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Sample records for actin filaments formed

  1. Bacterial actin MreB forms antiparallel double filaments

    PubMed Central

    van den Ent, Fusinita; Izoré, Thierry; Bharat, Tanmay AM; Johnson, Christopher M; Löwe, Jan

    2014-01-01

    Filaments of all actin-like proteins known to date are assembled from pairs of protofilaments that are arranged in a parallel fashion, generating polarity. In this study, we show that the prokaryotic actin homologue MreB forms pairs of protofilaments that adopt an antiparallel arrangement in vitro and in vivo. We provide an atomic view of antiparallel protofilaments of Caulobacter MreB as apparent from crystal structures. We show that a protofilament doublet is essential for MreB's function in cell shape maintenance and demonstrate by in vivo site-specific cross-linking the antiparallel orientation of MreB protofilaments in E. coli. 3D cryo-EM shows that pairs of protofilaments of Caulobacter MreB tightly bind to membranes. Crystal structures of different nucleotide and polymerisation states of Caulobacter MreB reveal conserved conformational changes accompanying antiparallel filament formation. Finally, the antimicrobial agents A22/MP265 are shown to bind close to the bound nucleotide of MreB, presumably preventing nucleotide hydrolysis and destabilising double protofilaments. DOI: http://dx.doi.org/10.7554/eLife.02634.001 PMID:24843005

  2. Cortical flow aligns actin filaments to form a furrow

    PubMed Central

    Reymann, Anne-Cecile; Staniscia, Fabio; Erzberger, Anna; Salbreux, Guillaume; Grill, Stephan W

    2016-01-01

    Cytokinesis in eukaryotic cells is often accompanied by actomyosin cortical flow. Over 30 years ago, Borisy and White proposed that cortical flow converging upon the cell equator compresses the actomyosin network to mechanically align actin filaments. However, actin filaments also align via search-and-capture, and to what extent compression by flow or active alignment drive furrow formation remains unclear. Here, we quantify the dynamical organization of actin filaments at the onset of ring assembly in the C. elegans zygote, and provide a framework for determining emergent actomyosin material parameters by the use of active nematic gel theory. We characterize flow-alignment coupling, and verify at a quantitative level that compression by flow drives ring formation. Finally, we find that active alignment enhances but is not required for ring formation. Our work characterizes the physical mechanisms of actomyosin ring formation and highlights the role of flow as a central organizer of actomyosin network architecture. DOI: http://dx.doi.org/10.7554/eLife.17807.001 PMID:27719759

  3. Structural complexity of filaments formed from the actin and tubulin folds

    PubMed Central

    Jiang, Shimin; Ghoshdastider, Umesh; Narita, Akihiro; Popp, David

    2016-01-01

    ABSTRACT From yeast to man, an evolutionary distance of 1.3 billion years, the F-actin filament structure has been conserved largely in line with the 94% sequence identity. The situation is entirely different in bacteria. In comparison to eukaryotic actins, the bacterial actin-like proteins (ALPs) show medium to low levels of sequence identity. This is extreme in the case of the ParM family of proteins, which often display less than 20% identity. ParMs are plasmid segregation proteins that form the polymerizing motors that propel pairs of plasmids to the extremities of a cell prior to cell division, ensuring faithful inheritance of the plasmid. Recently, exotic ParM filament structures have been elucidated that show ParM filament geometries are not limited to the standard polar pair of strands typified by actin. Four-stranded non-polar ParM filaments existing as open or closed nanotubules are found in Clostridium tetani and Bacillus thuringiensis, respectively. These diverse architectures indicate that the actin fold is capable of forming a large variety of filament morphologies, and that the conception of the “actin” filament has been heavily influenced by its conservation in eukaryotes. Here, we review the history of the structure determination of the eukaryotic actin filament to give a sense of context for the discovery of the new ParM filament structures. We describe the novel ParM geometries and predict that even more complex actin-like filaments may exist in bacteria. Finally, we compare the architectures of filaments arising from the actin and tubulin folds and conclude that the basic units possess similar properties that can each form a range of structures. Thus, the use of the actin fold in microfilaments and the tubulin fold for microtubules likely arose from a wider range of filament possibilities, but became entrenched as those architectures in early eukaryotes. PMID:28042378

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

    PubMed Central

    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

  5. Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation

    PubMed Central

    Jiang, Shimin; Narita, Akihiro; Popp, David; Ghoshdastider, Umesh; Lee, Lin Jie; Srinivasan, Ramanujam; Balasubramanian, Mohan K.; Oda, Toshiro; Koh, Fujiet; Larsson, Mårten; Robinson, Robert C.

    2016-01-01

    Here we report the discovery of a bacterial DNA-segregating actin-like protein (BtParM) from Bacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electron microscopy. The BtParM filament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. The BtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP. BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release by BtParM and paired two supercoiled BtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 Å and 145 Å, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosome-segregating microtubule. PMID:26873105

  6. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

    Siccardi, Stefano; Tuszynski, Jack A.; Adamatzky, Andrew

    2016-01-01

    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.

  7. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin.

    PubMed

    Chaudhry, Faisal; Breitsprecher, Dennis; Little, Kristin; Sharov, Grigory; Sokolova, Olga; Goode, Bruce L

    2013-01-01

    Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.

  8. Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation

    PubMed Central

    Periz, Javier; Whitelaw, Jamie; Harding, Clare; Gras, Simon; Del Rosario Minina, Mario Igor; Latorre-Barragan, Fernanda; Lemgruber, Leandro; Reimer, Madita Alice; Insall, Robert; Heaslip, Aoife; Meissner, Markus

    2017-01-01

    Apicomplexan actin is important during the parasite's life cycle. Its polymerization kinetics are unusual, permitting only short, unstable F-actin filaments. It has not been possible to study actin in vivo and so its physiological roles have remained obscure, leading to models distinct from conventional actin behaviour. Here a modified version of the commercially available actin-chromobody was tested as a novel tool for visualising F-actin dynamics in Toxoplasma gondii. Cb labels filamentous actin structures within the parasite cytosol and labels an extensive F-actin network that connects parasites within the parasitophorous vacuole and allows vesicles to be exchanged between parasites. In the absence of actin, parasites lack a residual body and inter-parasite connections and grow in an asynchronous and disorganized manner. Collectively, these data identify new roles for actin in the intracellular phase of the parasites lytic cycle and provide a robust new tool for imaging parasitic F-actin dynamics. DOI: http://dx.doi.org/10.7554/eLife.24119.001 PMID:28322189

  9. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    PubMed

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.

  10. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

    PubMed

    Niederman, R; Amrein, P C; Hartwig, J

    1983-05-01

    Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

  11. Self-assembly of Artificial Actin Filaments

    NASA Astrophysics Data System (ADS)

    Grosenick, Christopher; Cheng, Shengfeng

    Actin Filaments are long, double-helical biopolymers that make up the cytoskeleton along with microtubules and intermediate filaments. In order to further understand the self-assembly process of these biopolymers, a model to recreate actin filament geometry was developed. A monomer in the shape of a bent rod with vertical and lateral binding sites was designed to assemble into single or double helices. With Molecular Dynamics simulations, a variety of phases were observed to form by varying the strength of the binding sites. Ignoring lateral binding sites, we have found a narrow range of binding strengths that lead to long single helices via various growth pathways. When lateral binding strength is introduced, double helices begin to form. These double helices self-assemble into substantially more stable structures than their single helix counterparts. We have found double helices to form long filaments at about half the vertical binding strength of single helices. Surprisingly, we have found that triple helices occasionally form, indicating the importance of structural regulation in the self-assembly of biopolymers.

  12. Actin filament curvature biases branching direction

    NASA Astrophysics Data System (ADS)

    Wang, Evan; Risca, Viviana; Chaudhuri, Ovijit; Chia, Jia-Jun; Geissler, Phillip; Fletcher, Daniel

    2012-02-01

    Actin filaments are key components of the cellular machinery, vital for a wide range of processes ranging from cell motility to endocytosis. Actin filaments can branch, and essential in this process is a protein complex known as the Arp2/3 complex, which nucleate new ``daughter'' filaments from pre-existing ``mother'' filaments by attaching itself to the mother filament. Though much progress has been made in understanding the Arp2/3-actin junction, some very interesting questions remain. In particular, F-actin is a dynamic polymer that undergoes a wide range of fluctuations. Prior studies of the Arp2/3-actin junction provides a very static notion of Arp2/3 binding. The question we ask is how differently does the Arp2/3 complex interact with a straight filament compared to a bent filament? In this study, we used Monte Carlo simulations of a surface-tethered worm-like chain to explore possible mechanisms underlying the experimental observation that there exists preferential branch formation by the Arp2/3 complex on the convex face of a curved filament. We show that a fluctuation gating model in which Arp2/3 binding to the actin filament is dependent upon a rare high-local-curvature shape fluctuation of the filament is consistent with the experimental data.

  13. Force Generation, Polymerization Dynamics and Nucleation of Actin Filaments

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe

    We study force generation and actin filament dynamics using stochastic and deterministic methods. First, we treat force generation of bundled actin filaments by polymerization via molecular-level stochastic simulations. In the widely-used Brownian Ratchet model, actin filaments grow freely whenever the tip-obstacle gap created by thermal fluctuation exceeds the monomer size. We name this model the Perfect Brownian Ratchet (PBR) model. In the PBR model, actin monomer diffusion is treated implicitly. We perform a series of simulations based on the PBR, in which obstacle motion is treated explicitly; in most previous studies, obstacle motion has been treated implicitly. We find that the cooperativity of filaments is generally weak in the PBR model, meaning that more filaments would grow more slowly given the same force per filament. Closed-form formulas are also developed, which match the simulation results. These portable and accurate formulas provide guidance for experiments and upper and lower bounds for theoretical analyses. We also studied a variation of the PBR, called the Diffusing Brownian Ratchet (DBR) model, in which both actin monomer and obstacle diffusion are treated explicitly. We find that the growth rate of multiple filaments is even lower, compared with that in PBR. This finding challenges the widely-accepted PBR assumption and suggests that pushing the study of actin dynamics down to the sub-nanometer level yields new insights. We subsequently used a rate equation approach to model the effect of local depletion of actin monomers on the nucleation of actin filaments on biomimetic beads, and how the effect is regulated by capping protein (CP). We find that near the bead surface, a higher CP concentration increases local actin concentration, which leads to an enhanced activities of actin filaments' nucleation. Our model analysis matches the experimental results and lends support to an important but undervalued hypothesis proposed by Carlier and

  14. Reconstitution of a Minimal Actin Cortex by Coupling Actin Filaments to Reconstituted Membranes.

    PubMed

    Vogel, Sven K

    2016-01-01

    A thin layer of actin filaments in many eukaryotic cell types drives pivotal aspects of cell morphogenesis and is generally cited as the actin cortex. Myosin driven contractility and actin cytoskeleton membrane interactions form the basis of fundamental cellular processes such as cytokinesis, cell migration, and cortical flows. How the interplay between the actin cytoskeleton, the membrane, and actin binding proteins drives these processes is far from being understood. The complexity of the actin cortex in living cells and the hardly feasible manipulation of the omnipotent cellular key players, namely actin, myosin, and the membrane, are challenging in order to gain detailed insights about the underlying mechanisms. Recent progress in developing bottom-up in vitro systems where the actin cytoskeleton is combined with reconstituted membranes may provide a complementary route to reveal general principles underlying actin cortex properties. In this chapter the reconstitution of a minimal actin cortex by coupling actin filaments to a supported membrane is described. This minimal system may be very well suited to study for example protein interactions on membrane bound actin filaments in a very controlled and quantitative manner as it may be difficult to perform in living systems.

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

    PubMed

    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.

  16. Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites

    PubMed Central

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

    2011-01-01

    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. PMID:21998582

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

    SciTech Connect

    Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C.; Navaza, Jorge; Dominguez, Roberto

    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.

  18. Structure of a longitudinal actin dimer assembled by tandem w domains: implications for actin filament nucleation.

    PubMed

    Rebowski, Grzegorz; Namgoong, Suk; Boczkowska, Malgorzata; Leavis, Paul C; Navaza, Jorge; Dominguez, Roberto

    2010-10-15

    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 β4. 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 β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

  19. Demonstration of prominent actin filaments in the root columella

    NASA Technical Reports Server (NTRS)

    Collings, D. A.; Zsuppan, G.; Allen, N. S.; Blancaflor, E. B.; Brown, C. S. (Principal Investigator)

    2001-01-01

    The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

  20. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  1. Multiple crystal structures of actin dimers and their implications for interactions in the actin filament

    PubMed Central

    Sawaya, Michael R.; Kudryashov, D. S.; Pashkov, Inna; Adisetiyo, Helty; Reisler, Emil; Yeates, Todd O.

    2008-01-01

    The structure of actin in its monomeric form is known at high resolution, while the structure of filamentous F-actin is only understood at considerably lower resolution. Knowing pre­cisely how the monomers of actin fit together would lead to a deeper understanding of the dynamic behavior of the actin filament. Here, a series of crystal structures of actin dimers are reported which were prepared by cross-linking in either the longitudinal or the lateral direction in the filament state. Laterally cross-linked dimers, comprised of monomers belonging to different protofilaments, are found to adopt configurations in crystals that are not related to the native structure of filamentous actin. In contrast, multiple structures of longitudinal dimers consistently reveal the same interface between monomers within a single protofilament. The re­appearance of the same longitudinal interface in multiple crystal structures adds weight to arguments that the interface visualized is similar to that in actin filaments. Highly conserved atomic interactions involving residues 199–205 and 287–291 are highlighted. PMID:18391412

  2. Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss Physcomitrella patens.

    PubMed

    Yamashita, Hiroko; Sato, Yoshikatsu; Kanegae, Takeshi; Kagawa, Takatoshi; Wada, Masamitsu; Kadota, Akeo

    2011-02-01

    Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.

  3. Mechanism of Actin Filament Bundling by Fascin

    SciTech Connect

    Jansen, Silvia; Collins, Agnieszka; Yang, Changsong; Rebowski, Grzegorz; Svitkina, Tatyana; Dominguez, Roberto

    2013-03-07

    Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four {beta}-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in {beta}-trefoil domains 1 and 3. The site in {beta}-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in {beta}-trefoil-3 is related by pseudo-2-fold symmetry to that in {beta}-trefoil-1. The two sites are {approx}5 nm apart, resulting in a distance between actin filaments in the bundle of {approx}8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

  4. A Robust Actin Filaments Image Analysis Framework

    PubMed Central

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-01-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a ‘cartoon’ part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the ‘cartoon’ image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts

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

    PubMed Central

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

    2016-01-01

    A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. 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. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization. PMID:27760153

  6. Role of actin filaments in fusopod formation and osteoclastogenesis.

    PubMed

    Wang, Yongqiang; Brooks, Patricia Joyce; Jang, Janet Jinyoung; Silver, Alexandra Shade; Arora, Pamma D; McCulloch, Christopher A; Glogauer, Michael

    2015-07-01

    Cell fusion process is a critical, rate-limiting step in osteoclastogenesis but the mechanisms that regulate fusopod formation are not defined. We characterized fusopod generation in cultured pre-osteoclasts derived from cells stably transfected with a plasmid that expressed a short, actin filament binding peptide (Lifeact) fused to mEGFP that enables localization of actin filaments in living cells. Fusion was initiated at fusopods, which are cell extensions of width >2 μm and that are immunostained for myosin-X at the extension tips. Fusopods formed at the leading edge of larger migrating cells and from the tail of adjacent smaller cells, both of which migrated in the same direction. Staining for DC-STAMP was circumferential and did not localize to cell-cell fusion sites. Compared with wild-type cells, monocytes null for Rac1 exhibited 6-fold fewer fusopods and formed 4-fold fewer multinucleated osteoclasts. From time-lapse images we found that fusion was temporally related to the formation of coherent and spatially isolated bands of actin filaments that originated in cell bodies and extended into the fusopods. These bands of actin filaments were involved in cell fusion after approaching cells formed initial contacts. We conclude that the formation of fusopods is regulated by Rac1 to initiate intercellular contact during osteoclastogenesis. This step is followed by the tightly regulated assembly of bands of actin filaments in fusopods, which lead to closure of the intercellular gap and finally, cell fusion. These novel, actin-dependent processes are important for fusion processes in osteoclastogenesis.

  7. Formation and Destabilization of Actin Filaments with Tetramethylrhodamine-Modified Actin

    PubMed Central

    Kudryashov, Dmitry S.; Phillips, Martin; Reisler, Emil

    2004-01-01

    Actin labeling at Cys374 with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics. PMID:15298916

  8. Novel actin-like filament structure from Clostridium tetani.

    PubMed

    Popp, David; Narita, Akihiro; Lee, Lin Jie; Ghoshdastider, Umesh; Xue, Bo; Srinivasan, Ramanujam; Balasubramanian, Mohan K; Tanaka, Toshitsugu; Robinson, Robert C

    2012-06-15

    Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines.

  9. Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement.

    PubMed

    Wang, Xiu-Ling; Gao, Xin-Qi; Wang, Xue-Chen

    2011-08-01

    Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.

  10. Structural transition of actin filament in a cell-sized water droplet with a phospholipid membrane

    NASA Astrophysics Data System (ADS)

    Hase, M.; Yoshikawa, K.

    2006-03-01

    Actin filament, F-actin, is a semiflexible polymer with a negative charge, and is one of the main constituents of cell membranes. To clarify the effect of cross talk between a phospholipid membrane and actin filaments in cells, we conducted microscopic observations on the structural changes in actin filaments in a cell-sized (several tens of micrometers in diameter) water droplet coated with a phospholipid membrane such as phosphatidylserine (PS; negatively charged head group) or phosphatidylethanolamine (PE; neutral head group) as a simple model of a living cell membrane. With PS, actin filaments are distributed uniformly in the water phase without adsorption onto the membrane surface between 2 and 6mM Mg2+, while between 6 and 12mM Mg2+, actin filaments are adsorbed onto the inner membrane surface. With PE, the actin filaments are uniformly adsorbed onto the inner membrane surface between 2 and 12mM Mg2+. With both PS and PE membranes, at Mg2+ concentrations higher than 12mM, thick bundles are formed in the bulk water droplet accompanied by the dissolution of actin filaments from the membrane surface. The attraction between actin filaments and membrane is attributable to an increase in the translational entropy of counterions accompanied by the adsorption of actin filaments onto the membrane surface. These results suggest that a microscopic water droplet coated with phospholipid can serve as an easy-to-handle model of cell membranes.

  11. Aluminum modifies the viscosity of filamentous actin solutions as measured by optical displacement microviscometry.

    PubMed

    Arnoys, E J; Schindler, M

    2000-01-01

    A microtechnique has been developed that is capable of measuring the viscosity of filamentous actin (F-actin) solutions. This method, called optical displacement microviscometry (ODM), was utilized to determine the changes in viscosity of solutions of rabbit muscle, human platelet, and maize pollen actin when measured in the absence and presence of aluminum. Measurements demonstrated that the viscosity of the different actin solutions decreased with aluminum concentration. In contrast, increases in viscosity were observed when aluminum was added to F-actin solutions containing filamin (chicken gizzard), a protein that bundles actin filaments. Confocal fluorescence imaging of pure actin solutions in the presence of aluminum showed a disrupted actin network composed of fragmented actin filaments in the form of small aggregates. In contrast, in the presence of filamin, aluminum promoted the formation of thicker actin filaments. These measurements demonstrate that aluminum can affect actin filaments differentially depending on the presence of an actin-binding protein. In addition, a strong correlation is observed between the changes in viscosity as measured by ODM and the thickness and assembled state of bundles of actin filaments.

  12. Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments

    PubMed Central

    1995-01-01

    The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin- binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere. PMID:7822423

  13. Association of actin filaments with axonal microtubule tracts.

    PubMed

    Bearer, E L; Reese, T S

    1999-02-01

    Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 microm with some at least as long as 3.5 microm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.

  14. Addition of electrophilic lipids to actin alters filament structure

    SciTech Connect

    Gayarre, Javier; Sanchez, David; Sanchez-Gomez, Francisco J.; Terron, Maria C.; Llorca, Oscar; Perez-Sala, Dolores . E-mail: dperezsala@cib.csic.es

    2006-11-03

    Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-{delta}{sup 12,14}-PGJ{sub 2} (15d-PGJ{sub 2}) and PGA{sub 1} in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA{sub 1} and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ{sub 2} or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ{sub 2} at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles.

  15. Addition of electrophilic lipids to actin alters filament structure.

    PubMed

    Gayarre, Javier; Sánchez, David; Sánchez-Gómez, Francisco J; Terrón, María C; Llorca, Oscar; Pérez-Sala, Dolores

    2006-11-03

    Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and PGA(1) in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA(1) and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer-monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ(2) or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ(2) at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles.

  16. Regional orientation of actin filaments in the pericanalicular cytoplasm of rat hepatocytes.

    PubMed

    Ishii, M; Washioka, H; Tonosaki, A; Toyota, T

    1991-12-01

    To elucidate how actin filaments participate in bile formation, polarity of actin filaments in the pericanalicular cytoplasm was determined with myosin subfragment 1 by transmission electron microscopy of ultrathin sections and deep-etching replicas. Densely concentrated actin filaments were identified around the bile canaliculi in the forms of microvillous core filaments, pericanalicular web filaments, and filaments on the junctional complex. They bound subfragment 1 to form double-helical strands on the deep-etching replica or typical arrowheads on the ultrathin section. All microvillous core filaments showed their arrowheads pointing basally, suggesting the molecular growth occurring at their apical ends. In contrast, filaments of the pericanalicular web, running in parallel to the cell surface, showed unfixed polarities as indicated by their arrowheads. Furthermore, neighboring filament pairs often showed opposite polarities, an alignment necessary for filament sliding. The junctional complex had filaments with arrowheads pointed mostly at the cell center with a small number in opposite direction. In addition, a group of sporadic filaments appeared to be installed to link to both the canalicular membrane and coated vesicles. Such regionally specialized actin filaments are considered inclusively to form a cytoskeletal system that is in charge of (a) maintenance of length of the microvilli, (b) contraction of the canalicular walls, and (c) translocation of coated vesicles in the pericanalicular cytoplasm.

  17. Fission yeast IQGAP arranges actin filaments into the cytokinetic contractile ring

    PubMed Central

    Takaine, Masak; Numata, Osamu; Nakano, Kentaro

    2009-01-01

    The contractile ring (CR) consists of bundled actin filaments and myosin II; however, the actin-bundling factor remains elusive. We show that the fission yeast Schizosaccharomyces pombe IQGAP Rng2 is involved in the generation of CR F-actin and required for its arrangement into a ring. An N-terminal fragment of Rng2 is necessary for the function of Rng2 and is localized to CR F-actin. In vitro the fragment promotes actin polymerization and forms linear arrays of F-actin, which are resistant to the depolymerization induced by the actin-depolymerizing factor Adf1. Our findings indicate that Rng2 is involved in the generation of CR F-actin and simultaneously bundles the filaments and regulates its dynamics by counteracting the effects of Adf1, thus enabling the reconstruction of CR F-actin bundles, which provides an insight into the physical properties of the building blocks that comprise the CR. PMID:19713940

  18. Actin filaments as dynamic reservoirs for Drp1 recruitment

    PubMed Central

    Hatch, Anna L.; Ji, Wei-Ke; Merrill, Ronald A.; Strack, Stefan; Higgs, Henry N.

    2016-01-01

    Drp1 is a dynamin-family GTPase recruited to mitochondria and peroxisomes, where it oligomerizes and drives membrane fission. Regulation of mitochondrial Drp1 recruitment is not fully understood. We previously showed that Drp1 binds actin filaments directly, and actin polymerization is necessary for mitochondrial Drp1 oligomerization in mammals. Here we show the Drp1/actin interaction displays unusual properties that are influenced by several factors. At saturation, only a fraction Drp1 binds actin filaments, and the off-rate of actin-bound Drp1 is significantly increased by unbound Drp1. GDP and GTP accelerate and decelerate Drp1/actin binding dynamics, respectively. Actin has a biphasic effect on Drp1 GTP hydrolysis, increasing at low actin:Drp1 ratio but returning to baseline at high ratio. Drp1 also bundles filaments. Bundles have reduced dynamics but follow the same trends as single filaments. Drp1 preferentially incorporates into bundles at higher ionic strength. We measure Drp1 concentration to be ∼0.5 μM in U2OS cell cytosol, suggesting the actin-binding affinity measured here (Kd = 0.6 μM) is in the physiologically relevant range. The ability of Drp1 to bind actin filaments in a highly dynamic manner provides potential for actin filaments to serve as reservoirs of oligomerization-competent Drp1 that can be accessed for mitochondrial fission. PMID:27559132

  19. Geometrical and Mechanical Properties Control Actin Filament Organization

    PubMed Central

    Ennomani, Hajer; Théry, Manuel; Nedelec, Francois; Blanchoin, Laurent

    2015-01-01

    The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model. PMID:26016478

  20. Geometrical and mechanical properties control actin filament organization.

    PubMed

    Letort, Gaëlle; Politi, Antonio Z; Ennomani, Hajer; Théry, Manuel; Nedelec, Francois; Blanchoin, Laurent

    2015-05-01

    The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model.

