads to increased motility in breast myofibroblasts (Rønnov-Jessen L...... fibroblasts rapidly round up and stop migrating when filamentous -/-actin is reduced by short-term ADP-ribosylation, myofibroblasts maintain their flattened morphology and a basic low motility....

  20. Cofilin phosphorylation is elevated after F-actin disassembly induced by Rac1 depletion

    DEFF Research Database (Denmark)

    Liu, Linna; Li, Jing; Zhang, Liwang

    2015-01-01

    that actin filaments disassembled. In the epidermis of mice in which Rac1 was knocked out only in keratinocytes, cofilin phosphorylation was aberrantly elevated, corresponding to repression of the phosphatase slingshot1 (SSH1). These effects were independent of the signaling pathways for p21-activated kinase....../LIM kinase (Pak/LIMK), protein kinase C, or protein kinase D or generation of reactive oxygen species. Similarly, when actin polymerization was specifically inhibited or Rac1 was knocked down, cofilin phosphorylation was enhanced and SSH1 was repressed. Repression of SSH1 partially blocked actin...

  1. Externally refuelled optical filaments

    Science.gov (United States)

    Scheller, Maik; Mills, Matthew S.; Miri, Mohammad-Ali; Cheng, Weibo; Moloney, Jerome V.; Kolesik, Miroslav; Polynkin, Pavel; Christodoulides, Demetrios N.

    2014-04-01

    Plasma channels produced in air through femtosecond laser filamentation hold great promise for a number of applications, including remote sensing, attosecond physics and spectroscopy, channelling microwaves and lightning protection. In such settings, extended filaments are desirable, yet their longitudinal span is limited by dissipative processes. Although various techniques aiming to prolong this process have been explored, the substantial extension of optical filaments remains a challenge. Here, we experimentally demonstrate that the natural range of a plasma column can be enhanced by at least an order of magnitude when the filament is prudently accompanied by an auxiliary beam. In this arrangement, the secondary low-intensity `dressing' beam propagates linearly and acts as a distributed energy reservoir, continuously refuelling the optical filament. Our approach offers an efficient and viable route towards the generation of extended light strings in air without inducing premature wave collapse or an undesirable beam break-up into multiple filaments.

  2. Subcortical correlates of individual differences in aptitude.

    Science.gov (United States)

    Jung, Rex E; Ryman, Sephira G; Vakhtin, Andrei A; Carrasco, Jessica; Wertz, Chris; Flores, Ranee A

    2014-01-01

    The study of individual differences encompasses broad constructs including intelligence, creativity, and personality. However, substantially less research is devoted to the study of specific aptitudes in spite of their importance to educational, occupational, and avocational success. We sought to determine subcortical brain structural correlates of several broad aptitudes including Math, Vocabulary, Foresight, Paper Folding, and Inductive Reasoning in a large (N = 107), healthy, young (age range  = 16-29) cohort. Subcortical volumes were measured using an automated technique (FreeSurfer) across structures including bilateral caudate, putamen, globus pallidus, thalamus, nucleus accumbens, hippocampus, amygdala, and five equal regions of the corpus callosum. We found that performance on measures of each aptitude was predicted by different subcortical structures: Math--higher right nucleus accumbens volume; Vocabulary--higher left hippocampus volume; Paper Folding--higher right thalamus volume; Foresight--lower right thalamus and higher mid anterior corpus callosum volume; Inductive Reasoning--higher mid anterior corpus callosum volume. Our results support general findings, within the cognitive neurosciences, showing lateralization of structure-function relationships, as well as more specific relationships between individual structures (e.g., left hippocampus) and functions relevant to particular aptitudes (e.g., Vocabulary).

  3. Subcortical correlates of individual differences in aptitude.

    Directory of Open Access Journals (Sweden)

    Rex E Jung

    Full Text Available The study of individual differences encompasses broad constructs including intelligence, creativity, and personality. However, substantially less research is devoted to the study of specific aptitudes in spite of their importance to educational, occupational, and avocational success. We sought to determine subcortical brain structural correlates of several broad aptitudes including Math, Vocabulary, Foresight, Paper Folding, and Inductive Reasoning in a large (N = 107, healthy, young (age range  = 16-29 cohort. Subcortical volumes were measured using an automated technique (FreeSurfer across structures including bilateral caudate, putamen, globus pallidus, thalamus, nucleus accumbens, hippocampus, amygdala, and five equal regions of the corpus callosum. We found that performance on measures of each aptitude was predicted by different subcortical structures: Math--higher right nucleus accumbens volume; Vocabulary--higher left hippocampus volume; Paper Folding--higher right thalamus volume; Foresight--lower right thalamus and higher mid anterior corpus callosum volume; Inductive Reasoning--higher mid anterior corpus callosum volume. Our results support general findings, within the cognitive neurosciences, showing lateralization of structure-function relationships, as well as more specific relationships between individual structures (e.g., left hippocampus and functions relevant to particular aptitudes (e.g., Vocabulary.

  4. Clearance of a Hirano body-like F-actin aggresome generated by jasplakinolide.

    Science.gov (United States)

    Lázaro-Diéguez, Francisco; Knecht, Erwin; Egea, Gustavo

    2008-07-01

    We have reported in a variety of mammalian cells the reversible formation of a filamentous actin (F-actin)-enriched aggresome generated by the actin toxin jasplakinolide (Lázaro-Diéguez et al., J Cell Sci 2008; 121:1415-25). Notably, this F-actin aggresome (FAG) resembles in many aspects the pathological Hirano body, which frequently appears in some diseases such as Alzheimer's and alcoholism. Using selective inhibitors, we examined the molecular and subcellular mechanisms that participate in the clearance of the FAG. Chaperones, microtubules, proteasomes and autophagosomes all actively participate to eliminate the FAG. Here we compile and compare these results and discuss the involvement of each process. Because of its simplicity and high reproducibility, our cellular model could help to test pharmacological agents designed to interfere with the mechanisms involved in the clearance of intracellular bodies and, in particular, of those enriched in F-actin.

  5. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    Science.gov (United States)

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. A Diaphanous-related formin links Ras signaling directly to actin assembly in macropinocytosis and phagocytosis.

    Science.gov (United States)

    Junemann, Alexander; Filić, Vedrana; Winterhoff, Moritz; Nordholz, Benjamin; Litschko, Christof; Schwellenbach, Helena; Stephan, Till; Weber, Igor; Faix, Jan

    2016-11-22

    Phagocytosis and macropinocytosis are Ras-regulated and actin-driven processes that depend on the dynamic rearrangements of the plasma membrane that protrudes and internalizes extracellular material by cup-shaped structures. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis remain elusive. Here, we show that the Diaphanous-related formin G (ForG) from the professional phagocyte Dictyostelium discoideum localizes to endocytic cups. Biochemical analyses revealed that ForG is a rather weak nucleator but efficiently elongates actin filaments in the presence of profilin. Notably, genetic inactivation of ForG is associated with a strongly impaired endocytosis and a markedly diminished F-actin content at the base of the cups. By contrast, ablation of the Arp2/3 (actin-related protein-2/3) complex activator SCAR (suppressor of cAMP receptor) diminishes F-actin mainly at the cup rim, being consistent with its known localization. These data therefore suggest that ForG acts as an actin polymerase of Arp2/3-nucleated filaments to allow for efficient membrane expansion and engulfment of extracellular material. Finally, we show that ForG is directly regulated in large-scale endocytosis by RasB and RasG, which are highly related to the human proto-oncogene KRas.

  7. A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

    Science.gov (United States)

    Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

    2015-01-01

    Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division. DOI: http://dx.doi.org/10.7554/eLife.08828.001 PMID:26305500

  8. F-actin reorganization upon de- and rehydration in the aeroterrestrial green alga Klebsormidium crenulatum.

    Science.gov (United States)

    Blaas, Kathrin; Holzinger, Andreas

    2017-07-01

    Filamentous actin (F-actin) is a dynamic network involved in many cellular processes like cell division and cytoplasmic streaming. While many studies have addressed the involvement of F-actin in different cellular processes in cultured cells, little is known on the reactions to environmental stress scenarios, where this system might have essential regulatory functions. We investigated here the de- and rehydration kinetics of breakdown and reassembly of F-actin in the streptophyte green alga Klebsormidium crenulatum. Measurements of the chlorophyll fluorescence (effective quantum yield of photosystem II [ΔF/Fm']) via pulse amplitude modulation were performed as a measure for dehydration induced shut down of physiological activity, which ceased after 141±15min at ∼84% RH. We hypothesized that there is a link between this physiological parameter and the status of the F-actin system. Indeed, 20min of dehydration (ΔF/Fm'=0) leads to a breakdown of the fine cortical F-actin network as visualized by Atto 488 phalloidin staining, and dot-like structures remained. Already 10min after rehydration a beginning reassembly of F-actin is observed, after 25min the F-actin network appeared similar to untreated controls, indicating a full recovery. These results demonstrate the fast kinetics of F-actin dis- and reassembly likely contributing to cellular reorganization upon rehydration. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division.

    Science.gov (United States)

    Manor, Uri; Bartholomew, Sadie; Golani, Gonen; Christenson, Eric; Kozlov, Michael; Higgs, Henry; Spudich, James; Lippincott-Schwartz, Jennifer

    2015-08-25

    Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.

  10. Chronic Actinic Dermatitis

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    Bengü Çevirgen Cemil

    2017-06-01

    Full Text Available Chronic actinic dermatitis (CAD is characterized by persistent eczema-like lesions, mainly on sun-exposed sites, induced by ultraviolet B, sometimes ultraviolet A, and occasionally visible light. CAD is a rare photodermatitis. It is often associated with contact allergens including airborne allergens such as fragrances, plant antigens and topical medications. A 62 year old farmer is applied with eczematous lesions restricted to sun-exposed areas. Clinical findings and histopathologic features were consistent with the diagnosis of chronic actinic dermatitis. The patient also had contact allergy to multiple allergens. We present this case to emphasize the significance of patch test on CAD treatment and the success of topical tacrolimus and azathioprine.

  11. Hippocampal Dendritic Spines Are Segregated Depending on Their Actin Polymerization.

    Science.gov (United States)

    Domínguez-Iturza, Nuria; Calvo, María; Benoist, Marion; Esteban, José Antonio; Morales, Miguel

    2016-01-01

    Dendritic spines are mushroom-shaped protrusions of the postsynaptic membrane. Spines receive the majority of glutamatergic synaptic inputs. Their morphology, dynamics, and density have been related to synaptic plasticity and learning. The main determinant of spine shape is filamentous actin. Using FRAP, we have reexamined the actin dynamics of individual spines from pyramidal hippocampal neurons, both in cultures and in hippocampal organotypic slices. Our results indicate that, in cultures, the actin mobile fraction is independently regulated at the individual spine level, and mobile fraction values do not correlate with either age or distance from the soma. The most significant factor regulating actin mobile fraction was the presence of astrocytes in the culture substrate. Spines from neurons growing in the virtual absence of astrocytes have a more stable actin cytoskeleton, while spines from neurons growing in close contact with astrocytes show a more dynamic cytoskeleton. According to their recovery time, spines were distributed into two populations with slower and faster recovery times, while spines from slice cultures were grouped into one population. Finally, employing fast lineal acquisition protocols, we confirmed the existence of loci with high polymerization rates within the spine.

  12. Hippocampal Dendritic Spines Are Segregated Depending on Their Actin Polymerization

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    Nuria Domínguez-Iturza

    2016-01-01

    Full Text Available Dendritic spines are mushroom-shaped protrusions of the postsynaptic membrane. Spines receive the majority of glutamatergic synaptic inputs. Their morphology, dynamics, and density have been related to synaptic plasticity and learning. The main determinant of spine shape is filamentous actin. Using FRAP, we have reexamined the actin dynamics of individual spines from pyramidal hippocampal neurons, both in cultures and in hippocampal organotypic slices. Our results indicate that, in cultures, the actin mobile fraction is independently regulated at the individual spine level, and mobile fraction values do not correlate with either age or distance from the soma. The most significant factor regulating actin mobile fraction was the presence of astrocytes in the culture substrate. Spines from neurons growing in the virtual absence of astrocytes have a more stable actin cytoskeleton, while spines from neurons growing in close contact with astrocytes show a more dynamic cytoskeleton. According to their recovery time, spines were distributed into two populations with slower and faster recovery times, while spines from slice cultures were grouped into one population. Finally, employing fast lineal acquisition protocols, we confirmed the existence of loci with high polymerization rates within the spine.

  13. Challenge Integrity: The Cell-Penetrating Peptide BP100 Interferes with the Auxin-Actin Oscillator.

    Science.gov (United States)

    Eggenberger, Kai; Sanyal, Papia; Hundt, Svenja; Wadhwani, Parvesh; Ulrich, Anne S; Nick, Peter

    2017-01-01

    Actin filaments are essential for the integrity of the cell membrane. In addition to this structural role, actin can modulate signaling by altering polar auxin flow. On the other hand, the organization of actin filaments is modulated by auxin constituting a self-referring signaling hub. Although the function of this auxin–actin oscillator is not clear, there is evidence for a functional link with stress signaling activated by the NADPH oxidase Respiratory burst oxidase Homolog (RboH). In the current work, we used the cell-penetrating peptide BP100 to induce a mild and transient perturbation of membrane integrity. We followed the response of actin to the BP100 uptake in a green fluorescent protein (GFP)-tagged actin marker line of tobacco Bright Yellow 2 (BY-2) cells by spinning disc confocal microscopy. We observed that BP100 enters in a stepwise manner and reduces the extent of actin remodeling. This actin ‘freezing’ can be rescued by the natural auxin IAA, and mimicked by the auxin-efflux inhibitor 1-napthylphthalamic acid (NPA). We further tested the role of the membrane-localized NADPH oxidase RboH using the specific inhibitor diphenyl iodonium (DPI), and found that DPI acts antagonistically to BP100, although DPI alone can induce a similar actin ‘freezing’ as well. We propose a working model, where the mild violation of membrane integrity by BP100 stimulates RboH, and the resulting elevated levels of reactive oxygen species interfere with actin dynamicity. The mitigating effect of auxin is explained by competition of auxin- and RboH-triggered signaling for superoxide anions. This self-referring auxin–actin–RboH hub might be essential for integrity sensing.

  14. Multiple signal transduction pathways regulate TNF-induced actin reorganization in macrophages: inhibition of Cdc42-mediated filopodium formation by TNF

    NARCIS (Netherlands)

    Peppelenbosch, M.; Boone, E.; Jones, G. E.; van Deventer, S. J.; Haegeman, G.; Fiers, W.; Grooten, J.; Ridley, A. J.

    1999-01-01

    TNF is known to regulate macrophage (Mphi) migration, but the signaling pathways mediating this response have not been established. Here we report that stimulation of the 55-kDa TNF receptor (TNFR-1) induced an overall decrease in filamentous actin (F-actin), inhibited CSF-1- and Cdc42-dependent

  15. A Gly65Val substitution in an actin, GhACT_LI1, disrupts cell polarity and F-actin organization resulting in dwarf, lintless cotton plants.

    Science.gov (United States)

    Thyssen, Gregory N; Fang, David D; Turley, Rickie B; Florane, Christopher B; Li, Ping; Mattison, Christopher P; Naoumkina, Marina

    2017-04-01

    Actin polymerizes to form part of the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hirsutum) actin gene in the organization of actin filaments in lobed cotyledon pavement cells and the highly elongated single-celled trichomes that comprise cotton lint fibers. Using mapping-by-sequencing, virus-induced gene silencing, and molecular modeling, we identified the causative mutation of the dominant dwarf Ligon lintless Li1 short fiber mutant as a single Gly65Val amino acid substitution in a polymerization domain of an actin gene, GhACT_LI1 (Gh_D04G0865). We observed altered cell morphology and disrupted organization of F-actin in Li1 plant cells by confocal microscopy. Mutant leaf cells lacked interdigitation of lobes and F-actin did not uniformly decorate the nuclear envelope. While wild-type lint fiber trichome cells contained long longitudinal actin cables, the short Li1 fiber cells accumulated disoriented transverse cables. The polymerization-defective Gly65Val allele in Li1 plants likely disrupts processive elongation of F-actin, resulting in a disorganized cytoskeleton and reduced cell polarity, which likely accounts for the dominant gene action and diverse pleiotropic effects associated with the Li1 mutation. Lastly, we propose a model to account for these effects, and underscore the roles of actin organization in determining plant cell polarity, shape and plant growth. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  16. Tungsten Filament Fire

    Science.gov (United States)

    Ruiz, Michael J.; Perkins, James

    2016-01-01

    We safely remove the outer glass bulb from an incandescent lamp and burn up the tungsten filament after the glass is removed. This demonstration dramatically illustrates the necessity of a vacuum or inert gas for the environment surrounding the tungsten filament inside the bulb. Our approach has added historical importance since the incandescent…

  17. Proteomics of Filamentous Fungi

    NARCIS (Netherlands)

    Passel, van M.W.J.; Schaap, P.J.; Graaff, de L.H.

    2013-01-01

    Filamentous fungi, such as Aspergillus niger and Aspergillus oryzae traditionally have had an important role in providing enzymes and enzyme cocktails that are used in food industry. In recent years the genome sequences of many filamentous fungi have become available. This combined with

  18. Increased actin polymerization and stabilization interferes with neuronal function and survival in the AMPKγ mutant Loechrig.

    Directory of Open Access Journals (Sweden)

    Mandy Cook

    Full Text Available loechrig (loe mutant flies are characterized by progressive neuronal degeneration, behavioral deficits, and early death. The mutation is due to a P-element insertion in the gene for the γ-subunit of the trimeric AMP-activated protein kinase (AMPK complex, whereby the insertion affects only one of several alternative transcripts encoding a unique neuronal isoform. AMPK is a cellular energy sensor that regulates a plethora of signaling pathways, including cholesterol and isoprenoid synthesis via its downstream target hydroxy-methylglutaryl (HMG-CoA reductase. We recently showed that loe interferes with isoprenoid synthesis and increases the prenylation and thereby activation of RhoA. During development, RhoA plays an important role in neuronal outgrowth by activating a signaling cascade that regulates actin dynamics. Here we show that the effect of loe/AMPKγ on RhoA prenylation leads to a hyperactivation of this signaling pathway, causing increased phosphorylation of the actin depolymerizating factor cofilin and accumulation of filamentous actin. Furthermore, our results show that the resulting cytoskeletal changes in loe interfere with neuronal growth and disrupt axonal integrity. Surprisingly, these phenotypes were enhanced by expressing the Slingshot (SSH phosphatase, which during development promotes actin depolymerization by dephosphorylating cofilin. However, our studies suggest that in the adult SSH promotes actin polymerization, supporting in vitro studies using human SSH1 that suggested that SSH can also stabilize and bundle filamentous actin. Together with the observed increase in SSH levels in the loe mutant, our experiments suggest that in mature neurons SSH may function as a stabilization factor for filamentous actin instead of promoting actin depolymerization.

  19. Characterization of ring-like F-actin structure as a mechanical partner for spindle positioning in mitosis.

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    Huan Lu

    Full Text Available Proper spindle positioning and orientation are essential for accurate mitosis which requires dynamic interactions between microtubule and actin filament (F-actin. Although mounting evidence demonstrates the role of F-actin in cortical cytoskeleton dynamics, it remains elusive as to the structure and function of F-actin-based networks in spindle geometry. Here we showed a ring-like F-actin structure surrounding the mitotic spindle which forms since metaphase and maintains in MG132-arrested metaphase HeLa cells. This cytoplasmic F-actin structure is relatively isotropic and less dynamic. Our computational modeling of spindle position process suggests a possible mechanism by which the ring-like F-actin structure can regulate astral microtubule dynamics and thus mitotic spindle orientation. We further demonstrated that inhibiting Plk1, Mps1 or Myosin, and disruption of microtubules or F-actin polymerization perturbs the formation of the ring-like F-actin structure and alters spindle position and symmetric division. These findings reveal a previously unrecognized but important link between mitotic spindle and ring-like F-actin network in accurate mitosis and enables the development of a method to theoretically illustrate the relationship between mitotic spindle and cytoplasmic F-actin.

  20. Srv2/CAP is required for polarized actin cable assembly and patch internalization during clathrin-mediated endocytosis.

    Science.gov (United States)

    Toshima, Junko Y; Horikomi, Chika; Okada, Asuka; Hatori, Makiko N; Nagano, Makoto; Masuda, Atsushi; Yamamoto, Wataru; Siekhaus, Daria Elisabeth; Toshima, Jiro

    2016-01-15

    The dynamic assembly and disassembly of actin filaments is essential for the formation and transport of vesicles during endocytosis. In yeast, two types of actin structures, namely cortical patches and cytoplasmic cables, play a direct role in endocytosis, but how their interaction is regulated remains unclear. Here, we show that Srv2/CAP, an evolutionarily conserved actin regulator, is required for efficient endocytosis owing to its role in the formation of the actin patches that aid initial vesicle invagination and of the actin cables that these move along. Deletion of the SRV2 gene resulted in the appearance of aberrant fragmented actin cables that frequently moved past actin patches, the sites of endocytosis. We find that the C-terminal CARP domain of Srv2p is vitally important for the proper assembly of actin patches and cables; we also demonstrate that the N-terminal helical folded domain of Srv2 is required for its localization to actin patches, specifically to the ADP-actin rich region through an interaction with cofilin. These results demonstrate the in vivo roles of Srv2p in the regulation of the actin cytoskeleton during clathrin-mediated endocytosis. © 2016. Published by The Company of Biologists Ltd.

  1. Coordinated process of polarized growth in filamentous fungi.

    Science.gov (United States)

    Takeshita, Norio

    2016-09-01

    Filamentous fungi are extremely polarized organisms, exhibiting continuous growth at their hyphal tips. The hyphal form is related to their pathogenicity in animals and plants, and their high secretion ability for biotechnology. Polarized growth requires a sequential supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeleton. Therefore, the arrangement of the cytoskeleton is a crucial step to establish and maintain the cell polarity. This review summarizes recent findings unraveling the mechanism of polarized growth with special emphasis on the role of actin and microtubule cytoskeleton and polarity marker proteins. Rapid insertions of membranes via highly active exocytosis at hyphal tips could quickly dilute the accumulated polarity marker proteins. Recent findings by a super-resolution microscopy indicate that filamentous fungal cells maintain their polarity at the tips by repeating transient assembly and disassembly of polarity sites.

  2. Cytosolic SYT/SS18 isoforms are actin-associated proteins that function in matrix-specific adhesion.

    Directory of Open Access Journals (Sweden)

    Jaehong Kim

    2009-07-01

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

  3. Actin and Endocytosis in Budding Yeast

    Science.gov (United States)

    Goode, Bruce L.; Eskin, Julian A.; Wendland, Beverly

    2015-01-01

    Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. PMID:25657349

  4. Cell stress promotes the association of phosphorylated HspB1 with F-actin.

    Directory of Open Access Journals (Sweden)

    Joseph P Clarke

    Full Text Available Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.

  5. Regulation of the actin cytoskeleton by an interaction of IQGAP related protein GAPA with filamin and cortexillin I.

    Science.gov (United States)

    Mondal, Subhanjan; Burgute, Bhagyashri; Rieger, Daniela; Müller, Rolf; Rivero, Francisco; Faix, Jan; Schleicher, Michael; Noegel, Angelika A

    2010-11-10

    Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin.

  6. Regulation of the actin cytoskeleton by an interaction of IQGAP related protein GAPA with filamin and cortexillin I.

    Directory of Open Access Journals (Sweden)

    Subhanjan Mondal

    Full Text Available Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin.

  7. Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells’ osteogenic differentiation

    Science.gov (United States)

    Tai, I-Chun; Wang, Yao-Hsien; Chen, Chung-Hwan; Chuang, Shu-Chun; Chang, Je-Ken; Ho, Mei-Ling

    2015-01-01

    Recent studies have indicated that statins induce osteogenic differentiation both in vitro and in vivo. The molecular mechanism of statin-stimulated osteogenesis is unknown. Activation of RhoA signaling increases cytoskeletal tension, which plays a crucial role in the osteogenic differentiation of mesenchymal stem cells. We thus hypothesized that RhoA signaling is involved in simvastatin-induced osteogenesis in bone marrow mesenchymal stem cells. We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα. Simvastatin also increased the expression of osteogenic proteins, density of actin filament, the number of focal adhesions, and cellular tension. Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation. In vivo study also confirms that density of actin filament is increased in simvastatin-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeletons and enhancing cell rigidity are crucial in simvastatin-induced osteogenesis. The results suggested that simvastatin, which is an osteoinductive factor and acts by increasing actin filament organization and cell rigidity combined with osteoconductive biomaterials, may benefit stem-cell-based bone regeneration. PMID:26451103

  8. Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles

    Science.gov (United States)

    Boguslavsky, Shlomit; Chiu, Tim; Foley, Kevin P.; Osorio-Fuentealba, Cesar; Antonescu, Costin N.; Bayer, K. Ulrich; Bilan, Philip J.; Klip, Amira

    2012-01-01

    GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding–deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells. PMID:22918957

  9. The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri.

    Science.gov (United States)

    Sohn, Hae-Jin; Kim, Jong-Hyun; Shin, Myeong-Heon; Song, Kyoung-Ju; Shin, Ho-Joon

    2010-03-01

    Naegleria fowleri destroys target cells by trogocytosis, a phagocytosis mechanism, and a process of piecemeal ingestion of target cells by food-cups. Phagocytosis is an actin-dependent process that involves polymerization of monomeric G-actin into filamentous F-actin. However, despite the numerous studies concerning phagocytosis, its role in the N. fowleri food-cup formation related with trogocytosis has been poorly reported. In this study, we cloned and characterized an Nf-actin gene to elucidate the role of Nf-actin gene in N. fowleri pathogenesis. The Nf-actin gene is composed of 1,128-bp and produced a 54.1-kDa recombinant protein (Nf-actin). The sequence identity was 82% with nonpathogenic Naegleria gruberi but has no sequence identity with other mammals or human actin gene. Anti-Nf-actin polyclonal antibody was produced in BALB/c mice immunized with recombinant Nf-actin. The Nf-actin was localized on the cytoplasm, pseudopodia, and especially, food-cup structure (amoebastome) in N. fowleri trophozoites using immunofluorescence assay. When N. fowleri co-cultured with Chinese hamster ovary cells, Nf-actin was observed to localize around on phagocytic food-cups. We also observed that N. fowleri treated with cytochalasin D as actin polymerization inhibitor or transfected with antisense oligomer of Nf-actin gene had shown the reduced ability of food-cup formation and in vitro cytotoxicity. Finally, it suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri.

  10. Preserved episodic memory in subcortical band heterotopia.

    Science.gov (United States)

    Janzen, Laura; Sherman, Elisabeth; Langfitt, John; Berg, Michel; Connolly, Mary

    2004-05-01

    Neuropsychological profiles of four patients with subcortical band heterotopia (SBH) are presented to delineate further the phenotype of this disorder. Standardized, norm-referenced measures of cognitive functioning, including intelligence, processing speed, attention, language, visuomotor skills, memory, and fine motor ability were administered to four patients with magnetic resonance imaging evidence of SBH. Despite intellectual impairment and other severe cognitive deficits, all four patients displayed relatively intact episodic memory. This selective sparing of memory functions has not been previously reported in individuals with SBH and suggests that doublecortin does not play a role in the development of memory systems in the mesial temporal region, which tend to be spared in SBH.

  11. Titin-Actin Interaction: PEVK-Actin-Based Viscosity in a Large Animal

    Directory of Open Access Journals (Sweden)

    Charles S. Chung

    2011-01-01

    Full Text Available Titin exhibits an interaction between its PEVK segment and the actin filament resulting in viscosity, a speed dependent resistive force, which significantly influences diastolic filling in mice. While diastolic disease is clinically pervasive, humans express a more compliant titin (N2BA:N2B ratio ~0.5–1.0 than mice (N2BA:N2B ratio ~0.2. To examine PEVK-actin based viscosity in compliant titin-tissues, we used pig cardiac tissue that expresses titin isoforms similar to that in humans. Stretch-hold experiments were performed at speeds from 0.1 to 10 lengths/s from slack sarcomere lengths (SL to SL of 2.15 μm. Viscosity was calculated from the slope of stress-relaxation vs stretch speed. Recombinant PEVK was added to compete off native interactions and this found to reduce the slope by 35%, suggesting that PEVK-actin interactions are a strong contributor of viscosity. Frequency sweeps were performed at frequencies of 0.1–400 Hz and recombinant protein reduced viscous moduli by 40% at 2.15 μm and by 50% at 2.25 μm, suggesting a SL-dependent nature of viscosity that might prevent SL ``overshoot’’ at long diastolic SLs. This study is the first to show that viscosity is present at physiologic speeds in the pig and supports the physiologic relevance of PEVK-actin interactions in humans in both health and disease.

  12. Actin acting at the Golgi.

    Science.gov (United States)

    Egea, Gustavo; Serra-Peinado, Carla; Salcedo-Sicilia, Laia; Gutiérrez-Martínez, Enric

    2013-09-01

    The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in trafficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers.

  13. HLA typing in actinic prurigo.

    Science.gov (United States)

    Sheridan, D P; Lane, P R; Irvine, J; Martel, M J; Hogan, D J

    1990-06-01

    Thirty-two actinic prurigo patients of Cree ancestry underwent human lymphocyte antigen (HLA) typing and were compared with 32 control subjects of Cree ancestry. We found a significantly increased frequency of HLA-A24 and Cw4 antigens and a significant decrease in the frequency of the A3 antigen in actinic prurigo patients. These HLA associations may be helpful in determining whether actinic prurigo is a distinct disease or a variant of polymorphous light eruption.

  14. Phalloidin unzips nebulin from thin filaments in skeletal myofibrils.

    Science.gov (United States)

    Ao, X; Lehrer, S S

    1995-11-01

    Fluorescent phallotoxins such as rhodamine-phalloidin take hours to bind uniformly to thin filaments of skeletal myofibrils, after fast initial binding to both ends of thin filaments. Observation of this process in skeletal and cardiac myofibrils and of the resulting re-distribution of nebulin using anti-nebulin antibody showed that: (1) rhodamine-phalloidin binds uniformly to actin in cardiac myofibrils within minutes, in contrast to skeletal myofibrils; (2) overnight pre-incubation of skeletal myofibrils with phalloidin results in uniform initial binding of rhodamine-phalloidin and a changed nebulin localization; (3) pre-incubation of skeletal myofibrils with Ca(2+)-calmodulin results in uniform initial binding of rhodamine-phalloidin; (4) the binding of rhodamine-phalloidin to actin in skeletal myofibrils is unidirectional, i.e. the fluorescence of incorporated rhodamine-phalloidin moves from the pointed ends where it is bound initially toward the barbed end at the Z-band; (5) the unidirectional binding of rhodamine-phalloidin results in redistribution of nebulin, i.e. the initial fluorescent bands associated with the epitopes of bound nebulin antibody change to a single band located close to Z-line. These results indicate that nebulin inhibits rhodamine-phalloidin binding to actin and suggests that the unidirectional rhodamine-phalloidin binding may be due to cooperative competitive binding, i.e. phalloidin 'unzips' nebulin starting from the pointed ends of the thin filaments.

  15. Papaverine Prevents Vasospasm by Regulation of Myosin Light Chain Phosphorylation and Actin Polymerization in Human Saphenous Vein.

    Directory of Open Access Journals (Sweden)

    Kyle M Hocking

    Full Text Available Papaverine is used to prevent vasospasm in human saphenous veins (HSV during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation.HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6 and the vasodilator stimulated phosphoprotein (VASP, as well as decreased filamentous actin (F-actin levels suggesting depolymerization of actin.These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm.

  16. Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy

    Science.gov (United States)

    Shirai, Yuki M.; Tsunoyama, Taka A.; Hiramoto-Yamaki, Nao; Hirosawa, Koichiro M.; Shibata, Akihiro C. E.; Kondo, Kenichi; Tsurumune, Atsushi; Ishidate, Fumiyoshi; Kusumi, Akihiro

    2017-01-01

    Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named “actin-pl-clusters”. In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent “actin podosome-like clusters”. PMID:29190677

  17. Comparative neuropsychology of cortical and subcortical dementia.

    Science.gov (United States)

    Freedman, M; Oscar-Berman, M

    1986-11-01

    The terms "cortical" and "subcortical" dementia are controversial; however, the clinical distinction between them is real. For example, although Alzheimer's and Parkinson's disease (prototypical of cortical and subcortical dementia, respectively) share clinical features, they differ in the presence of aphasia, apraxia, and agnosia in Alzheimer's disease but not in Parkinson's dementia. We review our studies aimed at clarifying the mechanisms underlying the differences between these neurological disorders. Experimental paradigms adopted from animal models were used to study the functional anatomy and neuropsychological characteristics of Alzheimer's and Parkinson's disease. The tasks administered include delayed alternation (DA) and delayed response (DR), which are sensitive to frontal system damage, and tactile discrimination learning (TOL) and reversal (TRL) paradigms sensitive to parietal system damage. Alzheimer's patients were significantly impaired on all tasks whereas Parkinsonians with dementia were impaired only on DR and TRL. Consideration of neuroanatomical and neuropsychological mechanisms involved in DA, DR, TOL, and TRL appears to have sharpened the distinction between Alzheimer's and Parkinson's dementia. Dementia in Alzheimer's disease may involve dorsolateral frontal, orbitofrontal and parietal systems. In contrast, dementia in Parkinson's disease may involve prominent dorsolateral frontal system damage.

  18. Low-dimensional manifold of actin polymerization dynamics

    Science.gov (United States)

    Floyd, Carlos; Jarzynski, Christopher; Papoian, Garegin

    2017-12-01

    Actin filaments are critical components of the eukaryotic cytoskeleton, playing important roles in a number of cellular functions, such as cell migration, organelle transport, and mechanosensation. They are helical polymers with a well-defined polarity, composed of globular subunits that bind nucleotides in one of three hydrolysis states (ATP, ADP-Pi, or ADP). Mean-field models of the dynamics of actin polymerization have succeeded in, among other things, determining the nucleotide profile of an average filament and resolving the mechanisms of accessory proteins. However, these models require numerical solution of a high-dimensional system of nonlinear ordinary differential equations. By truncating a set of recursion equations, the Brooks–Carlsson (BC) model reduces dimensionality to 11, but it still remains nonlinear and does not admit an analytical solution, hence, significantly hindering understanding of its resulting dynamics. In this work, by taking advantage of the fast timescales of the hydrolysis states of the filament tips, we propose two model reduction schemes: the quasi steady-state approximation model is five-dimensional and nonlinear, whereas the constant tip (CT) model is five-dimensional and linear, resulting from the approximation that the tip states are not dynamic variables. We provide an exact solution of the CT model and use it to shed light on the dynamical behaviors of the full BC model, highlighting the relative ordering of the timescales of various collective processes, and explaining some unusual dependence of the steady-state behavior on initial conditions.

  19. NHERF1 regulates actin cytoskeleton organization through modulation of α-actinin-4 stability.

    Science.gov (United States)

    Sun, Licui; Zheng, Junfang; Wang, Qiqi; Song, Ran; Liu, Hua; Meng, Ran; Tao, Tao; Si, Yang; Jiang, Wenguo; He, Junqi

    2016-02-01

    The actin cytoskeleton is composed of a highly dynamic network of filamentous proteins, yet the molecular mechanism that regulates its organization and remodeling remains elusive. In this study, Na(+)/H(+) exchanger regulatory factor (NHERF)-1 loss-of-function and gain-of-function experiments reveal that polymerized actin cytoskeleton (F-actin) in HeLa cells is disorganized by NHERF1, whereas actin protein expression levels exhibit no detectable change. To elucidate the molecular mechanism underlying actin cytoskeleton disorganization by NHERF1, a combined 2-dimensional electrophoresis-matrix-assisted laser desorption/ionization-time of flight mass spectrometry approach was used to screen for proteins regulated by NHERF1 in HeLa cells. α-Actinin-4, an actin cross-linking protein, was identified. Glutathione S-transferase pull-down and coimmunoprecipitation studies showed the α-actinin-4 carboxyl-terminal region specifically interacted with the NHERF1 postsynaptic density 95/disc-large/zona occludens-1 domain. The NHERF1/α-actinin-4 interaction increased α-actinin-4 ubiquitination and decreased its expression levels, resulting in actin cytoskeleton disassembly. Our study identified α-actinin-4 as a novel NHERF1 interaction partner and provided new insights into the regulatory mechanism of the actin cytoskeleton by NHERF1. © FASEB.

  20. Direct interaction between two actin nucleators is required in Drosophila oogenesis.

    Science.gov (United States)

    Quinlan, Margot E

    2013-11-01

    Controlled actin assembly is crucial to a wide variety of cellular processes, including polarity establishment during early development. The recently discovered actin mesh, a structure that traverses the Drosophila oocyte during mid-oogenesis, is essential for proper establishment of the major body axes. Genetic experiments indicate that at least two proteins, Spire (Spir) and Cappuccino (Capu), are required to build this mesh. The spire and cappuccino genetic loci were first identified as maternal effect genes in Drosophila. Mutation in either locus results in the same phenotypes, including absence of the mesh, linking them functionally. Both proteins nucleate actin filaments. Spir and Capu also interact directly with each other in vitro, suggesting a novel synergistic mode of regulating actin. In order to understand how and why proteins with similar biochemical activity would be required in the same biological pathway, genetic experiments were designed to test whether a direct interaction between Spir and Capu is required during oogenesis. Indeed, data in this study indicate that Spir and Capu must interact directly with one another and then separate to function properly. Furthermore, these actin regulators are controlled by a combination of mechanisms, including interaction with one another, functional inhibition and regulation of their protein levels. Finally, this work demonstrates for the first time in a multicellular organism that the ability of a formin to assemble actin filaments is required for a specific structure.

  1. Redundant mechanisms recruit actin into the contractile ring in silkworm spermatocytes.

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2008-09-01

    Full Text Available Cytokinesis is powered by the contraction of actomyosin filaments within the newly assembled contractile ring. Microtubules are a spindle component that is essential for the induction of cytokinesis. This induction could use central spindle and/or astral microtubules to stimulate cortical contraction around the spindle equator (equatorial stimulation. Alternatively, or in addition, induction could rely on astral microtubules to relax the polar cortex (polar relaxation. To investigate the relationship between microtubules, cortical stiffness, and contractile ring assembly, we used different configurations of microtubules to manipulate the distribution of actin in living silkworm spermatocytes. Mechanically repositioned, noninterdigitating microtubules can induce redistribution of actin at any region of the cortex by locally excluding cortical actin filaments. This cortical flow of actin promotes regional relaxation while increasing tension elsewhere (normally at the equatorial cortex. In contrast, repositioned interdigitating microtubule bundles use a novel mechanism to induce local stimulation of contractility anywhere within the cortex; at the antiparallel plus ends of central spindle microtubules, actin aggregates are rapidly assembled de novo and transported laterally to the equatorial cortex. Relaxation depends on microtubule dynamics but not on RhoA activity, whereas stimulation depends on RhoA activity but is largely independent of microtubule dynamics. We conclude that polar relaxation and equatorial stimulation mechanisms redundantly supply actin for contractile ring assembly, thus increasing the fidelity of cleavage.

  2. Fluorescence single-molecule imaging of actin turnover and regulatory mechanisms.

    Science.gov (United States)

    Watanabe, Naoki

    2012-01-01

    Cells must rapidly remodel the actin filament network to achieve various cellular functions. Actin filament turnover is a dynamic process that plays crucial roles in cell adhesion, locomotion, cytokinesis, endocytosis, phagocytosis, tissue remodeling, etc., and is regulated by cell signaling cascades. Success in elucidating dynamic biological processes such as actin-based motility relies on the means enabling real time monitoring of the process. The invention of live-cell fluorescence single-molecule imaging has opened a window for direct viewing of various actin remodeling processes. In general, assembly and dissociation of actin and its regulators turned out to occur at the faster rates than previously estimated by biochemical and structural analyses. Cells undergo such fast continuous exchange of the components perhaps not only to drive actin remodeling but also to facilitate rapid response in many other cell mechanics and signaling cascades. This chapter describes how epifluorescence single-molecule imaging which visualizes deeper area than the TIRF microscopy is achieved in XTC cells, the currently best platform for this approach. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. An actin-dependent step in mitochondrial fission mediated by the ER-associated formin INF2.

    Science.gov (United States)

    Korobova, Farida; Ramabhadran, Vinay; Higgs, Henry N

    2013-01-25

    Mitochondrial fission is fundamentally important to cellular physiology. The dynamin-related protein Drp1 mediates fission, and interaction between mitochondrion and endoplasmic reticulum (ER) enhances fission. However, the mechanism for Drp1 recruitment to mitochondria is unclear, although previous results implicate actin involvement. Here, we found that actin polymerization through ER-localized inverted formin 2 (INF2) was required for efficient mitochondrial fission in mammalian cells. INF2 functioned upstream of Drp1. Actin filaments appeared to accumulate between mitochondria and INF2-enriched ER membranes at constriction sites. Thus, INF2-induced actin filaments may drive initial mitochondrial constriction, which allows Drp1-driven secondary constriction. Because INF2 mutations can lead to Charcot-Marie-Tooth disease, our results provide a potential cellular mechanism for this disease state.

  4. Femtosecond Laser Filamentation

    CERN Document Server

    Chin, See Leang

    2010-01-01

    Femtosecond Laser Filamentation gives a comprehensive review of the physics of propagation of intense femtosecond laser pulses in optical media (principally air) and the applications and challenges of this new technique. This book presents the modern understanding of the physics of femtosecond laser pulse propagation, including unusual new effects such as the self-transformation of the pulse into a white light laser pulse, intensity clamping, the physics of multiple filamentation and competition, and how filaments’ ability to melt glass leads to wave guide writing. The potential applications of laser filamentation in atmospheric sensing and the generation of other electromagnetic pulses from the UV to the radio frequency are treated, together with possible future challenges in the excitation of super-excited states of molecules. Exciting new phenomena such as filament induced ultrafast birefringence and the excitation of molecular rotational wave packets and their multiple revivals in air (gases) will also ...

  5. Bacterial intermediate filaments

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Cabeen, M.; Jacobs-Wagner, C.

    2009-01-01

    Crescentin, which is the founding member of a rapidly growing family of bacterial cytoskeletal proteins, was previously proposed to resemble eukaryotic intermediate filament (IF) proteins based on structural prediction and in vitro polymerization properties. Here, we demonstrate that crescentin...

  6. Identification of Actin-Binding Proteins from Maize Pollen

    Energy Technology Data Exchange (ETDEWEB)

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  7. Actin cytoskeleton stiffness grades metastatic potential of ovarian carcinoma Hey A8 cells via nanoindentation mapping.

    Science.gov (United States)

    Zhou, Z L; Sun, X X; Ma, J; Tong, M H; To, S K Y; Wong, A S T; Ngan, A H W

    2017-07-26

    Recent studies have indicated that the nanoindentation measured stiffness of carcinoma adherent cells is in general lower than normal cells, thus suggesting that cell stiffness may serve as a bio-marker for carcinoma. However, the proper establishment of such a conclusion would require biophysical understanding of the underlying mechanism of the cell stiffness. In this work, we compared the elastic moduli of the actin cytoskeletons of Hey A8 ovarian carcinoma cells with and without metastasis (HM and NM), as measured by 2D atomic force microscopy (AFM) with low-depth nanoindentation via a rate-jump method. The results indicate clearly that HM cells showed lower actin cytoskeleton stiffness atop of their nucleus position and higher actin cytoskeleton stiffness at their rims, compared to NM cells, suggesting that the local stiffness on the cytoskeleton can reflect actin filament distribution. Immunofluorescence staining and scanning electron microscopy (SEM) also indicated that the difference in stiffness in Hey A8 cells with different metastasis is associated with their F-actin rearrangement. Finite-element modelling (FEM) shows that a migrating cell would have its actin filaments bundled together to form stress fibers, which would exhibit lower indentation stiffness than the less aligned arrangement of filaments in a non-migrating cell. The results here indicate that the actin cytoskeleton stiffness can serve as a reliable marker for grading the metastasis of adherent carcinoma cells due to their cytoskeleton change and potentially predicting the migration direction of the cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. NMR assignments of actin depolymerizing factor (ADF) like UNC-60A and cofilin like UNC-60B proteins of Caenorhabditis elegans.

    Science.gov (United States)

    Shukla, Vaibhav Kumar; Kabra, Ashish; Yadav, Rahul; Ono, Shoichiro; Kumar, Dinesh; Arora, Ashish

    2015-10-01

    The actin filament dynamics in nematode, Caenorhabditis elegans, is regulated by differential activity of two proteins UNC-60A and UNC-60B. UNC-60A exhibits strong pointed end depolymerization on C. elegans actin (Ce-actin), strong inhibition of polymerization, strong monomer sequestering activity, weak severing activity, and low affinity for F-actin binding, while UNC-60B exhibits strong pointed end depolymerization on rabbit muscle actin, strong severing activity, and high affinity for F-actin binding. Structural characterization of these proteins will help to understand (1) molecular mechanism of actin dynamics regulation and (2) the differential activity of these proteins. Here, we report (1)H, (13)C, and (15)N chemical shift assignments of these two proteins as determined by heteronuclear NMR experiments (at pH 6.5 and temperature 298 K).

  9. Ring closure in actin polymers

    OpenAIRE

    Sinha, Supurna; Chattopadhyay, Sebanti

    2017-01-01

    We present an analysis for the ring closure probability of semiflexible polymers within the pure bend Worm Like Chain (WLC) model. The ring closure probability predicted from our analysis can be tested against fluorescent actin cyclization experiments.We also discuss the effect of ring closure on bend angle fluctuations in actin polymers.

  10. Actin Organization in Cells Responding to a Perforated Surface, Revealed by Live Imaging and Cryo-Electron Tomography.

    Science.gov (United States)

    Jasnin, Marion; Ecke, Mary; Baumeister, Wolfgang; Gerisch, Günther

    2016-07-06

    In a 3D environment, motile cells accommodate their protruding and retracting activities to geometrical cues. Dictyostelium cells migrating on a perforated film explored its holes by forming actin rings around their border and extending protrusions through the free space. The response was initiated when an actin wave passed a hole, and the rings persisted only in the PIP3-rich territories surrounded by a wave. To reconstruct actin structures from cryo-electron tomograms, actin rings were identified by cryo-correlative light and electron microscopy, and thin wedges of relevant regions were obtained by cryo-focused ion-beam milling. Retracting stages were distinguished from protruding ones by the accumulation of myosin-II. Early actin rings consisted of filaments pointing upright from the membrane, entangled with a meshwork of filaments close to the membrane. Branches identified at later stages suggested that formin-based nucleation of filaments was followed by Arp2/3-mediated network stabilization, which prevented buckling of the force-generating filaments. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array: evidence for a second binding site on monomeric actin.

    Science.gov (United States)

    Mannherz, Hans Georg; Ballweber, Edda; Galla, Marco; Villard, Sylvie; Granier, Claude; Steegborn, Clemens; Schmidtmann, Anja; Jaquet, Kornelia; Pope, Brian; Weeds, Alan G

    2007-02-23

    The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin

  12. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A

    2000-07-01

    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

  13. Gait and Equilibrium in Subcortical Vascular Dementia

    Directory of Open Access Journals (Sweden)

    Rita Moretti

    2011-01-01

    Full Text Available Subcortical vascular dementia is a clinical entity, widespread, even challenging to diagnose and correctly treat. Patients with this diagnosis are old, frail, often with concomitant pathologies, and therefore, with many drugs in therapy. We tried to diagnose and follow up for three years more than 600 patients. Study subjects were men and women, not bedridden, aged 68–94 years, outpatients, recruited from June, 1st 2007 to June, 1st 2010. We examined them clinically, neurologically, with specific consideration on drug therapies. Our aim has been to define gait and imbalance problem, if eventually coexistent with the pathology of white matter and/or with the worsening of the deterioration. Drug intake interference has been detected and considered.

  14. The actin depolymerizing factor (ADF)/cofilin signaling pathway and DNA damage responses in cancer.

    Science.gov (United States)

    Chang, Chun-Yuan; Leu, Jyh-Der; Lee, Yi-Jang

    2015-02-13

    The actin depolymerizing factor (ADF)/cofilin protein family is essential for actin dynamics, cell division, chemotaxis and tumor metastasis. Cofilin-1 (CFL-1) is a primary non-muscle isoform of the ADF/cofilin protein family accelerating the actin filamental turnover in vitro and in vivo. In response to environmental stimulation, CFL-1 enters the nucleus to regulate the actin dynamics. Although the purpose of this cytoplasm-nucleus transition remains unclear, it is speculated that the interaction between CFL-1 and DNA may influence various biological responses, including DNA damage repair. In this review, we will discuss the possible involvement of CFL-1 in DNA damage responses (DDR) induced by ionizing radiation (IR), and the implications for cancer radiotherapy.

  15. Captivating New Roles of F-Actin Cortex in Exocytosis and Bulk Endocytosis in Neurosecretory Cells.

    Science.gov (United States)

    Meunier, Frédéric A; Gutiérrez, Luis M

    2016-09-01

    The cortical actin network is a tight array of filaments located beneath the plasma membrane. In neurosecretory cells, secretory vesicles are recruited on this network via a small insert isoform of myosin VI in a Ca(2+)-dependent manner. Upon secretagogue stimulation, myosin II mediates a relaxation of the actin network leading to synchronous translocation of bound or caged vesicles to the plasma membrane where they undergo exocytosis. F-actin is also recruited to secretory sites, where structural changes are detected immediately preceding and following exocytic events. Here we examine the mechanism underpinning the astonishing multifunctionality of this network in the various stages of vesicular exocytosis and compensatory bulk endocytosis. We propose a theoretical framework incorporating critical roles of the actin network in coupling these processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

    Science.gov (United States)

    Lim, James; Danuser, Gaudenz

    2009-01-01

    In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM). We describe the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes. PMID:19684563

  17. Fluorescence microscopy of actin- and microtubule-associated septins in mammalian cells.

    Science.gov (United States)

    Spiliotis, E T; Karasmanis, E P; Dolat, L

    2016-01-01

    Septins are a major component of the mammalian cytoskeleton. Septins associate with filamentous actin (F-actin) and microtubules, but the nature and significance of these interactions are not well understood. Fluorescence microscopy of F-actin- and microtubule-associated septins in fixed and living cells has been instrumental in uncovering septin functions in cellular morphogenesis and cytoskeleton-dependent processes (eg, cell division, cell migration). Here, we provide a detailed methodology for the visualization of endogenous septins by immunofluorescence microscopy, discussing sample preparation and reagents that are critical for optimal staining. In addition, we review approaches for the construction and expression of fluorescent septins and their time-lapse imaging with F-actin and microtubules. The recommended methodology is adaptable for high- and superresolution imaging of mammalian cells with various instrumentation, including wide-field and confocal microscopy as well as total internal reflection fluorescence and structured illumination microscopy. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Combined effects of temperature, pressure, and co-solvents on the polymerization kinetics of actin.