  1. In vitro studies of actin filament and network dynamics

    PubMed Central

    Mullins, R Dyche; Hansen, Scott D

    2013-01-01

    Now that many genomes have been sequenced, a central concern of cell biology is to understand how the proteins they encode work together to create living matter. In vitro studies form an essential part of this program because understanding cellular functions of biological molecules often requires isolating them and reconstituting their activities. In particular, many elements of the actin cytoskeleton were first discovered by biochemical methods and their cellular functions deduced from in vitro experiments. We highlight recent advances that have come from in vitro studies, beginning with studies of actin filaments, and ending with multi-component reconstitutions of complex actin-based processes, including force-generation and cell spreading. We describe both scientific results and the technical innovations that made them possible. PMID:23267766

  2. Chlamydia trachomatis Tarp harbors distinct G and F actin binding domains that bundle actin filaments.

    PubMed

    Jiwani, Shahanawaz; Alvarado, Stephenie; Ohr, Ryan J; Romero, Adriana; Nguyen, Brenda; Jewett, Travis J

    2013-02-01

    All species of Chlamydia undergo a unique developmental cycle that transitions between extracellular and intracellular environments and requires the capacity to invade new cells for dissemination. A chlamydial protein called Tarp has been shown to nucleate actin in vitro and is implicated in bacterial entry into human cells. Colocalization studies of ectopically expressed enhanced green fluorescent protein (EGFP)-Tarp indicate that actin filament recruitment is restricted to the C-terminal half of the effector protein. Actin filaments are presumably associated with Tarp via an actin binding alpha helix that is also required for actin nucleation in vitro, but this has not been investigated. Tarp orthologs from C. pneumoniae, C. muridarum, and C. caviae harbor between 1 and 4 actin binding domains located in the C-terminal half of the protein, but C. trachomatis serovar L2 has only one characterized domain. In this work, we examined the effects of domain-specific mutations on actin filament colocalization with EGFP-Tarp. We now demonstrate that actin filament colocalization with Tarp is dependent on two novel F-actin binding domains that endow the Tarp effector with actin-bundling activity. Furthermore, Tarp-mediated actin bundling did not require actin nucleation, as the ability to bundle actin filaments was observed in mutant Tarp proteins deficient in actin nucleation. These data shed molecular insight on the complex cytoskeletal rearrangements required for C. trachomatis entry into host cells.

  3. Regulation of Sodium Channel Activity by Capping of Actin Filaments

    PubMed Central

    Shumilina, Ekaterina V.; Negulyaev, Yuri A.; Morachevskaya, Elena A.; Hinssen, Horst; Khaitlina, Sofia Yu

    2003-01-01

    Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of actin filaments and inactivated by G-actin under ionic conditions promoting rapid actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-actin, CapZ prevented actin-induced channel inactivation, and this effect occurred at CapZ/actin molar ratios from 1:5 to 1:100. When actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of actin filaments at the plasma membrane due to the modification of actin dynamics by CapZ. No effect on the channel activity was observed in response to F-actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that actin-capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells. PMID:12686620

  4. Mechanism of interaction of Dictyostelium severin with actin filaments

    PubMed Central

    1982-01-01

    Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2+ -dependent manner, was purified 500-fold to greater than 99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29 A and consists of a single polypeptide chain. It contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluoresence-labeled severin shows that this protein changes its conformation on binding Ca2+. Actin filaments are rapidly fragmented on addition of severin and Ca2+, but severin does not interact with actin filaments in the absence of Ca2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (t1/2 approximately equal to 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments with the disassembled actin pool (t1/2 approximately equal to 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin: severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo. PMID:6897549

  5. Direct Observation of Tropomyosin Binding to Actin Filaments

    PubMed Central

    Schmidt, William M.; Lehman, William; Moore, Jeffrey R.

    2015-01-01

    Tropomyosin is an elongated α-helical coiled-coil that binds to seven consecutive actin subunits along the long-pitch helix of actin filaments. Once bound, tropomyosin polymerizes end-to-end and both stabilizes F-actin and regulates access of various actin binding proteins including myosin in muscle and non-muscle cells. Single tropomyosin molecules bind weakly to F-actin with millimolar Kd, whereas the end-to-end linked tropomyosin associates with about a one thousand-fold greater affinity. Despite years of study, the assembly mechanism of tropomyosin onto actin filaments remains unclear. In the current study, we used total internal reflection fluorescence (TIRF) microscopy to directly monitor the cooperative binding of fluorescently labeled tropomyosin molecules to phalloidin-stabilized actin filaments. We find that tropomyosin molecules assemble from multiple growth sites following random low affinity binding of single molecules to actin. As the length of the tropomyosin chain increases, the probability of detachment decreases, which leads to further chain growth. Tropomyosin chain extension is linearly dependent on tropomyosin concentration, occurring at approximately 100 monomers/(μM*s). The random tropomyosin binding to F-actin leads to discontinuous end-to-end association where gaps in the chain continuity smaller than the required seven sequential actin monomers are available. Direct observation of tropomyosin detachment revealed the number of gaps in actin-bound tropomyosin, the time course of gap annealing, and the eventual filament saturation process. PMID:26033920

  6. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

    PubMed

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q P; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q P

    2016-10-20

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

  7. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin

    PubMed Central

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T.; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q. P.; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q. P.

    2016-01-01

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells. PMID:27762277

  8. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

    PubMed Central

    1996-01-01

    Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non- membrane-associated protein translation may be occurring in vivo. PMID:8947553

  9. Evidence for filamentous actin in ookinetes of a malarial parasite.

    PubMed

    Siden-Kiamos, Inga; Louis, Christos; Matuschewski, Kai

    2012-02-01

    Extracellular stages of apicomplexan parasites utilize their own actin myosin motor machinery for gliding locomotion, penetration of cell barriers, and host cell invasion. Thus far, filamentous actin could not be visualized by standard microscopic techniques in vivo. Here, we describe the generation of a novel peptide antibody against the divergent amino-terminal portion of the major Plasmodium isoform, actin I. We show that our antiserum, termed Ab-actinI-I, is conformation-specific. In motile ookinetes it recognizes actin in rod-like structures, which are sensitive to inhibitors interfering with actin polymerization. The average size of the rods is 600 nm, which is considerably longer than what has been detected in in vitro studies of actin filaments.

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

    PubMed Central

    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.

  11. Formation of actin filament bundles in the ring canals of developing Drosophila follicles

    PubMed Central

    1996-01-01

    Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay

  12. Mechanical Heterogeneity Favors Fragmentation of Strained Actin Filaments

    PubMed Central

    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

  13. Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity

    PubMed Central

    1992-01-01

    The organization and polarity of actin filaments in neuronal growth cones was studied with negative stain and freeze-etch EM using a permeabilization protocol that caused little detectable change in morphology when cultured nerve growth cones were observed by video- enhanced differential interference contrast microscopy. The lamellipodial actin cytoskeleton was composed of two distinct subpopulations: a population of 40-100-nm-wide filament bundles radiated from the leading edge, and a second population of branching short filaments filled the volume between the dorsal and ventral membrane surfaces. Together, the two populations formed the three- dimensional structural network seen within expanding lamellipodia. Interaction of the actin filaments with the ventral membrane surface occurred along the length of the filaments via membrane associated proteins. The long bundled filament population was primarily involved in these interactions. The filament tips of either population appeared to interact with the membrane only at the leading edge; this interaction was mediated by a globular Triton-insoluble material. Actin filament polarity was determined by decoration with myosin S1 or heavy meromyosin. Previous reports have suggested that the polarity of the actin filaments in motile cells is uniform, with the barbed ends toward the leading edge. We observed that the actin filament polarity within growth cone lamellipodia is not uniform; although the predominant orientation was with the barbed end toward the leading edge (47-56%), 22-25% of the filaments had the opposite orientation with their pointed ends toward the leading edge, and 19-31% ran parallel to the leading edge. The two actin filament populations display distinct polarity profiles: the longer filaments appear to be oriented predominantly with their barbed ends toward the leading edge, whereas the short filaments appear to be randomly oriented. The different length, organization and polarity of the two filament

  14. Contractile properties of thin (actin) filament-reconstituted muscle fibers.

    PubMed

    Ishiwata, S; Funatsu, T; Fujita, H

    1998-01-01

    Selective removal and reconstitution of the components of muscle fibers (fibrils) is a useful means of examining the molecular mechanism underlying the formation of the contractile apparatus. In addition, this approach is powerful for examining the structure-function relationship of a specific component of the contractile system. In previous studies, we have achieved the partial structural and functional reconstitution of thin filaments in the skeletal contractile apparatus and full reconstitution in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with plasma gelsolin, an actin filament-severing protein. Under these conditions, no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted by polymerization on the short actin fragments remaining at the Z line, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached as high as 30% of the original level in skeletal muscle, while it reached 140% in cardiac muscle. The augmentation of tension in cardiac muscle is mainly attributable to the elongation of reconstituted filaments, longer than the average length of thin filaments in an intact muscle. These results indicate that a muscle contractile apparatus with a high order structure and function can be constructed by the self-assembly of constituent proteins. Recently, we applied this reconstitution system to the study of the mechanism of spontaneous oscillatory contraction (SPOC) in thin (actin) filament-reconstituted cardiac muscle fibers. As a result, we found that SPOC occurs even in regulatory protein-free actin filament-reconstituted fibers (Fujita & Ishiwata, manuscript submitted), although the SPOC conditions were slightly different from the standard SPOC conditions. This result strongly suggests that spontaneous oscillation

  15. Clamped-filament elongation model for actin-based motors.

    PubMed Central

    Dickinson, Richard B; Purich, Daniel L

    2002-01-01

    Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility. PMID:11806905

  16. Actin Filament Stress Fibers in Vascular Endothelial Cells in vivo

    NASA Astrophysics Data System (ADS)

    Wong, Albert J.; Pollard, Thomas D.; Herman, Ira M.

    1983-02-01

    Fluorescence microscopy with 7-nitrobenz-2-oxa-3-diazole phallacidin was used to survey vertebrate tissues for actin filament bundles comparable to the stress fibers of cultured cells. Such bundles were found only in vascular endothelial cells. Like the stress fibers of cultured cells, these actin filament bundles were stained in a punctate pattern by fluorescent antibodies to both alpha-actinin and myosin. The stress fibers were oriented parallel to the direction of blood flow and were prominent in endothelial cells from regions exposed to high-velocity flow, such as the left ventricle, aortic valve, and aorta. Actin bundles may help the endothelial cell to withstand hemodynamic stress.

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

    PubMed

    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.

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

    PubMed Central

    Jasnin, Marion; Crevenna, Alvaro H.

    2016-01-01

    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. PMID:26497103

  19. Cofilin cooperates with fascin to disassemble filopodial actin filaments

    PubMed Central

    Breitsprecher, Dennis; Koestler, Stefan A.; Chizhov, Igor; Nemethova, Maria; Mueller, Jan; Goode, Bruce L.; Small, J. Victor; Rottner, Klemens; Faix, Jan

    2011-01-01

    Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin. PMID:21940796

  20. Functional synergy of actin filament cross-linking proteins.

    PubMed

    Tseng, Yiider; Schafer, Benjamin W; Almo, Steven C; Wirtz, Denis

    2002-07-12

    The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.

  1. Correlative nanoscale imaging of actin filaments and their complexes

    NASA Astrophysics Data System (ADS)

    Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E.; Reisler, Emil; Gimzewski, James K.

    2013-06-01

    Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

  2. F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments

    PubMed Central

    1996-01-01

    The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper. PMID:8947552

  3. The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model

    PubMed Central

    1991-01-01

    Three-dimensional (3-D) helical reconstructions computed from electron micrographs of negatively stained dispersed F-actin filaments invariably revealed two uninterrupted columns of mass forming the "backbone" of the double-helical filament. The contact between neighboring subunits along the thus defined two long-pitch helical strands was spatially conserved and of high mass density, while the intersubunit contact between them was of lower mass density and varied among reconstructions. In contrast, phalloidinstabilized F-actin filaments displayed higher and spatially more conserved mass density between the two long-pitch helical strands, suggesting that this bicyclic hepta-peptide toxin strengthens the intersubunit contact between the two strands. Consistent with this distinct intersubunit bonding pattern, the two long-pitch helical strands of unstabilized filaments were sometimes observed separated from each other over a distance of two to six subunits, suggesting that the intrastrand intersubunit contact is also physically stronger than the interstrand contact. The resolution of the filament reconstructions, extending to 2.5 nm axially and radially, enabled us to reproducibly "cut out" the F- actin subunit which measured 5.5 nm axially by 6.0 nm tangentially by 3.2 nm radially. The subunit is distinctly polar with a massive "base" pointing towards the "barbed" end of the filament, and a slender "tip" defining its "pointed" end (i.e., relative to the "arrowhead" pattern revealed after stoichiometric decoration of the filaments with myosin subfragment 1). Concavities running approximately parallel to the filament axis both on the inner and outer face of the subunit define a distinct cleft separating the subunit into two domains of similar size: an inner domain confined to radii less than or equal to 2.5-nm forms the uninterrupted backbone of the two long-pitch helical strands, and an outer domain placed at radii of 2-5-nm protrudes radially and thus predominantly

  4. The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

    PubMed Central

    Bane, Kartik S.; Singer, Mirko; Reinig, Miriam; Klug, Dennis; Heiss, Kirsten; Baum, Jake; Mueller, Ann-Kristin; Frischknecht, Friedrich

    2016-01-01

    Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics. PMID:27409081

  5. Regulation of actin filament length in erythrocytes and striated muscle.

    PubMed

    Fowler, V M

    1996-02-01

    Actin filaments polymerize in vitro to lengths which display an exponential distribution, yet in many highly differentiated cells they can be precisely maintained at uniform lengths in elaborate supramolecular structures. Recent results obtained using two classic model systems, the erythrocyte membrane cytoskeleton and the striated muscle sarcomere, reveal surprising similarities and instructive differences in the molecules and mechanisms responsible for determining and maintaining actin filament lengths in these two systems. Tropomodulin caps the slow-growing, pointed filament ends in muscle and in erythrocytes. CapZ caps the fast-growing, barbed filament ends in striated muscle, whereas a newly discovered barbed end capping protein, adducin, may cap the barbed filament ends in erythrocytes. The mechanisms responsible for specifying the characteristic filament lengths in these systems are more elusive and may include strict control of the relative amounts of actin filament capping proteins and side-binding proteins, molecular templates (e.g. tropomyosin and nebulin) and/or verniers (e.g. tropomyosin).

  6. Accelerated actin filament polymerization from microtubule plus-ends

    PubMed Central

    Henty-Ridilla, Jessica L.; Rankova, Aneliya; Eskin, Julian A.; Kenny, Katelyn; Goode, Bruce L.

    2016-01-01

    Microtubules govern actin network remodeling in a wide range of biological processes, yet the mechanisms underlying this cytoskeletal crosstalk have remained obscure. Here we used single-molecule fluorescence microscopy to show that the microtubule plus-end associated protein CLIP-170 binds tightly to formins to accelerate actin filament elongation. Furthermore, we observed mDia1 dimers and CLIP-170 dimers co-tracking growing filament ends for minutes. CLIP-170-mDia1 complexes promoted actin polymerization approximately 18 times faster than free barbed end growth, while simultaneously enhancing protection from capping protein. We used a microtubule-actin dynamics co-reconstitution system to observe CLIP-170-mDia1 complexes being recruited to growing microtubule ends by EB1. The complexes triggered rapid growth of actin filaments that remained attached to the microtubule surface. These activities of CLIP-170 were required in primary neurons for normal dendritic morphology. Thus, our results reveal a cellular mechanism whereby growing microtubule plus-ends direct rapid actin assembly. PMID:27199431

  7. Liquid crystal domains and thixotropy of filamentous actin suspensions.

    PubMed

    Kerst, A; Chmielewski, C; Livesay, C; Buxbaum, R E; Heidemann, S R

    1990-06-01

    The thixotropic properties of filamentous actin suspensions were examined by a step-function shearing protocol. Samples of purified filamentous actin were sheared at 0.2 sec-1 in a cone and plate rheometer. We noted a sharp stress overshoot upon the initiation of shear, indicative of a gel state, and a nearly instantaneous drop to zero stress upon cessation of shear. Stress-overshoot recovery was almost complete after 5 min of "rest" before samples were again sheared at 0.2 sec-1. Overshoot recovery increased linearly with the square root of rest time, suggesting that gel-state recovery is diffusion limited. Actin suspensions subjected to oscillatory shearing at frequencies from 0.003 to 30 radians/sec confirmed the existence of a 5-min time scale in the gel, similar to that for stress-overshoot recovery. Flow of filamentous actin was visualized by polarized light observations. Actin from 6 mg/ml to 20 mg/ml showed the "polycrystalline" texture of birefringence typical for liquid crystal structure. At shear rates less than 1 sec-1, flow occurred by the relative movement of irregular, roughly ellipsoidal actin domains 40-140 microns long; the appearance was similar to moving ice floes. At shear rates greater than 1 sec-1, domains decreased in size, possibly by frictional interactions among domains. Eventually domains flow in a "river" of actin aligned by the flow. Our observations confirm our previous domain-friction model for actin rheology. The similarities between the unusual flow properties of actin and cytoplasm argue that cytoplasm also may flow as domains.

  8. Probing the sliding interactions between bundled actin filaments

    NASA Astrophysics Data System (ADS)

    Ward, Andy; Dogic, Zvonimir

    2011-03-01

    Assemblies of filamentous biopolymers are hierarchical materials in which the properties of the overall assemblage are determined by structure and interactions between constituent particles at all hierarchical levels. For example, the overall bending rigidity of a two bundled filaments greatly depends on the bending rigidity of, and the adhesion strength between individual filaments. However, another property of importance is the ability for the filaments to slide freely against one another. Everyday experience indicates that it is much easier to bend a stack of papers in which individual sheets freely slide past each other than the same stack of papers in which all the sheets are irreversibly glued together. Similarly, in filamentous structures the ability for local re-arrangement is of the utmost importance in determining the properties of the structures observed. In order to study this phenomenon we create bundles of biopolymers by inducing attractive interactions between actin filaments via the depletion mechanism. We find that bundles of actin filaments to do not slide freely across one another. In order to characterize these sliding interactions, we perform active experiments using laser tweezers to pull one filament across the other at constant velocity.

  9. MARCKS actin-binding capacity mediates actin filament assembly during mitosis in human hepatic stellate cells.

    PubMed

    Rombouts, Krista; Mello, Tommaso; Liotta, Francesco; Galli, Andrea; Caligiuri, Alessandra; Annunziato, Francesco; Pinzani, Massimo

    2012-08-15

    Cross-linking between the actin cytoskeleton and plasma membrane actin-binding proteins is a key interaction responsible for the mechanical properties of the mitotic cell. Little is known about the identity, the localization, and the function of actin filament-binding proteins during mitosis in human hepatic stellate cells (hHSC). The aim of the present study was to identify and analyze the cross talk between actin and myristoylated alanine-rich kinase C substrate (MARCKS), an important PKC substrate and actin filament-binding protein, during mitosis in primary hHSC. Confocal analysis and chromosomal fraction analysis of mitotic hHSC demonstrated that phosphorylated (P)-MARCKS displays distinct phase-dependent localizations, accumulates at the perichromosomal layer, and is a centrosomal protein belonging to the chromosomal cytosolic fraction. Aurora B kinase (AUBK), an important mitotic regulator, β-actin, and P-MARCKS concentrate at the cytokinetic midbody during cleavage furrow formation. This localization is critical since MARCKS-depletion in hHSC is characterized by a significant loss in cytosolic actin filaments and cortical β-actin that induces cell cycle inhibition and dislocation of AUBK. A depletion of AUBK in hHSC affects cell cycle, resulting in multinucleation. Quantitative live cell imaging demonstrates that the actin filament-binding capacity of MARCKS is key to regulate mitosis since the cell cycle inhibitory effect in MARCKS-depleted cells caused abnormal cell morphology and an aberrant cytokinesis, resulting in a significant increase in cell cycle time. These findings implicate that MARCKS, an important PKC substrate, is essential for proper cytokinesis and that MARCKS and its partner actin are key mitotic regulators during cell cycle in hHSC.

  10. Diffusing wave spectroscopy microrheology of actin filament networks.

    PubMed Central

    Palmer, A; Xu, J; Kuo, S C; Wirtz, D

    1999-01-01

    Filamentous actin (F-actin), one of the constituents of the cytoskeleton, is believed to be the most important participant in the motion and mechanical integrity of eukaryotic cells. Traditionally, the viscoelastic moduli of F-actin networks have been measured by imposing a small mechanical strain and quantifying the resulting stress. The magnitude of the viscoelastic moduli, their concentration dependence and strain dependence, as well as the viscoelastic nature (solid-like or liquid-like) of networks of uncross-linked F-actin, have been the subjects of debate. Although this paper helps to resolve the debate and establishes the extent of the linear regime of F-actin networks' rheology, we report novel measurements of the high-frequency behavior of networks of F-actin, using a noninvasive light-scattering based technique, diffusing wave spectroscopy (DWS). Because no external strain is applied, our optical assay generates measurements of the mechanical properties of F-actin networks that avoid many ambiguities inherent in mechanical measurements. We observe that the elastic modulus has a small magnitude, no strain dependence, and a weak concentration dependence. Therefore, F-actin alone is not sufficient to generate the elastic modulus necessary to sustain the structural rigidity of most cells or support new cellular protrusions. Unlike previous studies, our measurements show that the mechanical properties of F-actin are highly dependent on the frequency content of the deformation. We show that the loss modulus unexpectedly dominates the elastic modulus at high frequencies, which are key for fast transitions. Finally, the measured mean square displacement of the optical probes, which is also generated by DWS measurements, offers new insight into the local bending fluctuations of the individual actin filaments and shows how they generate enhanced dissipation at short time scales. PMID:9916038

  11. The dynamics of semiflexible actin filaments in simple shear flow

    NASA Astrophysics Data System (ADS)

    Liu, Yanan; Lindner, Anke; Du Roure, Olivia

    2016-11-01

    The rheological properties of complex fluids made of particles in a suspended fluid depend on the behavior of microscopic particles in flow. A first step to understand this link is to investigate the individual particle dynamics in simple shear flows. A rigid rod will perform so-called Jeffery orbits, however when the rod becomes flexible and Brownian, the behavior in terms of deformation and migration is still to be fully understood. We chose here to address this situation by studying experimentally the behavior of semiflexible polymers. We use actin filaments and combine fluorescent labeling techniques, microfluidic devices to carry out controlled systematical experiments. Different dynamics are observed as a function of the elasto-viscous number, comparing viscous forces to elastic restoring forces ζ = (8 πηγ˙L4) / (LpkB T) . The bending modulus of the actin filaments is given by its persistence length Lp = 17 +/- 1 μm . When increasing the elasto-visous number we subsequently observe tumbling, buckling, and bending under flow. Those observations seem to be in good agreement with recent numerical simulations. At the same time, actin filaments fluctuate due to Brownian motion and these fluctuations can modify the individual dynamics of actin filaments. ERC PaDy No.682367.