    Science.gov (United States)

    Rosin, Christopher; Estel, Kathrin; Hälker, Jessica; Winter, Roland

    2015-05-18

    In vivo studies have shown that the cytoskeleton of cells is very sensitive to changes in temperature and pressure. In particular, actin filaments get depolymerized when pressure is increased up to several hundred bars, conditions that are easily encountered in the deep sea. We quantitatively evaluate the effects of temperature, pressure, and osmolytes on the kinetics of the polymerization reaction of actin by high-pressure stopped-flow experiments in combination with fluorescence detection and an integrative stochastic simulation of the polymerization process. We show that the compatible osmolyte trimethylamine-N-oxide is not only able to compensate for the strongly retarding effect of chaotropic agents, such as urea, on actin polymerization, it is also able to largely offset the deteriorating effect of pressure on actin polymerization, thereby allowing biological cells to better cope with extreme environmental conditions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Colocalization of synapsin and actin during synaptic vesicle recycling

    DEFF Research Database (Denmark)

    Bloom, Ona; Evergren, Emma; Tomilin, Nikolay

    2003-01-01

    activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin......-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known...

  20. A Role for Nuclear F-Actin Induction in Human Cytomegalovirus Nuclear Egress

    Science.gov (United States)

    Wilkie, Adrian R.; Lawler, Jessica L.

    2016-01-01

    ABSTRACT Herpesviruses, which include important pathogens, remodel the host cell nucleus to facilitate infection. This remodeling includes the formation of structures called replication compartments (RCs) in which herpesviruses replicate their DNA. During infection with the betaherpesvirus, human cytomegalovirus (HCMV), viral DNA synthesis occurs at the periphery of RCs within the nuclear interior, after which assembled capsids must reach the inner nuclear membrane (INM) for translocation to the cytoplasm (nuclear egress). The processes that facilitate movement of HCMV capsids to the INM during nuclear egress are unknown. Although an actin-based mechanism of alphaherpesvirus capsid trafficking to the INM has been proposed, it is controversial. Here, using a fluorescently-tagged, nucleus-localized actin-binding peptide, we show that HCMV, but not herpes simplex virus 1, strongly induced nuclear actin filaments (F-actin) in human fibroblasts. Based on studies using UV inactivation and inhibitors, this induction depended on viral gene expression. Interestingly, by 24 h postinfection, nuclear F-actin formed thicker structures that appeared by super-resolution microscopy to be bundles of filaments. Later in infection, nuclear F-actin primarily localized along the RC periphery and between the RC periphery and the nuclear rim. Importantly, a drug that depolymerized nuclear F-actin caused defects in production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization near the nuclear rim, without decreasing capsid accumulation in the nucleus. Thus, our results suggest that for at least one herpesvirus, nuclear F-actin promotes capsid movement to the nuclear periphery and nuclear egress. We discuss our results in terms of competing models for these processes. PMID:27555312

  1. The Plant Actin Cytoskeleton Responds to Signals from Microbe-Associated Molecular Patterns

    Energy Technology Data Exchange (ETDEWEB)

    Henty-Ridilla, Jessica L.; Shimono, Masaki; Li, Jiejie; Chang, Jeff H.; Day, Brad; Staiger, Christopher J.; Zhou, Jian-Min

    2013-04-04

    Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.

  2. A Bipolar Spindle of Antiparallel ParM Filaments Drives Bacterial Plasmid Segregation

    DEFF Research Database (Denmark)

    Gayathri, P; Fujii, T; Møller-Jensen, Jakob

    2012-01-01

    To ensure their stable inheritance by daughter cells during cell division, bacterial low copy-number plasmids make simple DNA segregating machines that use an elongating protein filament between sister plasmids. In the ParMRC system of Escherichia coli R1 plasmid, ParM, an actin-like protein, forms...

  3. Troponin-like regulation in muscle thin filaments of the mussel Crenomytilus grayanus (Bivalvia: Mytiloida).

    Science.gov (United States)

    Vyatchin, Ilya G; Shevchenko, Ulyana V; Lazarev, Stanislav S; Matusovsky, Oleg S; Shelud'ko, Nikolay S

    2015-10-01

    Muscles of bivalve molluscs have double calcium regulation--myosin-linked and actin-linked. While the mechanism of myosin-linked regulation is sufficiently studied, there is still no consensus on the mechanism of actin-linked regulation. Earlier we showed a high degree of Ca2+-sensitivity of thin filaments from the adductor muscle of the mussel Crenomytilus grayanus (Mytiloida). In order to elucidate the nature of this regulation, we isolated the fraction of minor proteins from the mussel thin filaments, which confers Ca2+-sensitivity to reconstituted actomyosin-tropomyosin. Proteins of this fraction, ABP-19, ABP-20, and ABP-28, were chromatographically purified and identified. According to the results of mass spectrometry and Western blot analysis, as well as by their functional properties, these mussel actin-binding proteins appeared to correspond to the troponin components from the skeletal muscles of vertebrates (TnC, TnI and TnT). The reconstituted mussel troponin complex confers to actomyosin-tropomyosin more than 80% Ca2+-sensitivity. The in vivo molar ratio of actin/tropomyosin/troponin was calculated to be 7:1:0.5, i.e., the content of troponin in mussel thin filaments is two times lower than in thin filaments of skeletal muscles of vertebrates. These data demonstrate that troponin-like regulation found in the catch muscle of the mussel C. grayanus is present at least in two suborders of bivalves: Pectinoida and Mytiloida. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Molecular mechanical differences between isoforms of contractile actin in the presence of isoforms of smooth muscle tropomyosin.

    Directory of Open Access Journals (Sweden)

    Lennart Hilbert

    2013-10-01

    Full Text Available The proteins involved in smooth muscle's molecular contractile mechanism - the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin - are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for [Formula: see text]-actin ([Formula: see text]A, [Formula: see text]-actin-tropomyosin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text], [Formula: see text]-actin-tropomyosin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text], [Formula: see text]-actin ([Formula: see text]A, [Formula: see text]-actin-tropomyosin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text], and [Formula: see text]-actin-tropomoysin-[Formula: see text] ([Formula: see text]A-Tm[Formula: see text]. Actin sliding analysis with our specifically developed video analysis software followed by statistical assessment (Bootstrapped Principal Component Analysis indicated that the in vitro motility of [Formula: see text]A, [Formula: see text]A, and [Formula: see text]A-Tm[Formula: see text] is not distinguishable. Compared to these three 'baseline conditions', statistically significant differences ([Formula: see text] were: [Formula: see text]A-Tm[Formula: see text] - actin sliding velocity increased 1.12-fold, [Formula: see text]A-Tm[Formula: see text] - motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, [Formula: see text]A-Tm[Formula: see text] - run time elevated 1.7-fold. We constructed a mathematical model, simulated actin sliding data, and adjusted the kinetic parameters so as to mimic the experimentally observed differences: [Formula: see text]A-Tm[Formula: see text] - myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, [Formula: see text

  5. Cell swelling activates cloned Ca(2+)-activated K(+) channels: a role for the F-actin cytoskeleton

    DEFF Research Database (Denmark)

    Jorgensen, Nanna K; Pedersen, Stine F; Rasmussen, Hanne B

    2003-01-01

    -induced activation of hIK channels was strongly inhibited by cytochalasin D (CD), in concentrations that caused depolymerization of F-actin filaments, indicating a role for the F-actin cytoskeleton in modulation of hIK by changes in cell volume. In conclusion, hIK and rSK3 channels are activated by cell swelling...... and inhibited by shrinkage. A role for the F-actin cytoskeleton in the swelling-induced activation of hIK channels is suggested....

  6. Filamin actin-binding and titin-binding fulfill distinct functions in Z-disc cohesion.

    Directory of Open Access Journals (Sweden)

    Nicanor González-Morales

    2017-07-01

    Full Text Available Many proteins contribute to the contractile properties of muscles, most notably myosin thick filaments, which are anchored at the M-line, and actin thin filaments, which are anchored at the Z-discs that border each sarcomere. In humans, mutations in the actin-binding protein Filamin-C result in myopathies, but the underlying molecular function is not well understood. Here we show using Drosophila indirect flight muscle that the filamin ortholog Cheerio in conjunction with the giant elastic protein titin plays a crucial role in keeping thin filaments stably anchored at the Z-disc. We identify the filamin domains required for interaction with the titin ortholog Sallimus, and we demonstrate a genetic interaction of filamin with titin and actin. Filamin mutants disrupting the actin- or the titin-binding domain display distinct phenotypes, with Z-discs breaking up in parallel or perpendicularly to the myofibril, respectively. Thus, Z-discs require filamin to withstand the strong contractile forces acting on them.

  7. p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging.

    Science.gov (United States)

    Tormos, Ana M; Rius-Pérez, Sergio; Jorques, María; Rada, Patricia; Ramirez, Lorena; Valverde, Ángela M; Nebreda, Ángel R; Sastre, Juan; Taléns-Visconti, Raquel

    2017-01-01

    Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation. Although the steady state levels of cyclin D1 in wild type and p38α knock out old livers remained unaffected, cyclin B1- a marker for G2/M transition- was significantly overexpressed in p38α knock out mice. Our findings suggest that hepatocytes do enter into S phase but they do not complete cell division upon p38α deficiency leading to cytokinesis failure and binucleation. Moreover, old liver-specific p38α MAPK knock out mice exhibited reduced F-actin polymerization and a dramatic loss of actin cytoskeleton. This was associated with abnormal hyperactivation of RhoA and Cdc42 GTPases. Long-term p38α deficiency drives to inactivation of HSP27, which seems to account for the impairment in actin cytoskeleton as Hsp27-silencing decreased the number and length of actin filaments in isolated hepatocytes. p38α MAPK is essential for actin dynamics with age in hepatocytes.

  8. AlphaE-catenin regulates actin dynamics independently of cadherin-mediated cell-cell adhesion.

    Science.gov (United States)

    Benjamin, Jacqueline M; Kwiatkowski, Adam V; Yang, Changsong; Korobova, Farida; Pokutta, Sabine; Svitkina, Tatyana; Weis, William I; Nelson, W James

    2010-04-19

    alphaE-catenin binds the cell-cell adhesion complex of E-cadherin and beta-catenin (beta-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of alphaE-catenin to the E-cadherin-beta-cat complex lowers alphaE-catenin affinity for F-actin, and alphaE-catenin alone can bind F-actin and inhibit Arp2/3 complex-mediated actin polymerization. In cells, to test whether alphaE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic alphaE-catenin pool was sequestered to mitochondria without affecting overall levels of alphaE-catenin or the cadherin-catenin complex. Sequestering cytosolic alphaE-catenin to mitochondria alters lamellipodia architecture and increases membrane dynamics and cell migration without affecting cell-cell adhesion. In contrast, sequestration of cytosolic alphaE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of alphaE-catenin regulates actin dynamics independently of cell-cell adhesion.

  9. αE-catenin regulates actin dynamics independently of cadherin-mediated cell–cell adhesion

    Science.gov (United States)

    Benjamin, Jacqueline M.; Kwiatkowski, Adam V.; Yang, Changsong; Korobova, Farida; Pokutta, Sabine; Svitkina, Tatyana

    2010-01-01

    αE-catenin binds the cell–cell adhesion complex of E-cadherin and β-catenin (β-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of αE-catenin to the E-cadherin–β-cat complex lowers αE-catenin affinity for F-actin, and αE-catenin alone can bind F-actin and inhibit Arp2/3 complex–mediated actin polymerization. In cells, to test whether αE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic αE-catenin pool was sequestered to mitochondria without affecting overall levels of αE-catenin or the cadherin–catenin complex. Sequestering cytosolic αE-catenin to mitochondria alters lamellipodia architecture and increases membrane dynamics and cell migration without affecting cell–cell adhesion. In contrast, sequestration of cytosolic αE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of αE-catenin regulates actin dynamics independently of cell–cell adhesion. PMID:20404114

  10. Autophagy in filamentous fungi.

    Science.gov (United States)

    Pollack, Judith K; Harris, Steven D; Marten, Mark R

    2009-01-01

    Autophagy is a ubiquitous, non-selective degradation process in eukaryotic cells that is conserved from yeast to man. Autophagy research has increased significantly in the last ten years, as autophagy has been connected with cancer, neurodegenerative disease and various human developmental processes. Autophagy also appears to play an important role in filamentous fungi, impacting growth, morphology and development. In this review, an autophagy model developed for the yeast Saccharomyces cerevisiae is used as an intellectual framework to discuss autophagy in filamentous fungi. Studies imply that, similar to yeast, fungal autophagy is characterized by the presence of autophagosomes and controlled by Tor kinase. In addition, fungal autophagy is apparently involved in protection against cell death and has significant effects on cellular growth and development. However, the only putative autophagy proteins characterized in filamentous fungi are Atg1 and Atg8. We discuss various strategies used to study and monitor fungal autophagy as well as the possible relationship between autophagy, physiology, and morphological development.

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

    Science.gov (United States)

    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

  12. Effects of keratin phosphorylation on the mechanical properties of keratin filaments in living cells.

    Science.gov (United States)

    Fois, Giorgio; Weimer, Michael; Busch, Tobias; Felder, Erika T; Oswald, Franz; von Wichert, Götz; Seufferlein, Thomas; Dietl, Paul; Felder, Edward

    2013-04-01

    Keratin filaments impart resilience against mechanical extension of the cell. Despite the pathophysiological relevance of this function, very little is known about the mechanical properties of intermediate filaments in living cells and how these properties are modulated. We used keratin mutants that mimic or abrogate phosphorylation of keratin 8-serine(431) and keratin 18-serine(52) and investigated their effect on keratin tortuousness after cell stretch release in squamous cell carcinoma cells. Cells transfected with the wild-type keratins were used as controls. We can show that keratin dephosphorylation alters the stretch response of keratin in living cells since keratin tortuousness was abolished when phosphorylation of keratin18-serine(52) was abrogated. Additional experiments demonstrate that keratin tortuousness is not simply caused by a plastic overextension of keratin filaments because tortuousness is reversible and requires an intact actin-myosin system. The role of actin in this process remains unclear, but we suggest anchorage of keratin filaments to actin during stretch that leads to buckling on stretch release. Dephosphorylated keratin18-serine(52) might strengthen the recoil force of keratin filaments and hence explain the abolished buckling. The almost exclusive immunolabeling for phosphorylated keratin18-serine (52) in the cell periphery points at a particular role of the peripheral keratin network in this regard.

  13. Filaments in Lupus I

    Science.gov (United States)

    Takahashi, Satoko; Rodon, J.; De Gregorio-Monsalvo, I.; Plunkett, A.

    2017-06-01

    The mechanisms behind the formation of sub-stellar mass sources are key to determine the populations at the low-mass end of the stellar distribution. Here, we present mapping observations toward the Lupus I cloud in C18O(2-1) and 13CO(2-1) obtained with APEX. We have identified a few velocity-coherent filaments. Each contains several substellar mass sources that are also identified in the 1.1mm continuum data (see also SOLA catalogue presentation). We will discuss the velocity structure, fragmentation properties of the identified filaments, and the nature of the detected sources.

  14. Aerogel-supported filament

    Science.gov (United States)

    Wuest, Craig R.; Tillotson, Thomas M.; Johnson, III, Coleman V.

    1995-01-01

    The present invention is a thin filament embedded in a low density aerogel for use in radiation detection instruments and incandescent lamps. The aerogel provides a supportive matrix that is thermally and electrically nonconductive, mechanically strong, highly porous, gas-permeable, and transparent to ionizing radiation over short distances. A low density, open-cell aerogel is cast around a fine filament or wire, which allows the wire to be positioned with little or no tension and keeps the wire in place in the event of breakage. The aerogel support reduces the stresses on the wire caused by vibrational, gravitational, electrical, and mechanical forces.

  15. Fundamentals of Filament Interaction

    Science.gov (United States)

    2017-05-19

    strictly to the research plan outlined in the original proposal. To our knowledge this was the first time the HEL JTO had used an MRI to advance the...Department Of Chemistry 2145 Sheridan Road • Evanston, Il 60208-3113 Phone 847-467-4979 • Fax 847-467-4996 • E-Mail T-Seideman@Northwestern.Edu...JTO had used an MRI to advance the understanding of optical filamentation in air by ultrashort laser pulses. Studies of filamentation involve many

  16. Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding.

    Science.gov (United States)

    Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael; Posern, Guido

    2016-05-15

    The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin-MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin-RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. Rapid transport of plasmid DNA into the nucleolus via actin depolymerization using the HVJ envelope vector.

    Science.gov (United States)

    Suvanasuthi, Saroj; Tamai, Katsuto; Kaneda, Yasufumi

    2007-01-01

    Although nuclear transport of therapeutic genes is an essential requirement of human gene therapy, factors required for nuclear entry of DNA remain to be elucidated. Non-viral vector systems have led to numerous improvements in the efficiency of delivery of exogenous DNA into cells. However, nuclear transport of plasmid is difficult to achieve. We examined nuclear translocation efficiency of Cy3-labeled plasmid DNA (Cy3-pDNA) delivered by the hemagglutinating virus of Japan envelope (HVJ-E) vector, Lipofectamine or microinjection. We also examined the effect of actin depolymerization on nuclear transport of Cy3-pDNA. Cy3-pDNA reached the nucleus, particularly in the nucleolus, in 30 min after fusion-mediated delivery using the HVJ-E vector, while the DNA was retained in the cytoplasm during the observed period after the delivery by cationic liposomes. HVJ-E treatment transiently depolymerized actin filaments, and acceleration of nucleolar entry of microinjected DNA was achieved when treated with either empty HVJ-E or cytochalasin D, an inhibitor of actin depolymerization, prior to microinjection. These results suggest that plasmid DNA can be transported rapidly from the cytoplasm to the nucleolus when actin filaments are depolymerized. Thus, the HVJ-E vector can accelerate the transport of DNA to the nucleolus by actin depolymerization. Copyright 2006 John Wiley & Sons, Ltd.

  18. The parafibromin tumor suppressor protein interacts with actin-binding proteins actinin-2 and actinin-3

    Directory of Open Access Journals (Sweden)

    Marx Stephen J

    2008-08-01

    Full Text Available Abstract Background Germline and somatic inactivating mutations in the HRPT2 gene occur in the inherited hyperparathyroidism-jaw tumor syndrome, in some cases of parathyroid cancer and in some cases of familial hyperparathyroidism. HRPT2 encodes parafibromin. To identify parafibromin interacting proteins we used the yeast two-hybrid system for screening a heart cDNA library with parafibromin as the bait. Results Fourteen parafibromin interaction positive preys representing 10 independent clones encoding actinin-2 were isolated. Parafibromin interacted with muscle alpha-actinins (actinin-2 and actinin-3, but not with non-muscle alpha-actinins (actinin-1 and actinin-4. The parafibromin-actinin interaction was verified by yeast two-hybrid, GST pull-down, and co-immunoprecipitation. Yeast two-hybrid analysis revealed that the N-terminal region of parafibromin interacted with actinins. In actin sedimentation assays parafibromin did not dissociate skeletal muscle actinins from actin filaments, but interestingly, parafibromin could also bundle/cross-link actin filaments. Parafibromin was predominantly nuclear in undifferentiated proliferating myoblasts (C2C12 cells, but in differentiated C2C12 myotubes parafibromin co-localized with actinins in the cytoplasmic compartment. Conclusion These data support a possible contribution of parafibromin outside the nucleus through its interaction with actinins and actin bundling/cross-linking. These data also suggest that actinins (and actin participate in sequestering parafibromin in the cytoplasmic compartment.

  19. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    Energy Technology Data Exchange (ETDEWEB)

    Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.; Tregear, R.T.; Ward, A.; Krzic, U.; Prince, K.M.; Perz-Edwards, R.J.; Reconditi, M.; Gore, D.; Irving, T.C.; Reedy, M.K. (IIT); (EMBL); (Scripps); (Duke); (Prince); (FSU); (MRC); (U. Florence)

    2008-09-03

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the 'steric blocking' mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca{sup 2+} with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca{sup 2+}], and stretch activation, at lower [Ca{sup 2+}], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored 'actin target zones.' Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca{sup 2+}] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca{sup 2+}], Vi-'paralyzed' fibers produce force substantially above passive response at pCa {approx} 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding 'brakes' by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

  20. Ring closure in actin polymers

    Energy Technology Data Exchange (ETDEWEB)

    Sinha, Supurna, E-mail: supurna@rri.res.in [Raman Research Institute, Bangalore 560080 (India); Chattopadhyay, Sebanti [Doon University, Dehradun 248001 (India)

    2017-03-18

    We present an analysis for the ring closure probability of semiflexible polymers within the pure bend Worm Like Chain (WLC) model. The ring closure probability predicted from our analysis can be tested against fluorescent actin cyclization experiments. We also discuss the effect of ring closure on bend angle fluctuations in actin polymers. - Highlights: • Ring closure of biopolymers. • Worm like chain model. • Predictions for experiments.

  1. Double Cortex Syndrome (Subcortical Band Heterotopia): A Case Report

    National Research Council Canada - National Science Library

    Momen, Ali Akbar; Momen, Mehdi

    2015-01-01

    .... Many congenital or acquired brain anomalies are revealed with MRIs. Although the majority of these abnormalities are sporadic but patients with subcortical band heterotopia or double cortex syndrome have sex-linked inheritance...

  2. Myosin Binding Protein C Interaction with Actin: CHARACTERIZATION AND MAPPING OF THE BINDING SITE*

    OpenAIRE

    Rybakova, Inna N.; Greaser, Marion L.; Moss, Richard L.

    2010-01-01

    Myosin binding protein C (MyBPC) is a multidomain protein associated with the thick filaments of striated muscle. Although both structural and regulatory roles have been proposed for MyBPC, its interactions with other sarcomeric proteins remain obscure. The current study was designed to examine the actin-binding properties of MyBPC and to define MyBPC domain regions involved in actin interaction. Here, we have expressed full-length mouse cardiac MyBPC (cMyBPC) in a baculovirus system and show...

  3. Quantity language speakers show enhanced subcortical processing.

    Science.gov (United States)

    Dawson, Caitlin; Aalto, Daniel; Šimko, Juraj; Putkinen, Vesa; Tervaniemi, Mari; Vainio, Martti

    2016-07-01

    The complex auditory brainstem response (cABR) can reflect language-based plasticity in subcortical stages of auditory processing. It is sensitive to differences between language groups as well as stimulus properties, e.g. intensity or frequency. It is also sensitive to the synchronicity of the neural population stimulated by sound, which results in increased amplitude of wave V. Finnish is a full-fledged quantity language, in which word meaning is dependent upon duration of the vowels and consonants. Previous studies have shown that Finnish speakers have enhanced behavioural sound duration discrimination ability and larger cortical mismatch negativity (MMN) to duration change compared to German and French speakers. The next step is to find out whether these enhanced duration discrimination abilities of quantity language speakers originate at the brainstem level. Since German has a complementary quantity contrast which restricts the possible patterns of short and long vowels and consonants, the current experiment compared cABR between nonmusician Finnish and German native speakers using seven short complex stimuli. Finnish speakers had a larger cABR peak amplitude than German speakers, while the peak onset latency was only affected by stimulus intensity and spectral band. The results suggest that early cABR responses are better synchronised for Finns, which could underpin the enhanced duration sensitivity of quantity language speakers. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Solar Features - Prominences and Filaments

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Prominences and filaments are two manifestations of the same phenomenon. Both prominences and filaments are features formed above the chromosphere by cool dense...

  5. Positrusion Filament Recycling System Project

    Data.gov (United States)

    National Aeronautics and Space Administration — TUI proposes a novel process to produce 3d printer feedstock filament out of scrap ABS on the ISS. Currently the plastic filament materials that most 3d printers use...

  6. Common genetic variants influence human subcortical brain structures

    OpenAIRE

    Hibar, Derrek P; Stein, Jason L; Renteria, Miguel E; Arias-Vasquez, Alejandro; Desrivières, Sylvane; Jahanshad, Neda; Toro, Roberto; Wittfeld, Katharina; Abramovic, Lucija; Andersson, Micael; Aribisala, Benjamin S; Armstrong, Nicola J; Bernard, Manon; Bohlken, Marc M; Boks, Marco P

    2015-01-01

    The highly complex structure of the human brain is strongly shaped by genetic influences. Subcortical brain regions form circuits with cortical areas to coordinate movement, learning, memory and motivation, and altered circuits can lead to abnormal behaviour and disease. To investigate how common genetic variants affect the structure of these brain regions, here we conduct genome-wide association studies of the volumes of seven subcortical regions and the intracranial volume derived from magn...

  7. Subcortical shape and volume abnormalities in an elderly HIV+ cohort

    Science.gov (United States)

    Wade, Benjamin S. C.; Valcour, Victor; Busovaca, Edgar; Esmaeili-Firidouni, Pardis; Joshi, Shantanu H.; Wang, Yalin; Thompson, Paul M.

    2015-03-01

    Over 50% of HIV+ individuals show significant impairment in psychomotor functioning, processing speed, working memory and attention [1, 2]. Patients receiving combination antiretroviral therapy may still have subcortical atrophy, but the profile of HIV-associated brain changes is poorly understood. With parametric surface-based shape analyses, we mapped the 3D profile of subcortical morphometry in 63 elderly HIV+ subjects (4 female; age=65.35 ± 2.21) and 31 uninfected elderly controls (2 female; age=64.68 ± 4.57) scanned with MRI as part of a San Francisco Bay Area study of elderly people with HIV. We also investigated whether morphometry was associated with nadir CD4+ (T-cell) counts, viral load and illness duration among HIV+ participants. FreeSurfer was used to segment the thalamus, caudate, putamen, pallidum, hippocampus, amygdala, accumbens, brainstem, callosum and ventricles from brain MRI scans. To study subcortical shape, we analyzed: (1) the Jacobian determinant (JD) indexed over structures' surface coordinates and (2) radial distances (RD) of structure surfaces from a medial curve. A JD less than 1 reflects regional tissue atrophy and greater than 1 reflects expansion. The volumes of several subcortical regions were found to be associated with HIV status. No regional volumes showed detectable associations with CD4 counts, viral load or illness duration. The shapes of numerous subcortical regions were significantly linked to HIV status, detectability of viral RNA and illness duration. Our results show subcortical brain differences in HIV+ subjects in both shape and volumetric domains.

  8. Capillary thinning of polymeric filaments

    DEFF Research Database (Denmark)

    Kolte, Mette Irene; Szabo, Peter; Hassager, Ole

    1998-01-01

    . Thereby the sample is stretched into a filament. Provided the filament is sufficiently long, surface tension will induce a thinningof the filament until breakage in finite time.This experiment was originally deviced by Bazilevsky et al. (1990). For the numerical simulations we employ a Lagrangian finite...

  9. Capillary thinning of polymeric filaments

    DEFF Research Database (Denmark)

    Kolte, Mette Irene; Szabo, Peter

    1999-01-01

    , the bottom plate is lowered under gravity to produce a specified strain. The sample is thereby stretched into a filament. Provided the filament is sufficiently long, surface tension will induce a thinning of the filament until breakup in finite time. The numerical simulations are performed with a Lagrangian...

  10. Interactions with the actin cytoskeleton are required for cell wall localization of barley stripe mosaic virus TGB proteins

    Science.gov (United States)

    The host cytoskeleton and membrane system are the main routes by which plant viruses move within or between cells. Barley stripe mosaic virus (BSMV) -induced actin filament thickening was visualized in the cytoskeleton of agroinfiltrated Nicotiana benthamiana epidermal cells expressing DsRed:Talin. ...

  11. Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy

    Science.gov (United States)

    Cohen, Shenhav; Zhai, Bo; Gygi, Steven P.

    2012-01-01

    During muscle atrophy, myofibrillar proteins are degraded in an ordered process in which MuRF1 catalyzes ubiquitylation of thick filament components (Cohen et al. 2009. J. Cell Biol. http://dx.doi.org/10.1083/jcb.200901052). Here, we show that another ubiquitin ligase, Trim32, ubiquitylates thin filament (actin, tropomyosin, troponins) and Z-band (α-actinin) components and promotes their degradation. Down-regulation of Trim32 during fasting reduced fiber atrophy and the rapid loss of thin filaments. Desmin filaments were proposed to maintain the integrity of thin filaments. Accordingly, we find that the rapid destruction of thin filament proteins upon fasting was accompanied by increased phosphorylation of desmin filaments, which promoted desmin ubiquitylation by Trim32 and degradation. Reducing Trim32 levels prevented the loss of both desmin and thin filament proteins. Furthermore, overexpression of an inhibitor of desmin polymerization induced disassembly of desmin filaments and destruction of thin filament components. Thus, during fasting, desmin phosphorylation increases and enhances Trim32-mediated degradation of the desmin cytoskeleton, which appears to facilitate the breakdown of Z-bands and thin filaments. PMID:22908310

  12. Filamentous Fungi Fermentation

    DEFF Research Database (Denmark)

    Nørregaard, Anders; Stocks, Stuart; Woodley, John

    2014-01-01

    Filamentous fungi (including microorganisms such as Aspergillus niger and Rhizopus oryzae) represent an enormously important platform for industrial fermentation. Two particularly valuable features are the high yield coefficients and the ability to secrete products. However, the filamentous...... morphology, together with non-Newtonian rheological properties (shear thinning), result in poor oxygen transfer unless sufficient energy is provided to the fermentation. While genomic research may improve the organisms, there is no doubt that to enable further application in future it will be necessary...... to match such research with studies of oxygen transfer and energy supply to high viscosity fluids. Hence, the implementation of innovative solutions (some of which in principle are already possible) will be essential to ensure the further development of such fermentations....

  13. SYP73 Anchors the ER to the Actin Cytoskeleton for Maintenance of ER Integrity and Streaming in Arabidopsis.

    Science.gov (United States)

    Cao, Pengfei; Renna, Luciana; Stefano, Giovanni; Brandizzi, Federica

    2016-12-05

    The endoplasmic reticulum (ER) is an essential organelle that spreads throughout the cytoplasm as one interconnected network of narrow tubules and dilated cisternae that enclose a single lumen. The ER network undergoes extensive remodeling, which critically depends on membrane-cytoskeleton interactions [1]. In plants, the ER is also highly mobile, and its streaming contributes significantly to the movement of other organelles [2, 3]. The remodeling and motility of the plant ER rely mainly on actin [4] and to a minor extent on microtubules [5]. Although a three-way interaction between the ER, cytosolic myosin-XI, and F-actin mediates the plant ER streaming [6], the mechanisms underlying stable interaction of the ER membrane with actin are unknown. Early electron microscopy studies suggested a direct attachment of the plant ER with actin filaments [7, 8], but it is plausible that yet-unknown proteins facilitate anchoring of the ER membrane with the cytoskeleton. We demonstrate here that SYP73, a member of the plant Syp7 subgroup of SNARE proteins [9] containing actin-binding domains, is a novel ER membrane-associated actin-binding protein. We show that overexpression of SYP73 causes a striking rearrangement of the ER over actin and that, similar to mutations of myosin-XI [4, 10, 11], loss of SYP73 reduces ER streaming and affects overall ER network morphology and plant growth. We propose a model for plant ER remodeling whereby the dynamic rearrangement and streaming of the ER network depend on the propelling action of myosin-XI over actin coupled with a SYP73-mediated bridging, which dynamically anchors the ER membrane with actin filaments. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Thymosin beta4 inhibits ADF/cofilin stimulated F-actin cycling and hela cell migration: reversal by active Arp2/3 complex.

    Science.gov (United States)

    Al Haj, Abdulatif; Mazur, Antonina Joanna; Buchmeier, Sabine; App, Christine; Theiss, Carsten; Silvan, Unai; Schoenenberger, Cora-Ann; Jockusch, Brigitte M; Hannappel, Ewald; Weeds, Alan G; Mannherz, Hans Georg

    2014-02-01

    F-actin treadmilling plays a key part in cell locomotion. Because immunofluorescence showed colocalisation of thymosin beta4 (Tβ4) with cofilin-1 and Arp2/3 complex in lamellipodia, we analyzed combinations of these proteins on F-actin-adenosine triphosphate (ATP)-hydrolysis, which provides a measure of actin treadmilling. Actin depolymerising factor (ADF)/cofilin stimulated treadmilling, while Tβ4 decreased treadmilling, presumably by sequestering monomers. Tβ4 added together with ADF/cofilin also inhibited the treadmilling, relative to cofilin alone, but both the rate and extent of depolymerization were markedly enhanced in the presence of both these proteins. Arp2/3 complex reversed the sequestering activity of Tβ4 when equimolar to actin, but not in the additional presence of cofilin-1 or ADF. Transfection experiments to explore the effects of changing the intracellular concentration of Tβ4 in HeLa cells showed that an increase in Tβ4 resulted in reduced actin filaments bundles and narrower lamellipodia, and a conspicuous decrease of cell migration as seen by two different assays. In contrast, cells transfected with a vector leading to Tβ4 knockdown by small interfering RNA (siRNA) displayed prominent actin filament networks within the lamellipodia and the leading lamella and enhanced migration. The experiments reported here demonstrate the importance of the interplay of these different classes of actin-binding proteins on cell behaviour. Copyright © 2013 Wiley Periodicals, Inc.

  15. ADF and Cofilin1 Control Actin Stress Fibers, Nuclear Integrity, and Cell Survival.

    Science.gov (United States)

    Kanellos, Georgios; Zhou, Jing; Patel, Hitesh; Ridgway, Rachel A; Huels, David; Gurniak, Christine B; Sandilands, Emma; Carragher, Neil O; Sansom, Owen J; Witke, Walter; Brunton, Valerie G; Frame, Margaret C

    2015-12-01

    Genetic co-depletion of the actin-severing proteins ADF and CFL1 triggers catastrophic loss of adult homeostasis in multiple tissues. There is impaired cell-cell adhesion in skin keratinocytes with dysregulation of E-cadherin, hyperproliferation of differentiated cells, and ultimately apoptosis. Mechanistically, the primary consequence of depleting both ADF and CFL1 is uncontrolled accumulation of contractile actin stress fibers associated with enlarged focal adhesions at the plasma membrane, as well as reduced rates of membrane protrusions. This generates increased intracellular acto-myosin tension that promotes nuclear deformation and physical disruption of the nuclear lamina via the LINC complex that normally connects regulated actin filaments to the nuclear envelope. We therefore describe a pathway involving the actin-severing proteins ADF and CFL1 in regulating the dynamic turnover of contractile actin stress fibers, and this is vital to prevent the nucleus from being damaged by actin contractility, in turn preserving cell survival and tissue homeostasis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Is chloroplast import of photosynthesis proteins facilitated by an actin-TOC-TIC-VIPP1 complex?

    Science.gov (United States)

    Jouhet, Juliette; Gray, John C

    2009-10-01

    Actin filaments are major components of the cytoskeleton that interact with chloroplast envelope membranes to allow chloroplast positioning and movement, stromule mobility and gravitropism perception. We recently reported that Toc159, a component of the TOC complex of the chloroplast protein import apparatus, interacts directly with actin. The interaction of Toc159 and actin was identified by co-immunoprecipitation and co-sedimentation experiments with detergent-solubilised pea chloroplast envelope membranes. In addition, many of the components of the TOC-TIC protein import apparatus and VIPP1 (vesicle-inducing protein in plastids 1) were identified by mass spectroscopy in the material co-immunoprecipitated with antibodies to actin. Toc159 is the receptor for the import of photosynthesis proteins and VIPP1 is involved in thylakoid membrane formation by inducing vesicle formation from the chloroplast inner envelope membrane, suggesting we may have identified an actin-TOC-TIC-VIPP1 complex that may provide a means of channeling cytosolic preproteins to the thylakoid membrane. The interaction of Toc159 with actin may facilitate exchange between the putative soluble and membrane forms of Toc159 and promote the interaction of cytosolic preproteins with the TOC complex.

  17. T lymphocyte migration: an action movie starring the actin and associated actors

    Directory of Open Access Journals (Sweden)

    Loïc eDupré

    2015-11-01

    Full Text Available The actin cytoskeleton is composed of a dynamic filament meshwork that builds the architecture of the cell to sustain its fundamental properties. This physical structure is characterized by a continuous remodeling, which allows cells to accomplish complex motility steps such as directed migration, crossing of biological barriers and interaction with other cells. T lymphocytes excel in these motility steps to ensure their immune surveillance duties. In particular, actin cytoskeleton remodeling is key to facilitate the journey of T lymphocytes through distinct tissue environments and to tune their stop and go behavior during the scanning of antigen-presenting cells. The molecular mechanisms controlling actin cytoskeleton remodeling during T lymphocyte motility have been only partially unraveled, since the function of many actin regulators has not yet been assessed in these cells. Our review aims to integrate the current knowledge into a comprehensive picture of how the actin cytoskeleton drives T lymphocyte migration. We will present the molecular actors that control actin cytoskeleton remodeling, as well as their role in the different T lymphocyte motile steps. We will also highlight which challenges remain to be addressed experimentally and which approaches appear promising to tackle them.

  18. Antibody against the actin-binding protein depactin attenuates Ca2+ signaling in starfish eggs.

    Science.gov (United States)

    Chun, Jong T; Vasilev, Filip; Santella, Luigia

    2013-11-15

    Being present in starfish oocytes, the cofilin/ADF (actin-depolymerizing factor) family protein depactin severs actin filaments. Previously, we reported that exogenous cofilin microinjected into starfish eggs significantly augmented the Ca(2+) release in response to inositol 1,4,5-trisphosphate (InsP3) or fertilizing sperm, raising the possibility that intracellular Ca(2+) signaling could be modulated by the actin cytoskeleton. In this communication, we have targeted the endogenous depactin by use of the specific antibody that was raised against its actin-binding domain. The anti-depactin antibody microinjected into the starfish oocytes and eggs effectively altered the structure of the actin cytoskeleton, and significantly delayed the meiotic progression induced by 1-methyladenine. When microinjected into the mature eggs, the anti-depactin antibody markedly reduced the amplitude of the Ca(2+) response in a dose-dependent manner, corroborating the results of our previous study with cofilin. In addition, the eggs microinjected with the anti-depactin antibody displayed reduced rate of successful elevation of the fertilization envelope and an elevated tendency of polyspermic interaction. Taken together, our data suggest that the actin cytoskeleton is implicated not only in meiotic maturation and intracellular Ca(2+) signaling, but also in the fine regulation of gametes interaction and cortical granules exocytosis. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Bacterial Actins? An Evolutionary Perspective

    Science.gov (United States)

    Doolittle, Russell F.; York, Amanda L.

    2003-01-01

    According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life. An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles. Two recent papers present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin. Sequence comparisons reveml that eukaryotic actin and the bacterial homolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories.

  20. Looking Down at Adsorption Dynamics: Filament Height Measurements with TIRFM

    Science.gov (United States)

    Welch, David; Dogic, Zvonimir

    2012-02-01

    Polymer adsorption is important in several contexts such as chromatography, colloid stabilization, and bio-fouling. Despite a cross-disciplinary interest in the subject, there are not many techniques to observe single-molecule absorption events in real time. We use TIRF microscopy to accomplish this using the biological polymer f-actin adsorbed to a microscope slide via the well-known depletion interaction. We find TIRFM is able to quantitatively measure filament height, and we compare our results to theoretical predictions and previous results obtained from other systems.

  1. Actin turnover-mediated gravity response in maize root apices: gravitropism of decapped roots implicates gravisensing outside of the root cap.

    Science.gov (United States)

    Mancuso, Stefano; Barlow, Peter W; Volkmann, Dieter; Baluska, Frantisek

    2006-03-01

    The dynamic actin cytoskeleton has been proposed to be linked to gravity sensing in plants but the mechanistic understanding of these processes remains unknown. We have performed detailed pharmacological analyses of the role of the dynamic actin cytoskeleton in gravibending of maize (Zea mays) root apices. Depolymerization of actin filaments with two drugs having different mode of their actions, cytochalasin D and latrunculin B, stimulated root gravibending. By contrast, drug-induced stimulation of actin polymerization and inhibition of actin turnover, using two different agents phalloidin and jasplakinolide, compromised the root gravibending. Importantly, all these actin drugs inhibited root growth to similar extents suggesting that high actin turnover is essential for the gravity-related growth responses rather than for the general growth process. Both latrunculin B and cytochalasin D treatments inhibited root growth but restored gravibending of the decapped root apices, indicating that there is a strong potential for effective actin-mediated gravity sensing outside the cap. This elusive gravity sensing outside the root cap is dependent not only on the high rate of actin turnover but also on weakening of myosin activities, as general inhibition of myosin ATPases induced stimulation of gravibending of the decapped root apices. Collectively, these data provide evidence for the actin turnover-mediated gravity sensing outside the root cap.

  2. The Intensity Of The 2.7nm Reflection As A Constraint For Models Of Myosin Docking To Actin

    Energy Technology Data Exchange (ETDEWEB)

    Reconditi, Massimo; Irving, Tom C.; (IIT); (U.Florence)

    2009-03-16

    Previous workers have proposed high resolution models for the docking of the myosin heads on actin on the basis of combined crystallographic and electron microscopy data (Mendelson and Morris, 1997 PNAS 94:8533; Holmes et al. 2003 Nature 425:423). We have used data from small angle X-ray fiber diffraction from living muscle to check the predictions of these models. Whole sartorius muscles from Rana pipiens were mounted in a chamber containing Ringer's solution at 10 C and at rest length at the BioCAT beamline (18 ID, Advanced Photon Source, Argonne, IL-U.S.A.). The muscles were activated by electrical stimulation and the force was recorded with a muscle lever system type 300B (Aurora Scientific). X-ray patterns were collected with 1s total exposures at rest and during isometric contraction out to 0.5 nm{sup -1} in reciprocal space, as the higher angle reflections are expected to be more sensitive to the arrangement of myosin heads on actin. We observed that during isometric contraction the meridional reflection originating from the 2.73nm repeat of the actin monomers along the actin filament increases its intensity by a factor 2.1 {+-} 0.2 relative to rest. Among the models tested, Holmes et al. fits the data when the actin filament is decorated with 30-40% the total available myosin heads, a fraction similar to that estimated with fast single fiber mechanics by Piazzesi et al. (2007, Cell 131:784). However, when the mismatch between the periodicities of actin and myosin filaments is taken into account, none of the models can reproduce the fiber diffraction data. We suggest that the fiber diffraction data should be used as a further constraint on new high resolution models for the docking of the myosin heads on actin.

  3. Subcortical biophysical abnormalities in patients with mood disorders.

    Science.gov (United States)

    Kumar, A; Yang, S; Ajilore, O; Wu, M; Charlton, R; Lamar, M

    2014-06-01

    Cortical-subcortical circuits have been implicated in the pathophysiology of mood disorders. Structural and biochemical abnormalities have been identified in patients diagnosed with mood disorders using magnetic resonance imaging-related approaches. In this study, we used magnetization transfer (MT), an innovative magnetic resonance approach, to study biophysical changes in both gray and white matter regions in cortical-subcortical circuits implicated in emotional regulation and behavior. Our study samples comprised 28 patients clinically diagnosed with major depressive disorder (MDD) and 31 non-depressed subjects of comparable age and gender. MT ratio (MTR), representing the biophysical integrity of macromolecular proteins within key components of cortical-subcortical circuits-the caudate, thalamic, striatal, orbitofrontal, anterior cingulate and dorsolateral regions-was the primary outcome measure. In our study, the MTR in the head of the right caudate nucleus was significantly lower in the MDD group when compared with the comparison group. MTR values showed an inverse relationship with age in both groups, with more widespread relationships observed in the MDD group. These data indicate that focal biophysical abnormalities in the caudate nucleus may be central to the pathophysiology of depression and critical to the cortical-subcortical abnormalities that underlie mood disorders. Depression may also accentuate age-related changes in the biophysical properties of cortical and subcortical regions. These observations have broad implications for the neuronal circuitry underlying mood disorders across the lifespan.

  4. Physical fitness and shapes of subcortical brain structures in children.

    Science.gov (United States)

    Ortega, Francisco B; Campos, Daniel; Cadenas-Sanchez, Cristina; Altmäe, Signe; Martínez-Zaldívar, Cristina; Martín-Matillas, Miguel; Catena, Andrés; Campoy, Cristina

    2017-03-27

    A few studies have recently reported that higher cardiorespiratory fitness is associated with higher volumes of subcortical brain structures in children. It is, however, unknown how different fitness measures relate to shapes of subcortical brain nuclei. We aimed to examine the association of the main health-related physical fitness components with shapes of subcortical brain structures in a sample of forty-four Spanish children aged 9·7 (sd 0·2) years from the NUtraceuticals for a HEALthier life project. Cardiorespiratory fitness, muscular strength and speed agility were assessed using valid and reliable tests (ALPHA-fitness test battery). Shape of the subcortical brain structures was assessed by MRI, and its relationship with fitness was examined after controlling for a set of potential confounders using a partial correlation permutation approach. Our results showed that all physical fitness components studied were significantly related to the shapes of subcortical brain nuclei. These associations were both positive and negative, indicating that a higher level of fitness in childhood is related to both expansions and contractions in certain regions of the accumbens, amygdala, caudate, hippocampus, pallidum, putamen and thalamus. Cardiorespiratory fitness was mainly associated with expansions, whereas handgrip was mostly associated with contractions in the structures studied. Future randomised-controlled trials will confirm or contrast our findings, demonstrating whether changes in fitness modify the shapes of brain structures and the extent to which those changes influence cognitive function.

  5. Creating biomolecular motors based on dynein and actin-binding proteins.

    Science.gov (United States)

    Furuta, Akane; Amino, Misako; Yoshio, Maki; Oiwa, Kazuhiro; Kojima, Hiroaki; Furuta, Ken'ya

    2017-03-01

    Biomolecular motors such as myosin, kinesin and dynein are protein machines that can drive directional movement along cytoskeletal tracks and have the potential to be used as molecule-sized actuators. Although control of the velocity and directionality of biomolecular motors has been achieved, the design and construction of novel biomolecular motors remains a challenge. Here we show that naturally occurring protein building blocks from different cytoskeletal systems can be combined to create a new series of biomolecular motors. We show that the hybrid motors-combinations of a motor core derived from the microtubule-based dynein motor and non-motor actin-binding proteins-robustly drive the sliding movement of an actin filament. Furthermore, the direction of actin movement can be reversed by simply changing the geometric arrangement of these building blocks. Our synthetic strategy provides an approach to fabricating biomolecular machines that work along artificial tracks at nanoscale dimensions.

  6. Actin cytoskeleton as a putative target of the neem limonoid Azadirachtin A.

    Science.gov (United States)

    Anuradha, Aritakula; Annadurai, Ramaswamy S; Shashidhara, L S

    2007-06-01

    Limonoids isolated from the Indian neem tree (Azadirachta indica) have been gaining global acceptance in agricultural applications and in contemporary medicine for their myriad but discrete properties. However, their mode of action is still not very well understood. We have studied the mode of action of Azadirachtin A, the major limonoid of neem seed extracts, using Drosophila melanogaster as the model system. Azadirachtin A induces moderate-to-severe phenotypes in different tissues in a dose-dependent manner. At the cellular level, Azadirachtin A induces depolymerization of Actin leading to arrest of cells and subsequently apoptosis in a caspase-independent manner. Azadirachtin A-induced phenotypes were rescued by the over-expression of Cyclin E in a tissue-dependent manner. Cyclin E, which caused global rescue of Azadirachtin A-induced phenotypes, also effected rearrangement of the actin filaments. These results suggest that probably actin is a target of Azadirachtin A activity.

  7. Initial stem cell adhesion on porous silicon surface: molecular architecture of actin cytoskeleton and filopodial growth

    Science.gov (United States)

    Collart-Dutilleul, Pierre-Yves; Panayotov, Ivan; Secret, Emilie; Cunin, Frédérique; Gergely, Csilla; Cuisinier, Frédéric; Martin, Marta

    2014-10-01

    The way cells explore their surrounding extracellular matrix (ECM) during development and migration is mediated by lamellipodia at their leading edge, acting as an actual motor pulling the cell forward. Lamellipodia are the primary area within the cell of actin microfilaments (filopodia) formation. In this work, we report on the use of porous silicon (pSi) scaffolds to mimic the ECM of mesenchymal stem cells from the dental pulp (DPSC) and breast cancer (MCF-7) cells. Our atomic force microscopy (AFM), fluorescence microscopy, and scanning electron microscopy (SEM) results show that pSi promoted the appearance of lateral filopodia protruding from the DPSC cell body and not only in the lamellipodia area. The formation of elongated lateral actin filaments suggests that pores provided the necessary anchorage points for protrusion growth. Although MCF-7 cells displayed a lower presence of organized actin network on both pSi and nonporous silicon, pSi stimulated the formation of extended cell protrusions.

  8. Actin polymerization is essential for myelin sheath fragmentation during Wallerian degeneration.

    Science.gov (United States)

    Jung, Junyang; Cai, Wenting; Lee, Hyun Kyoung; Pellegatta, Marta; Shin, Yoon Kyung; Jang, So Young; Suh, Duk Joon; Wrabetz, Lawrence; Feltri, M Laura; Park, Hwan Tae

    2011-02-09

    The mechanisms that trigger Wallerian degeneration (WD) of peripheral nerves after injury are not well understood. During the early period of WD, fragmentation of myelin into ovoid structures occurs near the Schmidt-Lantermann incisures (SLI), a noncompact region of the myelin sheath containing autotypical adherens junction. In this study, we found that new filamentous actin polymerization occurs in the SLI of mouse sciatic nerves after injury and that its inhibition prevented not only the degradation of E-cadherin in the SLI but also myelin ovoid formation. However, the inhibition of actin polymerization could not block Schwann cell dedifferentiation. The activation of Rac GTPase was observed in the distal stump of the injured nerves, and a specific Rac inhibitor, a dominant-negative Rac, and Rac1-RNA interference blocked myelin ovoid formation. Together, these findings suggest that dynamic changes in actin in the SLI are essential for initiation of demyelination after peripheral nerve injury.

  9. Leptin Elongates Hypothalamic Neuronal Cilia via Transcriptional Regulation and Actin Destabilization.

    Science.gov (United States)

    Kang, Gil Myoung; Han, Yu Mi; Ko, Hyuk Whan; Kim, Joon; Oh, Byung Chul; Kwon, Ijoo; Kim, Min-Seon

    2015-07-17

    Terminally differentiated neurons have a single, primary cilium. The primary cilia of hypothalamic neurons play a critical role in sensing metabolic signals. We recently showed that mice with leptin deficiency or resistance have shorter cilia in the hypothalamic neurons, and leptin treatment elongates cilia in hypothalamic neurons. Here, we investigated the molecular mechanisms by which leptin controls ciliary length in hypothalamic neurons. In N1 hypothalamic neuronal cells, leptin treatment increased the expression of intraflagellar transport proteins. These effects occurred via phosphatase and tensin homolog/glycogen synthase kinase-3β-mediated inhibition of the transcriptional factor RFX1. Actin filament dynamics were also involved in leptin-promoted ciliary elongation. Both leptin and cytochalasin-D treatment induced F-actin disruption and cilium elongation in hypothalamic neurons that was completely abrogated by co-treatment with the F-actin polymerizer phalloidin. Our findings suggest that leptin elongates hypothalamic neuronal cilia by stimulating the production of intraflagellar transport proteins and destabilizing actin filaments. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Dynamic Actin Structures Stabilized by Profilin

    National Research Council Canada - National Science Library

    Toren Finkel; Julie A. Theriot; Kirk R. Dise; Gordon F. Tomaselli; Pascal J. Goldschmidt-Clermont

    1994-01-01

    We describe the production and analysis of clonal cell lines in which we have overexpressed human profilin, a small ubiquitous actin monomer binding protein, to assess the role of profilin on actin function in vivo...