  12. Actin- and myosin-like filaments in rat brain pericytes.

    PubMed

    Le Beux, Y J; Willemot, J

    1978-04-01

    Heavy meromyosin (HMM) labeling was used to identify the nature of the filaments which form bundles in the cytoplasm of the pericytes in brain tissue. Rat brain tissue pieces were incubated in glycerol solutions at 4 degrees and then transferred into buffer (pH 7.0), (1) without HMM, (2) with HMM, (3) with HMM + 5 mM ATP, and (4) with HMM + 2.5 mM Na+ pyrophosphate. In pericytes from untreated tissue, smooth-surfaced microfilaments, averaging 6 nm in diameter, appear to branch and anastomose and to anchor on the plasma membrane. After exposure to HMM, the number and the density of the microfilaments are strikingly increased. These tightly-packed microfilaments are now heavily coated with exogeneous HMM thus increasing in width to 18-20 mm. They intertwine in closely-woven networks. After incubation in HMM solutions containing ATP or Na+ phosphate, they are no longer coated with thick sidearms. It can thus be concluded that these microfilaments are of actin-like nature. In addition, after incubation in ATP, they are intermingled with, and converge onto the surfaces of, thick, tapered filaments, which we have tentatively identified as of myosin-like nature. Thus, it appears that certain of the major elements necessary for contraction are present in brain pericytes.

  13. Single-filament kinetic studies provide novel insights into regulation of actin-based motility

    PubMed Central

    Shekhar, Shashank; Carlier, Marie-France

    2016-01-01

    Polarized assembly of actin filaments forms the basis of actin-based motility and is regulated both spatially and temporally. Cells use a variety of mechanisms by which intrinsically slower processes are accelerated, and faster ones decelerated, to match rates observed in vivo. Here we discuss how kinetic studies of individual reactions and cycles that drive actin remodeling have provided a mechanistic and quantitative understanding of such processes. We specifically consider key barbed-end regulators such as capping protein and formins as illustrative examples. We compare and contrast different kinetic approaches, such as the traditional pyrene-polymerization bulk assays, as well as more recently developed single-filament and single-molecule imaging approaches. Recent development of novel biophysical methods for sensing and applying forces will in future allow us to address the very important relationship between mechanical stimulus and kinetics of actin-based motility. PMID:26715420

  14. Liquid-like bundles of crosslinked actin filaments contract without motors

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly

    The actin cytoskeleton is a dynamic, structural material that drives cellular-scale deformations during processes such as cell migration and division. Motor proteins are responsible for actively driving many deformations by buckling and translocating actin filaments. However, there is evidence that deformations, such as the constriction of the actin bundle that drives the separation of cells during division, can occur without motors, mediated instead by crosslinker proteins. How might crosslinkers, independent of motors, drive contraction of a bundle? Using a model system of purified proteins, we show that crosslinkers, analogous to molecular cohesion, create an effective surface tension that induces bundle contraction. Crosslinked short actin filaments form micron-sized spindle-shaped bundles. Similar to tactoid granules found at the isotropic-nematic phase transition in liquid crystals, these bundles coarsen and coalesce like liquid droplets. In contrast, crosslinked long filaments coarsen into a steady state of bundles that are frozen in a solid-like network. Near the liquid-solid boundary, filaments of intermediate length initially form bundles that spontaneously contract into tactoid droplets. Our results, that crosslinked actin bundles are liquid-like with an effective surface tension, provide evidence for a mechanism of motor-independent contractility in biological materials.

  15. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

    PubMed Central

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. DOI: http://dx.doi.org/10.7554/eLife.06585.001 PMID:26295568

  16. Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy

    PubMed Central

    Ngo, Kien Xuan; Kodera, Noriyuki; Katayama, Eisaku; Ando, Toshio; Uyeda, Taro QP

    2015-01-01

    High-speed atomic force microscopy was employed to observe structural changes in actin filaments induced by cofilin binding. Consistent with previous electron and fluorescence microscopic studies, cofilin formed clusters along actin filaments, where the filaments were 2-nm thicker and the helical pitch was ∼25% shorter, compared to control filaments. Interestingly, the shortened helical pitch was propagated to the neighboring bare zone on the pointed-end side of the cluster, while the pitch on the barbed-end side was similar to the control. Thus, cofilin clusters induce distinctively asymmetric conformational changes in filaments. Consistent with the idea that cofilin favors actin structures with a shorter helical pitch, cofilin clusters grew unidirectionally toward the pointed-end of the filament. Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries. DOI: http://dx.doi.org/10.7554/eLife.04806.001 PMID:25642645

  17. Transportation of nanoscale cargoes by myosin propelled actin filaments.

    PubMed

    Persson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A Michael; Månsson, 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. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached") or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 µm) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10-50 streptavidin molecules, 1-10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.

  18. Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments.

    PubMed

    Beach, Jordan R; Bruun, Kyle S; Shao, Lin; Li, Dong; Swider, Zac; Remmert, Kirsten; Zhang, Yingfan; Conti, Mary A; Adelstein, Robert S; Rusan, Nasser M; Betzig, Eric; Hammer, John A

    2017-02-01

    The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

  19. Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear

    PubMed Central

    1982-01-01

    Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed. PMID:6890555

  20. Nano-assembly of nanodiamonds by conjugation to actin filaments.

    PubMed

    Bradac, Carlo; Say, Jana M; Rastogi, Ishan D; Cordina, Nicole M; Volz, Thomas; Brown, Louise J

    2016-03-01

    Fluorescent nanodiamonds (NDs) are remarkable objects. They possess unique mechanical and optical properties combined with high surface areas and controllable surface reactivity. They are non-toxic and hence suited for use in biological environments. NDs are also readily available and commercially inexpensive. Here, the exceptional capability of controlling and tailoring their surface chemistry is demonstrated. Small, bright diamond nanocrystals (size ˜30 nm) are conjugated to protein filaments of actin (length ˜3-7 µm). The conjugation to actin filaments is extremely selective and highly target-specific. These unique features, together with the relative simplicity of the conjugation-targeting method, make functionalised nanodiamonds a powerful and versatile platform in biomedicine and quantum nanotechnologies. Applications ranging from using NDs as superior biological markers to, potentially, developing novel bottom-up approaches for the fabrication of hybrid quantum devices that would bridge across the bio/solid-state interface are presented and discussed.

  1. Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles

    PubMed Central

    1995-01-01

    The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of uniform width. The spots and ribbons initially behaved as distinct structural entities but subsequently aligned with each other in a sarcomeric-like pattern. Electron microscopy established that the spots and ribbons consisted of bipolar minifilaments associated with each other at their head-containing ends and arranged in a single row in an "open" zig-zag conformation or as a "closed" parallel stack. Ribbons also contacted each other in a nonsarcomeric, network-like arrangement as described previously (Verkhovsky and Borisy, 1993. J. Cell Biol. 123:637-652). Myosin ribbons were particularly pronounced in REF 52 cells, but small ribbons and networks were found also in a range of other mammalian cells. At the edge of the cell, individual spots and open ribbons were associated with relatively disordered actin filaments. Further from the edge, myosin filament alignment increased in parallel with the development of actin bundles. In actin bundles, the actin cross-linking protein, alpha-actinin, was excluded from sites of myosin localization but concentrated in paired sites flanking each myosin ribbon, suggesting that myosin filament association may initiate a pathway for the formation of actin filament bundles. We propose that zig-zag assemblies of myosin II filaments induce the formation of actin bundles by pulling on an actin filament network and that co-alignment of actin and myosin filaments proceeds via folding of myosin II filament assemblies in an accordion-like fashion. PMID:7490299

  2. DNA Double-Strand Breaks Induce the Nuclear Actin Filaments Formation in Cumulus-Enclosed Oocytes but Not in Denuded Oocytes

    PubMed Central

    Sun, Ming-Hong; Yang, Mo; Xie, Feng-Yun; Wang, Wei; Zhang, Lili; Shen, Wei; Yin, Shen

    2017-01-01

    As a gamete, oocyte needs to maintain its genomic integrity and passes this haploid genome to the next generation. However, fully-grown mouse oocyte cannot respond to DNA double-strand breaks (DSBs) effectively and it is also unable to repair them before the meiosis resumption. To compensate for this disadvantage and control the DNA repair events, oocyte needs the cooperation with its surrounding cumulus cells. Recently, evidences have shown that nuclear actin filament formation plays roles in cellular DNA DSB repair. To explore whether these nuclear actin filaments are formed in the DNA-damaged oocytes, here, we labeled the filament actins in denuded oocytes (DOs) and cumulus-enclosed oocytes (CEOs). We observed that the nuclear actin filaments were formed only in the DNA-damaged CEOs, but not in DOs. Formation of actin filaments in the nucleus was an event downstream to the DNA damage response. Our data also showed that the removal of cumulus cells led to a reduction in the nuclear actin filaments in oocytes. Knocking down of the Adcy1 gene in cumulus cells did not affect the formation of nuclear actin filaments in oocytes. Notably, we also observed that the nuclear actin filaments in CEOs could be induced by inhibition of gap junctions. From our results, it was confirmed that DNA DSBs induce the nuclear actin filament formation in oocyte and which is controlled by the cumulus cells. PMID:28099474

  3. Force-Velocity Measurements of a Few Growing Actin Filaments

    PubMed Central

    Brangbour, Coraline; du Roure, Olivia; Helfer, Emmanuèle; Démoulin, Damien; Mazurier, Alexis; Fermigier, Marc; Carlier, Marie-France; Bibette, Jérôme; Baudry, Jean

    2011-01-01

    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. PMID:21541364

  4. Actin filaments growing against a barrier with fluctuating shape

    NASA Astrophysics Data System (ADS)

    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.

  5. Star-forming Filament Models

    NASA Astrophysics Data System (ADS)

    Myers, Philip C.

    2017-03-01

    New models of star-forming filamentary clouds are presented in order to quantify their properties and to predict their evolution. These 2D axisymmetric models describe filaments that have no core, one low-mass core, and one cluster-forming core. They are based on Plummer-like cylinders and spheroids that are bounded by a constant-density surface of finite extent. In contrast to 1D Plummer-like models, they have specific values of length and mass, they approximate observed column density maps, and their distributions of column density (N-pdfs) are pole-free. Each model can estimate the star-forming potential of a core-filament system by identifying the zone of gas dense enough to form low-mass stars and by counting the number of enclosed thermal Jeans masses. This analysis suggests that the Musca central filament may be near the start of its star-forming life, with enough dense gas to make its first ∼3 protostars, while the Coronet filament is near the midpoint of its star formation, with enough dense gas to add ∼8 protostars to its ∼20 known stars. In contrast, L43 appears to be near the end of its star-forming life, since it lacks enough dense gas to add any new protostars to the two young stellar objectsalready known.

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

    PubMed Central

    Simon, Dan N; Zastrow, Michael S

    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

  7. Arabidopsis FIM5 decorates apical actin filaments and regulates their organization in the pollen tube

    PubMed Central

    Zhang, Meng; Zhang, Ruihui; Qu, Xiaolu; Huang, Shanjin

    2016-01-01

    The actin cytoskeleton is increasingly recognized as a major regulator of pollen tube growth. Actin filaments have distinct distribution patterns and dynamic properties within different regions of the pollen tube. Apical actin filaments are highly dynamic and crucial for pollen tube growth. However, how apical actin filaments are generated and properly constructed remains an open question. Here we showed that Arabidopsis fimbrin5 (FIM5) decorates filamentous structures throughout the entire tube but is apically concentrated. Apical actin structures are disorganized to different degrees in the pollen tubes of fim5 loss-of-function mutants. Further observations suggest that apical actin structures are not constructed properly because apical actin filaments cannot be maintained at the cortex of fim5 pollen tubes. Actin filaments appeared to be more curved in fim5 pollen tubes and this was confirmed by measurements showing that the convolutedness and the rate of change of convolutedness of actin filaments was significantly increased in fim5 pollen tubes. This suggests that the rigidity of the actin filaments may be compromised in fim5 pollen tubes. Further, the apical cell wall composition is altered, implying that tip-directed vesicle trafficking events are impaired in fim5 pollen tubes. Thus, we found that FIM5 decorates apical actin filaments and regulates their organization in order to drive polarized pollen tube growth. PMID:27117336

  8. SWAP-70 identifies a transitional subset of actin filaments in motile cells.

    PubMed

    Hilpelä, Pirta; Oberbanscheidt, Pia; Hahne, Penelope; Hund, Martin; Kalhammer, Georg; Small, J Victor; Bähler, Martin

    2003-08-01

    Functionally different subsets of actin filament arrays contribute to cellular organization and motility. We report the identification of a novel subset of loose actin filament arrays through regulated association with the widely expressed protein SWAP-70. These loose actin filament arrays were commonly located behind protruding lamellipodia and membrane ruffles. Visualization of these loose actin filament arrays was dependent on lamellipodial protrusion and the binding of the SWAP-70 PH-domain to a 3'-phosphoinositide. SWAP-70 with a functional pleckstrin homology-domain lacking the C-terminal 60 residues was targeted to the area of the loose actin filament arrays, but it did not associate with actin filaments. The C-terminal 60 residues were sufficient for actin filament association, but they provided no specificity for the subset of loose actin filament arrays. These results identify SWAP-70 as a phosphoinositide 3-kinase signaling-dependent marker for a distinct, hitherto unrecognized, array of actin filaments. Overexpression of SWAP-70 altered the actin organization and lamellipodial morphology. These alterations were dependent on a proper subcellular targeting of SWAP-70. We propose that SWAP-70 regulates the actin cytoskeleton as an effector or adaptor protein in response to agonist stimulated phosphatidylinositol (3,4)-bisphosphate production and cell protrusion.

  9. Actin-filament disassembly: it takes two to shrink them fast.

    PubMed

    Winterhoff, Moritz; Faix, Jan

    2015-06-01

    Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level.

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

    PubMed Central

    Appaduray, Mark A.; Masedunskas, Andrius; Lucas, Christine A.; Warren, Sean C.; Timpson, Paul; Stear, Jeffrey H.

    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. PMID:27977753

  11. Multiple supramolecular structures formed by interaction of actin with protamine

    PubMed Central

    Grazi, Enrico; Magri, Ermes; Pasquali-Ronchetti, Ivonne

    1982-01-01

    When protamine is added to actin, different supramolecular structures are formed depending on the molar ratio of the two proteins and of the ionic strength of the medium. At low ionic strength, and going from a molar ratio of protamine to G-actin of 4:1, 2:1 and 1:1, globular aggregates are first converted into extended structures and then to long threads in which the constituent ATP–G-actin is rapidly exchangeable with the actin of the medium. At high ionic strength {Tyrode [(1910) Arch. Int. Pharmacodyn. Ther. 20, 205–212] solution}, starting from G-actin and protamine in the 1:1 molar ratio, long ropes are formed that can be resolved into intertwining filaments of 4–5nm diameter. The addition of protamine in a 1:1 molar ratio to a solution of F-actin in Tyrode solution causes the breakage of the actin filaments, which is also revealed by the decrease of the viscosity of the solution and the formation of ordered latero-lateral aggregates. The structures formed by reaction of protamine with G-actin can be separated from free G-actin and protamine by filtration through 0.45μm-pore-size Millipore filters. This technique has been exploited to study the exchange reaction between free actin and the actin–protamine complexes. For these studies the 1:1 actin–protamine complex formed at low ionic strength and the 2:1 actin–protamine complex formed in the presence of 23nm-free Mg2+ have been selected. In the first case the exchange reaction is practically complete in the dead time of the experiment (20s). In the second case, where the complex operates like a true ATPase, the rate of the exchange is initially comparable with the rate of the ATP cleavage. Later on, however, the complex undergoes a change and the rate of the exchange between free actin and the actin bound to protamine becomes lower than the rate of the ATPase reaction. It is proposed that the ATP exchanges for ADP directly on the G-actin bound in the complex. ImagesPLATE 1PLATE 2PLATE 3 PMID

  12. Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth[W

    PubMed Central

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

    2013-01-01

    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. PMID:24058157

  13. Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.

    PubMed

    Li, Hailong; Aksenova, Marina; Bertrand, Sarah J; Mactutus, Charles F; Booze, Rosemarie

    2016-02-10

    Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

  14. Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

    PubMed Central

    Bertrand, Sarah J.; Mactutus, Charles F.; Booze, Rosemarie

    2016-01-01

    Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments. PMID:26889716

  15. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells

    PubMed Central

    1975-01-01

    The association of actin filaments with membranes is now recognized as an important parameter in the motility of nonmuscle cells. We have investigated the organization of one of the most extensive and highly ordered actin filament-membrane complexes in nature, the brush border of intestinal epithelial cells. Through the analysis of isolated, demembranated brush borders decorated with the myosin subfragment, S1, we have determined that all the microvillar actin filaments have the same polarity. The S1 arrowhead complexes point away from the site of attachment of actin filaments at the apical tip of the microvillar membrane. In addition to the end-on attachment of actin filaments at the tip of the microvillus, these filaments are also connected to the plasma membrane all along their lengths by periodic (33 nm) cross bridges. These bridges were best observed in isolated brush borders incubated in high concentrations of Mg++. Their visibility is attributed to the induction of actin paracrystals in the filament bundles of the microvilli. Finally, we present evidence for the presence of myosinlike filaments in the terminal web region of the brush border. A model for the functional organization of actin and myosin in the brush border is presented. PMID:1202021

  16. Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner*

    PubMed Central

    Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

    2016-01-01

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. PMID:26747606

  17. Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube growth by severing actin filaments.

    PubMed

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament-severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca(2+), in vitro. Analysis of a mutant that bears a point mutation at the Ca(2+) binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca(2+) level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament-severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.

  18. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments

    PubMed Central

    Heaslip, Aoife T.; Nelson, Shane R.; Warshaw, David M.

    2016-01-01

    The survival of Toxoplasma gondii within its host cell requires protein release from secretory vesicles, called dense granules, to maintain the parasite’s intracellular replicative niche. Despite the importance of DGs, nothing is known about the mechanisms underlying their transport. In higher eukaryotes, secretory vesicles are transported to the plasma membrane by molecular motors moving on their respective cytoskeletal tracks (i.e., microtubules and actin). Because the organization of these cytoskeletal structures differs substantially in T. gondii, the molecular motor dependence of DG trafficking is far from certain. By imaging the motions of green fluorescent protein–tagged DGs in intracellular parasites with high temporal and spatial resolution, we show through a combination of molecular genetics and chemical perturbations that directed DG transport is independent of microtubules and presumably their kinesin/dynein motors. However, directed DG transport is dependent on filamentous actin and a unique class 27 myosin, TgMyoF, which has structural similarity to myosin V, the prototypical cargo transporter. Actomyosin DG transport was unexpected, since filamentous parasite actin has yet to be visualized in vivo due in part to the prevailing model that parasite actin forms short, unstable filaments. Thus our data uncover new critical roles for these essential proteins in the lytic cycle of this devastating pathogen. PMID:27146112

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

    PubMed

    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.

  20. Effects of filament rigidity in myosin II-induced actin network contractility and dynamics

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Gardel, Margaret

    2014-03-01

    Cells change shape, deforming to move and divide. The dynamic protein scaffold that shapes the cell is the cortex, a disordered, thin network of actin filaments. Random, local stresses generated by myosin II in the network create cellular-scale deformations. Myosin induced buckling and severing of actin filaments has been shown to underlie the contractility of two-dimensional disordered actin networks. This non-linear elastic response of actin filaments is thought to be an essential symmetry breaking mechanism to produce robust contractility in disordered actomyosin networks. To test this idea, we explore the effects of an actin bundling protein fascin, a crosslinker which induces polarity specific bundling of actin filaments, to create a network of F-actin bundles. We investigate myosin-induced stresses in a network of randomly oriented actin filaments, confined to a thin sheet at a supported lipid bilayer surface through a crowding agent. We find fascin-bundled filaments are less prone to filament buckling and show increased filament sliding, causing the myosin activity to induce network reorganization rather than contraction. Thus, changes in the filament bending rigidity in motor-filament systems can drive the system between distinct states with unique dynamic and mechanical signatures.

  1. Multi-photon Imaging of Actin Filament Formation and Mitochondrial Energetics of Human ACBT Gliomas

    PubMed Central

    Hwang, Yu-Jer; Kolettis, Nomiki; Yang, Miso; Gillard, Elizabeth R.; Sanchez, Edgar; Sun, Chung-ho; Tromberg, Bruce J.; Krasieva, Tatiana B.; Lyubovitsky, Julia G.

    2011-01-01

    We studied the three-dimensional (3D) distribution of actin filaments and mitochondria in relation to ACBT glioblastoma cells migration. We embedded the cells in the spheroid form within collagen hydrogels and imaged them by in-situ multi-photon microscopy (MPM). The static 3D overlay of the distribution of actin filaments and mitochondria provided a greater understanding of cell-to-cell and cell-to-substrate interactions and morphology. While imaging mitochondria to obtain ratiometric redox index based on cellular fluorescence from reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) we observed differential sensitivity of the migrating ACBT glioblastoma cells to femtosecond laser irradiation employed in MPM. We imaged actin-GFP fluorescence in live ACBT glioma cells and for the first time observed dynamic modulation of the pools of actin during migration in 3D. The MPM imaging, which probes cells directly within the 3D cancer models, could potentially aid in working out a link between the functional performance of mitochondria, actin distribution and cancer invasiveness. PMID:21143483

  2. Site-specific cation release drives actin filament severing by vertebrate cofilin

    PubMed Central

    Kang, Hyeran; Bradley, Michael J.; Cao, Wenxiang; Zhou, Kaifeng; Grintsevich, Elena E.; Michelot, Alphée; Sindelar, Charles V.; Hochstrasser, Mark; De La Cruz, Enrique M.

    2014-01-01

    Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this “stiffness cation” unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics. PMID:25468977

  3. Mammalian CARMIL Inhibits Actin Filament Capping by Capping Protein

    PubMed Central

    Yang, Changsong; Pring, Martin; Wear, Martin A.; Huang, Minzhou; Cooper, John A.; Svitkina, Tatyana M.; Zigmond, Sally H.

    2009-01-01

    Summary Actin polymerization in cells occurs via filament elongation at the barbed end. Proteins that cap the barbed end terminate this elongation. Heterodimeric capping protein (CP) is an abundant and ubiquitous protein that caps the barbed end. We find that the mouse homolog of the adaptor protein CARMIL (mCARMIL) binds CP with high affinity and decreases its affinity for the barbed end. Addition of mCARMIL to cell extracts increases the rate and extent of Arp2/3 or spectrin-actin seed-induced polymerization. In cells, GFP-mCARMIL concentrates in lamellipodia and increases the fraction of cells with large lamellipodia. Decreasing mCARMIL levels by siRNA transfection lowers theF-actin level and slows cell migration through a mechanism that includes decreased lamellipodia protrusion. This phenotype is reversed by full-length mCARMIL but not mCARMIL lacking the domain that binds CP. Thus, mCARMIL is a key regulator of CP and has profound effects on cell behavior. PMID:16054028

  4. Fetal akinesia caused by a novel actin filament aggregate myopathy skeletal muscle actin gene (ACTA1) mutation.

    PubMed

    Stenzel, Werner; Prokop, Stefan; Kress, Wolfram; Huppmann, Stephanie; Loui, Andrea; Sarioglu, Nanette M E; Laing, Nigel G; Sparrow, John C; Heppner, Frank L; Goebel, Hans H

    2010-08-01

    We report a female newborn, diagnosed with fetal akinesia in utero, who died one hour after birth. Post-mortem muscle biopsy demonstrated actin-filament myopathy based on immunolabelling for sarcomeric actin, and large areas of filaments, without rod formation, ultrastructurally. Analysis of DNA extracted from the muscle disclosed a novel de novo heterozygous c.44G>A, GGC>GAC, 'p.Gly15Asp' mutation in the ACTA1 gene. Analysis of the location of the mutated amino-acid in the actin molecule suggests the mutation most likely causes abnormal nucleotide binding, and consequent pathological actin polymerization. This case emphasizes the association of fetal akinesia with actin-filament myopathy.

  5. Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.

    PubMed

    Pang, K M; Lee, E; Knecht, D A

    1998-03-26

    Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

  6. Preparation of filamentous actin for polarized total internal reflection fluorescence microscopy (polTIRFM) motility assays.

    PubMed

    Beausang, John F; Sun, Yujie; Quinlan, Margot E; Forkey, Joseph N; Goldman, Yale E

    2012-05-01

    Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. In this protocol, filamentous actin (F-actin) is polymerized from purified, monomeric actin (G-actin) for use in polTIRFM motility assays in which actin interacts with myosin. The procedures include (1) the preparation of unlabeled F-actin from G-actin; (2) the preparation of F-actin that is sparsely labeled with 6'-IATR (6'-iodoacetamidotetramethylrhodamine); and (3) the preparation of F-actin with a combination of unlabeled, biotinylated, and rhodamine-labeled monomers. Rhodamine-phalloidin actin, also used in polTIRFM assays, can be prepared using a procedure similar to the one for unlabeled actin.