  11. Demonstration in vivo of the role of Arabidopsis PLIM2 actin-binding proteins during pollination.

    Science.gov (United States)

    Sudo, Keisuke; Park, Jong-In; Sakazono, Satomi; Masuko-Suzuki, Hiromi; Osaka, Masaaki; Kawagishi, Mizuho; Fujita, Kotomi; Maruoka, Mayumi; Nanjo, Hikaru; Suzuki, Go; Suwabe, Keita; Watanabe, Masao

    2013-01-01

    In plant reproduction, pollination is the initial key process in bringing together the male and female gametophytes. When a pollen grain lands on the surface of the stigma, information is exchanged between the pollen and stigmatic cell to determine whether the pollen grain will be accepted or rejected. If it is accepted, the stigmatic papilla cell supplies water and other resources to the pollen for germination and pollen tube elongation. Cellular processes involving actin are essential for pollen germination and tube growth, and actin-binding proteins regulate these processes by interacting with actin filaments to assemble cytoskeletal structures and actin networks. LIM proteins, which belong to a subfamily of cysteine-rich proteins, are a family of actin-binding proteins in plants, and are considered to be important for formation of the actin cytoskeleton and maintenance of its dynamics. Although the physiological and biochemical characteristics of LIMs have been elucidated in vitro in a variety of cell types, their exact role in pollen germination and pollen tube growth during pollination remained unclear. In this manuscript, we focus on the pollen-specific LIM proteins, AtPLIM2a and AtPLIM2c, and define their biological function during pollination in Arabidopsis thaliana. The atplim2a/atplim2c double knockdown RNAi plants showed a reduced pollen germination, approximately one-fifth of wild type, and slower pollen tube growth in the pistil, that is 80.4 μm/hr compared to 140.8 μm/hr in wild type. These defects led to an occasional unfertilized ovule at the bottom of the silique in RNAi plants. Our data provide direct evidence of the biological function of LIM proteins during pollination as actin-binding proteins, modulating cytoskeletal structures and actin networks, and their consequent importance in seed production.

  12. Is sarcomere lattice geometry optimal? Analysis of several potential virtual polygon cross-sectional patterns for actin and myosin myofilaments in muscle.

    Science.gov (United States)

    Kepner, Gordon R

    2014-09-01

    The hexagonal arrangement of actin filaments in skeletal muscle is not the fundamental geometrical or functioning myofilament unit. This analysis of several possible sarcomere lattice geometries for the arrangement of the actin and myosin filaments identifies several geometrical constraints that can be compared for their effect on muscle sarcomere functioning and efficiency. Three distinct virtual polygons, with myosins at their vertices and that tessellate the plane, are compared for both centered actin and perimeter actin arrangements. The analysis evaluates the optimal ratio of myosin to actin filaments, the packing density, and the effect on new myofilament formation in muscle hypertrophy for the various lattice geometries. The results support the view that no single measure of geometrical effectiveness can evaluate definitively the efficiency of any particular arrangement of the myofilaments. The analysis provides quantitative measures of several parameters that, taken overall, support the effectiveness of the myofilament arrangement in Nature. It provides a new definition of the fundamental myofilament unit (FMU). It is possible to calculate the number of actin and myosin myofilaments that need to be added to each polygon arrangement of the myofilaments to create a new FMU for that specific geometry. This leads to useful conclusions about the biochemical efficiency involved in where such units arise in the course of muscle hypertrophy. It supports the idea that the evolutionary endpoint for optimizing muscle's force-generating function can be better understood via the concepts of a FMU and the polygon arrangement of the sarcomere lattice geometry. © 2014 Wiley Periodicals, Inc.

  13. Arabidopsis Actin-Depolymerizing Factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics.

    Directory of Open Access Journals (Sweden)

    Katie Porter

    Full Text Available The primary role of Actin-Depolymerizing Factors (ADFs is to sever filamentous actin, generating pointed ends, which in turn are incorporated into newly formed filaments, thus supporting stochastic actin dynamics. Arabidopsis ADF4 was recently shown to be required for the activation of resistance in Arabidopsis following infection with the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst expressing the effector protein AvrPphB. Herein, we demonstrate that the expression of RPS5, the cognate resistance protein of AvrPphB, was dramatically reduced in the adf4 mutant, suggesting a link between actin cytoskeletal dynamics and the transcriptional regulation of R-protein activation. By examining the PTI (PAMP Triggered Immunity response in the adf4 mutant when challenged with Pst expressing AvrPphB, we observed a significant reduction in the expression of the PTI-specific target gene FRK1 (Flg22-Induced Receptor Kinase 1. These data are in agreement with recent observations demonstrating a requirement for RPS5 in PTI-signaling in the presence of AvrPphB. Furthermore, MAPK (Mitogen-Activated Protein Kinase-signaling was significantly reduced in the adf4 mutant, while no such reduction was observed in the rps5-1 point mutation under similar conditions. Isoelectric focusing confirmed phosphorylation of ADF4 at serine-6, and additional in planta analyses of ADF4's role in immune signaling demonstrates that nuclear localization is phosphorylation independent, while localization to the actin cytoskeleton is linked to ADF4 phosphorylation. Taken together, these data suggest a novel role for ADF4 in controlling gene-for-gene resistance activation, as well as MAPK-signaling, via the coordinated regulation of actin cytoskeletal dynamics and R-gene transcription.

  14. Chorein Sensitivity of Actin Polymerization, Cell Shape and Mechanical Stiffness of Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ioana Alesutan

    2013-09-01

    Full Text Available Background/Aims: Endothelial cell stiffness plays a key role in endothelium-dependent control of vascular tone and arterial blood pressure. Actin polymerization and distribution of microfilaments is essential for mechanical cell stiffness. Chorein, a protein encoded by the VPS13A gene, defective in chorea-acanthocytosis (ChAc, is involved in neuronal cell survival as well as cortical actin polymerization of erythrocytes and blood platelets. Chorein is expressed in a wide variety of further cells, yet nothing is known about the impact of chorein on cells other than neurons, erythrocytes and platelets. The present study explored whether chorein is expressed in human umbilical vein endothelial cells (HUVECs and addressed the putative role of chorein in the regulation of cytoskeletal architecture, stiffness and survival of those cells. Methods: In HUVECs with or without silencing of the VPS13A gene, VPS13A mRNA expression was determined utilizing quantitative RT-PCR, cytoskeletal organization visualized by confocal microscopy, G/F actin ratio and phosphorylation status of focal adhesion kinase quantified by western blotting, cell death determined by flow cytometry, mechanical properties studied by atomic force microscopy (AFM and cell morphology analysed by scanning ion conductance microscopy (SICM. Results: VPS13A mRNA expression was detectable in HUVECs. Silencing of the VPS13A gene attenuated the filamentous actin network, decreased the ratio of soluble G-actin over filamentous F-actin, reduced cell stiffness and changed cell morphology as compared to HUVECs silenced with negative control siRNA. These effects were paralleled by a significant decrease in FAK phosphorylation following VPS13A silencing. Moreover, silencing of the VPS13A gene increased caspase 3 activity and induced necrosis in HUVECs. Conclusions: Chorein is a novel regulator of cytoskeletal architecture, cell shape, mechanical stiffness and survival of vascular endothelial cells.

  15. Direct Interaction of Chivosazole F with Actin Elicits Cell Responses Similar to Latrunculin A but Distinct from Chondramide.

    Science.gov (United States)

    Filipuzzi, Ireos; Thomas, Jason Ray; Pries, Verena; Estoppey, David; Salcius, Michael; Studer, Christian; Schirle, Markus; Hoepfner, Dominic

    2017-09-15

    The microbial metabolite Chivosazole F has been described to affect the cytoskeleton and to inhibit actin polymerization in vitro. Applying orthogonal genomic and proteomics approaches, we now show for the first time that Chivosazole F exerts its effect by directly interacting with actin and demonstrate the cellular impact of Chivosazole F in an unbiased, genome-wide context in yeast and in mammalian cells. Furthermore, mutation-based resistance mapping identifies two SNPs located in the putative Chivosazole F binding site of actin. Comparing chemogenomic profiles and responses to the Chivosazole F-resistant SNPs shows a partially conserved mechanism of action for Chivosazole F and Latrunculin A, but clear divergence from Chondramide. In addition, C14orf80 is an evolutionarily highly conserved ORF, lacking any functional annotation. As editing of C14orf80 leads to Chivosazole F hyper-resistance, we propose a function for this gene product in counteracting perturbation of actin filaments.

  16. Erythrocyte membrane fractions contain free barbed filament ends despite sufficient concentrations of retained capper(s) to prevent barbed end growth.

    Science.gov (United States)

    DiNubile, M J

    1999-01-01

    Many cellular functions depend on rapid cytoskeletal rearrangements localized to specific cytoplasmic domains. Tight regulation of the submembranous microfilament network is accomplished in large part in erythrocytes and granulocytes by actin binding proteins that cap the fast-growing barbed filament ends. Study of this dynamic system is necessarily hampered by the confounding perturbations of cell lysis and dilution. In this paper, we characterize the functional properties of the membrane-associated spectrin-actin complex from human erythrocytes as it exists after hypotonic lysis. Purified spectrin-actin "seeds" extracted from erythrocyte membranes effectively nucleated actin elongation from their barbed ends. However, polymerization from spectrin-actin complexes associated with the membrane fraction prematurely slowed despite the presence of G-actin in great excess of the critical monomer concentration. The addition of cytochalasin B decreased (rather than augmented) the slowing of elongation attributable to the membrane fraction, indicating that capping of barbed filament ends (not monomer sequestration) was the major mechanism underlying this effect. The paradoxical implication of our findings is that, despite the presence of excess capper(s) in the membrane fraction, the membrane-associated spectrin-actin seeds were not capped until after dilution into physiological ionic strength buffer containing monomeric actin. Furthermore, by comparing the degrees of contamination of the extracted and membrane-associated spectrin-actin preparations, it appeared that recognized capping proteins (including gelsolin and capping protein beta2) were not the predominant cappers found in the membrane pellet after hypotonic lysis. We hypothesize that the barbed ends of membrane-associated spectrin-actin complexes, while not excluding actin monomers, may be selectively inaccessible to certain cappers (perhaps simply as the result of steric hindrance). Growth from such complexes in

  17. Chaperonin filaments: The archael cytoskeleton

    Energy Technology Data Exchange (ETDEWEB)

    Trent, J.D.; Kagawa, H.K.; Yaoi, Takuro; Olle, E.; Zaluzec, N.J.

    1997-08-01

    Chaperonins are multi-subunit double-ring complexed composed of 60-kDa proteins that are believed to mediate protein folding in vivo. The chaperonins in the hyperthermophilic archaeon Sulfolobus shibatae are composed of the organism`s two most abundant proteins, which represent 4% of its total protein and have an intracellular concentration of {ge} 3.0 mg/ml. At concentrations of 1.0 mg/ml, purified chaperonin proteins aggregate to form ordered filaments. Filament formation, which requires Mg{sup ++} and nucleotide binding (not hydrolysis), occurs at physiological temperatures under conditions suggesting filaments may exist in vivo. If the estimated 4,600 chaperonins per cell, formed filaments in vivo, they could create a matrix of filaments that would span the diameter of an average S. shibatae cell 100 times. Direct observations of unfixed, minimally treated cells by intermediate voltage electron microscopy (300 kV) revealed an intracellular network of filaments that resembles chaperonin filaments produced in vitro. The hypothesis that the intracellular network contains chaperonins is supported by immunogold analyses. The authors propose that chaperonin activity may be regulated in vivo by filament formation and that chaperonin filaments may serve a cytoskeleton-like function in archaea and perhaps in other prokaryotes.

  18. The effect of Cytochalasin D on F-Actin behavior of single-cell electroendocytosis using multi-chamber micro cell chip

    KAUST Repository

    Lin, Ran

    2012-03-01

    Electroendocytosis (EED) is a pulsed-electric-field (PEF) induced endocytosis, facilitating cells uptake molecules through nanometer-sized EED vesicles. We herein investigate the effect of a chemical inhibitor, Cytochalasin D (CD) on the actin-filaments (F-Actin) behavior of single-cell EED. The CD concentration (C CD) can control the depolymerization of F-actin. A multi-chamber micro cell chip was fabricated to study the EED under different conditions. Large-scale single-cell data demonstrated EED highly depends on both electric field and C CD. © 2012 IEEE.

  19. Cytoskeleton in motion: the dynamics of keratin intermediate filaments in epithelia

    Science.gov (United States)

    Windoffer, Reinhard; Beil, Michael; Magin, Thomas M.

    2011-01-01

    Epithelia are exposed to multiple forms of stress. Keratin intermediate filaments are abundant in epithelia and form cytoskeletal networks that contribute to cell type–specific functions, such as adhesion, migration, and metabolism. A perpetual keratin filament turnover cycle supports these functions. This multistep process keeps the cytoskeleton in motion, facilitating rapid and protein biosynthesis–independent network remodeling while maintaining an intact network. The current challenge is to unravel the molecular mechanisms underlying the regulation of the keratin cycle in relation to actin and microtubule networks and in the context of epithelial tissue function. PMID:21893596

  20. Disturbances in the positioning, proliferation and apoptosis of neural progenitors contribute to subcortical band heterotopia formation.

    Science.gov (United States)

    Fitzgerald, M P; Covio, M; Lee, K S

    2011-03-10

    Cortical malformations are commonly associated with intractable epilepsy and other developmental disorders. Our studies utilize the tish rat, a spontaneously occurring genetic model of subcortical band heterotopia (SBH) associated with epilepsy, to evaluate the developmental events underlying SBH formation in the neocortex. Our results demonstrate that Pax6(+) and Tbr2(+) progenitors are mislocalized in tish(+/-) and tish(-/-)- neocortex throughout neurogenesis. In addition, mislocalized tish(-/-) progenitors possess a longer cell cycle than wild type or normally-positioned tish(-/-) progenitors, owing to a lengthened G(2)+M+G(1) time. This mislocalization is not associated with adherens junction breakdown or loss of radial glial polarity in the ventricular zone (VZ), as assessed by immunohistochemistry against phalloidin (to identify F-actin), aPKC-λ and Par3. However, vimentin immunohistochemistry indicates that the radial glial scaffold is disrupted in the region of the tish(-/-) heterotopia. Moreover, lineage tracing experiments using in utero electroporation in tish(-/-) neocortex demonstrate that mislocalized progenitors do not retain contact with the ventricular surface and that ventricular/subventricular zone (SVZ) progenitors produce neurons that migrate into both the heterotopia and cortical plate (CP). Taken together, these findings define a series of developmental errors contributing to SBH formation that differs fundamentally from a primary error in neuronal migration. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  1. The circular F-actin bundles provide a track for turnaround and bidirectional movement of mitochondria in Arabidopsis root hair.

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    Full Text Available The movement of organelles in root hairs primarily occurs along the actin cytoskeleton. Circulation and "reverse fountain" cytoplasmic streaming constitute the typical forms by which most organelles (such as mitochondria and the Golgi apparatus in plant root hair cells engage in bidirectional movement. However, there remains a lack of in-depth research regarding the relationship between the distribution of the actin cytoskeleton and turnaround organelle movement in plant root hair cells.In this paper, Arabidopsis seedlings that had been stably transformed with a GFP-ABD2-GFP (green fluorescent protein-actin-binding domain 2-green fluorescent protein construct were utilized to study the distribution of bundles of filamentous (F-actin and the directed motion of mitochondria along these bundles in root hairs. Observations with a confocal laser scanning microscope revealed that there were widespread circular F-actin bundles in the epidermal cells and root hairs of Arabidopsis roots. In root hairs, these circular bundles primarily start at the sub-apical region, which is the location where the turnaround movement of organelles occurs. MitoTracker probes were used to label mitochondria, and the dynamic observation of root hair cells with a confocal laser scanning microscope indicated that turnaround mitochondrial movement occurred along circular F-actin bundles.Relevant experimental results demonstrated that the circular F-actin bundles provide a track for the turnaround and bidirectional movement of mitochondria.

  2. ADF/cofilin is not essential but is critically important for actin activities during phagocytosis in Tetrahymena thermophila.

    Science.gov (United States)

    Shiozaki, Nanami; Nakano, Kentaro; Kushida, Yasuharu; Noguchi, Taro Q P; Uyeda, Taro Q P; Wloga, Dorota; Dave, Drashti; Vasudevan, Krishna Kumar; Gaertig, Jacek; Numata, Osamu

    2013-08-01

    ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.

  3. Design, synthesis, and biological evaluation of simplified side chain hybrids of the potent actin binding polyketides rhizopodin and bistramide.

    Science.gov (United States)

    Herkommer, Daniel; Dreisigacker, Sandra; Sergeev, Galina; Sasse, Florenz; Gohlke, Holger; Menche, Dirk

    2015-03-01

    The natural products rhizopodin and bistramide belong to an elite class of highly potent actin binding agents. They show powerful antiproliferative activities against a range of tumor cell lines, with IC50 values in the low-nanomolar range. At the molecular level they disrupt the actin cytoskeleton by binding specifically to a few critical sites of G-actin, resulting in actin filament stabilization. The important biological properties of rhizopodin and bistramide, coupled with their unique and intriguing molecular architectures, render them attractive compounds for further development. However, this is severely hampered by the structural complexity of these metabolites. We initiated an interdisciplinary approach at the interface between molecular modeling, organic synthesis, and chemical biology to support further biological applications. We also wanted to expand structure-activity relationship studies with the goal of accessing simplified analogues with potent biological properties. We report computational analyses of actin-inhibitor interactions involving molecular docking, validated on known actin binding ligands, that show a close match between the crystal and modeled structures. Based on these results, the ligand shape was simplified, and more readily accessible rhizopodin-bistramide mimetics were designed. A flexible and modular strategy was applied for the synthesis of these compounds, enabling diverse access to dramatically simplified rhizopodin-bistramide hybrids. This novel analogue class was analyzed for its antiproliferative and actin binding properties. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Nuclear Actin in Development and Transcriptional Reprogramming.

    Science.gov (United States)

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin's roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation.

  5. Mutations to the formin homology 2 domain of INF2 protein have unexpected effects on actin polymerization and severing.

    Science.gov (United States)

    Ramabhadran, Vinay; Gurel, Pinar S; Higgs, Henry N

    2012-10-05

    INF2 (inverted formin 2) is a formin protein with unusual biochemical characteristics. As with other formins, the formin homology 2 (FH2) domain of INF2 accelerates actin filament assembly and remains at the barbed end, modulating elongation. The unique feature of INF2 is its ability to sever filaments and enhance depolymerization, which requires the C-terminal region. Physiologically, INF2 acts in the secretory pathway and is mutated in two human diseases, focal and segmental glomerulosclerosis and Charcot-Marie-Tooth disease. In this study, we investigate the effects of mutating two FH2 residues found to be key in other formins: Ile-643 and Lys-792. Surprisingly, neither mutation abolishes barbed end binding, as judged by pyrene-actin and total internal reflection (TIRF) microscopy elongation assays. The I643A mutation causes tight capping of a subset of filaments, whereas K792A causes slow elongation of all filaments. The I643A mutation has a minor inhibitory effect on polymerization activity but causes almost complete abolition of severing and depolymerization activity. The K792A mutation has relatively small effects on polymerization, severing, and depolymerization. In cells, the K792A mutant causes actin accumulation around the endoplasmic reticulum to a similar extent as wild type, whereas the I643A mutant causes no measurable polymerization. The inability of I643A to induce actin polymerization in cells is explained by its inability to promote robust actin polymerization in the presence of capping protein. These results highlight an important point: it is dangerous to assume that mutation of conserved FH2 residues will have equivalent effects in all formins. The work also suggests that both mutations have effects on the mechanism of processive elongation.

  6. Mutations to the Formin Homology 2 Domain of INF2 Protein Have Unexpected Effects on Actin Polymerization and Severing*

    Science.gov (United States)

    Ramabhadran, Vinay; Gurel, Pinar S.; Higgs, Henry N.

    2012-01-01

    INF2 (inverted formin 2) is a formin protein with unusual biochemical characteristics. As with other formins, the formin homology 2 (FH2) domain of INF2 accelerates actin filament assembly and remains at the barbed end, modulating elongation. The unique feature of INF2 is its ability to sever filaments and enhance depolymerization, which requires the C-terminal region. Physiologically, INF2 acts in the secretory pathway and is mutated in two human diseases, focal and segmental glomerulosclerosis and Charcot-Marie-Tooth disease. In this study, we investigate the effects of mutating two FH2 residues found to be key in other formins: Ile-643 and Lys-792. Surprisingly, neither mutation abolishes barbed end binding, as judged by pyrene-actin and total internal reflection (TIRF) microscopy elongation assays. The I643A mutation causes tight capping of a subset of filaments, whereas K792A causes slow elongation of all filaments. The I643A mutation has a minor inhibitory effect on polymerization activity but causes almost complete abolition of severing and depolymerization activity. The K792A mutation has relatively small effects on polymerization, severing, and depolymerization. In cells, the K792A mutant causes actin accumulation around the endoplasmic reticulum to a similar extent as wild type, whereas the I643A mutant causes no measurable polymerization. The inability of I643A to induce actin polymerization in cells is explained by its inability to promote robust actin polymerization in the presence of capping protein. These results highlight an important point: it is dangerous to assume that mutation of conserved FH2 residues will have equivalent effects in all formins. The work also suggests that both mutations have effects on the mechanism of processive elongation. PMID:22879592

  7. On the mechanisms of cortical actin flow and its role in cytoskeletal organisation of fibroblasts.

    Science.gov (United States)

    Heath, J P; Holifield, B F

    1993-01-01

    In this review we discuss the organization of F-actin in motile fibroblasts in relation to the phenomenon of cortical actin flow; we review some of the mechanisms proposed to drive this process and then relate some of our new findings on the interactions of cortical flow and substratum adhesions in fibroblasts. It is our thesis that the centripetal flow of F-actin through the lamellipodium and leading lamella is the major determining factor organising the polarized stress fiber system and associated cell-substratum adhesions in crawling fibroblastic cells. The broad flattened region of cytoplasm anterior to the nucleus, called the leading lamella, is fringed with much thinner structures, the lamellipodia, which are the primary protrusive organelles of motile cells. Lamellipodia are filled with a criss-crossed network of actin filaments interspersed with small bundles or ribs. In the leading lamella, the actin cytoskeleton is largely confined to two cortical layers beneath the dorsal and ventral cell surfaces. The ventral cortex is engaged in cell adhesion; the dorsal cortex is made up of a circumferentially orientated sheet of actin filaments and bundles which we team the dorsal cortical microfilament sheath (DCMS). Stress fibers insert into the ventral adhesions and pass back through the lamella rising up to meet the DCMS. Cortical flow appears to be a constitutive process in most types of cells when they become motile. In fibroblasts a continuous centripetal flux of structure is seen flowing through the lamellipodium and into the more central regions of the lamella at approximately 0.1 micron per second. Some of the structures engaged in the flux pass back and merge into the DCMS which is also moving rearward at a slower rate of 1 to 5 microns per minute. We find that the formation of a stress fibre is a direct consequence of cortical flow. Initially, stress fibre formation involves the establishment of a focal adhesion between an F-actin bundle in the

  8. Phonemic Characteristics of Apraxia of Speech Resulting from Subcortical Hemorrhage

    Science.gov (United States)

    Peach, Richard K.; Tonkovich, John D.