  7. The ATP binding cassette transporter, ABCG1, localizes to cortical actin filaments

    PubMed Central

    Pandzic, Elvis; Gelissen, Ingrid C.; Whan, Renee; Barter, Philip J.; Sviridov, Dmitri; Gaus, Katharina; Rye, Kerry-Anne; Cochran, Blake J.

    2017-01-01

    The ATP-binding cassette sub-family G member 1 (ABCG1) exports cellular cholesterol to high-density lipoproteins (HDL). However, a number of recent studies have suggested ABCG1 is predominantly localised to intracellular membranes. In this study, we found that ABCG1 was organized into two distinct cellular pools: one at the plasma membrane and the other associated with the endoplasmic reticulum (ER). The plasma membrane fraction was organized into filamentous structures that were associated with cortical actin filaments. Inhibition of actin polymerization resulted in complete disruption of ABCG1 filaments. Cholesterol loading of the cells increased the formation of the filamentous ABCG1, the proximity of filamentous ABCG1 to actin filaments and the diffusion rate of membrane associated ABCG1. Our findings suggest that the actin cytoskeleton plays a critical role in the plasma membrane localization of ABCG1. PMID:28165022

  8. Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein

    PubMed Central

    Brzoska, Anthony J.; Jensen, Slade O.; Barton, Deborah A.; Davies, Danielle S.; Overall, Robyn L.; Skurray, Ronald A.; Firth, Neville

    2016-01-01

    Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments. PMID:27310470

  9. Phosphatidylserine liposomes can be tethered by caldesmon to actin filaments.

    PubMed Central

    Makuch, R; Zasada, A; Mabuchi, K; Krauze, K; Wang, C L; Dabrowska, R

    1997-01-01

    Rotary shadowing electron microscopy revealed that attachment of caldesmon to phosphatidylserine (PS) liposomes was mainly through its C-terminal end. To determine the PS-binding sites of caldesmon, we have made use of synthetic peptides covering the two C-terminal calmodulin binding sites and a recombinant fragment corresponding to the N-terminal end of the C-terminal domain that contains an amphipathic helix. Interactions of these peptides with the PS liposomes were studied by nondenaturing gel electrophoresis and fluorescence spectroscopy. The results showed that both calmodulin-binding sites of caldesmon were able to interact with PS. The affinity (Kd) of PS for these sites was in the range of 1.8-14.3 x 10(-5) M, compared to 0.69 x 10(-5) M for the whole caldesmon molecule. Fragments located outside of calmodulin-binding sites bound PS weakly (3.85 x 10(-4) M) and thus may contain a second class of lipid-binding sites. Binding of PS induced conformational changes in regions other than the C-terminal PS-binding sites, as evidenced by the changes in the susceptibility to proteolytic cleavages. Most significantly, the presence of caldesmon greatly increased binding of PS to F-actin, suggesting that caldesmon may tether PS liposomes to actin filaments. These results raise the possibility that caldesmon-lipid interactions could play a functionally important role in the assembly of contractile filaments near the membranes. Images FIGURE 2 FIGURE 4 FIGURE 6 PMID:9284327

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

    PubMed Central

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

    2016-01-01

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

  11. Profilin Interaction with Actin Filament Barbed End Controls Dynamic Instability, Capping, Branching, and Motility

    PubMed Central

    Pernier, Julien; Shekhar, Shashank; Jegou, Antoine; Guichard, Bérengère; Carlier, Marie-France

    2016-01-01

    Summary Cell motility and actin homeostasis depend on the control of polarized growth of actin filaments. Profilin, an abundant regulator of actin dynamics, supports filament assembly at barbed ends by binding G-actin. Here, we demonstrate how, by binding and destabilizing filament barbed ends at physiological concentrations, profilin also controls motility, cell migration, and actin homeostasis. Profilin enhances filament length fluctuations. Profilin competes with Capping Protein at barbed ends, which generates a lower amount of profilin-actin than expected if barbed ends were tightly capped. Profilin competes with barbed end polymerases, such as formins and VopF, and inhibits filament branching by WASP-Arp2/3 complex by competition for filament barbed ends, accounting for its as-yet-unknown effects on motility and metastatic cell migration observed in this concentration range. In conclusion, profilin is a major coordinator of polarized growth of actin filaments, controlled by competition between barbed end cappers, trackers, destabilizers, and filament branching machineries. PMID:26812019

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

    PubMed

    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.

  13. Covalent attachment of actin filaments to Tween 80 coated polystyrene beads for cargo transportation.

    PubMed

    Kaur, Harsimran; Das, Tapan; Kumar, Rajesh; Ajore, Ram; Bharadwaj, Lalit M

    2008-04-01

    In this manuscript, a new strategy has been reported for circumscribed covalent attachment of barbed and pointed ends of actin filaments to polystyrene beads. A comparative study of attachment of actin filaments to polystyrene beads was performed by blocking functionally active sites on polystyrene beads with nonionic detergents such as Tween 20, Tween 80 and polyethylene glycol (PEG). Effective blocking of active sites was obtained with Tween 80 at 0.1% concentration. Attachment of single bundle of actin filament to bead was assessed by rotational motion of bead tailed actin in front and lateral view. Velocity of actin filaments attached to different size of beads in in-vitro motility assay was calculated to ascertain their attachments. Velocity of actin attached to 1.0 and 3.0 microm polystyrene beads was reduced to 3.0-4.0 and 0.0-1.0 microm/s, respectively as compared to free actin velocity of 4.0-6.0 microm/s. Single point attachment of actin filaments to different size of beads was assessed by decrease in sliding velocity. Present study provides insight into the actin-myosin based molecular motor systems for drug delivery and biosensors applications.

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

    PubMed Central

    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 experiments diffusion was slower through short (< or = 1.0 micron) filaments, although this result was not consistently reproducible. Measured diffusion coefficients through unregulated F-actin and filaments of lengths > 1.0 micron were more rapid than predicted by theory for tracer diffusion through rigid, random networks, which was consistent with some degree of actin bundling. Avidin-induced cross-linking of biotinylated F-actin did not affect diffusion through unregulated F-actin, but in cases where diffusion was slower through short filaments this cross-linking method resulted in enhanced tracer diffusion rates indistinguishable from unregulated F-actin. This finding, in conjunction with increased turbidity of 1.0-micron filaments upon avidin cross-linking, indicated that this cross-linking method induces F-actin bundling. By contrast, ABP-280 cross-linking retarded diffusion through unregulated F-actin and decreased turbidity. Tracer diffusion under these conditions was well approximated by the diffusion theory. Both cross-linking procedures resulted in gel formation as determined by falling ball viscometry. These results demonstrate that network microscopic geometry is dependent on the cross-linking method, although both methods markedly increase F-actin macroscopic viscosity. PMID:8913611

  15. Viral infectivity and intracellular distribution of matrix (M) protein of canine distemper virus are affected by actin filaments.

    PubMed

    Klauschies, F; Gützkow, T; Hinkelmann, S; von Messling, V; Vaske, B; Herrler, G; Haas, L

    2010-09-01

    To investigate the role of cytoskeletal components in canine distemper virus (CDV) replication, various agents were used that interfere with turnover of actin filaments and microtubules. Only inhibition of actin filaments significantly reduced viral infectivity. Analysis of the intracellular localization of the viral matrix (M) protein revealed that it aligned along actin filaments. Treatment with actin filament-disrupting drugs led to a marked intracellular redistribution of M protein during infection as well as transfection. In contrast, the localization of the CDV fusion (F) protein was not significantly changed during transfection. Thus, a M protein-actin filament interaction appears to be important for generation of infectious CDV.

  16. Characterization and regulation of an additional actin-filament-binding site in large isoforms of the stereocilia actin-bundling protein espin.

    PubMed

    Zheng, Lili; Beeler, Dina M; Bartles, James R

    2014-03-15

    The espin actin-bundling proteins, which are produced as isoforms of different sizes from a single gene, are required for the growth of hair cell stereocilia. We have characterized an additional actin-filament-binding site present in the extended amino-termini of large espin isoforms. Constitutively active in espin 2, the site increased the size of actin bundles formed in vitro and inhibited actin fluorescence recovery in microvilli. In espin 1, which has an N-terminal ankyrin repeat domain, the site was autoinhibited by binding between the ankyrin repeat domain and a peptide near the actin-binding site. Deletion of this peptide from espin 1 activated its actin-binding site. The peptide resembled tail homology domain I of myosin III, a ligand of the ankyrin repeat domain localized with espin 1 at the tip of stereocilia. A myosin III tail homology domain I peptide, but not scrambled control peptides, inhibited internal binding of the ankyrin repeat domain and released the espin 1 actin-binding site from autoinhibition. Thus, this regulation could result in local activation of the additional actin-binding site of espin 1 by myosin III in stereocilia.

  17. Structural basis of thymosin-β4/profilin exchange leading to actin filament polymerization.

    PubMed

    Xue, Bo; Leyrat, Cedric; Grimes, Jonathan M; Robinson, Robert C

    2014-10-28

    Thymosin-β4 (Tβ4) and profilin are the two major sequestering proteins that maintain the pool of monomeric actin (G-actin) within cells of higher eukaryotes. Tβ4 prevents G-actin from joining a filament, whereas profilin:actin only supports barbed-end elongation. Here, we report two Tβ4:actin structures. The first structure shows that Tβ4 has two helices that bind at the barbed and pointed faces of G-actin, preventing the incorporation of the bound G-actin into a filament. The second structure displays a more open nucleotide binding cleft on G-actin, which is typical of profilin:actin structures, with a concomitant disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, show that the exchange of bound actin between Tβ4 and profilin involves both steric and allosteric components. The sensitivity of profilin to the conformational state of actin indicates a similar allosteric mechanism for the dissociation of profilin during filament elongation.

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

    PubMed

    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.

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

    PubMed Central

    Yamagishi, Yuka; Abe, Hiroshi

    2015-01-01

    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. PMID:26424802

  20. Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

    PubMed

    Fujita, H; Ishiwata, S

    1998-09-01

    Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself.

  1. Spontaneous oscillatory contraction without regulatory proteins in actin filament-reconstituted fibers.

    PubMed Central

    Fujita, H; Ishiwata, S

    1998-01-01

    Skinned skeletal and cardiac muscle fibers exhibits spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi)1 but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307-2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca(2+)-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself. PMID:9726945

  2. Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of LysosomesV⃞

    PubMed Central

    Cordonnier, Marie-Neige; Dauzonne, Daniel; Louvard, Daniel; Coudrier, Evelyne

    2001-01-01

    An earlier report suggested that actin and myosin I alpha (MMIα), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIα were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIα. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIα impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIα contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors. PMID:11739797

  3. F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling

    PubMed Central

    1995-01-01

    Transverse sections though Drosophila bristles reveal 7-11 nearly round, plasma membrane-associated bundles of actin filaments. These filaments are hexagonally packed and in a longitudinal section they show a 12-nm periodicity in both the 1.1 and 1.0 views. From earlier studies this periodicity is attributable to cross-links and indicates that the filaments are maximally cross-linked, singed mutants also have 7-11 bundles, but the bundles are smaller, flattened, and the filaments within the bundles are randomly packed (not hexagonal); no periodicity can be detected in longitudinal sections. Another mutant, forked (f36a), also has 7-11 bundles but even though the bundles are very small, the filaments within them are hexagonally packed and display a 12-nm periodicity in longitudinal section. The singed-forked double mutant lacks filament bundles. Thus there are at least two species of cross-links between adjacent actin filaments. Hints of why two species of cross-links are necessary can be gleaned by studying bristle formation. Bristles sprout with only microtubules within them. A little later in development actin filaments appear. At early stages the filaments in the bundles are randomly packed. Later the filaments in the bundles become hexagonally packed and maximally cross-linked. We consider that the forked proteins may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together by fascin (the singed gene product). PMID:7622563

  4. Possible association of actin filaments with chloroplasts of spinach mesophyll cells in vivo and in vitro.

    PubMed

    Kumatani, T; Sakurai-Ozato, N; Miyawaki, N; Yokota, E; Shimmen, T; Terashima, I; Takagi, S

    2006-11-01

    In palisade mesophyll cells of spinach (Spinacia oleracea L.) kept under low-intensity white light, chloroplasts were apparently immobile and seemed to be surrounded by fine bundles of actin filaments. High-intensity blue light induced actin-dependent chloroplast movement concomitant with the appearance of a couple of long, straight bundles of actin filaments in each cell, whereas high-intensity red light was essentially ineffective in inducing these responses. The actin organization observed under low-intensity white light has been postulated to function in anchoring chloroplasts at proper intracellular positions through direct interaction with the chloroplasts. Intact chloroplasts, which retained their outer envelopes, were isolated after homogenization of leaves and Percoll centrifugation. No endogenous actin was detected by immunoblotting in the final intact-chloroplast fraction prepared from the leaves kept under low-intensity white light or in darkness. In cosedimentation assays with exogenously added skeletal muscle filamentous actin, however, actin was detected in the intact-chloroplast fraction precipitated after low-speed centrifugation. The association of actin with chloroplasts was apparently dependent on incubation time and chloroplast density. After partial disruption of the outer envelope of isolated chloroplasts by treatment with trypsin, actin was no longer coprecipitated. The results suggest that chloroplasts in spinach leaves can directly interact with actin, and that this interaction may be involved in the regulation of intracellular positioning of chloroplasts.

  5. Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro.

    PubMed

    Defeu Soufo, Hervé Joël; Reimold, Christian; Breddermann, Hannes; Mannherz, Hans G; Graumann, Peter L

    2015-04-24

    EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro and was active in dynamic light-scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein.

  6. High-speed depolymerization at actin filament ends jointly catalysed by Twinfilin and Srv2/CAP.

    PubMed

    Johnston, Adam B; Collins, Agnieszka; Goode, Bruce L

    2015-11-01

    Purified actin filaments depolymerize slowly, and cytosolic conditions strongly favour actin assembly over disassembly, which has left our understanding of how actin filaments are rapidly turned over in vivo incomplete. One mechanism for driving filament disassembly is severing by factors such as Cofilin. However, even after severing, pointed-end depolymerization remains slow and unable to fully account for observed rates of actin filament turnover in vivo. Here we describe a mechanism by which Twinfilin and Cyclase-associated protein work in concert to accelerate depolymerization of actin filaments by 3-fold and 17-fold at their barbed and pointed ends, respectively. This mechanism occurs even under assembly conditions, allowing reconstitution and direct visualization of individual filaments undergoing tunable, accelerated treadmilling. Further, we use specific mutations to demonstrate that this activity is critical for Twinfilin function in vivo. These findings fill a major gap in our knowledge of cellular disassembly mechanisms, and suggest that depolymerization and severing may be deployed separately or together to control the dynamics and architecture of distinct actin networks.

  7. High Speed Depolymerization at Actin Filament Ends Jointly Catalyzed by Twinfilin and Srv2/CAP

    PubMed Central

    Johnston, Adam B.; Collins, Agnieszka; Goode, Bruce L.

    2015-01-01

    Purified actin filaments depolymerize slowly, and cytosolic conditions strongly favor actin assembly over disassembly, which has left our understanding of how actin filaments are rapidly turned over in vivo incomplete 1,2. One mechanism for driving filament disassembly is severing by factors such as Cofilin. However, even after severing, pointed end depolymerization remains slow and unable to fully account for observed rates of actin filament turnover in vivo. Here we describe a mechanism by which Twinfilin and Cyclase-associated protein work in concert to accelerate depolymerization of actin filaments by 3-fold and 17-fold at their barbed and pointed ends, respectively. This mechanism occurs even under assembly conditions, allowing reconstitution and direct visualization of individual filaments undergoing tunable, accelerated treadmilling. Further, we use specific mutations to demonstrate that this activity is critical for Twinfilin function in vivo. These findings fill a major gap in our knowledge of mechanisms, and suggest that depolymerization and severing may be deployed separately or together to control the dynamics and architecture of distinct actin networks. PMID:26458246

  8. The effects of formins on the conformation of subdomain 1 in actin filaments.

    PubMed

    Ujfalusi, Zoltán; Barkó, Szilvia; Hild, Gábor; Nyitrai, Miklós

    2010-01-21

    In this study we investigated the effects of formins on the conformation of actin filaments by using the method of fluorescence quenching. Actin was labelled with IAEDANS at Cys(374) and the quencher was acrylamide. The results showed that formin binding induced structural changes in the subdomain 1 of actin protomers which were reflected by greater quenching constants (K(SV)). Simultaneously the fraction of the fluorophore population accessible for the quencher (alpha) decreased. These observations suggest that the conformational distribution characteristic for the actin protomers became broader after the binding of formins, for which the structural framework was provided by a more flexible protein matrix in the microenvironment of the label. The effects of formins depended on the formin:actin molar ratio, and also on the ionic strength of the medium. These observations are in agreement with previous results and underline the importance of the intramolecular conformational changes induced by formins in the structure of actin filaments.

  9. ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays

    PubMed Central

    Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M.; Lowe, Martin; Vartiainen, Maria K.; Jokitalo, Eija

    2014-01-01

    The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network. PMID:24523293

  10. Simultaneous quantification of actin monomer and filament dynamics with modeling-assisted analysis of photoactivation

    PubMed Central

    Kapustina, Maryna; Read, Tracy-Ann

    2016-01-01

    ABSTRACT Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three-dimensional cellular environment. We use this method to determine differences in the dynamic behavior of β- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that β-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the β-actin monomer pool. Finally, we used modeling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half-time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments. PMID:27831495

  11. ER sheet persistence is coupled to myosin 1c-regulated dynamic actin filament arrays.

    PubMed

    Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M; Lowe, Martin; Vartiainen, Maria K; Jokitalo, Eija

    2014-04-01

    The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.

  12. Actin-crosslinking protein regulation of filament movement in motility assays: a theoretical model.

    PubMed Central

    Janson, L W; Taylor, D L

    1994-01-01

    The interaction of single actin filaments on a myosin-coated coverslip has been modeled by several authors. One model adds a component of "frictional drag" by myosin heads that oppose movement of the actin filaments. We have extended this concept by including the resistive drag from actin crosslinking proteins to understand better the relationship among crosslinking number, actin-myosin force generation, and motility. The validity of this model is supported by agreement with the experimental results from a previous study in which crosslinking proteins were added with myosin molecules under otherwise standard motility assay conditions. The theoretical relationship provides a means to determine many physical parameters that characterize the interaction between a single actin filament and a single actin-crosslinking molecule (various types). In particular, the force constant of a single filamin molecule is calculated as 1.105 pN, approximately 3 times less than a driving myosin head (3.4 pN). Knowledge of this parameter and others derived from this model allows a better understanding of the interaction between myosin and the actin/actin-binding protein cytoskeleton and the role of actin-binding proteins in the regulation and modulation of motility. PMID:7811954

  13. A novel form of actin in Leishmania: molecular characterisation, subcellular localisation and association with subpellicular microtubules.

    PubMed

    Sahasrabuddhe, Amogh A; Bajpai, Virendra K; Gupta, Chhitar M

    2004-03-01

    To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 10(6) copies per cell of this protein (M(r) 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network. These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed.

  14. Single-Molecule Discrimination within Dendritic Spines of Discrete Perisynaptic Sites of Actin Filament Assembly Driving Postsynaptic Reorganization

    NASA Astrophysics Data System (ADS)

    Blanpied, Thomas A.

    2013-03-01

    In the brain, the strength of synaptic transmission between neurons is principally set by the organization of proteins within the receptive, postsynaptic cell. Synaptic strength at an individual site of contact can remain remarkably stable for months or years. However, it also can undergo diverse forms of plasticity which change the strength at that contact independent of changes to neighboring synapses. Such activity-triggered neural plasticity underlies memory storage and cognitive development, and is disrupted in pathological physiology such as addiction and schizophrenia. Much of the short-term regulation of synaptic plasticity occurs within the postsynaptic cell, in small subcompartments surrounding the synaptic contact. Biochemical subcompartmentalization necessary for synapse-specific plasticity is achieved in part by segregation of synapses to micron-sized protrusions from the cell called dendritic spines. Dendritic spines are heavily enriched in the actin cytoskeleton, and regulation of actin polymerization within dendritic spines controls both basal synaptic strength and many forms of synaptic plasticity. However, understanding the mechanism of this control has been difficult because the submicron dimensions of spines limit examination of actin dynamics in the spine interior by conventional confocal microscopy. To overcome this, we developed single-molecule tracking photoactivated localization microscopy (smtPALM) to measure the movement of individual actin molecules within living spines. This revealed inward actin flow from broad areas of the spine plasma membrane, as well as a dense central core of heterogeneous filament orientation. The velocity of single actin molecules along filaments was elevated in discrete regions within the spine, notably near the postsynaptic density but surprisingly not at the endocytic zone which is involved in some forms of plasticity. We conclude that actin polymerization is initiated at many well-separated foci within

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

    PubMed

    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.

  16. Peritubular myoid cells from rat seminiferous tubules contain actin and myosin filaments distributed in two independent layers.

    PubMed

    Losinno, Antonella D; Morales, Alfonsina; Fernández, Dario; Lopez, Luis A

    2012-05-01

    In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.

  17. Distribution pattern changes of actin filaments during chloroplast movement in Adiantum capillus-veneris.

    PubMed

    Tsuboi, Hidenori; Wada, Masamitsu

    2012-05-01

    Chloroplasts change their positions in a cell in response to light intensities. The photoreceptors involved in chloroplast photo-relocation movements and the behavior of chloroplasts during their migration were identified in our previous studies, but the mechanism of movement has yet to be clarified. In this study, the behavior of actin filaments under various light conditions was observed in Adiantum capillus-veneris gametophytes. In chloroplasts staying in one place under a weak light condition and not moving, circular structures composed of actin filaments were observed around the chloroplast periphery. In contrast, short actin filaments were observed at the leading edge of moving chloroplasts induced by partial cell irradiation. In the dark, the circular structures found under the weak light condition disappeared and then reappeared around the moving chloroplasts. Mutant analyses revealed that the disappearance of the circular actin structure was mediated by the blue light photoreceptor, phototropin2.

  18. Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport

    PubMed Central

    Kumar, Saroj; ten Siethoff, Lasse; Persson, Malin; Lard, Mercy; te Kronnie, Geertruy; Linke, Heiner; Månsson, Alf

    2012-01-01

    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. PMID:23056279

  19. Actin filaments align into hollow comets for rapid VASP-mediated propulsion.

    PubMed

    Plastino, Julie; Olivier, Stéphane; Sykes, Cécile

    2004-10-05

    For cells, the growth of a dense array of branched actin filaments organized by the actin-related proteins 2 and 3 (Arp2/3) complex at the plasma membrane offers an explanation as to how movement is produced, and this arrangement is considered to be optimal for motility. Here, we challenged this assumption by using an in vitro system of polystyrene beads in cell extracts that contained a complex mix of actin polymerization proteins as in vivo. We employed the surface of the bead as a reactor where we mixed two different actin polymerization-activating factors, the Arp2/3 complex and the vasodilator-stimulated phosphoprotein (VASP), to examine their contribution to actin-based movement and filament organization. We varied the coating of the bead surface but left the extracts identical for all assays. We found that the degree of filament alignment in the actin comet tails depended on the surface ratio of VASP to Arp2/3. Alignment of actin filaments parallel to the direction of bead movement in the presence of VASP was accompanied by an abrupt 7-fold increase in velocity that was independent of bead size and by hollowing out of the comets. The actin filament-bundling proteins fimbrin and fascin did not appear to play a role in this transformation. Together with the idea that VASP enhances filament detachment and with the presence of pulling forces at the rear of the bead, a mesoscopic analysis of movement provides a possible explanation for our results.

  20. Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament.

    PubMed

    Masuda, Tadashi

    2013-09-01

    Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the "Driven by Detachment (DbD)" mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated.