    2004-01-01

    Reports describing subcortical apraxia of speech (AOS) have received little consideration in the development of recent speech processing models because the speech characteristics of patients with this diagnosis have not been described precisely. We describe a case of AOS with aphasia secondary to basal ganglia hemorrhage. Speech-language symptoms…

  9. Common genetic variants influence human subcortical brain structures

    NARCIS (Netherlands)

    Hibar, Derrek P.; Stein, Jason L.; Renteria, Miguel E.; Arias-Vasquez, Alejandro; Desrivieres, Sylvane; Jahanshad, Neda; Toro, Roberto; Wittfeld, Katharina; Abramovic, Lucija; Andersson, Micael; Aribisala, Benjamin S.; Armstrong, Nicola J.; Bernard, Manon; Bohlken, Marc M.; Boks, Marco P.; Bralten, Janita; Brown, Andrew A.; Chakravarty, M. Mallar; Chen, Qiang; Ching, Christopher R. K.; Cuellar-Partida, Gabriel; den Braber, Anouk; Giddaluru, Sudheer; Goldman, Aaron L.; Grimm, Oliver; Guadalupe, Tulio; Hass, Johanna; Woldehawariat, Girma; Holmes, Avram J.; Hoogman, Martine; Janowitz, Deborah; Jia, Tianye; Kim, Sungeun; Klein, Marieke; Kraemer, Bernd; Lee, Phil H.; Loohuis, Loes M. Olde; Luciano, Michelle; Macare, Christine; Mather, Karen A.; Mattheisen, Manuel; Milaneschi, Yuri; Nho, Kwangsik; Papmeyer, Martina; Ramasamy, Adaikalavan; Risacher, Shannon L.; Roiz-Santianez, Roberto; Rose, Emma J.; Salami, Alireza; Saemann, Philipp G.; Schmaal, Lianne; Schork, Andrew J.; Shin, Jean; Strike, Lachlan T.; Teumer, Alexander; van Donkelaar, Marjolein M. J.; van Eijk, Kristel R.; Walters, Raymond K.; Westlye, Lars T.; Whelan, Christopher D.; Winkler, Anderson M.; Zwiers, Marcel P.; Alhusaini, Saud; Athanasiu, Lavinia; Ehrlich, Stefan; Hakobjan, Marina M. H.; Hartberg, Cecilie B.; Haukvik, Unn K.; Heister, Angelien J. G. A. M.; Hoehn, David; Kasperaviciute, Dalia; Liewald, David C. M.; Lopez, Lorna M.; Makkinje, Remco R. R.; Matarin, Mar; Naber, Marlies A. M.; McKay, D. Reese; Needham, Margaret; Nugent, Allison C.; Puetz, Benno; Royle, Natalie A.; Shen, Li; Sprooten, Emma; Trabzuni, Daniah; van der Marel, Saskia S. L.; van Hulzen, Kimm J. E.; Walton, Esther; Wolf, Christiane; Almasy, Laura; Ames, David; Arepalli, Sampath; Assareh, Amelia A.; Bastin, Mark E.; Brodaty, Henry; Bulayeva, Kazima B.; Carless, Melanie A.; Cichon, Sven; Corvin, Aiden; Curran, Joanne E.; Czisch, Michael; de Zubicaray, Greig I.; Dillman, Allissa; Duggirala, Ravi; Dyer, Thomas D.; Erk, Susanne; Fedko, Iryna O.; Ferrucci, Luigi; Foroud, Tatiana M.; Fox, Peter T.; Fukunaga, Masaki; Gibbs, J. Raphael; Goering, Harald H. H.; Green, Robert C.; Guelfi, Sebastian; Hansell, Narelle K.; Hartman, Catharina A.; Hegenscheid, Katrin; Heinz, Andreas; Hernandez, Dena G.; Heslenfeld, Dirk J.; Hoekstra, Pieter J.; Holsboer, Florian; Homuth, Georg; Hottenga, Jouke-Jan; Ikeda, Masashi; Jack, Clifford R.; Jenkinson, Mark; Johnson, Robert; Kanai, Ryota; Keil, Maria; Kent, Jack W.; Kochunov, Peter; Kwok, John B.; Lawrie, Stephen M.; Liu, Xinmin; Longo, Dan L.; McMahon, Katie L.; Meisenzah, Eva; Melle, Ingrid; Mahnke, Sebastian; Montgomery, Grant W.; Mostert, Jeanette C.; Muehleisen, Thomas W.; Nalls, Michael A.; Nichols, Thomas E.; Nilsson, Lars G.; Noethen, Markus M.; Ohi, Kazutaka; Olvera, Rene L.; Perez-Iglesias, Rocio; Pike, G. Bruce; Potkin, Steven G.; Reinvang, Ivar; Reppermund, Simone; Rietschel, Marcella; Romanczuk-Seiferth, Nina; Rosen, Glenn D.; Rujescu, Dan; Schnell, Knut; Schofield, Peter R.; Smith, Colin; Steen, Vidar M.; Sussmann, Jessika E.; Thalamuthu, Anbupalam; Toga, Arthur W.; Traynor, Bryan J.; Troncoso, Juan; Turner, Jessica A.; Valdes Hernandez, Maria C.; van't Ent, Dennis; van der Brug, Marcel; van der Wee, Nic J. A.; van Tol, Marie-Jose; Veltman, Dick J.; Wassink, Thomas H.; Westman, Eric; Zielke, Ronald H.; Zonderman, Alan B.; Ashbrook, David G.; Hager, Reinmar; Lu, Lu; McMahon, Francis J.; Morris, Derek W.; Williams, Robert W.; Brunner, Han G.; Buckner, Randy L.; Buitelaar, Jan K.; Cahn, Wiepke; Calhoun, Vince D.; Cavalleri, Gianpiero L.; Crespo-Facorro, Benedicto; Dale, Anders M.; Davies, Gareth E.; Delanty, Norman; Depondt, Chantal; Djurovic, Srdjan; Drevets, Wayne C.; Espeseth, Thomas; Gollub, Randy L.; Ho, Beng-Choon; Hoffman, Wolfgang; Hosten, Norbert; Kahn, Rene S.; Le Hellard, Stephanie; Meyer-Lindenberg, Andreas; Mueller-Myhsok, Bertram; Nauck, Matthias; Nyberg, Lars; Pandolfo, Massimo; Penninx, Brenda W. J. H.; Roffman, Joshua L.; Sisodiya, Sanjay M.; Smoller, Jordan W.; van Bokhoven, Hans; van Haren, Neeltje E. M.; Voelzke, Henry; Walter, Henrik; Weiner, Michael W.; Wen, Wei; White, Tonya; Agartz, Ingrid; Andreassen, Ole A.; Blangero, John; Boomsma, Dorret I.; Brouwer, Rachel M.; Cannon, Dara M.; Cookson, Mark R.; de Geus, Eco J. C.; Deary, Ian J.; Donohoe, Gary; Fernandez, Guillen; Fisher, Simon E.; Francks, Clyde; Glahn, David C.; Grabe, Hans J.; Gruber, Oliver; Hardy, John; Hashimoto, Ryota; Pol, Hilleke E. Hulshoff; Joensson, Erik G.; Kloszewska, Iwona; Lovestone, Simon; Mattay, Venkata S.; Mecocci, Patrizia; McDonald, Colm; McIntosh, Andrew M.; Ophoff, Roel A.; Paus, Tomas; Pausova, Zdenka; Ryten, Mina; Sachdev, Perminder S.; Saykin, Andrew J.; Simmons, Andy; Singleton, Andrew; Soininen, Hilkka; Wardlaw, Joanna M.; Weale, Michael E.; Weinberger, Daniel R.; Adams, Hieab H. H.; Launer, Lenore J.; Seiler, Stephan; Schmidt, Reinhold; Chauhan, Ganesh; Satizabal, Claudia L.; Becker, James T.; Yanek, Lisa; van der Lee, Sven J.; Ebling, Maritza; Fischl, Bruce; Longstreth, W. T.; Greve, Douglas; Schmidt, Helena; Nyquist, Paul; Vinke, Louis N.; van Duijn, Cornelia M.; Xue, Luting; Mazoyer, Bernard; Bis, Joshua C.; Gudnason, Vilmundur; Seshadri, Sudha; Ikram, M. Arfan; Martin, Nicholas G.; Wright, Margaret J.; Schumann, Gunter; Franke, Barbara; Thompson, Paul M.; Medland, Sarah E.

    2015-01-01

    The highly complex structure of the human brain is strongly shaped by genetic influences(1). Subcortical brain regions form circuits with cortical areas to coordinate movement(2), learning, memory(3) and motivation(4), and altered circuits can lead to abnormal behaviour and disease(5). To

  10. Common genetic variants influence human subcortical brain structures

    NARCIS (Netherlands)

    D.P. Hibar (Derrek); J.L. Stein; M.E. Rentería (Miguel); A. Arias-Vásquez (Alejandro); S. Desrivières (Sylvane); N. Jahanshad (Neda); R. Toro (Roberto); K. Wittfeld (Katharina); L. Abramovic (Lucija); M. Andersson (Micael); B. Aribisala (Benjamin); N.J. Armstrong (Nicola J.); M. Bernard (Manon); M.M. Bohlken (Marc M.); M.P.M. Boks (Marco); L.B.C. Bralten (Linda); A.A. Brown (Andrew); M.M. Chakravarty (M. Mallar); Q. Chen (Qiang); C.R.K. Ching (Christopher); G. Cuellar-Partida (Gabriel); A. den Braber (Anouk); S. Giddaluru (Sudheer); A.L. Goldman (Aaron L.); O. Grimm (Oliver); T. Guadalupe (Tulio); J. Hass (Johanna); G. Woldehawariat (Girma); A.J. Holmes (Avram); M. Hoogman (Martine); D. Janowitz (Deborah); T. Jia (Tianye); S. Kim (Shinseog); M. Klein (Marieke); B. Kraemer (Bernd); P.H. Lee (Phil H.); L.M. Olde Loohuis (Loes M.); M. Luciano (Michelle); C. MacAre (Christine); R. Mather; M. Mattheisen (Manuel); Y. Milaneschi (Yuri); K. Nho (Kwangsik); M. Papmeyer (Martina); A. Ramasamy (Adaikalavan); S.L. Risacher (Shannon); R. Roiz-Santiañez (Roberto); E.J. Rose (Emma); A. Salami (Alireza); P.G. Sämann (Philipp); L. Schmaal (Lianne); N.J. Schork (Nicholas); J. Shin (Jean); V.M. Strike (Vanessa); A. Teumer (Alexander); M.M.J. Van Donkelaar (Marjolein M. J.); K.R. van Eijk (Kristel); R.K. Walters (Raymond); L.T. Westlye (Lars); C.D. Whelan (Christopher); A.M. Winkler (Anderson); M.P. Zwiers (Marcel); S. Alhusaini (Saud); L. Athanasiu (Lavinia); S.M. Ehrlich (Stefan); M. Hakobjan (Marina); C.B. Hartberg (Cecilie B.); U.K. Haukvik (Unn); A.J.G.A.M. Heister (Angelien J. G. A. M.); D. Hoehn (David); D. Kasperaviciute (Dalia); D.C. Liewald (David C.); L.M. Lopez (Lorna); R.R.R. Makkinje (Remco R. R.); M. Matarin (Mar); M.A.M. Naber (Marlies A. M.); D. Reese McKay; M. Needham (Margaret); A.C. Nugent (Allison); B. Pütz (Benno); N.A. Royle (Natalie); L. Shen (Li); R. Sprooten (Roy); D. Trabzuni (Danyah); S.S.L. Van Der Marel (Saskia S. L.); K.J.E. Van Hulzen (Kimm J. E.); E. Walton (Esther); A. Björnsson (Asgeir); L. Almasy (Laura); D.J. Ames (David); S. Arepalli (Sampath); A.A. Assareh; M.E. Bastin (Mark); H. Brodaty (Henry); K. Bulayeva (Kazima); M.A. Carless (Melanie); S. Cichon (Sven); A. Corvin (Aiden); J.E. Curran (Joanne); M. Czisch (Michael); G.I. de Zubicaray (Greig); A. Dillman (Allissa); A. Duggirala (Aparna); M.D. Dyer (Matthew); S. Erk; I. Fedko (Iryna); L. Ferrucci (Luigi); T. Foroud (Tatiana); P.T. Fox (Peter); M. Fukunaga (Masaki); J. Raphael Gibbs; H.H.H. Göring (Harald H.); R.C. Green (Robert C.); S. Guelfi (Sebastian); N.K. Hansell (Narelle); C.A. Hartman (Catharina); K. Hegenscheid (Katrin); J. Heinz (Judith); D.G. Hernandez (Dena); D.J. Heslenfeld (Dirk); P.J. Hoekstra (Pieter); F. Holsboer; G. Homuth (Georg); J.J. Hottenga (Jouke Jan); M. Ikeda (Masashi); C.R. Jack Jr. (Clifford); S. Jenkinson (Sarah); R. Johnson (Robert); R. Kanai (Ryota); M. Keil (Maria); J.W. Kent (Jack W.); P. Kochunov (Peter); J.B. Kwok (John B.); S. Lawrie (Stephen); X. Liu (Xinmin); D.L. Longo (Dan L.); K.L. Mcmahon (Katie); E. Meisenzahl (Eva); I. Melle (Ingrid); S. Mohnke (Sebastian); G.W. Montgomery (Grant); J.C. Mostert (Jeanette C.); T.W. Mühleisen (Thomas); M.A. Nalls (Michael); T.E. Nichols (Thomas); L.G. Nilsson; M.M. Nöthen (Markus); K. Ohi (Kazutaka); R.L. Olvera (Rene); R. Perez-Iglesias (Rocio); G. Bruce Pike; S.G. Potkin (Steven); I. Reinvang (Ivar); S. Reppermund; M. Rietschel (Marcella); N. Seiferth (Nina); G.D. Rosen (Glenn D.); D. Rujescu (Dan); K. Schnell (Kerry); C.J. Schofield (Christopher); C. Smith (Colin); V.M. Steen (Vidar); J. Sussmann (Jessika); A. Thalamuthu (Anbupalam); A.W. Toga (Arthur W.); B. Traynor (Bryan); J.C. Troncoso (Juan); J. Turner (Jessica); M.C. Valdés Hernández (Maria); D. van 't Ent (Dennis); M.P. van der Brug (Marcel); N.J. van der Wee (Nic); M.J.D. van Tol (Marie-José); D.J. Veltman (Dick); A.M.J. Wassink (Annemarie); E. Westman (Eric); R.H. Zielke (Ronald H.); A.B. Zonderman (Alan B.); D.G. Ashbrook (David G.); R. Hager (Reinmar); L. Lu (Lu); F.J. Mcmahon (Francis J); D.W. Morris (Derek W); R.W. Williams (Robert W.); H.G. Brunner; M. Buckner; J.K. Buitelaar (Jan K.); W. Cahn (Wiepke); V.D. Calhoun Vince D. (V.); G. Cavalleri (Gianpiero); B. Crespo-Facorro (Benedicto); A.M. Dale (Anders); G.E. Davies (Gareth); N. Delanty; C. Depondt (Chantal); S. Djurovic (Srdjan); D.A. Drevets (Douglas); T. Espeseth (Thomas); R.L. Gollub (Randy); B.C. Ho (Beng ); W. Hoffmann (Wolfgang); N. Hosten (Norbert); R. Kahn (René); S. Le Hellard (Stephanie); A. Meyer-Lindenberg; B. Müller-Myhsok (B.); M. Nauck (Matthias); L. Nyberg (Lars); M. Pandolfo (Massimo); B.W.J.H. Penninx (Brenda); J.L. Roffman (Joshua); S.M. Sisodiya (Sanjay); J.W. Smoller; H. van Bokhoven (Hans); N.E.M. van Haren (Neeltje E.); H. Völzke (Henry); H.J. Walter (Henrik); M.W. Weiner (Michael); W. Wen (Wei); T.J.H. White (Tonya); I. Agartz (Ingrid); O.A. Andreassen (Ole); J. Blangero (John); D.I. Boomsma (Dorret); R.M. Brouwer (Rachel); D.M. Cannon (Dara); M.R. Cookson (Mark); E.J.C. de Geus (Eco); I.J. Deary (Ian J.); D.J. Donohoe (Dennis); G. Fernandez (Guillén); S.E. Fisher (Simon); C. Francks (Clyde); D.C. Glahn (David); H.J. Grabe (Hans Jörgen); O. Gruber (Oliver); J. Hardy (John); R. Hashimoto (Ryota); H.E. Hulshoff Pol (Hilleke); E.G. Jönsson (Erik); I. Kloszewska (Iwona); S. Lovestone (Simon); V.S. Mattay (Venkata S.); P. Mecocci (Patrizia); C. McDonald (Colm); A.M. McIntosh (Andrew); R.A. Ophoff (Roel); T. Paus (Tomas); Z. Pausova (Zdenka); M. Ryten (Mina); P.S. Sachdev (Perminder); A.J. Saykin (Andrew); A. Simmons (Andrew); A. Singleton (Andrew); H. Soininen (H.); J.M. Wardlaw (J.); M.E. Weale (Michael); D.R. Weinberger (Daniel); H.H.H. Adams (Hieab); L.J. Launer (Lenore); S. Seiler (Stephan); R. Schmidt (Reinhold); G. Chauhan (Ganesh); C.L. Satizabal (Claudia L.); J.T. Becker (James); L.R. Yanek (Lisa); S.J. van der Lee (Sven); M. Ebling (Maritza); B. Fischl (Bruce); W.T. Longstreth Jr; D. Greve (Douglas); R. Schmidt (Reinhold); P. Nyquist (Paul); L.N. Vinke (Louis N.); C.M. van Duijn (Cornelia); L. Xue (Luting); B. Mazoyer (Bernard); J.C. Bis (Joshua); V. Gudnason (Vilmundur); S. Seshadri (Sudha); M.A. Ikram (Arfan); N.G. Martin (Nicholas); M.J. Wright (Margaret); G. Schumann (Gunter); B. Franke (Barbara); P.M. Thompson (Paul); S.E. Medland (Sarah Elizabeth)

    2015-01-01

    textabstractThe highly complex structure of the human brain is strongly shaped by genetic influences. Subcortical brain regions form circuits with cortical areas to coordinate movement, learning, memory and motivation, and altered circuits can lead to abnormal behaviour and disease. To investigate

  11. Automated localization of periventricular and subcortical white matter lesions

    Science.gov (United States)

    van der Lijn, Fedde; Vernooij, Meike W.; Ikram, M. Arfan; Vrooman, Henri A.; Rueckert, Daniel; Hammers, Alexander; Breteler, Monique M. B.; Niessen, Wiro J.

    2007-03-01

    It is still unclear whether periventricular and subcortical white matter lesions (WMLs) differ in etiology or clinical consequences. Studies addressing this issue would benefit from automated segmentation and localization of WMLs. Several papers have been published on WML segmentation in MR images. Automated localization however, has not been investigated as much. This work presents and evaluates a novel method to label segmented WMLs as periventricular and subcortical. The proposed technique combines tissue classification and registration-based segmentation to outline the ventricles in MRI brain data. The segmented lesions can then be labeled into periventricular WMLs and subcortical WMLs by applying region growing and morphological operations. The technique was tested on scans of 20 elderly subjects in which neuro-anatomy experts manually segmented WMLs. Localization accuracy was evaluated by comparing the results of the automated method with a manual localization. Similarity indices and volumetric intraclass correlations between the automated and the manual localization were 0.89 and 0.95 for periventricular WMLs and 0.64 and 0.89 for subcortical WMLs, respectively. We conclude that this automated method for WML localization performs well to excellent in comparison to the gold standard.

  12. Neuropsychological Profile of Children with Subcortical Band Heterotopia

    Science.gov (United States)

    Spencer-Smith, Megan; Leventer, Richard; Jacobs, Rani; De Luca, Cinzia; Anderson, Vicki

    2009-01-01

    Aim: Subcortical band heterotopia (SBH) or "double cortex" is a malformation of cortical development resulting from impaired neuronal migration. So far, research has focused on the neurological, neuroimaging, and genetic correlates of SBH. More recently, clinical reports and small sample studies have documented neuropsychological dysfunction in…

  13. Childhood adversity impacts on brain subcortical structures relevant to depression

    NARCIS (Netherlands)

    Frodl, Thomas; Janowitz, Deborah; Schmaal, Lianne; Tozzi, Leonardo; Dobrowolny, Henrik; Stein, Dan J.; Veltman, Dick. J.; Wittfeld, Katharina; van Erp, Theo G. M.; Jahanshad, Neda; Block, Andrea; Hegenscheid, Katrin; Voelzke, Henry; Lagopoulos, Jim; Hatton, Sean N.; Hickie, Ian B.; Frey, Eva Maria; Carballedo, Angela; Brooks, Samantha J; Vuletic, Daniella; Uhlmann, Anne; Veer, Ilya M.; Walter, Henrik; Schnell, Knut; Grotegerd, Dominik; Arolt, Volker; Kugel, Harald; Schramm, Elisabeth; Konrad, Carsten; Zurowski, Bartosz; Baune, Bernhard T; van der Wee, Nic J. A.; van Tol, Marie-Jose; Penninx, Brenda W. J. H.; Thompson, Paul M.; Hibar, Derrek P.; Dannlowski, Udo; Grabe, Hans J.

    Childhood adversity plays an important role for development of major depressive disorder (MDD). There are differences in subcortical brain structures between patients with MDD and healthy controls, but the specific impact of childhood adversity on such structures in MDD remains unclear. Thus, aim of

  14. Arp2/3- and cofilin-coordinated actin dynamics is required for insulin-mediated GLUT4 translocation to the surface of muscle cells.

    Science.gov (United States)

    Chiu, Tim Ting; Patel, Nish; Shaw, Alisa E; Bamburg, James R; Klip, Amira

    2010-10-15

    GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.

  15. The unrelated surface proteins ActA of Listeria monocytogenes and IcsA of Shigella flexneri are sufficient to confer actin-based motility on Listeria innocua and Escherichia coli respectively.

    Science.gov (United States)

    Kocks, C; Marchand, J B; Gouin, E; d'Hauteville, H; Sansonetti, P J; Carlier, M F; Cossart, P

    1995-11-01

    Listeria monocytogenes and Shigella flexneri are two unrelated facultative intracellular pathogens which spread from cell to cell by using a similar mode of intracellular movement based on continuous actin assembly at one pole of the bacterium. This process requires the asymmetrical expression of the ActA surface protein in L. monocytogenes and the IcsA (VirG) surface protein in S. flexneri. ActA and IcsA share no sequence homology. To assess the role of the two proteins in the generation of actin-based movement, we expressed them in the genetic context of two non-actin polymerizing, non-pathogenic bacterial species, Listeria innocua and Escherichia coli. In the absence of any additional bacterial pathogenicity determinants, both proteins induced actin assembly and propulsion of the bacteria in cytoplasmic extracts from Xenopus eggs, as visualized by the formation of characteristic actin comet tails. E. coli expressing IcsA moved about two times faster than Listeria and displayed longer actin tails. However, actin dynamics (actin filament distribution and filament half-lives) were similar in IcsA- and ActA-induced actin tails suggesting that by using unrelated surface molecules, L. monocytogenes and S. flexneri move intracellularly by interacting with the same host cytoskeleton components or by interfering with the same host cell signal transduction pathway.

  16. Shear-induced reorganization of renal proximal tubule cell actin cytoskeleton and apical junctional complexes.

    Science.gov (United States)

    Duan, Yi; Gotoh, Nanami; Yan, Qingshang; Du, Zhaopeng; Weinstein, Alan M; Wang, Tong; Weinbaum, Sheldon

    2008-08-12

    In this study, we demonstrate that fluid shear stress (FSS)-induced actin cytoskeletal reorganization and junctional formation in renal epithelial cells are nearly completely opposite the corresponding changes in vascular endothelial cells (ECs) [Thi MM et al. (2004) Proc Natl Acad Sci USA 101:16483-16488]. Mouse proximal tubule cells (PTCs) were subjected to 5 h of FSS (1 dyn/cm(2)) to investigate the dynamic responses of the cytoskeletal distribution of filamentous actin (F-actin), ZO-1, E-cadherin, vinculin, and paxillin to FSS. Immunofluorescence analysis revealed that FSS caused basal stress fiber disruption, more densely distributed peripheral actin bands (DPABs), and the formation of both tight junctions (TJs) and adherens junctions (AJs). A dramatic reinforcement of vinculin staining was found at the cell borders as well as the cell interior. These responses were abrogated by the actin-disrupting drug, cytochalasin D. To interpret these results, we propose a "junctional buttressing" model for PTCs in which FSS enables the DPABs, TJs, and AJs to become more tightly connected. In contrast, in the "bumper-car" model for ECs, all junctional connections were severely disrupted by FSS. This "junctional buttressing" model explains why a FSS of only 1/10 of that used in the EC study can cause a similarly dramatic, cytoskeletal response in these tall, cuboidal epithelial cells; and why junctional buttressing between adjacent cells may benefit renal epithelium in maximizing flow-activated, brush border-dependent, transcellular salt and water reabsorption.

  17. Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions.

    Science.gov (United States)

    Swaminathan, Vinay; Kalappurakkal, Joseph Mathew; Mehta, Shalin B; Nordenfelt, Pontus; Moore, Travis I; Koga, Nobuyasu; Baker, David A; Oldenbourg, Rudolf; Tani, Tomomi; Mayor, Satyajit; Springer, Timothy A; Waterman, Clare M

    2017-10-03

    Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.

  18. Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

    Science.gov (United States)

    Swaminathan, Vinay; Kalappurakkal, Joseph Mathew; Moore, Travis I.; Koga, Nobuyasu; Baker, David A.; Oldenbourg, Rudolf; Tani, Tomomi; Springer, Timothy A.; Waterman, Clare M.

    2017-01-01

    Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven “retrograde flow” of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues. PMID:29073038

  19. Actin-based mechanism of holospora obtusa trafficking in Paramecium caudatum.

    Science.gov (United States)

    Sabaneyeva, Elena V; Derkacheva, M E; Benken, K A; Fokin, Sergei I; Vainio, Seppo; Skovorodkin, Ilya N

    2009-05-01

    Holospora obtusa, an alpha-proteobacterium, is an obligate endonuclear pathogen of the ciliate Paramecium caudatum. It is engulfed by the host cell in the course of phagocytosis but soon escapes from the phagosome and is transported across the host cell cytoplasm to the paramecium macronucleus. Electron microscopy reveals a comet-like tail resembling that of Listeria trailing after H. obtusa in the host cytoplasm. In this study we investigated the role of the host cell actin and Arp3 in the process of infection with Holospora. Cytochalasin D treatment significantly reduced the rate of nuclear infection. Using immunocytochemistry and experimental infection of GFP-actin-transfected paramecia we demonstrated that the Paramecium actin1-1 took part in the bacterial escape from the phagosome, its trafficking in the cytoplasm and entry into the host macronucleus. Rapid assembly/disassembly of actin filaments in P. caudatum led to quick loss of actin1-1 from the trails left by H. obtusa. Immunocytochemistry using anti-bovine Arp3 antibodies demonstrated the presence of Arp3 in these trails. Our data indicate that details of H. obtusa infection are rather similar to those of Listeria and Rickettsia.

  20. 21 CFR 882.5840 - Implanted intracerebral/subcortical stimulator for pain relief.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Implanted intracerebral/subcortical stimulator for... Devices § 882.5840 Implanted intracerebral/subcortical stimulator for pain relief. (a) Identification. An implanted intracerebral/subcortical stimulator for pain relief is a device that applies electrical current...