  1. Dendritic Actin Filament Nucleation Causes Traveling Waves and Patches

    PubMed Central

    Carlsson, Anders E

    2010-01-01

    The polymerization of actin via branching at a cell membrane containing nucleation-promoting factors (NPFs) is simulated using a stochastic-growth methodology. The polymerized-actin distribution displays three types of behavior: a) traveling waves, b) moving patches, and c) random fluctuations. Increasing actin concentration causes a transition from patches to waves. The waves and patches move by a treadmilling mechanism which does not require myosin II. The effects of downregulation of key proteins on actin wave behavior are evaluated. PMID:20867207

  2. Synthetic chondramide A analogues stabilize filamentous actin and block invasion by Toxoplasma gondii.

    PubMed

    Ma, Christopher I; Diraviyam, Karthikeyan; Maier, Martin E; Sept, David; Sibley, L David

    2013-09-27

    Apicomplexan parasites such as Toxoplasma gondii rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of T. gondii in vitro. Structural modeling of chondramide A (2) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (2b-k) with substitutions in the β-tyrosine moiety blocked parasite growth on host cell monolayers with EC₅₀ values that ranged from 0.3 to 1.3 μM. In vitro polymerization assays using highly purified recombinant actin from T. gondii verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites.

  3. 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

    PubMed Central

    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

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

    PubMed

    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.

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

    PubMed

    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.

  6. A mechanical model for the motility of actin filaments on myosin

    NASA Astrophysics Data System (ADS)

    Nicolau, Dan V., Jr.; Fulga, Florin; Nicolau, Dan V.

    2004-03-01

    The interaction of actin filaments with myosin is crucial to cell motility, muscular contraction, cell division and other processes. The in vitro motility assay involves the motion of actin filaments on a substrate coated with myosin, and is used extensively to investigate the dynamics of the actomyosin system. Following on from previous work, we propose a new mechanical model of actin motility on myosin, wherein a filament is modeled as a chain of beads connected by harmonic springs. This imposes a limitation on the "stretching" of the filament. The rotation of one bead with respect to its neighbours is also constrained in similar way. We implemented this model and used Monte Carlo simulations to determine whether it can predict the directionality of filament motion. The principal advantages of this model over our previous one are that we have removed the empirically correct but artificial assumption that the filament moves like a "worm" i.e. the head determines the direction of movement and the rest of the filament "follows" the head as well as the inclusion of dependencies on experimental rate constants (and so also on e.g. ATP concentration) via the cross-bridge cycle.

  7. Phototropin-dependent biased relocalization of cp-actin filaments can be induced even when chloroplast movement is inhibited.

    PubMed

    Yamada, Noboru; Suetsugu, Noriyuki; Wada, Masamitsu; Kadota, Akeo

    2011-11-01

    In a recent publication using an actin-visualized line of Arabidopsis (Ichikawa et al. 2011, ref. 11), we reported a detailed analysis with higher time resolution on the dynamics of chloroplast actin filaments (cp-actin filaments) during chloroplast avoidance movement and demonstrated a good correlation between the biased configuration of cp-actin filaments and chloroplast movement. However, we could not conclusively determine whether the reorganization of cp-actin filaments into a biased configuration preceded actual chloroplast movement (and, thus, whether it could be a cause of the movement). In this report, we present clear evidence that the reorganization of cp-actin filaments into a biased distribution is induced even in the absence of the actual movement of chloroplasts. When the cells were treated with 2,3-butanedione monoxime (BDM), a potent inhibitor of myosin ATPase, chloroplast motility was completely suppressed. Nevertheless, the disappearance and biased relocalization of cp-actin filaments toward the side of the prospective movement direction were induced by irradiation with a strong blue light microbeam. The results definitively indicate that the reorganization of cp-actin filaments is not an effect of chloroplast movement; however, it is feasible that the biased localization of cp-actin filaments is an event leading to chloroplast movement.

  8. Rapid severing and motility of chloroplast-actin filaments are required for the chloroplast avoidance response in Arabidopsis.

    PubMed

    Kong, Sam-Geun; Arai, Yoshiyuki; Suetsugu, Noriyuki; Yanagida, Toshio; Wada, Masamitsu

    2013-02-01

    Phototropins (phot1 and phot2 in Arabidopsis thaliana) relay blue light intensity information to the chloroplasts, which move toward weak light (the accumulation response) and away from strong light (the avoidance response). Chloroplast-actin (cp-actin) filaments are vital for mediating these chloroplast photorelocation movements. In this report, we examine in detail the cp-actin filament dynamics by which the chloroplast avoidance response is regulated. Although stochastic dynamics of cortical actin fragments are observed on the chloroplasts, the basic mechanisms underlying the disappearance (including severing and turnover) of the cp-actin filaments are regulated differently from those of cortical actin filaments. phot2 plays a pivotal role in the strong blue light-induced severing and random motility of cp-actin filaments, processes that are therefore essential for asymmetric cp-actin formation for the avoidance response. In addition, phot2 functions in the bundling of cp-actin filaments that is induced by dark incubation. By contrast, the function of phot1 is dispensable for these responses. Our findings suggest that phot2 is the primary photoreceptor involved in the rapid reorganization of cp-actin filaments that allows chloroplasts to change direction rapidly and control the velocity of the avoidance movement according to the light's intensity and position.

  9. Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement.

    PubMed

    Ichikawa, Satoshi; Yamada, Noboru; Suetsugu, Noriyuki; Wada, Masamitsu; Kadota, Akeo

    2011-08-01

    The phototropin (phot)-dependent intracellular relocation of chloroplasts is a ubiquitous phenomenon in plants. We have previously revealed the involvement of a short cp-actin (chloroplast actin) filament-based mechanism in this movement. Here, the reorganization of cp-actin filaments during the avoidance movement of chloroplasts was analyzed in higher time resolution under blue GFP (green fluorescent protein) excitation light in an actin filament-visualized line of Arabidopsis thaliana. Under standard background red light of 89 μmol m(-2) s(-1), cp-actin filaments transiently disappeared at approximately 30 s and reappeared in a biased configuration on chloroplasts approximately 70 s after blue excitation light irradiation. The timing of biased cp-actin reappearance was delayed under the background of strong red light or in the absence of red light. Consistently, chloroplast movement was delayed under these conditions. In phot1 mutants, acceleration of both the disappearance and reappearance of cp-actin filaments occurred, indicating an inhibitory action of phot1 on reorganization of cp-actin filaments. Avoidance movements began sooner in phot1 than in wild-type plants. No reorganization of cp-actin filaments was seen in phot2 or phot1phot2 mutants lacking phot2, which is responsible for avoidance movements. Surprisingly, jac1 (j-domain protein required for chloroplast accumulation response 1) mutants, lacking the accumulation response, showed no avoidance movements under the whole-cell irradiation condition for GFP observation. Cp-actin filaments in jac1 did not show a biased distribution, with a small or almost no transient decrease in the number. These results indicate a close association between the biased distribution of cp-actin filaments and chloroplast movement. Further, JAC1 is suggested to function in the biased cp-actin filament distribution by regulating their appearance and disappearance.

  10. Probing the sliding interactions between bundled actin filaments

    NASA Astrophysics Data System (ADS)

    Ward, Andy; Dogic, Zvonimir

    2012-02-01

    Assemblies of filamentous biopolymers are hierarchical materials in which the properties of the overall assemblage are determined by structure and interactions between constituent particles at all hierarchical levels. For example, the overall bending rigidity of a two bundled filaments greatly depends on the bending rigidity of, and the adhesion strength between individual filaments. However, another property of importance is the ability for the filaments to slide freely against one another. Everyday experience indicates that it is much easier to bend a stack of papers in which individual sheets freely slide past each other than the same stack of papers in which all the sheets are irreversibly glued together. Similarly, in filamentous structures the ability for local re-arrangement is of the utmost importance in determining the properties of the structures observed. We have developed a method to directly measure the frictional interactions between a pair of aligned filaments in a well-defined and controllable configuration. This enables us to systematically investigate the role of adhesion strength, filament orientation, length, and surface structure.

  11. Role of the DNase-I-binding loop in dynamic properties of actin filament.

    PubMed

    Khaitlina, Sofia Yu; Strzelecka-Gołaszewska, Hanna

    2002-01-01

    Effects of proteolytic modifications of the DNase-I-binding loop (residues 39-51) in subdomain 2 of actin on F-actin dynamics were investigated by measuring the rates of the polymer subunit exchange with the monomer pool at steady state and of ATP hydrolysis associated with it, and by determination of relative rate constants for monomer addition to and dissociation from the polymer ends. Cleavage of actin between Gly-42 and Val-43 by protease ECP32 resulted in enhancement of the turnover rate of polymer subunits by an order of magnitude or more, in contrast to less than a threefold increase produced by subtilisin cleavage between Met-47 and Gly-48. Probing the structure of the modified actins by limited digestion with trypsin revealed a correlation between the increased F-actin dynamics and a change in the conformation of subdomain 2, indicating a more open state of the filament subunits relative to intact F-actin. The cleavage with trypsin and steady-state ATPase were cooperatively inhibited by phalloidin, with half-maximal effects at phalloidin to actin molar ratio of 1:8 and full inhibition at a 1:1 ratio. The results support F-actin models in which only the N-terminal segment of loop 39-51 is involved in monomer-monomer contacts, and suggest a possibility of regulation of actin dynamics in the cell through allosteric effects on this segment of the actin polypeptide chain.

  12. The "Le Chatelier's principle"-governed response of actin filaments to osmotic stress.

    PubMed

    Ito, Tadanao; Yamazaki, Masahito

    2006-07-13

    Actin filaments inhibit osmotic stress-driven water flow across a semipermeable membrane in proportion to the filament concentration (Ito, T.; Zaner, K. S.; Stossel, T. P. Biophys. J. 1987, 51, 745). When the filaments are cross-linked by F-actin binding protein, filamin A, this flow is stopped completely (Ito, T.; Suzuki, A.; Stossel, T. P. Biophys. J. 1992, 61, 1301). No conventional theory accurately accounts for these results. Here, this response is analyzed by formulating the entropy of the system under osmotic stress. Results demonstrate that the response of the actin filaments to osmotic stress is governed by the Le Chatelier's principle, which states that an external interaction that disturbs the equilibrium brings about processes in the body that tend to reduce the effects of this interaction. In the present case, disrupting equilibrium by osmotic stress brings about a reaction that decreases the chemical potential of water in the F-actin solution, reducing the effect of the applied osmotic disturbance. This decrease in the chemical potential of the water in the F-actin solution is caused by an increase in the chemical potential of F-actin, which is induced by isothermal absorption of heat by F-actin aided by work done by osmotic stress. As a result, F-actin has an inhibitory effect on the osmotic stress-driven water flow, and can even completely stop the flow when it is cross-linked. This is the first report demonstrating that the Le Chatelier's principle applies to the reaction of biopolymers against equilibrium disturbances such as osmotic stress.

  13. Flexural rigidity of microtubules and actin filaments measured from thermal fluctuations in shape

    PubMed Central

    1993-01-01

    Microtubules are long, proteinaceous filaments that perform structural functions in eukaryotic cells by defining cellular shape and serving as tracks for intracellular motor proteins. We report the first accurate measurements of the flexural rigidity of microtubules. By analyzing the thermally driven fluctuations in their shape, we estimated the mean flexural rigidity of taxol-stabilized microtubules to be 2.2 x 10(-23) Nm2 (with 6.4% uncertainty) for seven unlabeled microtubules and 2.1 x 10(-23) Nm2 (with 4.7% uncertainty) for eight rhodamine-labeled microtubules. These values are similar to earlier, less precise estimates of microtubule bending stiffness obtained by modeling flagellar motion. A similar analysis on seven rhodamine-phalloidin- labeled actin filaments gave a flexural rigidity of 7.3 x 10(-26) Nm2 (with 6% uncertainty), consistent with previously reported results. The flexural rigidity of these microtubules corresponds to a persistence length of 5,200 microns showing that a microtubule is rigid over cellular dimensions. By contrast, the persistence length of an actin filament is only approximately 17.7 microns, perhaps explaining why actin filaments within cells are usually cross-linked into bundles. The greater flexural rigidity of a microtubule compared to an actin filament mainly derives from the former's larger cross-section. If tubulin were homogeneous and isotropic, then the microtubule's Young's modulus would be approximately 1.2 GPa, similar to Plexiglas and rigid plastics. Microtubules are expected to be almost inextensible: the compliance of cells is due primarily to filament bending or sliding between filaments rather than the stretching of the filaments themselves. PMID:8432732

  14. Effects of solution crowding on actin polymerization reveal the energetic basis for nucleotide-dependent filament stability

    PubMed Central

    Frederick, Kendra B.; Sept, David; De La Cruz, Enrique M.

    2008-01-01

    Actin polymerization is a fundamental cellular process involved in cell structure maintenance, force generation, and motility. Phosphate release from filament subunits following ATP hydrolysis destabilizes the filament lattice and increases the critical concentration (Cc) for assembly. The structural differences between ATP- and ADP-actin are still debated, as well as the energetic factors that underlie nucleotide-dependent filament stability, particularly under crowded intracellular conditions. Here, we investigate the effect of crowding agents on ATP- and ADP-actin polymerization, and find that ATP-actin polymerization is largely unaffected by solution crowding, while crowding agents lower the Cc of ADP-actin in a concentration-dependent manner. The stabilities of ATP- and ADP-actin filaments are comparable in the presence of physiological amounts (~30% w/v) and types (sorbitol) of low molecular weight crowding agents. Crowding agents act to stabilize ADP-F-actin by slowing subunit dissociation. These observations suggest that nucleotide hydrolysis and phosphate release per se do not introduce intrinsic differences in the in vivo filament stability. Rather, the preferential disassembly of ADP-actin filaments in cells is driven through interactions with regulatory proteins. Interpretation of the experimental data according to osmotic stress theory implicates water as an allosteric regulator of actin activity and hydration as the molecular basis for nucleotide-dependent filament stability. PMID:18374941

  15. Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments[W

    PubMed Central

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca2+, in vitro. Analysis of a mutant that bears a point mutation at the Ca2+ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca2+ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth. PMID:24424096

  16. Live Cell Imaging of Actin Dynamics in the Filamentous Fungus Aspergillus nidulans.

    PubMed

    Schultzhaus, Zachary; Quintanilla, Laura; Hilton, Angelyn; Shaw, Brian D

    2016-04-01

    Hyphal cells of filamentous fungi grow at their tips in a method analogous to pollen tube and root hair elongation. This process, generally referred to as tip growth, requires precise regulation of the actin cytoskeleton, and characterizing the various actin structures in these cell types is currently an active area of research. Here, the actin marker Lifeact was used to document actin dynamics in the filamentous fungus Aspergillus nidulans. Contractile double rings were observed at septa, and annular clusters of puncta were seen subtending growing hyphal tips, corresponding to the well-characterized subapical endocytic collar. However, Lifeact also revealed two additional structures. One, an apical array, was dynamic on the face opposite the tip, while a subapical web was dynamic on the apical face and was located several microns behind the growth site. Each was observed turning into the other over time, implying that they could represent different localizations of the same structure, although hyphae with a subapical web grew faster than those exhibiting an apical array. The subapical web has not been documented in any filamentous fungus to date, and is separate from the networks of F-actin seen in other tip-growing organisms surrounding septa or stationary along the plasmalemma.

  17. Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites.

    PubMed

    Ji, Wei-ke; Hatch, Anna L; Merrill, Ronald A; Strack, Stefan; Higgs, Henry N

    2015-11-26

    While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites.

  18. Direct interaction of actin filaments with F-BAR protein pacsin2

    PubMed Central

    Kostan, Julius; Salzer, Ulrich; Orlova, Albina; Törö, Imre; Hodnik, Vesna; Senju, Yosuke; Zou, Juan; Schreiner, Claudia; Steiner, Julia; Meriläinen, Jari; Nikki, Marko; Virtanen, Ismo; Carugo, Oliviero; Rappsilber, Juri; Lappalainen, Pekka; Lehto, Veli-Pekka; Anderluh, Gregor; Egelman, Edward H; Djinović-Carugo, Kristina

    2014-01-01

    Two mechanisms have emerged as major regulators of membrane shape: BAR domain-containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F-BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N-BAR and F-BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes. PMID:25216944

  19. Extracellular Inhibitors, Repellents, and Semaphorin/Plexin/MICAL-mediated Actin Filament Disassembly

    PubMed Central

    Hung, Ruei-Jiun; Terman, Jonathan R.

    2011-01-01

    Multiple extracellular signals have been identified that regulate actin dynamics within motile cells, but how these instructive cues present on the cell surface exert their precise effects on the internal actin cytoskeleton is still poorly understood. One particularly interesting class of these cues is a group of extracellular proteins that negatively alter the movement of cells and their processes. Over the years, these types of events have been described using a variety of terms and herein we provide an overview of inhibitory/repulsive cellular phenomena and highlight the largest known protein family of repulsive extracellular cues, the Semaphorins. Specifically, the Semaphorins (Semas) utilize Plexin cell-surface receptors to dramatically collapse the actin cytoskeleton and we summarize what is known of the direct molecular and biochemical mechanisms of Sema-triggered actin filament (F-actin) disassembly. We also discuss new observations from our lab that reveal that the multi-domain oxidoreductase (Redox) enzyme MICAL, an important mediator of Sema/Plexin repulsion, is a novel F-actin disassembly factor. Our results indicate that MICAL triggers Sema/Plexin-mediated reorganization of the F-actin cytoskeleton and suggest a role for specific Redox signaling events in regulating actin dynamics. PMID:21800438

  20. Real-Time Measurements of Actin Filament Polymerization by Total Internal Reflection Fluorescence Microscopy

    PubMed Central

    Kuhn, Jeffrey R.; Pollard, Thomas D.

    2005-01-01

    Understanding the mechanism of actin polymerization and its regulation by associated proteins requires an assay to monitor polymerization dynamics and filament topology simultaneously. The only assay meeting these criteria is total internal reflection fluorescence microscopy (Amann and Pollard, 2001; Fujiwara et al., 2002). The fluorescence signal is fourfold stronger with actin labeled on Cys-374 with Oregon green rather than rhodamine. To distinguish growth at barbed and pointed ends we used image drift correction and maximum intensity projections to reveal points where single N-ethylmaleimide inactivated myosins attach filaments to the glass coverslip. We estimated association rates at high actin concentrations and dissociation rates near and below the critical actin concentration. At the barbed end, the association rate constant for Mg-ATP-actin is 7.4 μM−1 s−1 and the dissociation rate constant is 0.89 s−1. At the pointed end the association and dissociation rate constants are 0.56 μM−1 s−1 and 0.19 s−1. When vitamin D binding protein sequesters all free monomers, ADP-actin dissociates from barbed ends at 1.4 s−1 and from pointed ends at 0.16 s−1 regardless of buffer nucleotide. PMID:15556992

  1. Control of actin filament dynamics at barbed ends by WH2 domains: from capping to permissive and processive assembly.

    PubMed

    Carlier, Marie-France; Pernier, Julien; Avvaru, Balendu Sankara

    2013-10-01

    WH2 domains are multifunctional regulators of actin assembly that can either sequester G-actin or allow polarized barbed end growth. They all bind similarly to a hydrophobic pocket at the barbed face of actin. Depending on their electrostatic environment, WH2 domains can nucleate actin assembly by facilitating the formation of prenuclei dimers along the canonical spontaneous assembly pathway. They also modulate filament barbed end dynamics in a versatile fashion, acting either as barbed end cappers or assisting barbed end growth like profilin or uncapping barbed ends and potentially mediating processive elongation like formins when they are dimerized. Tandem repeats of WH2 domains can sever filaments and either remain bound to created barbed ends like gelsolin, or strip off an ADP-actin subunit from the severed polymer end, depending on their relative affinity for terminal ADP-F-actin or ADP-G-actin. In summary, WH2 domains recapitulate all known elementary regulatory functions so far found in individual actin-binding proteins. By combining different discrete sets of these multifunctional properties, they acquire specific functions in various actin-based processes, and participate in activities as diverse as filament branching, filopodia extension, or actin remodeling in ciliogenesis and asymmetric meiotic division. They also integrate these functions with other actin-binding motifs present either in the same protein or in a complex with another protein, expanding the range of complexity in actin regulation. The details of their molecular mechanisms and the underlying structural basis provide exciting avenues in actin research.

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

    PubMed Central

    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

  3. Direct observation of motion of single F-actin filaments in the presence of myosin

    NASA Astrophysics Data System (ADS)

    Yanagida, Toshio; Nakase, Michiyuki; Nishiyama, Katsumi; Oosawa, Fumio

    1984-01-01

    Actin is found in almost all kinds of non-muscle cells where it is thought to have an important role in cell motility. A proper understanding of that role will only be possible when reliable in vitro systems are available for investigating the interaction of cellular actin and myosin. A start has been made on several systems1-4, most recently by Sheetz and Spudich who demonstrated unidirectional movement of HMM-coated beads along F-actin cables on arrays of chloroplasts exposed by dissection of a Nitella cell5. As an alternative approach, we report here the direct observation by fluorescence microscopy of the movements of single F-actin filaments interacting with soluble myosin fragments energized by Mg2+-ATP.

  4. Tropomyosin-dependent filament formation by a polymerization-defective mutant yeast actin (V266G,L267G).

    PubMed

    Wen, K K; Kuang, B; Rubenstein, P A

    2000-12-22

    A major function of tropomyosin (TPM) in nonmuscle cells may be stabilization of F-actin by binding longitudinally along the actin filament axis. However, no clear evidence exists in vitro that TPM can significantly affect the critical concentration of actin. We previously made a polymerization-defective mutant actin, GG (V266G, L267G). This actin will not polymerize alone at 25 degrees C but will in the presence of phalloidin or beryllium fluoride. With beryllium fluoride, but not phalloidin, this polymerization rescue is cold-sensitive. We show here that GG-actin polymerizability was restored by cardiac tropomyosin and yeast TPM1 and TPM2 at 25 degrees C with rescue efficiency inversely proportional to TPM length (TPM2 > TPM1 > cardiac tropomyosin), indicating the importance of the ends in polymerization rescue. In the presence of TPM, the apparent critical concentration of actin is 5.5 microm, 10-15-fold higher than that of wild type actin but well below that of the GG-actin alone (>20 microm). Non N-acetylated TPMs did not rescue GG-actin polymerization. The TPMs did not prevent cold-induced depolymerization of GG F-actin. TPM-dependent GG-actin polymerization did not occur at temperatures below 20 degrees C. Polymerization rescue may depend initially on the capture of unstable GG-F-actin oligomers by the TPM, resulting in the strengthening of actin monomer-monomer contacts along the filament axis.

  5. The ultrastructural organization of actin and myosin II filaments in the contractile ring: new support for an old model of cytokinesis.

    PubMed

    Henson, John H; Ditzler, Casey E; Germain, Aphnie; Irwin, Patrick M; Vogt, Eric T; Yang, Shucheng; Wu, Xufeng; Shuster, Charles B

    2017-03-01

    Despite recent advances in our understanding of the components and spatial regulation of the contractile ring (CR), the precise ultrastructure of actin and myosin II within the animal cell CR remains an unanswered question. We used superresolution light microscopy and platinum replica transmission electron microscopy (TEM) to determine the structural organization of actin and myosin II in isolated cortical cytoskeletons prepared from dividing sea urchin embryos. Three-dimensional structured illumination microscopy indicated that within the CR, actin and myosin II filaments were organized into tightly packed linear arrays oriented along the axis of constriction and restricted to a narrow zone within the furrow. In contrast, myosin II filaments in earlier stages of cytokinesis were organized into small, discrete, and regularly spaced clusters. TEM showed that actin within the CR formed a dense and anisotropic array of elongate, antiparallel filaments, whereas myosin II was organized into laterally associated, head-to-head filament chains highly reminiscent of mammalian cell stress fibers. Together these results not only support the canonical "purse-string" model for contractile ring constriction, but also suggest that the CR may be derived from foci of myosin II filaments in a manner similar to what has been demonstrated in fission yeast.

  6. Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins.

    PubMed

    Blanchoin, L; Amann, K J; Higgs, H N; Marchand, J B; Kaiser, D A; Pollard, T D

    2000-04-27

    Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.

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

    PubMed

    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.

  8. Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

    PubMed Central

    Yehl, Jenna; Kudryashova, Elena; Reisler, Emil; Kudryashov, Dmitri; Polenova, Tatyana

    2017-01-01

    Cellular actin dynamics is an essential element of numerous cellular processes, such as cell motility, cell division and endocytosis. Actin’s involvement in these processes is mediated by many actin-binding proteins, among which the cofilin family plays unique and essential role in accelerating actin treadmilling in filamentous actin (F-actin) in a nucleotide-state dependent manner. Cofilin preferentially interacts with older filaments by recognizing time-dependent changes in F-actin structure associated with the hydrolysis of ATP and release of inorganic phosphate (Pi) from the nucleotide cleft of actin. The structure of cofilin on F-actin and the details of the intermolecular interface remain poorly understood at atomic resolution. Here we report atomic-level characterization by magic angle spinning (MAS) NMR of the muscle isoform of human cofilin 2 (CFL2) bound to F-actin. We demonstrate that resonance assignments for the majority of atoms are readily accomplished and we derive the intermolecular interface between CFL2 and F-actin. The MAS NMR approach reported here establishes the foundation for atomic-resolution characterization of a broad range of actin-associated proteins bound to F-actin. PMID:28303963

  9. Mechanical output of myosin II motors is regulated by myosin filament size and actin network mechanics

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Alberts, Jonathan; Gardel, Margaret; Munro, Edwin

    2013-03-01

    The interactions of bipolar myosin II filaments with actin arrays are a predominate means of generating forces in numerous physiological processes including muscle contraction and cell migration. However, how the spatiotemporal regulation of these forces depends on motor mechanochemistry, bipolar filament size, and local actin mechanics is unknown. Here, we simulate myosin II motors with an agent-based model in which the motors have been benchmarked against experimental measurements. Force generation occurs in two distinct regimes characterized either by stable tension maintenance or by stochastic buildup and release; transitions between these regimes occur by changes to duty ratio and myosin filament size. The time required for building force to stall scales inversely with the stiffness of a network and the actin gliding speed of a motor. Finally, myosin motors are predicted to contract a network toward stiffer regions, which is consistent with experimental observations. Our representation of myosin motors can be used to understand how their mechanical and biochemical properties influence their observed behavior in a variety of in vitro and in vivo contexts.

  10. MicroFilament Analyzer identifies actin network organizations in epidermal cells of Arabidopsis thaliana roots

    PubMed Central

    Jacques, Eveline; Lewandowski, Michal; Buytaert, Jan; Fierens, Yves; Verbelen, Jean-Pierre; Vissenberg, Kris

    2013-01-01

    The plant cytoskeleton plays a crucial role in the cells’ growth and development during different developmental stages and it undergoes many rearrangements. In order to describe the arrangements of the F-actin cytoskeleton in root epidermal cells of Arabidopsis thaliana, the recently developed software MicroFilament Analyzer (MFA) was exploited. This software enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way. Using confocal microscopy and transgenic GFP-FABD2-GFP plants the actin cytoskeleton was visualized in the root epidermis. MFA analysis revealed that during the early stages of cell development F-actin is organized in a mainly random pattern. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. In the evolution from young to old cells, an approximately even distribution of transverse, oblique or combined orientations is always present besides the switch from random to a longitudinal oriented actin cytoskeleton. PMID:23656865

  11. Impact of actin filament stabilization on adult hippocampal and olfactory bulb neurogenesis.

    PubMed

    Kronenberg, Golo; Gertz, Karen; Baldinger, Tina; Kirste, Imke; Eckart, Sarah; Yildirim, Ferah; Ji, Shengbo; Heuser, Isabella; Schröck, Helmut; Hörtnagl, Heide; Sohr, Reinhard; Djoufack, Pierre Chryso; Jüttner, René; Glass, Rainer; Przesdzing, Ingo; Kumar, Jitender; Freyer, Dorette; Hellweg, Rainer; Kettenmann, Helmut; Fink, Klaus Benno; Endres, Matthias

    2010-03-03

    Rearrangement of the actin cytoskeleton is essential for dynamic cellular processes. Decreased actin turnover and rigidity of cytoskeletal structures have been associated with aging and cell death. Gelsolin is a Ca(2+)-activated actin-severing protein that is widely expressed throughout the adult mammalian brain. Here, we used gelsolin-deficient (Gsn(-/-)) mice as a model system for actin filament stabilization. In Gsn(-/-) mice, emigration of newly generated cells from the subventricular zone into the olfactory bulb was slowed. In vitro, gelsolin deficiency did not affect proliferation or neuronal differentiation of adult neural progenitors cells (NPCs) but resulted in retarded migration. Surprisingly, hippocampal neurogenesis was robustly induced by gelsolin deficiency. The ability of NPCs to intrinsically sense excitatory activity and thereby implement coupling between network activity and neurogenesis has recently been established. Depolarization-induced [Ca(2+)](i) increases and exocytotic neurotransmitter release were enhanced in Gsn(-/-) synaptosomes. Importantly, treatment of Gsn(-/-) synaptosomes with mycotoxin cytochalasin D, which, like gelsolin, produces actin disassembly, decreased enhanced Ca(2+) influx and subsequent exocytotic norepinephrine release to wild-type levels. Similarly, depolarization-induced glutamate release from Gsn(-/-) brain slices was increased. Furthermore, increased hippocampal neurogenesis in Gsn(-/-) mice was associated with a special microenvironment characterized by enhanced density of perfused vessels, increased regional cerebral blood flow, and increased endothelial nitric oxide synthase (NOS-III) expression in hippocampus. Together, reduced filamentous actin turnover in presynaptic terminals causes increased Ca(2+) influx and, subsequently, elevated exocytotic neurotransmitter release acting on neural progenitors. Increased neurogenesis in Gsn(-/-) hippocampus is associated with a special vascular niche for neurogenesis.

  12. High expression of Lifeact in Arabidopsis thaliana reduces dynamic reorganization of actin filaments but does not affect plant development.

    PubMed

    van der Honing, Hannie S; van Bezouwen, Laura S; Emons, Anne Mie C; Ketelaar, Tijs

    2011-10-01

    Lifeact is a novel probe that labels actin filaments in a wide range of organisms. We compared the localization and reorganization of Lifeact:Venus-labeled actin filaments in Arabidopsis root hairs and root epidermal cells of lines that express different levels of Lifeact: Venus with that of actin filaments labeled with GFP:FABD2, a commonly used probe in plants. Unlike GFP:FABD2, Lifeact:Venus labeled the highly dynamic fine F-actin in the subapical region of tip-growing root hairs. Lifeact:Venus expression at varying levels was not observed to affect plant development. However, at expression levels comparable to those of GFP:FABD2 in a well-characterized marker line, Lifeact:Venus reduced reorganization rates of bundles of actin filaments in root epidermal cells. Reorganization rates of cytoplasmic strands, which reflect the reorganization of the actin cytoskeleton, were also reduced in these lines. Moreover, in the same line, Lifeact:Venus-decorated actin filaments were more resistant to depolymerization by latrunculin B than those in an equivalent GFP:FABD2-expressing line. In lines where Lifeact: Venus is expressed at lower levels, these effects are less prominent or even absent. We conclude that Lifeact: Venus reduces remodeling of the actin cytoskeleton in Arabidopsis in a concentration-dependent manner. Since this reduction occurs at expression levels that do not cause defects in plant development, selection of normally growing plants is not sufficient to determine optimal Lifeact expression levels. When correct expression levels of Lifeact have been determined, it is a valuable probe that labels dynamic populations of actin filaments such as fine F-actin, better than FABD2 does.

  13. Electric field modulation of the motility of actin filaments on myosin-functionalised surfaces

    NASA Astrophysics Data System (ADS)

    Ramsey, L. C.; Aveyard, J.; van Zalinge, H.; Persson, M.; Mânsson, A.; Nicolau, D. V.

    2013-02-01

    We investigated the difference in electrically guided acto-myosin motility on two surfaces. Rabbit skeletal muscle heavy meromyosin (HMM) was absorbed onto surfaces coated with Nitrocellulose (NC) and Poly(butyl methacrylate) (PBMA). A modified in vitro motility assay with sealed chambers for the insertion of electrodes allowed an electrical field to be applied across the flow cell. On all surfaces a small increase in velocity and general guidance of the actin filaments towards the positive electrode is seen at field strengths in the range of ~3000 - 4000Vm-1. A large increase in velocity was observed at ~5000Vm-1 and a significant change in the velocity of the actin filaments present in field strengths higher than this. NC supported the highest percentage of motile filaments and at a field of 8000Vm-1 reached ~66%. PBMA however supported the least percentage of motile filaments and irregular motility was observed even at higher fields where guidance was expected to be strong. The change in velocity in the range of fields tested varied significantly on the surfaces with NC displaying a 46% increase from 0 to 8000Vm-1 whereas on PBMA this value was just 37%.

  14. Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells.

    PubMed

    Laine, R O; Phaneuf, K L; Cunningham, C C; Kwiatkowski, D; Azuma, T; Southwick, F S

    1998-08-01

    The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed

  15. Visualization and force measurement of branching by Arp2/3 complex and N-WASP in actin filament.

    PubMed

    Fujiwara, Ikuko; Suetsugu, Shiro; Uemura, Sotaro; Takenawa, Tadaomi; Ishiwata, Shin'ichi

    2002-05-24

    To determine whether the Arp2/3 complex activated by N-WASP (VCA) branches actin filaments at the side (side branching), or at the barbed (B-)end (end branching) of the mother filaments, we have directly observed the branching process of actin filaments and examined single-molecule unbinding under optical microscope. We found that side branching was predominant, though not exclusive. At the initial stage of polymerization, the branching at the B-end occurred and subsequently the side branching started to occur. In either type of branching, the mother and daughter filaments elongated at nearly the same rate (growing type). Independently of the stage of polymerization, branching due to the direct coupling of filaments with an acute angle to the mother filaments (a coupling type) occurred. Phalloidin suppressed the growing type of branching but not the coupling type, implying that actin monomers are required for the former but not the latter. We found, by single molecule measurements using optical tweezers, that the Arp2/3 complex attaches to the side of actin filaments and the N-WASP appears to detach from the actin-Arp2/3 complex at 6-7 pN.

  16. Calcium-induced movement of troponin-I relative to actin in skeletal muscle thin filaments.

    PubMed

    Tao, T; Gong, B J; Leavis, P C

    1990-03-16

    The role of troponin-I (the inhibitory subunit of troponin) in the regulation by Ca2+ of skeletal muscle contraction was investigated with resonance energy transfer and photo cross-linking techniques. The effect of Ca2+ on the proximity of troponin-I to actin in reconstituted rabbit skeletal thin filaments was determined. The distance between the cysteine residue at position 133 (Cys133) of troponin-I and Cys374 of actin increases by approximately 15 angstroms on binding of Ca2+ to troponin-C. Also, troponin-I labeled at Cys133 with benzophenone-4-maleimide could be photo cross-linked to actin in the absence of Ca2+, but not in its presence. These results suggest that troponin-I is attached to actin in the Ca2(+)-free or relaxed state of muscle, and that it detaches from actin on Ca2+ activation of contraction. Thus, troponin-I may function as a Ca2(+)-dependent molecular switch in regulation of skeletal muscle contraction.

  17. Mesoscopic model for filament orientation in growing actin networks: the role of obstacle geometry

    NASA Astrophysics Data System (ADS)

    Weichsel, Julian; Schwarz, Ulrich S.

    2013-03-01

    Propulsion by growing actin networks is a universal mechanism used in many different biological systems, ranging from the sheet-like lamellipodium of crawling animal cells to the actin comet tails induced by certain bacteria and viruses in order to move within their host cells. Although the core molecular machinery for actin network growth is well preserved in all of these cases, the geometry of the propelled obstacle varies considerably. During recent years, filament orientation distribution has emerged as an important observable characterizing the structure and dynamical state of the growing network. Here we derive several continuum equations for the orientation distribution of filaments growing behind stiff obstacles of various shapes and validate the predicted steady state orientation patterns by stochastic computer simulations based on discrete filaments. We use an ordinary differential equation approach to demonstrate that for flat obstacles of finite size, two fundamentally different orientation patterns peaked at either ±35° or +70°/0°/ - 70° exhibit mutually exclusive stability, in agreement with earlier results for flat obstacles of very large lateral extension. We calculate and validate phase diagrams as a function of model parameters and show how this approach can be extended to obstacles with piecewise straight contours. For curved obstacles, we arrive at a partial differential equation in the continuum limit, which again is in good agreement with the computer simulations. In all cases, we can identify the same two fundamentally different orientation patterns, but only within an appropriate reference frame, which is adjusted to the local orientation of the obstacle contour. Our results suggest that two fundamentally different network architectures compete with each other in growing actin networks, irrespective of obstacle geometry, and clarify how simulated and electron tomography data have to be analyzed for non-flat obstacle geometries.

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

    PubMed Central

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

    2013-01-01

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

  19. ForC lacks canonical formin activity but bundles actin filaments and is required for multicellular development of Dictyostelium cells.

    PubMed

    Junemann, Alexander; Winterhoff, Moritz; Nordholz, Benjamin; Rottner, Klemens; Eichinger, Ludwig; Gräf, Ralph; Faix, Jan

    2013-01-01

    Diaphanous-related formins (DRFs) drive the nucleation and elongation of linear actin filaments downstream of Rho GTPase signalling pathways. Dictyostelium formin C (ForC) resembles a DRF, except that it lacks a genuine formin homology domain 1 (FH1), raising the questions whether or not ForC can nucleate and elongate actin filaments. We found that a recombinant ForC-FH2 fragment does not nucleate actin polymerization, but moderately decreases the rate of spontaneous actin assembly and disassembly, although the barbed-end elongation rate in the presence of the formin was not markedly changed. However, the protein bound to and crosslinked actin filaments into loose bundles of mixed polarity. Furthermore, ForC is an important regulator of morphogenesis since ForC-null cells are severely impaired in development resulting in the formation of aberrant fruiting bodies. Immunoblotting revealed that ForC is absent during growth, but becomes detectable at the onset of early aggregation when cells chemotactically stream together to form a multicellular organism, and peaks around the culmination stage. Fluorescence microscopy of cells ectopically expressing a GFP-tagged, N-terminal ForC fragment showed its prominent accumulation in the leading edge, suggesting that ForC may play a role in cell migration. In agreement with its expression profile, no defects were observed in random migration of vegetative mutant cells. Notably, chemotaxis of starved cells towards a source of cAMP was severely impaired as opposed to control. This was, however, largely due to a marked developmental delay of the mutant, as evidenced by the expression profile of the early developmental marker csA. In line with this, chemotaxis was almost restored to wild type levels after prolonged starvation. Finally, we observed a complete failure of phototaxis due to abolished slug formation and a massive reduction of spores consistent with forC promoter-driven expression of β-galactosidase in prespore cells

  20. As functional nuclear actin comes into view, is it globular, filamentous, or both?

    PubMed Central

    Pederson, Thoru

    2008-01-01

    The idea that actin may have an important function in the nucleus has undergone a rapid transition from one greeted with skepticism to a now rapidly advancing research field. Actin has now been implicated in transcription by all three RNA polymerases, but the structural form it adopts in these processes remains unclear. Recently, a claim was made that monomeric nuclear actin plays a role in signal transduction, while a just-published study of RNA polymerase I transcription has implicated polymeric actin, consorting with an isoform of its classical partner myosin. Both studies are critically discussed here, and although there are several issues to be resolved, it now seems reasonable to start thinking about functions for both monomeric and assembled actin in the nucleus. PMID:18347069

  1. The unique organization of filamentous actin in the medullary canal of the medulla oblongata.

    PubMed

    Tan, Bai-Hong; Guo, Chun-Yan; Xiong, Tian-Qing; Chen, Ling-Meng; Li, Yan-Chao

    2017-01-24

    In the central canal, F-actin is predominantly localized in the apical region, forming a ring-like structure around the circumference of the lumen. However, an exception is found in the medulla oblongata, where the apical F-actin becomes interrupted in the ventral aspect of the canal. To clarify the precise localization of F-actin, the fluorescence signals for F-actin were converted to the peroxidase/DAB reaction products in this study by a phalloidin-based ultrastructural technique, which demonstrated that F-actin is located mainly in the microvilli and terminal webs in the ependymocytes. It is because the ventrally oriented ependymocytes do not possess well-developed microvilli or terminal web that led to a discontinuous labeling of F-actin in the medullary canal. Since spinal motions can change the shape and size of the central canal, we next examined the cytoskeletons in the medullary canal in both rats and monkeys, because these two kinds of animals show different kinematics at the atlanto-occipital articulation. Our results first demonstrated that the apical F-actin in the medullary canal is differently organized in the animals with different head-neck kinemics, which suggests that the mechanic stretching of spinal motions is capable of inducing F-actin reorganization and the subsequent cell-shape changes in the central canal.

  2. Abundance of actin filaments in the preprophase band and mitotic spindle of brick1 Zea mays mutant.

    PubMed

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Tzioutziou, Nickoleta A

    2009-07-01

    The preprophase band and mitotic spindle of dividing protodermal cells of wild-type Zea mays leaves include few actin filaments. Surprisingly, abundant actin filaments were observed in the above arrays, in dividing protodermal cells in the leaves of the brick1 mutant. The same abundance was observed in the spindle of Taxol-treated brick1 mitotic protodermal cells. Apart from the above difference, the relevant arrays displayed normal microtubule organization in both wild type and mutant cells, as far as can be discerned by immunofluorescence microscopy. Accordingly, the abundance of actin filaments in the preprophase band and spindle of brick1 mitotic cells seems not to influence the structure of the above arrays and might be a non-functional "side-effect" of defective F-actin organization in this mutant.

  3. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments.

    PubMed

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-10-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H(+)-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation.

  4. Myosin-Va binds to and mechanochemically couples microtubules to actin filaments.

    PubMed

    Cao, Tracy T; Chang, Wakam; Masters, Sarah E; Mooseker, Mark S

    2004-01-01

    Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.

  5. Closed membrane shapes with attached BAR domains subject to external force of actin filaments.

    PubMed

    Mesarec, Luka; Góźdź, Wojciech; Iglič, Veronika Kralj; Kralj, Samo; Iglič, Aleš

    2016-05-01

    Membrane deformations induced by attached BAR superfamily domains could trigger or facilitate the growth of plasma membrane protrusions. The BAR domain family consists of BAR, F-BAR and I-BAR domains, each enforcing a different local curvature when attached to the membrane surface. Our theoretical study mainly focuses on the role of I-BAR in the membrane tubular deformations generated or stabilised by actin filaments. The influence of the area density of membrane attached BAR domains and their intrinsic curvature on the closed membrane shapes (vesicles) was investigated numerically. We derived an analytical approximative expression for the critical relative area density of BARs at which the membrane tubular protrusions on vesicles are most prominent. We have shown that the BARs with a higher intrinsic curvature induce thinner and longer cylindrical protrusions. The average orientation of the membrane attached BARs is altered when the vesicle shape is subjected to external force of growing actin rod-like structure inside a vesicle. The average orientation angle of membrane attached BARs may indicate whether the actin filaments are just stabilising the protrusion or generating it by stretching the vesicle.

  6. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments

    PubMed Central

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C.; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-01-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H+-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation. PMID:26272553

  7. Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction.

    PubMed

    Butkevich, Eugenia; Bodensiek, Kai; Fakhri, Nikta; von Roden, Kerstin; Schaap, Iwan A T; Majoul, Irina; Schmidt, Christoph F; Klopfenstein, Dieter R

    2015-07-06

    Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling.

  8. Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments.

    PubMed

    Billington, Neil; Beach, Jordan R; Heissler, Sarah M; Remmert, Kirsten; Guzik-Lendrum, Stephanie; Nagy, Attila; Takagi, Yasuharu; Shao, Lin; Li, Dong; Yang, Yi; Zhang, Yingfan; Barzik, Melanie; Betzig, Eric; Hammer, John A; Sellers, James R

    2015-03-30

    Class-18 myosins are most closely related to conventional class-2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse, and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, suggesting that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the longest coiled coil of any myosin outside of the class-2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any of the conditions tested. Instead, M18A formed an ∼ 65-nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.

  9. The role of cyclase-associated protein in regulating actin filament dynamics - more than a monomer-sequestration factor.

    PubMed

    Ono, Shoichiro

    2013-08-01

    Dynamic reorganization of the actin cytoskeleton is fundamental to a number of cell biological events. A variety of actin-regulatory proteins modulate polymerization and depolymerization of actin and contribute to actin cytoskeletal reorganization. Cyclase-associated protein (CAP) is a conserved actin-monomer-binding protein that has been studied for over 20 years. Early studies have shown that CAP sequesters actin monomers; recent studies, however, have revealed more active roles of CAP in actin filament dynamics. CAP enhances the recharging of actin monomers with ATP antagonistically to ADF/cofilin, and also promotes the severing of actin filaments in cooperation with ADF/cofilin. Self-oligomerization and binding to other proteins regulate activities and localization of CAP. CAP has crucial roles in cell signaling, development, vesicle trafficking, cell migration and muscle sarcomere assembly. This Commentary discusses the recent advances in our understanding of the functions of CAP and its implications as an important regulator of actin cytoskeletal dynamics, which are involved in various cellular activities.

  10. Mammalian CAP (Cyclase-associated protein) in the world of cell migration: Roles in actin filament dynamics and beyond.

    PubMed

    Zhou, Guo-Lei; Zhang, Haitao; Field, Jeffrey

    2014-01-01

    Cell migration is essential for a variety of fundamental biological processes such as embryonic development, wound healing, and immune response. Aberrant cell migration also underlies pathological conditions such as cancer metastasis, in which morphological transformation promotes spreading of cancer to new sites. Cell migration is driven by actin dynamics, which is the repeated cycling of monomeric actin (G-actin) into and out of filamentous actin (F-actin). CAP (Cyclase-associated protein, also called Srv2) is a conserved actin-regulatory protein, which is implicated in cell motility and the invasiveness of human cancers. It cooperates with another actin regulatory protein, cofilin, to accelerate actin dynamics. Hence, knockdown of CAP1 slows down actin filament turnover, which in most cells leads to reduced cell motility. However, depletion of CAP1 in HeLa cells, while causing reduction in dynamics, actually led to increased cell motility. The increases in motility are likely through activation of cell adhesion signals through an inside-out signaling. The potential to activate adhesion signaling competes with the negative effect of CAP1 depletion on actin dynamics, which would reduce cell migration. In this commentary, we provide a brief overview of the roles of mammalian CAP1 in cell migration, and highlight a likely mechanism underlying the activation of cell adhesion signaling and elevated motility caused by depletion of CAP1.

  11. Gas7b (growth arrest specific protein 7b) regulates neuronal cell morphology by enhancing microtubule and actin filament assembly.

    PubMed

    Gotoh, Aina; Hidaka, Masafumi; Hirose, Keiko; Uchida, Takafumi

    2013-11-29

    Neurons undergo several morphological changes as a part of normal neuron maturation process. Alzheimer disease is associated with increased neuroproliferation and impaired neuronal maturation. In this study, we demonstrated that Gas7b (growth arrest specific protein 7b) expression in a neuronal cell line, Neuro 2A, induces cell maturation by facilitating formation of dendrite-like processes and/or filopodia projections and that Gas7b co-localizes with neurite microtubules. Molecular analysis was performed to evaluate whether Gas7b associates with actin filaments and microtubules, and the data revealed two novel roles of Gas7b in neurite outgrowth: we showed that Gas7b enhances bundling of several microtubule filaments and connects microtubules with actin filaments. These results suggest that Gas7b governs neural cell morphogenesis by enhancing the coordination between actin filaments and microtubules. We conclude that lower neuronal Gas7b levels may impact Alzheimer disease progression.

  12. Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins

    PubMed Central

    1994-01-01

    Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells. PMID:8051203

  13. Cargo Transport by Two Coupled Myosin Va Motors on Actin Filaments and Bundles.

    PubMed

    Ali, M Yusuf; Vilfan, Andrej; Trybus, Kathleen M; Warshaw, David M

    2016-11-15

    Myosin Va (myoVa) is a processive, actin-based molecular motor essential for intracellular cargo transport. When a cargo is transported by an ensemble of myoVa motors, each motor faces significant physical barriers and directional challenges created by the complex actin cytoskeleton, a network of actin filaments and actin bundles. The principles that govern the interaction of multiple motors attached to the same cargo are still poorly understood. To understand the mechanical interactions between multiple motors, we developed a simple in vitro model in which two individual myoVa motors labeled with different-colored Qdots are linked via a third Qdot that acts as a cargo. The velocity of this two-motor complex was reduced by 27% as compared to a single motor, whereas run length was increased by only 37%, much less than expected from multimotor transport models. Therefore, at low ATP, which allowed us to identify individual motor steps, we investigated the intermotor dynamics within the two-motor complex. The randomness of stepping leads to a buildup of tension in the linkage between motors-which in turn slows down the leading motor-and increases the frequency of backward steps and the detachment rate. We establish a direct relationship between the velocity reduction and the distribution of intermotor distances. The analysis of run lengths and dwell times for the two-motor complex, which has only one motor engaged with the actin track, reveals that half of the runs are terminated by almost simultaneous detachment of both motors. This finding challenges the assumptions of conventional multimotor models based on consecutive motor detachment. Similar, but even more drastic, results were observed with two-motor complexes on actin bundles, which showed a run length that was even shorter than that of a single motor.