  1. Morphologic characteristics of subcortical heterotopia: MR imaging study.

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    Barkovich, A J

    2000-02-01

    Gray matter heterotopia have been divided into three groups based on clinical and imaging characteristics: subependymal, subcortical, and band heterotopia. Nonetheless, subcortical heterotopia can have variable morphologic findings. The purpose of this study was to perform a morphologic analysis of a series of cases of subcortical heterotopia based on MR images, to correlate the morphologic appearance with clinical characteristics, and to speculate about the embryologic implications of our results. The MR imaging studies and clinical records of 24 patients with subcortical heterotopia were retrospectively reviewed. The morphologic findings of the heterotopia were recorded along with presence and type of associated malformations. These results were correlated with available data on development and neurologic status. Analysis revealed that, in six cases, the heterotopia were composed exclusively of multiple nodules, in 13, they appeared primarily as curvilinear ribbons of cortex extending into the white matter, and in five, they had deep nodular regions with curvilinear areas more peripherally. All of the curvilinear regions were contiguous with the cerebral cortex in at least two locations. In eight cases, curvilinear heterotopia contained curvilinear areas of flow void that were thought to be blood vessels; in 10, they contained fluid resembling CSF. No difference in developmental or neurologic manifestations was noted among patients with heterotopia of different morphologic appearances. Subcortical heterotopia can have nodular or curvilinear morphologic appearances. Although no difference was found in the clinical conditions of the patients with differing morphologic appearances, additional analysis of these patients or studies of animal models of these malformations may further our understanding of normal and abnormal brain development.

  2. Two distinct mechanisms for actin capping protein regulation--steric and allosteric inhibition.

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    Takeda, Shuichi; Minakata, Shiho; Koike, Ryotaro; Kawahata, Ichiro; Narita, Akihiro; Kitazawa, Masashi; Ota, Motonori; Yamakuni, Tohru; Maéda, Yuichiro; Nitanai, Yasushi

    2010-07-06

    The actin capping protein (CP) tightly binds to the barbed end of actin filaments, thus playing a key role in actin-based lamellipodial dynamics. V-1 and CARMIL proteins directly bind to CP and inhibit the filament capping activity of CP. V-1 completely inhibits CP from interacting with the barbed end, whereas CARMIL proteins act on the barbed end-bound CP and facilitate its dissociation from the filament (called uncapping activity). Previous studies have revealed the striking functional differences between the two regulators. However, the molecular mechanisms describing how these proteins inhibit CP remains poorly understood. Here we present the crystal structures of CP complexed with V-1 and with peptides derived from the CP-binding motif of CARMIL proteins (CARMIL, CD2AP, and CKIP-1). V-1 directly interacts with the primary actin binding surface of CP, the C-terminal region of the alpha-subunit. Unexpectedly, the structures clearly revealed the conformational flexibility of CP, which can be attributed to a twisting movement between the two domains. CARMIL peptides in an extended conformation interact simultaneously with the two CP domains. In contrast to V-1, the peptides do not directly compete with the barbed end for the binding surface on CP. Biochemical assays revealed that the peptides suppress the interaction between CP and V-1, despite the two inhibitors not competing for the same binding site on CP. Furthermore, a computational analysis using the elastic network model indicates that the interaction of the peptides alters the intrinsic fluctuations of CP. Our results demonstrate that V-1 completely sequesters CP from the barbed end by simple steric hindrance. By contrast, CARMIL proteins allosterically inhibit CP, which appears to be a prerequisite for the uncapping activity. Our data suggest that CARMIL proteins down-regulate CP by affecting its conformational dynamics. This conceptually new mechanism of CP inhibition provides a structural basis for the

  3. Three-dimensional cryo-electron microscopy on intermediate filaments.

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    Kirmse, Robert; Bouchet-Marquis, Cédric; Page, Cynthia; Hoenger, Andreas

    2010-01-01

    Together with microtubules and actin filaments (F-actin), intermediate filaments (IFs) form the cytoskeleton of metazoan cells. However, unlike the other two entities that are extremely conserved, IFs are much more diverse and are grouped into five different families. In contrast to microtubules and F-actin, IFs do not exhibit a polarity, which may be the reason that no molecular motors travel along them. The molecular structure of IFs is less well resolved than that of the other cytoskeletal systems. This is partially due to their functional variability, tissue-specific expression, and their intrinsic structural properties. IFs are composed mostly of relatively smooth protofibrils formed by antiparallel arranged α-helical coiled-coil bundles flanked by small globular domains at either end. These features make them difficult to study by various electron microscopy methods or atomic force microscopy (AFM). Furthermore, the elongated shape of monomeric or dimeric IF units interferes with the formation of highly ordered three-dimensional (3-D) crystals suitable for atomic resolution crystallography. So far, most of the data we currently have on IF macromolecular structures come from electron microscopy of negatively stained samples, and fragmented α-helical coiled-coil units solved by X-ray diffraction. In addition, AFM allows the observation of the dynamic states of IFs in solution and delivers a new view into the assembly properties of IFs. Here, we discuss the applicability of cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) for the field. Both methods are strongly related and have only recently been applied to IFs. However, cryo-EM revealed distinct new features within IFs that have not been seen before, and cryo-ET adds a 3-D view of IFs revealing the path and number of protofilaments within the various IF assemblies. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Molecular Characterization of abLIM, a Novel Actin-binding and Double Zinc Finger Protein

    Science.gov (United States)

    Roof, Dorothy J.; Hayes, Annmarie; Adamian, Michael; Chishti, Athar H.; Li, Tiansen

    1997-01-01

    Molecules that couple the actin-based cytoskeleton to intracellular signaling pathways are central to the processes of cellular morphogenesis and differentiation. We have characterized a novel protein, the actin-binding LIM (abLIM) protein, which could mediate such interactions between actin filaments and cytoplasmic targets. abLIM protein consists of a COOH-terminal cytoskeletal domain that is fused to an NH2-terminal domain consisting of four double zinc finger motifs. The cytoskeletal domain is ∼50% identical to erythrocyte dematin, an actin-bundling protein of the red cell membrane skeleton, while the zinc finger domains conform to the LIM motif consensus sequence. In vitro expression studies demonstrate that abLIM protein can bind to F-actin through the dematin-like domain. Transcripts corresponding to three distinct isoforms have a widespread tissue distribution. However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments. Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners. The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments. PMID:9245787

  5. Cell protrusion and retraction driven by fluctuations in actin polymerization: A two-dimensional model.

    Science.gov (United States)

    Ryan, Gillian L; Holz, Danielle; Yamashiro, Sawako; Taniguchi, Daisuke; Watanabe, Naoki; Vavylonis, Dimitrios

    2017-07-28

    Animal cells that spread onto a surface often rely on actin-rich lamellipodial extensions to execute protrusion. Many cell types recently adhered on a two-dimensional substrate exhibit protrusion and retraction of their lamellipodia, even though the cell is not translating. Travelling waves of protrusion have also been observed, similar to those observed in crawling cells. These regular patterns of protrusion and retraction allow quantitative analysis for comparison to mathematical models. The periodic fluctuations in leading edge position of XTC cells have been linked to excitable actin dynamics using a one-dimensional model of actin dynamics, as a function of arc-length along the cell. In this work we extend this earlier model of actin dynamics into two dimensions (along the arc-length and radial directions of the cell) and include a model membrane that protrudes and retracts in response to the changing number of free barbed ends of actin filaments near the membrane. We show that if the polymerization rate at the barbed ends changes in response to changes in their local concentration at the leading edge and/or the opposing force from the cell membrane, the model can reproduce the patterns of membrane protrusion and retraction seen in experiment. We investigate both Brownian ratchet and switch-like force-velocity relationships between the membrane load forces and actin polymerization rate. The switch-like polymerization dynamics recover the observed patterns of protrusion and retraction as well as the fluctuations in F-actin concentration profiles. The model generates predictions for the behavior of cells after local membrane tension perturbations. © 2017 Wiley Periodicals, Inc.

  6. Actin fringes of polar cell growth.

    Science.gov (United States)

    Stephan, Octavian O H

    2017-06-15

    The eukaryotic actin cytoskeleton is a highly dynamic framework that is involved in many biological processes, such as cell growth, division, morphology, and motility. G-actin polymerizes into microfilaments that associate into bundles, patches, and networks, which, in turn, organize into higher order structures that are fundamental for the course of important physiological events. Actin rings are an example for such higher order actin entities, but this term represents an actually diverse set of subcellular structures that are involved in various processes. This review especially sheds light on a crucial type of non-constricting ring-like actin networks, and categorizes them under the term 'actin fringe'. These 'actin fringes' are visualized as highly dynamic and yet steady structures in the tip of various polarized growing cells. The present comprehensive overview compares the actin fringe characteristics of rapidly elongating pollen tubes with several related actin arrays in other cell types of diverse species. The current state of knowledge about various actin fringe functions is summarized, and the key role of this structure in the polar growth process is discussed. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. Fascin regulates nuclear actin during Drosophila oogenesis.

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    Kelpsch, Daniel J; Groen, Christopher M; Fagan, Tiffany N; Sudhir, Sweta; Tootle, Tina L

    2016-10-01

    Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5-9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved. © 2016 Kelpsch 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).

  8. CADM1 Controls Actin Cytoskeleton Assembly and Regulates Extracellular Matrix Adhesion in Human Mast Cells

    Science.gov (United States)

    Moiseeva, Elena P.; Straatman, Kees R.; Leyland, Mark L.; Bradding, Peter

    2014-01-01

    CADM1 is a major receptor for the adhesion of mast cells (MCs) to fibroblasts, human airway smooth muscle cells (HASMCs) and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM). Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion. PMID:24465823

  9. CADM1 controls actin cytoskeleton assembly and regulates extracellular matrix adhesion in human mast cells.

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    Elena P Moiseeva

    Full Text Available CADM1 is a major receptor for the adhesion of mast cells (MCs to fibroblasts, human airway smooth muscle cells (HASMCs and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM. Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion.

  10. A Multimodular Tensegrity Model of an Actin Stress Fiber

    Science.gov (United States)

    Luo, Yaozhi; Xu, Xian; Lele, Tanmay; Kumar, Sanjay; Ingber, Donald E.

    2008-01-01

    Stress fibers are contractile bundles in the cytoskeleton that stabilize cell structure by exerting traction forces on extracellular matrix. Individual stress fibers are molecular bundles composed of parallel actin and myosin filaments linked by various actin-binding proteins, which are organized end-on-end in a sarcomere-like pattern within an elongated three-dimensional network. While measurements of single stress fibers in living cells show that they behave like tensed viscoelastic fibers, precisely how this mechanical behavior arises from this complex supramolecular arrangement of protein components remains unclear. Here we show that computationally modeling a stress fiber as a multi-modular tensegrity network can predict several key behaviors of stress fibers measured in living cells, including viscoelastic retraction, fiber splaying after severing, non-uniform contraction, and elliptical strain of a puncture wound within the fiber. The tensegrity model also can explain how they simultaneously experience passive tension and generate active contraction forces; in contrast, a tensed cable net model predicts some, but not all, of these properties. Thus, tensegrity models may provide a useful link between molecular and cellular scale mechanical behaviors, and represent a new handle on multi-scale modeling of living materials. PMID:18632107

  11. Actin-dependent light-induced translocation of mitochondria and ER cisternae in the photoreceptor cells of the locust Schistocerca gregaria.

    Science.gov (United States)

    Stürmer, K; Baumann, O; Walz, B

    1995-06-01

    Light-dependent changes in the positioning of organelles in photoreceptor cells of arthropods are a well-known phenomenon. In this study, we examine the role of the cytoskeleton in these light-dependent antagonistic movements. In dark-adapted photoreceptor cells of the locust Schistocerca gregaria, prominent sacs of smooth endoplasmic reticulum (ER) oppose the bases of the photoreceptive microvilli. Light stimulation causes a translocation of the ER elements towards the main cell body, and an aggregation of mitochondria adjacent to the microvilli. Immunofluorescence studies and electron-microscopic examination of chemically fixed or high-pressure-frozen, freeze-substituted specimens demonstrate a lack of microtubules in the submicrovillar region. However, numerous filament bundles are aligned in close association with mitochondria and ER elements, along the track of their movement. Fluorescent phallotoxins and monoclonal anti-actin antibodies label filament bundles in the submicrovillar region, indicating that they are composed of F-actin. Finally, depolymerization of the submicrovillar actin filaments by incubation with cytochalasin B results in a blockade of the movement of mitochondria and ER cisternae towards the rhabdom. These results suggest that the light-dependent translocation of both ER cisternae and mitochondria occurs along actin filaments.

  12. Chordate muscle actins differ distinctly from invertebrate muscle actins. The evolution of the different vertebrate muscle actins.

    Science.gov (United States)

    Vandekerckhove, J; Weber, K

    1984-11-05

    A total of 30 actins from various chordate and invertebrate muscle sources were either characterized by full amino acid sequence data or typed by those partial sequences in the NH2-terminal tryptic peptide which are known to be specific markers for different actin isoforms. The results show that most, if not all, invertebrate muscle actins are homologous to each other and to the isoforms recognized as vertebrate cytoplasmic actins. In contrast the actin forms typically found in muscle cells of warm-blooded vertebrates are noticeably different from invertebrate muscle actins and seem to have appeared in evolution already with the origin of chordates. During subsequent vertebrate evolution there has been a high degree of sequence conservation similar or stronger than that seen in histone H4. Urochordates, Cephalochordates and probably also Agnathes express only one type of muscle actin. Two types, a striated muscle-specific form and a smooth muscle form, are already observed in Chondrichthyes and Osteichthyes. Later in evolution, with the origin of reptiles, both muscle actins seem to have duplicated again; the striated muscle type branched into a skeletal- and cardiac-specific form, while the smooth muscle form duplicated into a vascular- and stomach-specific type. These findings support the hypothesis that each of the four muscle actins of warm-blooded vertebrates are coded for by a small number and possibly only one functional gene.

  13. BipC, a Predicted Burkholderia pseudomallei Type 3 Secretion System Translocator Protein with Actin Binding Activity

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    Charles W. Vander Broek

    2017-07-01

    Full Text Available Burkholderia pseudomallei is an intracellular bacterial pathogen and the causative agent of melioidosis, a severe disease of humans and animals. Like other clinically important Gram-negative bacteria, fundamental to B. pseudomallei pathogenesis is the Bsa Type III Secretion System. The Bsa system injects bacterial effector proteins into the cytoplasm of target host cells subverting cellular pathways for the benefit of the bacteria. It is required for invasion of non-phagocytic host cells, escape from the endocytic compartment into the host cell cytoplasm, and for virulence in murine models of melioidosis. We have recently described the repertoire of effector proteins secreted by the B. pseudomallei Bsa system, however the functions of many of these effector proteins remain an enigma. One such protein is BipC, a homolog of the translocator/effector proteins SipC and IpaC from Salmonella spp. and Shigella flexneri respectively. SipC and IpaC each have separate and distinct roles acting both as translocators, involved in creating a pore in the eukaryotic cell membrane through which effector proteins can transit, and as effectors by interacting with and polymerizing host cell actin. In this study, pull-down assays demonstrate an interaction between BipC and actin. Furthermore, we show that BipC directly interacts with actin, preferentially with actin polymers (F-actin and has the ability to polymerize actin in a similar manner as that described for SipC. Yet unlike SipC, BipC does not stabilize F-actin filaments, indicating a functionally distinct interaction with actin. Expression of Myc-tagged BipC in HeLa cells induces the formation of pseudopodia similar to that seen for IpaC. This study explores the effector function of BipC and reveals that actin interaction is conserved within the BipC/SipC/IpaC family of translocator/effector proteins.

  14. The Intriguing Dual Lattices of the Myosin Filaments in Vertebrate Striated Muscles: Evolution and Advantage

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    Pradeep K. Luther

    2014-12-01

    Full Text Available Myosin filaments in vertebrate striated muscle have a long roughly cylindrical backbone with cross-bridge projections on the surfaces of both halves except for a short central bare zone. In the middle of this central region the filaments are cross-linked by the M-band which holds them in a well-defined hexagonal lattice in the muscle A-band. During muscular contraction the M-band-defined rotation of the myosin filaments around their long axes influences the interactions that the cross-bridges can make with the neighbouring actin filaments. We can visualise this filament rotation by electron microscopy of thin cross-sections in the bare-region immediately adjacent to the M-band where the filament profiles are distinctly triangular. In the muscles of teleost fishes, the thick filament triangular profiles have a single orientation giving what we call the simple lattice. In other vertebrates, for example all the tetrapods, the thick filaments have one of two orientations where the triangles point in opposite directions (they are rotated by 60° or 180° according to set rules. Such a distribution cannot be developed in an ordered fashion across a large 2D lattice, but there are small domains of superlattice such that the next-nearest neighbouring thick filaments often have the same orientation. We believe that this difference in the lattice forms can lead to different contractile behaviours. Here we provide a historical review, and when appropriate cite recent work related to the emergence of the simple and superlattice forms by examining the muscles of several species ranging back to primitive vertebrates and we discuss the functional differences that the two lattice forms may have.

  15. Cross-correlated TIRF/AFM reveals asymmetric distribution of force-generating heads along self-assembled, "synthetic" myosin filaments.

    Science.gov (United States)

    Brown, André E X; Hategan, Alina; Safer, Daniel; Goldman, Yale E; Discher, Dennis E

    2009-03-04

    Myosin-II's rod-like tail drives filament assembly with a head arrangement that is often considered to be a symmetric bipole that generates equal and opposite contractile forces on actin. Self-assembled myosin filaments are shown here to be asymmetric in physiological buffer based on cross-correlated images from both atomic force microscopy and total internal reflection fluorescence. Quantitative cross-correlation of these orthogonal methods produces structural information unavailable to either method alone in showing that fluorescence intensity along the filament length is proportional to height. This implies that myosin heads form a shell around the filament axis, consistent with F-actin binding. A motor density of approximately 50-100 heads/micrometer is further estimated but with an average of 32% more motors on one half of any given filament compared to the other, regardless of length. A purely entropic pyramidal lattice model is developed and mapped onto the Dyck paths problem that qualitatively captures this lack of length dependence and the distribution of filament asymmetries. Such strongly asymmetric bipoles are likely to produce an unbalanced contractile force in cells and in actin-myosin gels and thereby contribute to motility as well as cytoskeletal tension.

  16. Intestinal, segmented, filamentous bacteria.

    Science.gov (United States)

    Klaasen, H L; Koopman, J P; Poelma, F G; Beynen, A C

    1992-06-01

    Segmented, filamentous bacteria (SFBs) are autochthonous, apathogenic bacteria, occurring in the ileum of mice and rats. Although the application of formal taxonomic criteria is impossible due to the lack of an in vitro technique to culture SFBs, microbes with a similar morphology, found in the intestine of a wide range of vertebrate and invertebrate host species, are considered to be related. SFBs are firmly attached to the epithelial cells of the distal ileal mucosa, their preferential ecological niche being the epithelium covering the Peyer's patches. Electron microscopic studies have demonstrated a considerable morphological diversity of SFBs, which may relate to different stages of a life cycle. Determinants of SFB colonization in vivo are host species, genotypical and phenotypical characteristics of the host, diet composition, environmental stress and antimicrobial drugs. SFBs can survive in vitro incubation, but do not multiply. On the basis of their apathogenic character and intimate relationship with the host, it is suggested that SFBs contribute to development and/or maintenance of host resistance to enteropathogens.

  17. Centromeres of filamentous fungi

    Science.gov (United States)

    Smith, Kristina M.; Galazka, Jonathan M.; Phatale, Pallavi A.; Connolly, Lanelle R.; Freitag, Michael

    2012-01-01

    How centromeres are assembled and maintained remains one of the fundamental questions in cell biology. Over the past 20 years the idea of centromeres as precise genetic loci has been replaced by the realization that it is predominantly the protein complement that defines centromere localization and function. Thus, placement and maintenance of centromeres are excellent examples of epigenetic phenomena in the strict sense. In contrast, the highly derived “point centromeres” of the budding yeast Saccharomyces cerevisiae and its close relatives are counterexamples for this general principle of centromere maintenance. While we have learned much in the past decade, it remains unclear if mechanisms for epigenetic centromere placement and maintenance are shared amongst various groups of organisms. For that reason it seems prudent to examine species from many different phylogenetic groups with the aim to extract comparative information that will yield a more complete picture of cell division in all eukaryotes. This review addresses what has been learned by studying the centromeres of filamentous fungi, a large, heterogeneous group of organisms that includes important plant, animal and human pathogens, saprobes and symbionts that fulfill essential roles in the biosphere, as well as a growing number of taxa that have become indispensable for industrial use. PMID:22752455

  18. Pharmacological treatment of actinic keratosis

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    Ewa Zwierzyńska

    2016-09-01

    Full Text Available Actinic keratosis (AK is a disease characterized by hyperkeratotic lesions on skin damaged by ultraviolet. radiation. These lesions may progress to squamous cell or basal cell carcinoma. Currently pharmacotherapy and different surgical procedures are used in AK therapy. The most common treatment options are 5-fluorouracil, imiquimod, diclofenac, ingenol mebutate, and first and third generation retinoids (retinol, adapalene, tazarotene. Furthermore, research is being carried out in order to test new medications including nicotinamide, resiquimod, piroxicam, potassium dobesilate and oleogel based on a triterpene extract (betulin, betulinic acid. Recently, the preventive effect of acetylsalicylic acid and celecoxib has also been investigated.

  19. Bioinformatics study of the mangrove actin genes

    Science.gov (United States)

    Basyuni, M.; Wasilah, M.; Sumardi

    2017-01-01

    This study describes the bioinformatics methods to analyze eight actin genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, subcellular localization, similarity, and phylogenetic. The physical and chemical properties of eight mangroves showed variation among the genes. The percentage of the secondary structure of eight mangrove actin genes followed the order of a helix > random coil > extended chain structure for BgActl, KcActl, RsActl, and A. corniculatum Act. In contrast to this observation, the remaining actin genes were random coil > extended chain structure > a helix. This study, therefore, shown the prediction of secondary structure was performed for necessary structural information. The values of chloroplast or signal peptide or mitochondrial target were too small, indicated that no chloroplast or mitochondrial transit peptide or signal peptide of secretion pathway in mangrove actin genes. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove actin genes. To clarify the relationship among the mangrove actin gene, a phylogenetic tree was constructed. Three groups of mangrove actin genes were formed, the first group contains B. gymnorrhiza BgAct and R. stylosa RsActl. The second cluster which consists of 5 actin genes the largest group, and the last branch consist of one gene, B. sexagula Act. The present study, therefore, supported the previous results that plant actin genes form distinct clusters in the tree.

  20. Dynamic Actin Gene Family Evolution in Primates

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    Liucun Zhu

    2013-01-01

    Full Text Available Actin is one of the most highly conserved proteins and plays crucial roles in many vital cellular functions. In most eukaryotes, it is encoded by a multigene family. Although the actin gene family has been studied a lot, few investigators focus on the comparison of actin gene family in relative species. Here, the purpose of our study is to systematically investigate characteristics and evolutionary pattern of actin gene family in primates. We identified 233 actin genes in human, chimpanzee, gorilla, orangutan, gibbon, rhesus monkey, and marmoset genomes. Phylogenetic analysis showed that actin genes in the seven species could be divided into two major types of clades: orthologous group versus complex group. Codon usages and gene expression patterns of actin gene copies were highly consistent among the groups because of basic functions needed by the organisms, but much diverged within species due to functional diversification. Besides, many great potential pseudogenes were found with incomplete open reading frames due to frameshifts or early stop codons. These results implied that actin gene family in primates went through “birth and death” model of evolution process. Under this model, actin genes experienced strong negative selection and increased the functional complexity by reproducing themselves.

  1. Dynamic Actin Gene Family Evolution in Primates

    Science.gov (United States)

    Zhu, Liucun; Zhang, Ying; Hu, Yijun; Wen, Tieqiao; Wang, Qiang

    2013-01-01

    Actin is one of the most highly conserved proteins and plays crucial roles in many vital cellular functions. In most eukaryotes, it is encoded by a multigene family. Although the actin gene family has been studied a lot, few investigators focus on the comparison of actin gene family in relative species. Here, the purpose of our study is to systematically investigate characteristics and evolutionary pattern of actin gene family in primates. We identified 233 actin genes in human, chimpanzee, gorilla, orangutan, gibbon, rhesus monkey, and marmoset genomes. Phylogenetic analysis showed that actin genes in the seven species could be divided into two major types of clades: orthologous group versus complex group. Codon usages and gene expression patterns of actin gene copies were highly consistent among the groups because of basic functions needed by the organisms, but much diverged within species due to functional diversification. Besides, many great potential pseudogenes were found with incomplete open reading frames due to frameshifts or early stop codons. These results implied that actin gene family in primates went through “birth and death” model of evolution process. Under this model, actin genes experienced strong negative selection and increased the functional complexity by reproducing themselves. PMID:23841080

  2. Common behavioral clusters and subcortical anatomy in stroke

    Science.gov (United States)

    Corbetta, Maurizio; Ramsey, Lenny; Callejas, Alicia; Baldassarre, Antonello; Hacker, Carl D.; Siegel, Joshua S.; Astafiev, Serguei V.; Rengachary, Jennifer; Zinn, Kristina; Lang, Catherine E.; Connor, Lisa Tabor; Fucetola, Robert; Strube, Michael; Carter, Alex R.; Shulman, Gordon L.

    2015-01-01

    SUMMARY A long-held view is that stroke causes many distinct neurological syndromes due to damage of specialized cortical and subcortical centers. However, it is unknown if a syndrome-based description is helpful in characterizing behavioral deficits across a large number of patients. We studied a large prospective sample of first-time stroke patients with heterogeneous lesions at 1–2 weeks post-stroke. We measured behavior over multiple domains and lesion anatomy with structural MRI and a probabilistic atlas of white matter pathways. Multivariate methods estimated the percentage of behavioral variance explained by structural damage. A few clusters of behavioral deficits spanning multiple functions explained neurological impairment. Stroke topography was predominantly subcortical, and disconnection of white matter tracts critically contributed to behavioral deficits and their correlation. The locus of damage explained more variance for motor and language than memory or attention deficits. Our findings highlight the need for better models of white matter damage on cognition. PMID:25741721

  3. [The CADASIL syndrome: a model of subcortical-cortical disconnection].