  14. Brownian Ratchets in Biophysics: from Diffusing Phospholipids to Polymerizing Actin Filaments

    NASA Astrophysics Data System (ADS)

    van Oudenaarden, Alexander

    2000-03-01

    In the 'Feynman Lectures on Physics' Feynman introduces a mechanical ratchet and pawl subjected to thermal fluctuations to demonstrate the impossibility to violate the second law of thermodynamics. Since this introduction the Brownian ratchet has evolved from Gedanken experiments to real experiments in the interdisciplinary sciences such as biophysics and biochemistry. In this symposium I will present two experiments in which the concept Brownian ratchet is of key importance. The first experiment addresses a so-called geometrical Brownian ratchet [1]. This ratchet consists of a two-dimensional microfabricated periodic array of asymmetric diffusion barriers. As an experimental realization of a two-dimensional fluid of Brownian particles, a bilayer of phospholipid molecules is used. I will demonstrate that the geometrical Brownian ratchet can be used as a molecular sieve to separate mixtures of membrane molecules without the need to extract them from the membrane. In the second experiment I explore the spontaneous symmetry breaking of polymerizing actin networks [2]. Small submicron size beads coated uniformly with a protein that catalyzes actin polymerization, are initially surrounded by a symmetrical cloud of actin filaments. This symmetry can be broken spontaneously after which the beads undergo directional motion with constant velocity. I will present a simple stochastic theory, in which each filament is modeled as an elastic Brownian ratchet that qualitatively reproduces the experimental results. The presence of the bead couples the dynamics of different filaments which results in a complex collective system of interacting Brownian ratchets that exhibits an emergent symmetry breaking behavior. [1] A. van Oudenaarden and S. G. Boxer, Science 285, 1046 (1999). [2] A. van Oudenaarden and J. A. Theriot, Nature Cell Biology 1, 493 (1999).

  15. Effects of asymmetric nanostructures on the extinction difference properties of actin biomolecules and filaments

    PubMed Central

    Khoo, E. H.; Leong, Eunice S. P.; Wu, S. J.; Phua, W. K.; Hor, Y. L.; Liu, Y. J.

    2016-01-01

    In this paper, symmetric and asymmetric tapering on the arms of the gammadion nanostructure is proposed to enhance both local field distribution and extinction difference (ED). The asymmetric tapered gammadion with tapering fraction (TF) of 0.67 is seen to have the largest ED and spatial local field distribution, producing a large wavelength shift of more than 50 percent as compared to the untapered gammadion nanostructures when immersed in a solution of actin molecules and filaments. The optical chirality, ζ shows that the larger local field amplitudes produced by the asymmetric designs increases the rate of chiral molecules excitation. This enhanced field is strongly rotating and highly sensitive to single molecules and larger filaments. Here, we show that the ED, optical chirality, sensitivity and rate of chiral molecules excitation can be improved by incorporating asymmetric designs into chiral gammadion nanostructures through tapering. PMID:26792371

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

    SciTech Connect

    Stall, Richard; Ramos, Joseph; Kent Fulcher, F.; Patel, Yashomati M.

    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.

  17. Regulation of structure and function of sarcomeric actin filaments in striated muscle of the nematode Caenorhabditis elegans

    PubMed Central

    Ono, Shoichiro

    2014-01-01

    The nematode Caenorhabditis elegans has been used as a valuable system to study structure and function of striated muscle. The body wall muscle of C. elegans is obliquely striated muscle with highly organized sarcomeric assembly of actin, myosin, and other accessary proteins. Genetic and molecular biological studies in C. elegans have identified a number of genes encoding structural and regulatory components for the muscle contractile apparatuses, and many of them have counterparts in mammalian cardiac and skeletal muscles or striated muscles in other invertebrates. Applicability of genetics, cell biology, and biochemistry has made C. elegans an excellent system to study mechanisms of muscle contractility and assembly and maintenance of myofibrils. This review focuses on the regulatory mechanisms of structure and function of actin filaments in the C. elegans body wall muscle. Sarcomeric actin filaments in C. elegans muscle are associated with the troponin-tropomyosin system that regulates the actin-myosin interaction. Proteins that bind to the side and ends of actin filaments support ordered assembly of thin filaments. Furthermore, regulators of actin dynamics play important roles in initial assembly, growth, and maintenance of sarcomeres. The knowledge acquired in C. elegans can serve as bases to understand the basic mechanisms of muscle structure and function. PMID:25125169

  18. Thermal activation energy for bidirectional movement of actin along bipolar tracks of myosin filaments.

    PubMed

    Okubo, Hiroyuki; Iwai, Masanori; Iwai, Sosuke; Chaen, Shigeru

    2010-05-28

    Previous in vitro motility assays using bipolar myosin thick filaments demonstrated that actin filaments were capable of moving in both directions along the myosin filament tracks. The movements; however, were slower in the direction leading away from the central bare zone than towards it. To understand the mechanism underlying these different direction-dependent motilities, we have examined the effects of temperature on the velocities of the bidirectional movements along reconstituted myosin filaments. Activation energies of the movements were determined by Arrhenius plots at high and low concentrations of ATP. As a result, the thermal activation energy of the movement away from the central bare zone was significantly higher than that of the movement toward the zone. Given that the backward movement away from the central bare zone would cause the myosin heads to be constrained and the stiffness of the cross-bridges to increase, these results suggest that elastic energy required for the cross-bridge transition is supplied by thermal fluctuations.

  19. Impaired tropomyosin-troponin interactions reduce activation of the actin thin filament.

    PubMed

    Robaszkiewicz, Katarzyna; Ostrowska, Zofia; Cyranka-Czaja, Anna; Moraczewska, Joanna

    2015-05-01

    Tropomyosin and troponin are bound to the actin filament to control the contraction of striated muscle in the Ca-dependent manner. The interactions between both regulatory proteins important for the regulation process are not fully understood. To gain more insight into the mechanisms of the thin filament regulation by skeletal α-tropomyosin and troponin, we analyzed effects of seven myopathy-related substitutions: Leu99Met, Ala155Thr, Arg167Gly, Arg167Cys, Arg167His, Lys168Glu, and Arg244Gly. All substitutions reduced Ca-dependent activation of the actomyosin ATPase. The effects of mutations in Arg167 and Lys168 were the most severe. The amino acid substitutions did not significantly affect troponin binding to the whole filament, but reduced 1.2-2.8 fold the affinity of troponin to tropomyosin alone. The excimer fluorescence of N-(1-pyrene)iodoacetamide, a probe attached to the central Cys190, demonstrated that substitutions located near the troponin core domain-binding region strongly affected conformational changes accompanying the tropomyosin-troponin interactions. The thermal stability of all tropomyosin mutants was lower than the stability of the wild type tropomyosin, with TM reduced by 5.3-8.5°C. Together the analyses demonstrated that the myopathy-causing mutations affected tropomyosin structure and led to changes in interactions between tropomyosin and troponin, which impaired the transition of the thin filament from the inactive off to the active on state.

  20. Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis

    PubMed Central

    1994-01-01

    The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers. PMID:8034744

  1. Migration of Nucleocapsids in Vesicular Stomatitis Virus-Infected Cells Is Dependent on both Microtubules and Actin Filaments

    PubMed Central

    Yacovone, Shalane K.; Smelser, Amanda M.; Macosko, Jed C.; Holzwarth, George; Ornelles, David A.

    2016-01-01

    ABSTRACT The distribution of vesicular stomatitis virus (VSV) nucleocapsids in the cytoplasm of infected cells was analyzed by scanning confocal fluorescence microscopy using a newly developed quantitative approach called the border-to-border distribution method. Nucleocapsids were located near the cell nucleus at early times postinfection (2 h) but were redistributed during infection toward the edges of the cell. This redistribution was inhibited by treatment with nocodazole, colcemid, or cytochalasin D, indicating it is dependent on both microtubules and actin filaments. The role of actin filaments in nucleocapsid mobility was also confirmed by live-cell imaging of fluorescent nucleocapsids of a virus containing P protein fused to enhanced green fluorescent protein. However, in contrast to the overall redistribution in the cytoplasm, the incorporation of nucleocapsids into virions as determined in pulse-chase experiments was dependent on the activity of actin filaments with little if any effect on inhibition of microtubule function. These results indicate that the mechanisms by which nucleocapsids are transported to the farthest reaches of the cell differ from those required for incorporation into virions. This is likely due to the ability of nucleocapsids to follow shorter paths to the plasma membrane mediated by actin filaments. IMPORTANCE Nucleocapsids of nonsegmented negative-strand viruses like VSV are assembled in the cytoplasm during genome RNA replication and must migrate to the plasma membrane for assembly into virions. Nucleocapsids are too large to diffuse in the cytoplasm in the time required for virus assembly and must be transported by cytoskeletal elements. Previous results suggested that microtubules were responsible for migration of VSV nucleocapsids to the plasma membrane for virus assembly. Data presented here show that both microtubules and actin filaments are responsible for mobility of nucleocapsids in the cytoplasm, but that actin filaments

  2. Correlation of expression of the actin filament-bundling protein espin with stereociliary bundle formation in the developing inner ear.

    PubMed

    Li, Huawei; Liu, Hong; Balt, Steve; Mann, Sabine; Corrales, C Eduardo; Heller, Stefan

    2004-01-01

    The vertebrate hair cell is named for its stereociliary bundle or hair bundle that protrudes from the cell's apical surface. Hair bundles mediate mechanosensitivity, and their highly organized structure plays a critical role in mechanoelectrical transduction and amplification. The prototypical hair bundle is composed of individual stereocilia, 50-300 in number, depending on the animal species and on the type of hair cell. The assembly of stereocilia, in particular, the formation during development of individual rows of stereocilia with descending length, has been analyzed in great morphological detail. Electron microscopic studies have demonstrated that stereocilia are filled with actin filaments that are rigidly cross-linked. The growth of individual rows of stereocilia is associated with the addition of actin filaments and with progressively increasing numbers of cross-bridges between actin filaments. Recently, a mutation in the actin filament-bundling protein espin has been shown to underlie hair bundle degeneration in the deaf jerker mouse, subsequently leading to deafness. Our study was undertaken to investigate the appearance and developmental expression of espin in chicken inner ear sensory epithelia. We found that the onset of espin expression correlates with the initiation and growth of stereocilia bundles in vestibular and cochlear hair cells. Intense espin immunolabeling of stereocilia was colocalized with actin filament staining in all types of hair cells at all developmental stages and in adult animals. Our analysis of espin as a molecular marker for actin filament cross-links in stereocilia is in full accordance with previous morphological studies and implicates espin as an important structural component of hair bundles from initiation of bundle assembly to mature chicken hair cells.

  3. Zyxin regulates endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules

    PubMed Central

    Han, Xiaofan; Li, Pin; Yang, Zhenghao; Huang, Xiaoshuai; Wei, Guoqin; Sun, Yujie; Kang, Xuya; Hu, Xueting; Deng, Qiuping; Chen, Liangyi; He, Aibin; Huo, Yingqing; Li, Dong; Betzig, Eric; Luo, Jincai

    2017-01-01

    Endothelial exocytosis of Weibel–Palade body (WPB) is one of the first lines of defence against vascular injury. However, the mechanisms that control WPB exocytosis in the final stages (including the docking, priming and fusion of granules) are poorly understood. Here we show that the focal adhesion protein zyxin is crucial in this process. Zyxin downregulation inhibits the secretion of von Willebrand factor (VWF), the most abundant cargo in WPBs, from human primary endothelial cells (ECs) induced by cAMP agonists. Zyxin-deficient mice exhibit impaired epinephrine-stimulated VWF release, prolonged bleeding time and thrombosis, largely due to defective endothelial secretion of VWF. Using live-cell super-resolution microscopy, we visualize previously unappreciated reorganization of pre-existing actin filaments around WPBs before fusion, dependent on zyxin and an interaction with the actin crosslinker α-actinin. Our findings identify zyxin as a physiological regulator of endothelial exocytosis through reorganizing local actin network in the final stage of exocytosis. PMID:28256511

  4. Malaria parasites form filamentous cell-to-cell connections during reproduction in the mosquito midgut.

    PubMed

    Rupp, Ingrid; Sologub, Ludmilla; Williamson, Kim C; Scheuermayer, Matthias; Reininger, Luc; Doerig, Christian; Eksi, Saliha; Kombila, Davy U; Frank, Matthias; Pradel, Gabriele

    2011-04-01

    Physical contact is important for the interaction between animal cells, but it can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of > 100 μm and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive "nanotubes" in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut.

  5. The rearrangement of filamentous actin in mossy fiber synapses in pentylenetetrazol-kindled C57BL/6 mice.

    PubMed

    Zhang, Yan-Feng; Li, Shu-Lei; Xiong, Tian-Qing; Yang, Li-Bin; Li, Yong-Nan; Tan, Bai-Hong; Liu, Qun; Li, Yan-Chao

    2014-01-01

    Chemical kindling, as an experimental model of epileptogenesis, is induced by repetitive administration of subconvulsive amount of excitatory drugs. Kindled mice do not typically display spontaneous recurrent seizures, but are instead characterized by enhanced seizure susceptibility to convulsive stimulations. In order to provide insights into the aberrant synaptic plasticity during kindling, this study investigated the effect of pentylenetetrazol (PTZ) kindling on filamentous actin (F-actin) in mossy fiber synapses in C57BL/6 mice. Phalloidin labeling of F-actin showed that F-actin puncta were increased in number in the stratum lucidum of CA3 region in the hippocampus after kindling. The rearrangement of F-actin seemed to occur presynaptically, since synapsin I, a specific marker for mossy fiber terminals, was also up-regulated. Such subtle structural modifications occurring in the synapses are thought to contribute to the long-lasting increased sensitivity in the PTZ-kindled C57BL/6 mice.

  6. Reaction-diffusion waves of actin filament polymerization/depolymerization in Dictyostelium pseudopodium extension and cell locomotion.

    PubMed

    Vicker, M G

    2000-04-14

    Cell surface movements and the intracellular spatial patterns and dynamics of actin filament (F-actin) were investigated in living and formalin-fixed cells of Dictyostelium discoideum by confocal microscopy. Excitation waves of F-actin assembly developed and propagated several micrometers at up to 26 microm/min in cells which had been intracellularly loaded with fluorescently labeled actin monomer. Wave propagation and extinction corresponded with the initiation and attenuation of pseudopodium extension and cell advance, respectively. The identification of chemical waves was supported by the ring, sphere, spiral and scroll wave patterns, which were observed in the extensions of fixed cells stained with phalloidin-rhodamine, and by the similar, asymmetrical [F-actin] distribution in wavefronts in living and fixed cells. These F-actin patterns and dynamics in Dictyostelium provide evidence for a new supramolecular state of actin, which propagates as a self-organized, reaction-diffusion wave of reversible F-actin assembly and affects pseudopodium extension. Actin's properties of oscillation and self-organization might also fundamentally determine the nature of the eukaryotic cell's reactions of adaptation, timing and signal response.

  7. Mammalian homolog of the yeast cyclase associated protein, CAP/Srv2p, regulates actin filament assembly.

    PubMed

    Freeman, N L; Field, J

    2000-02-01

    Control of cell shape and motility requires rearrangements of the actin cytoskeleton. One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56). CAP was first isolated from yeast as an adenylyl cyclase associated protein required for RAS regulation of cAMP signaling. In addition, CAP also regulates the actin cytoskeleton primarily through an actin monomer binding activity. CAP homologs are found in many eukaryotes, including mammals where they also bind actin, but little is known about their biological function. We, therefore, designed experiments to address CAP1 regulation of the actin cytoskeleton. CAP1 localized to membrane ruffles and actin stress fibers in fixed cells of various types. To address localization in living cells, we constructed GFP-CAP1 fusion proteins and found that fusion proteins lacking the actin-binding region localized like the wild type protein. We also performed microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts and found that the antibodies attenuated serum stimulation of stress fibers. Finally, CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulated the formation of stress fiber-like filaments when it was microinjected into serum-starved Swiss 3T3 cells. Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton.

  8. Kinetic heterogeneity of F-actin polymers. Further evidence that the elongation reaction may occur through condensation of the actin filaments with small aggregates.

    PubMed Central

    Grazi, E; Magri, E

    1987-01-01

    We have shown that F-actin, polymerized in 50 mM-KCl at 20 degrees C and pH 8.0, can be resolved by centrifugation into two polymer populations, which differ morphologically as well as kinetically. The first population represents about 10% of the overall polymer and is composed of small amorphous aggregates. It rapidly exchanges the bound nucleotide with free ATP in the medium, either directly or through the monomers. The second population is composed of long actin filaments. These are labelled by free ATP in the medium only through condensation with labelled small amorphous aggregates. Images Fig. 1. PMID:3435480

  9. Growth and Division of Filamentous Forms of Escherichia coli.

    PubMed

    Adler, H I; Hardigree, A A

    1965-07-01

    Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), and Alice A. Hardigree. Growth and division of filamentous forms of Escherichia coli. J. Bacteriol. 90:223-226. 1965.-Cells of certain mutant strains of Escherichia coli grow into long multinucleate filaments after exposure to radiation. Deoxyribonucleic acid, ribonucleic acid, and protein synthesis proceed, but cytokinesis does not occur. Cytokinesis (cross-septation) can be initiated by exposure of the filaments to pantoyl lactone or a temperature of 42 C. If growing filaments are treated with mitomycin C, nuclear division does not occur, and nuclear material is confined to the central region of the filament. Cytokinesis cannot be induced in mitomycin C-treated filaments by pantoyl lactone or treatment at 42 C.

  10. Direct observation of the uncapping of capping protein-capped actin filaments by CARMIL homology domain 3.

    PubMed

    Fujiwara, Ikuko; Remmert, Kirsten; Hammer, John A

    2010-01-22

    Bulk solution assays have shown that the isolated CARMIL homology 3 (CAH3) domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with capping protein (CP). To demonstrate this putative uncapping activity directly, we used total internal reflection microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). The addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric green fluorescent protein (mGFP-CP). Restoration of polymerization upon the addition of mCAH3 was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of approximately 90% at approximately 250 nm mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from approximately 30 min without mCAH3 to approximately 10 s with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL (and presumably the intact molecule as well) possesses the ability to uncap CP-capped actin filaments.

  11. Direct Observation of the Uncapping of Capping Protein-capped Actin Filaments by CARMIL Homology Domain 3*

    PubMed Central

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

    2010-01-01

    Bulk solution assays have shown that the isolated CARMIL homology 3 (CAH3) domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with capping protein (CP). To demonstrate this putative uncapping activity directly, we used total internal reflection microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). The addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric green fluorescent protein (mGFP-CP). Restoration of polymerization upon the addition of mCAH3 was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of ∼90% at ∼250 nm mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from ∼30 min without mCAH3 to ∼10 s with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL (and presumably the intact molecule as well) possesses the ability to uncap CP-capped actin filaments. PMID:19926785

  12. Actin-based movement of Listeria monocytogenes: actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface

    PubMed Central

    1995-01-01

    The thermodynamic basis for actin-based motility of Listeria monocytogenes has been investigated using cytoplasmic extracts of Xenopus eggs, initially developed by Theriot et al. (Theriot, J. A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J. Mitchison. 1994. Cell. 76:505-517) as an in vitro cell-free system. A large proportion (75%) of actin was found unpolymerized in the extracts. The amount of unassembled actin (12 microM) is accounted for by the sequestering functions of T beta 4Xen (20 microM) and profilin (5 microM), the barbed ends being capped. Movement of Listeria was not abolished by depletion of over 99% of the endogenous profilin. The proline-rich sequences of ActA are unlikely to be the target of profilin. All data support the view that actin assembly at the rear of Listeria results from a local shift in steady state due to a factor, keeping filaments uncapped, bound to the surface of the bacterium, while barbed ends are capped in the bulk cytoplasm. Movement is controlled by the energetic difference (i.e., the difference in critical concentration) between the two ends of the filaments, hence a constant ATP supply and the presence of barbed end capped F-actin in the medium are required to buffer free G-actin at a high concentration. The role of membrane components is demonstrated by the facts that: (a) Listeria movement can be reconstituted in the resuspended pellets of high speed-centrifuged extracts that are enriched in membranes; (b) Actin-based motility of endogenous vesicles, exhibiting the same rocketing movement as Listeria, can be observed in the extracts. PMID:7615635

  13. Specific Transformation of Assembly with Actin Filaments and Molecular Motors in a Cell-Sized Self-Emerged Liposome

    NASA Astrophysics Data System (ADS)

    Takiguchi, Kingo; Negishi, Makiko; Tanaka-Takiguchi, Yohko; Hayashi, Masahito; Yoshikawa, Kenichi

    2014-12-01

    Eukaryotes, by the same combination of cytoskeleton and molecular motor, for example actin filament and myosin, can generate a variety of movements. For this diversity, the organization of biological machineries caused by the confinement and/or crowding effects of internal living cells, may play very important roles.

  14. Vimentin intermediate filaments control actin stress fiber assembly through GEF-H1 and RhoA

    PubMed Central

    Peränen, Johan; Schaible, Niccole; Cheng, Fang; Eriksson, John E.; Krishnan, Ramaswamy

    2017-01-01

    ABSTRACT The actin and intermediate filament cytoskeletons contribute to numerous cellular processes, including morphogenesis, cytokinesis and migration. These two cytoskeletal systems associate with each other, but the underlying mechanisms of this interaction are incompletely understood. Here, we show that inactivation of vimentin leads to increased actin stress fiber assembly and contractility, and consequent elevation of myosin light chain phosphorylation and stabilization of tropomyosin-4.2 (see Geeves et al., 2015). The vimentin-knockout phenotypes can be rescued by re-expression of wild-type vimentin, but not by the non-filamentous ‘unit length form’ vimentin, demonstrating that intact vimentin intermediate filaments are required to facilitate the effects on the actin cytoskeleton. Finally, we provide evidence that the effects of vimentin on stress fibers are mediated by activation of RhoA through its guanine nucleotide exchange factor GEF-H1 (also known as ARHGEF2). Vimentin depletion induces phosphorylation of the microtubule-associated GEF-H1 on Ser886, and thereby promotes RhoA activity and actin stress fiber assembly. Taken together, these data reveal a new mechanism by which intermediate filaments regulate contractile actomyosin bundles, and may explain why elevated vimentin expression levels correlate with increased migration and invasion of cancer cells. PMID:28096473

  15. The ALP-Enigma protein ALP-1 functions in actin filament organization to promote muscle structural integrity in Caenorhabditis elegans.

    PubMed

    Han, Hsiao-Fen; Beckerle, Mary C

    2009-05-01

    Mutations that affect the Z-disk-associated ALP-Enigma proteins have been linked to human muscular and cardiac diseases. Despite their clear physiological significance for human health, the mechanism of action of ALP-Enigma proteins is largely unknown. In Caenorhabditis elegans, the ALP-Enigma protein family is encoded by a single gene, alp-1; thus C. elegans provides an excellent model to study ALP-Enigma function. Here we present a molecular and genetic analysis of ALP-Enigma function in C. elegans. We show that ALP-1 and alpha-actinin colocalize at dense bodies where actin filaments are anchored and that the proper localization of ALP-1 at dense bodies is dependent on alpha-actinin. Our analysis of alp-1 mutants demonstrates that ALP-1 functions to maintain actin filament organization and participates in muscle stabilization during contraction. Reducing alpha-actinin activity enhances the actin filament phenotype of the alp-1 mutants, suggesting that ALP-1 and alpha-actinin function in the same cellular process. Like alpha-actinin, alp-1 also interacts genetically with a connectin/titin family member, ketn-1, to provide mechanical stability for supporting body wall muscle contraction. Taken together, our data demonstrate that ALP-1 and alpha-actinin function together to stabilize actin filaments and promote muscle structural integrity.