    Science.gov (United States)

    Blanco Menéndez, R; Aguado Balsas, A M; Blanco, E; Lobo Rodríguez, B; Vera De La Puente, E

    CADASIL syndrome (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarts and Leukoencephalopathy) includes some neurological signs and symptoms (gait disturbances, epileptic seizures, pseudobulbar palsy, migraines, etc.), as well as neuropsychological dysfunctions (cognitive and executive impairment, emotional disorders and, frequently, dementia). This syndrome is a good model of white matter damage and potential disconnection syndromes. In this article, the neuropsychological profile of a 47 year-old woman diagnosed of CADASIL is investigated and thoroughly discussed. Results show the presence of a moderate cognitive and executive impairment, specially of memory, psychomotor abilities, executive functions and verbal fluency, but not dementia, overall suggesting the presence of a temporal-frontal-subcortical disfunction. This clinical pattern is an illuminating example of the neuropsychological consequences of the partial disconnection of prefrontal cortex from the thalamus and basal ganglia.

  4. The implication of subcortical motor centers in voluntary human activities

    OpenAIRE

    Queralt Blasco, Ana

    2009-01-01

    The main objective of the present dissertation was to analyse the subcortical implications in the preparation and execution of complex voluntary movements. Three different tasks were selected on purpose. They all are everyday activities which although functionally related have differential characteristics. The first task was the sit-to-stand manoeuvre. Simple ballistic movements are executed faster in reaction time task paradigms when the imperative signal is accompanied by a startling audito...

  5. Double Cortex Syndrome (Subcortical Band Heterotopia): A Case Report

    OpenAIRE

    Momen, Ali Akbar; Momen, Mehdi

    2015-01-01

    AbstractObjectiveApproximately 5–10% of preschool age children are considered developmentally disabled. Brain Magnetic Resonance Imaging (MRI) plays a key role in the diagnostic evaluation in these children. Many congenital or acquired brain anomalies are revealed with MRIs. Although the majority of these abnormalities are sporadic but patients with subcortical band heterotopia or double cortex syndrome have sex-linked inheritance. We are going to present the first case in Iran from Ahvaz cit...

  6. Electron cryomicroscopy of E. coli reveals filament bundles involved in plasmid DNA segregation.

    Science.gov (United States)

    Salje, Jeanne; Zuber, Benoît; Löwe, Jan

    2009-01-23

    Bipolar elongation of filaments of the bacterial actin homolog ParM drives movement of newly replicated plasmid DNA to opposite poles of a bacterial cell. We used a combination of vitreous sectioning and electron cryotomography to study this DNA partitioning system directly in native, frozen cells. The diffraction patterns from overexpressed ParM bundles in electron cryotomographic reconstructions were used to unambiguously identify ParM filaments in Escherichia coli cells. Using a low-copy number plasmid encoding components required for partitioning, we observed small bundles of three to five intracellular ParM filaments that were situated close to the edge of the nucleoid. We propose that this may indicate the capture of plasmid DNA within the periphery of this loosely defined, chromosome-containing region.

  7. Mapping abnormal subcortical brain morphometry in an elderly HIV+ cohort

    Directory of Open Access Journals (Sweden)

    Benjamin S.C. Wade

    2015-01-01

    Full Text Available Over 50% of HIV+ individuals exhibit neurocognitive impairment and subcortical atrophy, but the profile of brain abnormalities associated with HIV is still poorly understood. Using surface-based shape analyses, we mapped the 3D profile of subcortical morphometry in 63 elderly HIV+ participants and 31 uninfected controls. The thalamus, caudate, putamen, pallidum, hippocampus, amygdala, brainstem, accumbens, callosum and ventricles were segmented from high-resolution MRIs. To investigate shape-based morphometry, we analyzed the Jacobian determinant (JD and radial distances (RD defined on each region's surfaces. We also investigated effects of nadir CD4+ T-cell counts, viral load, time since diagnosis (TSD and cognition on subcortical morphology. Lastly, we explored whether HIV+ participants were distinguishable from unaffected controls in a machine learning context. All shape and volume features were included in a random forest (RF model. The model was validated with 2-fold cross-validation. Volumes of HIV+ participants' bilateral thalamus, left pallidum, left putamen and callosum were significantly reduced while ventricular spaces were enlarged. Significant shape variation was associated with HIV status, TSD and the Wechsler adult intelligence scale. HIV+ people had diffuse atrophy, particularly in the caudate, putamen, hippocampus and thalamus. Unexpectedly, extended TSD was associated with increased thickness of the anterior right pallidum. In the classification of HIV+ participants vs. controls, our RF model attained an area under the curve of 72%.

  8. Subcortical auditory structures in the Mongolian gerbil: I. Golgi architecture.

    Science.gov (United States)

    Mylius, Judith; Brosch, Michael; Scheich, Henning; Budinger, Eike

    2013-04-15

    By means of the Golgi-Cox and Nissl methods we investigated the cyto- and fiberarchitecture as well as the morphology of neurons in the subcortical auditory structures of the Mongolian gerbil (Meriones unguiculatus), a frequently used animal model in auditory neuroscience. We describe the divisions and subdivisions of the auditory thalamus including the medial geniculate body, suprageniculate nucleus, and reticular thalamic nucleus, as well as of the inferior colliculi, nuclei of the lateral lemniscus, superior olivary complex, and cochlear nuclear complex. In this study, we 1) confirm previous results about the organization of the gerbil's subcortical auditory pathway using other anatomical staining methods (e.g., Budinger et al. [2000] Eur J Neurosci 12:2452-2474); 2) add substantially to the knowledge about the laminar and cellular organization of the gerbil's subcortical auditory structures, in particular about the orientation of their fibrodendritic laminae and about the morphology of their most distinctive neuron types; and 3) demonstrate that the cellular organization of these structures, as seen by the Golgi technique, corresponds generally to that of other mammalian species, in particular to that of rodents. Copyright © 2012 Wiley Periodicals, Inc.

  9. Pigmentary mosaicism, subcortical band heterotopia, and brain cystic lesions.

    Science.gov (United States)

    Ruggieri, Martino; Roggini, Mario; Spalice, Alberto; Addis, Maria; Iannetti, Paola

    2009-05-01

    A 10-year-old boy presented with a severe and diffuse mosaic skin hypopigmentation running (in narrow bands) along the lines of Blaschko associated with mosaic areas of alopecia, facial dysmorphism with midface hypoplasia, bilateral punctate cataract, microretrognathia, short neck, pectus excavatum, joint hypermobility, mild muscular hypotonia, generalized seizures, and mild mental retardation. Cranial magnetic resonance imaging revealed hypoplastic corpus callosum (primarily posterior), subcortical band heterotopia, and diffuse subcortical, periventricular cystic-like lesions. Similar dysmorphic features were observed in the child's mother, but with no imaging abnormalities. The facial phenotype coupled with the cysts in the brain was strongly reminiscent of the oculocerebrorenal Lowe syndrome. Full chromosome studies in the parents and the proband and mutation analysis on peripheral blood lymphocytes (and on skin cultured fibroblasts from affected and unaffected skin areas in the child) in the genes for subcortical band heterotopia (DCX (Xq22.3-q23)], lissencephaly (PAFAH1B1, alias LIS1, at 17p13.3), and oculocerebrorenal syndrome of Lowe (OCRL at Xq23-q24)] were unrevealing. This constellation of multiple congenital anomalies including skin hypopigmentation and eye, musculoskeletal, and nervous system abnormalities was sufficiently characterized to be regarded as a novel example of pigmentary mosaicism of the Ito type (i.e., hypomelanosis of Ito).

  10. Actin-binding and Cell Proliferation Activities of Angiomotin Family Members Are Regulated by Hippo Pathway-mediated Phosphorylation*

    Science.gov (United States)

    Chan, Siew Wee; Lim, Chun Jye; Guo, Fusheng; Tan, Ivan; Leung, Thomas; Hong, Wanjin

    2013-01-01

    Whether the Hippo pathway has downstream targets other than YAP and TAZ is unknown. In this report, we have identified angiomotin (Amot) family members as novel substrates of Hippo core kinases. The N-terminal regions of Amot proteins contain a conserved HXRXXS consensus site for LATS1/2-mediated phosphorylation. Phospho-specific antibodies showed that Hippo core kinases could mediate phosphorylation of endogenous as well as exogenous Amot family members. Knockdown of LATS1 and LATS2 endogenously reduced the phosphorylation of Amots detected by the phospho-specific antibodies. Mutation of the serine to alanine within this HXRXXS site in Amot and AmotL2 established that this site was essential for Hippo core kinase-mediated phosphorylation. Wild-type and non-phosphorylated Amot (Amot-S175A) were targeted to actin filaments, whereas phospho-mimic Amot (Amot-S175D) failed to be localized with actin. Overexpression of LATS2 caused dissociation of Amot from actin but not Amot-S175A. Mapping of the actin-binding site of Amot showed that serine 175 of Amot was important for the actin-binding activity. Amot-S175A promoted, whereas Amot and Amot-S175D inhibited, cell proliferation. These results collectively suggest that the Hippo pathway negatively regulates the actin-binding activity of Amot family members through direct phosphorylation. PMID:24225952

  11. Actin-binding and cell proliferation activities of angiomotin family members are regulated by Hippo pathway-mediated phosphorylation.

    Science.gov (United States)

    Chan, Siew Wee; Lim, Chun Jye; Guo, Fusheng; Tan, Ivan; Leung, Thomas; Hong, Wanjin

    2013-12-27

    Whether the Hippo pathway has downstream targets other than YAP and TAZ is unknown. In this report, we have identified angiomotin (Amot) family members as novel substrates of Hippo core kinases. The N-terminal regions of Amot proteins contain a conserved HXRXXS consensus site for LATS1/2-mediated phosphorylation. Phospho-specific antibodies showed that Hippo core kinases could mediate phosphorylation of endogenous as well as exogenous Amot family members. Knockdown of LATS1 and LATS2 endogenously reduced the phosphorylation of Amots detected by the phospho-specific antibodies. Mutation of the serine to alanine within this HXRXXS site in Amot and AmotL2 established that this site was essential for Hippo core kinase-mediated phosphorylation. Wild-type and non-phosphorylated Amot (Amot-S175A) were targeted to actin filaments, whereas phospho-mimic Amot (Amot-S175D) failed to be localized with actin. Overexpression of LATS2 caused dissociation of Amot from actin but not Amot-S175A. Mapping of the actin-binding site of Amot showed that serine 175 of Amot was important for the actin-binding activity. Amot-S175A promoted, whereas Amot and Amot-S175D inhibited, cell proliferation. These results collectively suggest that the Hippo pathway negatively regulates the actin-binding activity of Amot family members through direct phosphorylation.

  12. A second actin-like MamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island.

    Directory of Open Access Journals (Sweden)

    Jean-Baptiste Rioux

    Full Text Available Magnetotactic bacteria are able to swim navigating along geomagnetic field lines. They synthesize ferromagnetic nanocrystals that are embedded in cytoplasmic membrane invaginations forming magnetosomes. Regularly aligned in the cytoplasm along cytoskeleton filaments, the magnetosome chain effectively forms a compass needle bestowing on bacteria their magnetotactic behaviour. A large genomic island, conserved among magnetotactic bacteria, contains the genes potentially involved in magnetosome formation. One of the genes, mamK has been described as encoding a prokaryotic actin-like protein which when it polymerizes forms in the cytoplasm filamentous structures that provide the scaffold for magnetosome alignment. Here, we have identified a series of genes highly similar to the mam genes in the genome of Magnetospirillum magneticum AMB-1. The newly annotated genes are clustered in a genomic islet distinct and distant from the known magnetosome genomic island and most probably acquired by lateral gene transfer rather than duplication. We focused on a mamK-like gene whose product shares 54.5% identity with the actin-like MamK. Filament bundles of polymerized MamK-like protein were observed in vitro with electron microscopy and in vivo in E. coli cells expressing MamK-like-Venus fusions by fluorescence microscopy. In addition, we demonstrate that mamK-like is transcribed in AMB-1 wild-type and DeltamamK mutant cells and that the actin-like filamentous structures observed in the DeltamamK strain are probably MamK-like polymers. Thus MamK-like is a new member of the prokaryotic actin-like family. This is the first evidence of a functional mam gene encoded outside the magnetosome genomic island.

  13. Insight into the assembly properties and functional organisation of the magnetotactic bacterial actin-like homolog, MamK.

    Directory of Open Access Journals (Sweden)

    Sanjiv Sonkaria

    Full Text Available Magnetotactic bacteria (MTB synthesize magnetosomes, which are intracellular vesicles comprising a magnetic particle. A series of magnetosomes arrange themselves in chains to form a magnetic dipole that enables the cell to orient itself along the Earth's magnetic field. MamK, an actin-like homolog of MreB has been identified as a central component in this organisation. Gene deletion, fluorescence microscopy and in vitro studies have yielded mechanistic differences in the filament assembly of MamK with other bacterial cytoskeletal proteins within the cell. With little or no information on the structural and behavioural characteristics of MamK outside the cell, the mamK gene from Magnetospirillium gryphiswaldense was cloned and expressed to better understand the differences in the cytoskeletal properties with its bacterial homologues MreB and acitin. Despite the low sequence identity shared between MamK and MreB (22% and actin (18%, the behaviour of MamK monitored by light scattering broadly mirrored that of its bacterial cousin MreB primarily in terms of its pH, salt, divalent metal-ion and temperature dependency. The broad size variability of MamK filaments revealed by light scattering studies was supported by transmission electron microscopy (TEM imaging. Filament morphology however, indicated that MamK conformed to linearly orientated filaments that appeared to be distinctly dissimilar compared to MreB suggesting functional differences between these homologues. The presence of a nucleotide binding domain common to actin-like proteins was demonstrated by its ability to function both as an ATPase and GTPase. Circular dichroism and structural homology modelling showed that MamK adopts a protein fold that is consistent with the 'classical' actin family architecture but with notable structural differences within the smaller domains, the active site region and the overall surface electrostatic potential.

  14. Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells

    Directory of Open Access Journals (Sweden)

    Bhanu Neupane

    2015-09-01

    Full Text Available Stimulated emission depletion (STED microscopy provides a new opportunity to study fine sub-cellular structures and highly dynamic cellular processes, which are challenging to observe using conventional optical microscopy. Using actin as an example, we explored the feasibility of using a continuous wave (CW-STED microscope to study the fine structure and dynamics in fixed and live cells. Actin plays an important role in cellular processes, whose functioning involves dynamic formation and reorganization of fine structures of actin filaments. Frequently used confocal fluorescence and STED microscopy dyes were employed to image fixed PC-12 cells (dyed with phalloidin- fluorescein isothiocyante and live rat chondrosarcoma cells (RCS transfected with actin-green fluorescent protein (GFP. Compared to conventional confocal fluorescence microscopy, CW-STED microscopy shows improved spatial resolution in both fixed and live cells. We were able to monitor cell morphology changes continuously; however, the number of repetitive analyses were limited primarily by the dyes used in these experiments and could be improved with the use of dyes less susceptible to photobleaching. In conclusion, CW-STED may disclose new information for biological systems with a proper characteristic length scale. The challenges of using CW-STED microscopy to study cell structures are discussed.

  15. Caldesmon regulates actin dynamics to influence cranial neural crest migration in Xenopus.

    Science.gov (United States)

    Nie, Shuyi; Kee, Yun; Bronner-Fraser, Marianne

    2011-09-01

    Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells to migrate a significantly shorter distance, prevents their segregation into distinct migratory streams, and later results in severe defects in cartilage formation. Demonstrating specificity, these effects are rescued by adding back exogenous CaD. Interestingly, CaD proteins with mutations in the Ca(2+)-calmodulin-binding sites or ErK/Cdk1 phosphorylation sites fail to rescue the knockdown phenotypes, whereas mutation of the PAK phosphorylation site is able to rescue them. Analysis of neural crest explants reveals that CaD is required for the dynamic arrangements of actin and, thus, for cell shape changes and process formation. Taken together, these results suggest that the actin-modulating activity of CaD may underlie its critical function and is regulated by distinct signaling pathways during normal neural crest migration.

  16. An ancient Sec10-formin fusion provides insights into actin-mediated regulation of exocytosis.

    Science.gov (United States)

    van Gisbergen, Peter A C; Wu, Shu-Zon; Chang, Mingqin; Pattavina, Kelli A; Bartlett, Madelaine E; Bezanilla, Magdalena

    2018-01-26

    Exocytosis, facilitated by the exocyst, is fundamentally important for remodeling cell walls and membranes. Here, we analyzed For1F, a novel gene that encodes a fusion of an exocyst subunit (Sec10) and an actin nucleation factor (formin). We showed that the fusion occurred early in moss evolution and has been retained for more than 170 million years. In Physcomitrella patens, For1F is essential, and the expressed protein is a fusion of Sec10 and formin. Reduction of For1F or actin filaments inhibits exocytosis, and For1F dynamically associates with Sec6, another exocyst subunit, in an actin-dependent manner. Complementation experiments demonstrate that constitutive expression of either half of the gene or the paralogous Sec10b rescues loss of For1F, suggesting that fusion of the two domains is not essential, consistent with findings in yeast, where formin and the exocyst are linked noncovalently. Although not essential, the fusion may have had selective advantages and provides a unique opportunity to probe actin regulation of exocytosis. © 2018 van Gisbergen et al.

  17. Correlated light and electron microscopy observations of the uterine epithelial cell actin cytoskeleton using fluorescently labeled resin-embedded sections.

    Science.gov (United States)

    Moore, Chad L; Cheng, Delfine; Shami, Gerald J; Murphy, Christopher R

    2016-05-01

    In order to perform correlative light and electron microscopy (CLEM) more precisely, we have modified existing specimen preparation protocols allowing fluorescence retention within embedded and sectioned tissue, facilitating direct observation across length scales. We detail a protocol which provides a precise correlation accuracy using accessible techniques in biological specimen preparation. By combining a pre-embedding uranyl acetate staining step with the progressive lowering of temperature (PLT) technique, a methacrylate embedded tissue specimen is ultrathin sectioned and mounted onto a TEM finder grid for immediate viewing in the confocal and electron microscope. In this study, the protocol is applied to rat uterine epithelial cells in vivo during early pregnancy. Correlative overlay data was used to track changes in filamentous actin that occurs in these cells from fertilization (Day 1) to implantation on Day 6 as part of the plasma membrane transformation, a process essential in the development of uterine receptivity in the rat. CLEM confirmed that the actin cytoskeleton is disrupted as apical microvilli are progressively lost toward implantation, and revealed the thick and continuous terminal web is replaced by a thinner and irregular actin band, with individually distinguishable filaments connecting actin meshworks which correspond with remaining plasma membrane protrusions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Specification of Architecture and Function of Actin Structures by Actin Nucleation Factors.

    Science.gov (United States)

    Skau, Colleen T; Waterman, Clare M

    2015-01-01

    The actin cytoskeleton is essential for diverse processes in mammalian cells; these processes range from establishing cell polarity to powering cell migration to driving cytokinesis to positioning intracellular organelles. How these many functions are carried out in a spatiotemporally regulated manner in a single cytoplasm has been the subject of much study in the cytoskeleton field. Recent work has identified a host of actin nucleation factors that can build architecturally diverse actin structures. The biochemical properties of these factors, coupled with their cellular location, likely define the functional properties of actin structures. In this article, we describe how recent advances in cell biology and biochemistry have begun to elucidate the role of individual actin nucleation factors in generating distinct cellular structures. We also consider how the localization and orientation of actin nucleation factors, in addition to their kinetic properties, are critical to their ability to build a functional actin cytoskeleton.

  19. A method for rapidly screening functionality of actin mutants and tagged actins

    Directory of Open Access Journals (Sweden)

    Rommelaere Heidi

    2004-01-01

    Full Text Available Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three &bgr;-actin mutants that have been associated with diseases.

  20. Molecular dissection of the mechanisms of substrate recognition and F-actin-mediated activation of cofilin-phosphatase Slingshot-1.

    Science.gov (United States)

    Kurita, Souichi; Watanabe, Yosuke; Gunji, Emi; Ohashi, Kazumasa; Mizuno, Kensaku

    2008-11-21

    Slingshot-1 (SSH1), a member of a dual-specificity protein phosphatase family, regulates actin dynamics by dephosphorylating and reactivating cofilin, an actin-depolymerizing factor. SSH1 has the SSH family-specific, N-terminal, noncatalytic (SSH-N) domain, consisting of the A and B subdomains. SSH1 is activated by binding to actin filaments. In this study, we examined the mechanisms of SSH1 substrate recognition of phospho-cofilin (P-cofilin) and SSH1 activation by F-actin. We found that P-cofilin binds to a phosphatase-inactive mutant, SSH1(CS), in which the catalytic Cys-393 is replaced by Ser. Using a series of deletion mutants, we provided evidence that both the phosphatase (P) domain and the adjacent B domain are indispensable for P-cofilin binding of SSH1(CS) and cofilin-phosphatase activity of SSH1. In contrast, the A domain is required for the F-actin-mediated activation of SSH1, but not for P-cofilin binding or basal cofilin-phosphatase activity. The P domain alone is sufficient for the phosphatase activity toward p-nitrophenyl phosphate (pNPP), indicating that the SSH-N domain is not essential for the basal phosphatase activity of SSH1. Addition of F-actin increased the cofilin-phosphatase activity of SSH1 more than 1200-fold, but the pNPP-phosphatase activity only 2.2-fold, which suggests that F-actin principally affects the cofilin-specific phosphatase activity of SSH1. When expressed in cultured cells, SSH1, but not its mutant deleted of SSH-N, accumulated in the rear of the lamellipodium. Together, these findings suggest that the conserved SSH-N domain plays critical roles in P-cofilin recognition, F-actin-mediated activation, and subcellular localization of SSH1.

  1. Actin-based motility of Listeria: Right-handed helical trajectories

    Science.gov (United States)

    Rangarajan, Murali

    2012-06-01

    Bacteria such as Listeria monocytogenes recruit cellular machinery to move in and between cells. Understanding the mechanism of motility, including force and torque generation and the resultant displacements, holds keys to numerous applications in medicine and biosensing. In this work, a simple back-of-the-envelope calculation is presented to illustrate that a biomechanical model of actin-based motility of a rigid surface through persistently attached filaments propelled by affinity-modulated molecular motors can produce a right-handed helical trajectory consistent with experimental observations. The implications of the mechanism to bacterial motility are discussed.

  2. Buckling of Actin-Coated Membranes under Application of a Local Force

    Energy Technology Data Exchange (ETDEWEB)

    Helfer, E.; Harlepp, S.; Bourdieu, L.; Robert, J.; MacKintosh, F. C.; Chatenay, D.

    2001-08-20

    The mechanical properties of composite membranes obtained by self-assembly of actin filaments with giant fluid vesicles are studied by micromanipulation with optical tweezers. These complexes exhibit typical mechanical features of a solid shell, including a finite in-plane shear elastic modulus ({approx}10{sup -6} N /m). A buckling instability is observed when a localized force of the order of 0.5pN is applied perpendicular to the membrane plane. Although predicted for polymerized vesicles, this is the first evidence of such an instability.

  3. Aspects of Subcortical Ischaemic Vascular Disease : Early clinical manifestations and associations with Type 2 diabetes mellitus

    OpenAIRE

    Harten, van, B.

    2006-01-01

    Summary Subcortical ischaemic vascular disease (SIVD) is an important cause of cognitive impairment in elderly patients. Screening and diagnostic tests are needed to identify these patients. The HIV dementia scale (HDS) is a reliable and quantitative scale for identifying HIV dementia1. The cognitive profile of HIV dementia has subcortical features that resemble subcortical ischaemic vascular disease (SIVD). The clinical syndrome is characterized by early impairment of attention and executive...

  4. Nuclear Actin and Myosins in Adenovirus Infection

    Science.gov (United States)

    Fuchsova, Beata; Serebryannyy, Leonid A.; de Lanerolle, Primal

    2015-01-01

    Adenovirus serotypes have been shown to cause drastic changes in nuclear organization, including the transcription machinery, during infection. This ability of adenovirus to subvert transcription in the host cell facilitates viral replication. Because nuclear actin and nuclear myosin I, myosin V and myosin VI have been implicated as direct regulators of transcription and important factors in the replication of other viruses, we sought to determine how nuclear actin and myosins are involved in adenovirus infection. We first confirmed reorganization of the host’s transcription machinery to viral replication centers. We found that nuclear actin also reorganizes to sites of transcription through the intermediate but not the advanced late phase of viral infection. Furthermore, nuclear myosin I localized with nuclear actin and sites of transcription in viral replication centers. Intriguingly, nuclear myosins V and VI, which also reorganized to viral replication centers, exhibited different localization patterns, suggesting specialized roles for these nuclear myosins. Finally, we assessed the role of actin in adenovirus infection and found both cytoplasmic and nuclear actin likely play roles in adenovirus infection and replication. Together our data suggest the involvement of actin and multiple myosins in the nuclear replication and late viral gene expression of adenovirus. PMID:26226218

  5. Erbium laser resurfacing for actinic cheilitis.

    Science.gov (United States)

    Cohen, Joel L

    2013-11-01

    Actinic cheilitis is a precancerous condition characterized by grayish-whitish area(s) of discoloration on the mucosal lip, often blunting the demarcation between mucosa and cutaneous lip. Actinic cheilitis is considered to be an early part of the spectrum of squamous cell carcinoma. Squamous cell carcinoma specifically of the lip has a high rate of recurrence and metastasis through the oral cavity leading to a poor overall survival. Risk factors for the development of actinic cheilitis include chronic solar irradiation, increasing age, male gender, light skin complexion, immunosuppression, and possibly tobacco and alcohol consumption. Treatment options include topical pharmacotherapy (eg, fluorouracil, imiquimod) or procedural interven