  16. Demonstration of actin filament stress fibers in microvascular endothelial cells in situ.

    PubMed

    Nehls, V; Drenckhahn, D

    1991-07-01

    We have developed a method for immunostaining the microvascular tree of rat mesenteric windows in situ. The procedure consists of three steps, i.e., mild fixation with formaldehyde, controlled proteolytic digestion of the mesothelial layer, and permeabilization with acetone. Discrimination between different microvascular segments was possible by double-fluorescent staining with antibodies to the smooth muscle isoform of alpha-actin and to nonmuscle myosin from platelets. Antibodies to nonmuscle myosin labeled numerous longitudinally oriented cables in endothelial cells of all microvascular segments (arterioles, metarterioles, pre-, mid-, and postcapillaries, small venules). Occasionally, the myosin-containing cables displayed the interrupted sarcomere-like staining pattern that is diagnostic for stress fibers. In contrast, staining of actin filaments with phalloidin-rhodamin resulted in a noninterrupted, continuous fluorescence of the stress fibers. A possible functional role of microvascular endothelial stress fibers is to serve as a tensile cytoskeletal scaffold that stabilizes the tubular, three-dimensional geometry of microvessels and, in addition, to help the endothelium resist the shear forces created by blood flow and by collision with red and white blood cells.

  17. Oxygen radicals production and actin filament disruption in bivalve haemocytes treated with benzo(a)pyrene.

    PubMed

    Gómez-Mendikute, Amagoia; Etxeberria, Ainhoa; Olabarrieta, Igor; Cajaraville, Miren P

    2002-01-01

    Haemocytes play an essential role in the internal defence of molluscs. It has been reported that organic xenobiotics commonly found as pollutants in the marine environment impair defence capabilities of haemocytes. The purpose of the present study was to investigate the effects of benzo(a)pyrene [B(a)P] on the integrity of the actin cytoskeleton and on endocytosis in haemocytes and to see if these effects are related to generation of reactive oxygen species. Haemocytes were exposed in vitro to B(a)P (0.5-40 microg/ml) for 1 h. Cell viability (using 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide or XTT assay) indicated that selected doses were sublethal. Uptake of neutral red was significantly decreased in a dose-dependent manner in B(a)P-treated haemocytes. Distribution of actin filaments, labeled with rhodamine-conjugated phalloidin, was altered in haemocytes treated with 20 or 40 microg/ml B(a)P. These effects could be related to an increased production of superoxide anion during B(a)P metabolism, as detected by the nitroblue tetrazolium (NBT) reduction assay in haemocytes treated with > or = 10 microg/ml B(a)P.

  18. Observation of Cell Shortening and Dynamic Changes of Actin Filaments during Cell Detachment from Thermoresponsive-Gelatin-Coated Substrate

    NASA Astrophysics Data System (ADS)

    Nagayama, Kazuaki; Matsumoto, Takeo

    We observed cell shortening and dynamic changes of actin filaments during detachment from the substrate by using a thermoresponsive-gelatin. The thermoresponsive-gelatin, a mixture of the poly(N-isopropylacrylamide) grafted gelatin (PNIPAAm-grafted-gelatin) and PNIPAAm, was coated on the glass bottom culture dishes. Rat aortic smooth muscle cells (SMC) expressed GFP-actin were cultured on the thermoresponsive-gelatin-coated dishes filled with serum free Dulbecco’s Modified Eagle’s medium (DMEM) or Ca2 + -Mg2 + -free Hank’s balanced salt solution (HBSS(-)). They adhered normally at 37°C, and became shortened and detached from the dishes when the ambient temperature was dropped below 34°C due to melting of the gelatin substrate. The shortening of SMCs was larger in DMEM (51.0 ±3.0%, mean ± SEM, n=14) than in HBSS(-) (35.8 ±3.6%, n=14). Actin filaments remained straight during detachment in HBSS(-), while in DMEM, they locally concentrated and disappeared at cell periphery. The shortening of SMCs upon detachment from ordinary plastic culture dishes by trypsinization was more than for both media. No significant difference was observed between the two, indicating detrimental effects of the trypsin. The present method was useful to study the cell contraction and the actin filament behavior during cell detachment for this causes minimal damage to cells.

  19. The Interaction of Arp2/3 Complex with Actin: Nucleation, High Affinity Pointed End Capping, and Formation of Branching Networks of Filaments

    NASA Astrophysics Data System (ADS)

    Dyche Mullins, R.; Heuser, John A.; Pollard, Thomas D.

    1998-05-01

    The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μ M. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

  20. Why is Actin Patchy?

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2009-03-01

    The intracellular protein actin, by reversibly polymerizing into filaments, generates forces for motion and shape changes of many types of biological cells. Fluorescence imaging studies show that actin often occurs in the form of localized patches of size roughly one micrometer at the cell membrane. Patch formation is most prevalent when the free-actin concentration is low. I investigate possible mechanisms for the formation of actin patches by numerically simulating the ``dendritic nucleation'' model of actin network growth. The simulations include filament growth, capping, branching, severing, and debranching. The attachment of membrane-bound activators to actin filaments, and subsequent membrane diffusion of unattached activators, are also included. It is found that as the actin concentration increases from zero, the actin occurs in patches at lower actin concentrations, and the size of the patches increases with increasing actin concentration. At a critical value of the actin concentration, the system undergoes a transition to complete coverage. The results are interpreted within the framework of reaction-diffusion equations in two dimensions.

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

    PubMed Central

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

    2015-01-01

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

  2. A variational approach to the growth dynamics of pre-stressed actin filament networks

    NASA Astrophysics Data System (ADS)

    John, Karin; Stöter, Thomas; Misbah, Chaouqi

    2016-09-01

    In order to model the growth dynamics of elastic bodies with residual stresses a thermodynamically consistent approach is needed such that the cross-coupling between growth and mechanics can be correctly described. In the present work we apply a variational principle to the formulation of the interfacial growth dynamics of dendritic actin filament networks growing from biomimetic beads, an experimentally well studied system, where the buildup of residual stresses governs the network growth. We first introduce the material model for the network via a strain energy density for an isotropic weakly nonlinear elastic material and then derive consistently from this model the dynamic equations for the interfaces, i.e. for a polymerizing internal interface in contact with the bead and a depolymerizing external interface directed towards the solvent. We show that (i) this approach automatically preserves thermodynamic symmetry-properties, which is not the case for the often cited ‘rubber-band-model’ (Sekimoto et al 2004 Eur. Phys. J. E 13 247-59, Plastino et al 2004 Eur. Biophys. J. 33 310-20) and (ii) leads to a robust morphological instability of the treadmilling network interfaces. The nature of the instability depends on the interplay of the two dynamic interfaces. Depending on the biochemical conditions the network envelope evolves into a comet-like shape (i.e. the actin envelope thins out at one side and thickens on the opposite side of the bead) via a varicose instability or it breaks the symmetry via higher order zigzag modes. We conclude that morphological instabilities due to mechano-chemical coupling mechanisms and the presences of mechancial pre-stresses can play a major role in locally organizing the cytoskeleton of living cells.

  3. 7-forming, superconducting filaments through bicomponent dry spinning

    DOEpatents

    Tuominen, Olli P.; Morgan, Carol W.; Burlone, Dominick A.; Blankenship, Keith V.

    2001-01-01

    Fibers which contain potentially superconducting material are dry spun by the steps of preparing a suspension of potentially superconducting powder in a thickened solvent; preparing a solution of fiber-forming polymer; supplying the suspension and the solution to a spinning apparatus; in the spinning apparatus, arranging the solution and the suspension in a bicomponent arrangement; extruding the arranged solution and suspension from a spinneret as a bicomponent filament; and removing the solvent from the filament.

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

    PubMed

    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.

  5. Trimers of the fibronectin cell adhesion domain localize to actin filament bundles and undergo rearward translocation.

    PubMed

    Coussen, Françoise; Choquet, Daniel; Sheetz, Michael P; Erickson, Harold P

    2002-06-15

    Previous studies have shown that small beads coated with FN7-10, a four-domain cell adhesion fragment of fibronectin, bind to cell surfaces and translocate rearward. Here we investigate whether soluble constructs containing two to five FN7-10 units might be sufficient for activity. We have produced a monomer, three forms of dimers, a trimer and a pentamer of FN7-10, on the end of spacer arms. These oligomers could bind small clusters of up to five integrins. Fluorescence microscopy showed that the trimer and pentamer bound strongly to the cell surface, and within 5 minutes were prominently localized to actin fiber bundles. Monomers and dimers showed only diffuse localization. Beads coated with a low concentration (probably one complex per bead) of trimer or pentamer showed prolonged binding and rearward translocation, presumably with the translocating actin cytskeleton. Beads containing monomer or dimer showed only brief binding and diffusive movements. We conclude that clusters of three integrin-binding ligands are necessary and sufficient for coupling to and translocating with the actin cytoskeleton.

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

    PubMed

    Fang, Kefeng; Gao, Sai; Zhang, Weiwei; Xing, Yu; Cao, Qingqin; Qin, Ling

    2016-01-01

    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.

  7. Myosin IIIa boosts elongation of stereocilia by transporting espin 1 to the plus ends of actin filaments.

    PubMed

    Salles, Felipe T; Merritt, Raymond C; Manor, Uri; Dougherty, Gerard W; Sousa, Aurea D; Moore, Judy E; Yengo, Christopher M; Dosé, Andréa C; Kachar, Bechara

    2009-04-01

    Two proteins implicated in inherited deafness, myosin IIIa, a plus-end-directed motor, and espin, an actin-bundling protein containing the actin-monomer-binding motif WH2, have been shown to influence the length of mechanosensory stereocilia. Here we report that espin 1, an ankyrin repeat-containing isoform of espin, colocalizes with myosin IIIa at stereocilia tips and interacts with a unique conserved domain of myosin IIIa. We show that combined overexpression of these proteins causes greater elongation of stereocilia, compared with overexpression of either myosin IIIa alone or espin 1 alone. When these two proteins were co-expressed in the fibroblast-like COS-7 cell line they induced a tenfold elongation of filopodia. This extraordinary filopodia elongation results from the transport of espin 1 to the plus ends of F-actin by myosin IIIa and depends on espin 1 WH2 activity. This study provides the basis for understanding the role of myosin IIIa and espin 1 in regulating stereocilia length, and presents a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments.

  8. Emerging roles of sumoylation in the regulation of actin, microtubules, intermediate filaments, and septins

    PubMed Central

    Alonso, Annabel; Greenlee, Matt; Matts, Jessica; Kline, Jake; Davis, Kayla J.

    2015-01-01

    Sumoylation is a powerful regulatory system that controls many of the critical processes in the cell, including DNA repair, transcriptional regulation, nuclear transport, and DNA replication. Recently, new functions for SUMO have begun to emerge. SUMO is covalently attached to components of each of the four major cytoskeletal networks, including microtubule‐associated proteins, septins, and intermediate filaments, in addition to nuclear actin and actin‐regulatory proteins. However, knowledge of the mechanisms by which this signal transduction system controls the cytoskeleton is still in its infancy. One story that is beginning to unfold is that SUMO may regulate the microtubule motor protein dynein by modification of its adaptor Lis1. In other instances, cytoskeletal elements can both bind to SUMO non‐covalently and also be conjugated by it. The molecular mechanisms for many of these new functions are not yet clear, but are under active investigation. One emerging model links the function of MAP sumoylation to protein degradation through SUMO‐targeted ubiquitin ligases, also known as STUbL enzymes. Other possible functions for cytoskeletal sumoylation are also discussed. © 2015 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc. PMID:26033929

  9. Controlling RNA self-assembly to form filaments.

    PubMed

    Nasalean, Lorena; Baudrey, Stéphanie; Leontis, Neocles B; Jaeger, Luc

    2006-01-01

    Fundamental control over supra-molecular self-assembly for organization of matter on the nano-scale is a major objective of nanoscience and nanotechnology. 'RNA tectonics' is the design of modular RNA units, called tectoRNAs, that can be programmed to self-assemble into novel nano- and meso-scopic architectures of desired size and shape. We report the three-dimensional design of tectoRNAs incorporating modular 4-way junction (4WJ) motifs, hairpin loops and their cognate loop-receptors to create extended, programmable interaction interfaces. Specific and directional RNA-RNA interactions at these interfaces enable conformational, topological and orientational control of tectoRNA self-assembly. The interacting motifs are precisely positioned within the helical arms of the 4WJ to program assembly from only one helical stacking conformation of the 4WJ. TectoRNAs programmed to assemble with orientational compensation produce micrometer-scale RNA filaments through supra-molecular equilibrium polymerization. As visualized by transmission electron microscopy, these RNA filaments resemble actin filaments from the protein world. This work emphasizes the potential of RNA as a scaffold for designing and engineering new controllable biomaterials mimicking modern cytoskeletal proteins.

  10. Crystallization of fluorescent quantum dots within a three-dimensional bio-organic template of actin filaments and lipid membranes.

    PubMed

    Henry, Etienne; Dif, Aurélien; Schmutz, Marc; Legoff, Loic; Amblard, François; Marchi-Artzner, Valérie; Artzner, Franck

    2011-12-14

    Biological molecules and molecular self-assemblies are promising templates to organize well-defined inorganic nanostructures. We demonstrate the ability of a self-assembled three-dimensional crystal template of helical actin protein filaments and lipids bilayers to generate a hierarchical self-assembly of quantum dots. Functionnalized tricystein peptidic quantum dots (QDs) are incorporated during the dynamical self-assembly of this actin/lipid template resulting in the formation of crystalline fibers. The crystal parameters, 26.5×18.9×35.5 nm3, are imposed by the membrane thickness, the diameter, and the pitch of the actin self-assembly. This process ensures the high quality of the crystal and results in unexpected fluorescence properties. This method of preparation offers opportunities to generate crystals with new symmetries and a large range of distance parameters.

  11. Cooperative regulation of myosin-S1 binding to actin filaments by a continuous flexible Tm-Tn chain.

    PubMed

    Mijailovich, Srboljub M; Kayser-Herold, Oliver; Li, Xiaochuan; Griffiths, Hugh; Geeves, Michael A

    2012-12-01

    The regulation of striated muscle contraction involves cooperative interactions between actin filaments, myosin-S1 (S1), tropomyosin (Tm), troponin (Tn), and calcium. These interactions are modeled by treating overlapping tropomyosins as a continuous flexible chain (CFC), weakly confined by electrostatic interactions with actin. The CFC is displaced locally in opposite directions on the actin surface by the binding of either S1 or Troponin I (TnI) to actin. The apparent rate constants for myosin and TnI binding to and detachment from actin are then intrinsically coupled via the CFC model to the presence of neighboring bound S1s and TnIs. Monte Carlo simulations at prescribed values of the CFC stiffness, the CFC's degree of azimuthal confinement, and the angular displacements caused by the bound proteins were able to predict the stopped-flow transients of S1 binding to regulated F-actin. The transients collected over a large range of calcium concentrations could be well described by adjusting a single calcium-dependent parameter, the rate constant of TnI detachment from actin, k(-I). The resulting equilibrium constant K(B) ≡ 1/K(I) varied sigmoidally with the free calcium, increasing from 0.12 at low calcium (pCa >7) to 12 at high calcium (pCa <5.5) with a Hill coefficient of ~2.15. The similarity of the curves for excess-actin and excess-myosin data confirms their allosteric relationship. The spatially explicit calculations confirmed variable sizes for the cooperative units and clustering of bound myosins at low calcium concentrations. Moreover, inclusion of negative cooperativity between myosin units predicted the observed slowing of myosin binding at excess-myosin concentrations.

  12. Actin filament-associated protein 1 is required for cSrc activity and secretory activation in the lactating mammary gland

    PubMed Central

    Cunnick, Jess M; Kim, Stephanie; Hadsell, James; Collins, Stephen; Cerra, Carmine; Reiser, Patti; Flynn, Daniel C; Cho, Youngjin

    2014-01-01

    Actin filament-associated protein 1 (AFAP1) is an adaptor protein of cSrc that binds to filamentous actin and regulates the activity of this tyrosine kinase to affect changes to the organization of the actin cytoskeleton. In breast and prostate cancer cells, AFAP1 has been shown to regulate cellular responses requiring actin cytoskeletal changes such as adhesion, invadopodia formation and invasion. However, a normal physiological role for AFAP1 has remained elusive. In this study, we generated an AFAP1 knockout mouse model that establishes a novel physiological role for AFAP1 in lactation. Specifically, these animals displayed a defect in lactation that resulted in an inability to efficiently nurse. Histologically, the mammary glands of the lactating knockout mice were distinguished by the accumulation of large cytoplasmic lipid droplets in the alveolar epithelial cells. There was a reduction in lipid synthesis and the expression of lipogenic genes without a corresponding reduction in the production of beta-casein, a milk protein. Furthermore, these defects were associated with histological and biochemical signs of precocious involution. This study also demonstrated that AFAP1 responds to prolactin, a lactogenic hormone, by forming a complex with cSrc and becoming tyrosine phosphorylated. Together, these observations pointed to a defect in secretory activation. Certain characteristics of this phenotype mirrored the defect in secretory activation in the cSrc knockout mouse, but most importantly, the activity of cSrc in the mammary gland was reduced during early lactation in the AFAP1 null mouse and the localization of active cSrc at the apical surface of luminal epithelial cells during lactation was selectively lost in the absence of AFAP1. These data define, for the first time, the requirement of AFAP1 for the spatial and temporal regulation of cSrc activity in the normal breast, specifically for milk production. PMID:25043309

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

    PubMed

    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.

  14. Septal Junctions in Filamentous Heterocyst-Forming Cyanobacteria.

    PubMed

    Flores, Enrique; Herrero, Antonia; Forchhammer, Karl; Maldener, Iris

    2016-02-01

    In the filaments of heterocyst-forming cyanobacteria, septal junctions that traverse the septal peptidoglycan join adjacent cells, allowing intercellular communication. Perforations in the septal peptidoglycan have been observed, and proteins involved in the formation of such perforations and putative protein components of the septal junctions have been identified, but their relationships are debated.

  15. Lifeact: a versatile marker to visualize F-actin.

    PubMed

    Riedl, Julia; Crevenna, Alvaro H; Kessenbrock, Kai; Yu, Jerry Haochen; Neukirchen, Dorothee; Bista, Michal; Bradke, Frank; Jenne, Dieter; Holak, Tad A; Werb, Zena; Sixt, Michael; Wedlich-Soldner, Roland

    2008-07-01

    Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.

  16. Structural Differences Explain Diverse Functions of Plasmodium Actins

    PubMed Central

    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

  17. Measuring the flexural rigidity of actin filaments and microtubules from their thermal fluctuating shapes: A new perspective

    NASA Astrophysics Data System (ADS)

    Jia, Kangyu; Liu, Xiaohu

    Actin filaments and microtubules are important components of cytoskeletal networks and show both active and passive dynamic mechanical behaviors. Measuring the mechanical properties of individual filament can not only help us understand the mechanisms behind the complex dynamic behaviors, but also provide parameters that are needed to calibrate biological piconewton forcemeters. Although many methods have been proposed, the values of flexural rigidity reported in literature are still quite different for both actin filaments and microtubules. In this paper, a new formulation based on mode analysis of the thermal fluctuating shapes and principle of virtual work has been proposed, where both the linear and nonlinear assumptions are considered. What's more, following previous inspiring works, both the effects of sampling time interval and hydrodynamics are taken into account in our model. When applied to the experiment data in literature and the simulation data generated by finite element method software, our method gives good results and show an advantage over the previous methods. Besides, we suggest that the inconformity of the flexural rigidity in literature might be caused by the different sampling time intervals and hydrodynamic wall effects in experiments.

  18. Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes.

    PubMed Central

    Omata, W; Shibata, H; Li, L; Takata, K; Kojima, I

    2000-01-01

    Actin-based cytoskeletons have been implicated in insulin-stimulated glucose transport and translocation of the insulin-regulated glucose transporter, GLUT4, from the intracellular pool to the plasma membrane. However, most previous studies were done using adherent cell systems such as L6 myotubes and 3T3-L1 adipocytes, and very little information is available on the significance of the actin filaments to the insulin action in isolated adipocytes, a widely used experimental system. In the present study, we investigated the physiological role of actin filaments in the subcellular trafficking of GLUT4 in isolated rat adipocytes. We first compared the effects of two actin-disrupting reagents, latrunculin A and cytochalasin D, on the organization of the actin filaments as well as on the insulin action on glucose transport by laser confocal microscopy combined with biochemical analysis of the insulin action. Treatment of the cells with latrunculin A induced dose- and time-dependent disappearance of the filamentous actin, which correlated very well with inhibition of the insulin effect on glucose transport. Although cytochalasin D at 50 microM significantly inhibited insulin-stimulated glucose transport, it was not effective in disassembly of the actin filaments; rather, many intense punctate signals were observed in cytochalasin D-treated cells. In the actin-disrupted adipocytes treated with latrunculin A, insulin-induced GLUT4 translocation was inhibited completely. In addition, latrunculin A remarkably inhibited both insulin-induced glucose transport and GLUT4 translocation in the presense of D(k)-(62-85), a potent inhibitor of GLUT4 endocytosis, suggesting that intactness of the actin filaments was necessary for insulin-induced exocytosis of the GLUT4-containing vesicles. On the other hand, latrunculin A showed little inhibitory effect on either endocytosis of the trypsin-cleaved 35-kDa fragment of GLUT4 or decay of the glucose transport activity after addition of

  19. Drosophila Homologues of Adenomatous Polyposis Coli (APC) and the Formin Diaphanous Collaborate by a Conserved Mechanism to Stimulate Actin Filament Assembly*

    PubMed Central

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

    2013-01-01

    Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad range of animal phyla. PMID:23558679

  20. Nucleocapsid of Tomato spotted wilt tospovirus forms mobile particles that traffic on an actin/endoplasmic reticulum network driven by myosin XI-K.

    PubMed

    Feng, Zhike; Chen, Xiaojiao; Bao, Yiqun; Dong, Jiahong; Zhang, Zhongkai; Tao, Xiaorong

    2013-12-01

    A number of viral proteins from plant viruses, other than movement proteins, have been shown to traffic intracellularly along actin filaments and to be involved in viral infection. However, there has been no report that a viral capsid protein may traffic within a cell by utilizing the actin/endoplasmic reticulum (ER) network. We used Tomato spotted wilt tospovirus (TSWV) as a model virus to study the cell biological properties of a nucleocapsid (N) protein. We found that TSWV N protein was capable of forming highly motile cytoplasmic inclusions that moved along the ER and actin network. The disruption of actin filaments by latrunculin B, an actin-depolymerizing agent, almost stopped the intracellular movement of N inclusions, whereas treatment with a microtubule-depolymerizing reagent, oryzalin, did not. The over-expression of a myosin XI-K tail, functioning in a dominant-negative manner, completely halted the movement of N inclusions. Latrunculin B treatment strongly inhibited the formation of TSWV local lesions in Nicotiana tabacum cv Samsun NN and delayed systemic infection in N. benthamiana. Collectively, our findings provide the first evidence that the capsid protein of a plant virus has the novel property of intracellular trafficking. The findings add capsid protein as a new class of viral protein that traffics on the actin/ER system.

  1. Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth

    PubMed Central

    Zhou, Zhenzhen; Shi, Haifan; Chen, Binqing; Zhang, Ruihui; Huang, Shanjin; Fu, Ying

    2015-01-01

    Pollen tubes deliver sperms to the ovule for fertilization via tip growth. The rapid turnover of F-actin in pollen tube tips plays an important role in this process. In this study, we demonstrate that Arabidopsis thaliana RIC1, a member of the ROP-interactive CRIB motif-containing protein family, regulates pollen tube growth via its F-actin severing activity. Knockout of RIC1 enhanced pollen tube elongation, while overexpression of RIC1 dramatically reduced tube growth. Pharmacological analysis indicated that RIC1 affected F-actin dynamics in pollen tubes. In vitro biochemical assays revealed that RIC1 directly bound and severed F-actin in the presence of Ca2+ in addition to interfering with F-actin turnover by capping F-actin at the barbed ends. In vivo, RIC1 localized primarily to the apical plasma membrane (PM) of pollen tubes. The level of RIC1 at the apical PM oscillated during pollen tube growth. The frequency of F-actin severing at the apex was notably decreased in ric1-1 pollen tubes but was increased in pollen tubes overexpressing RIC1. We propose that RIC1 regulates F-actin dynamics at the apical PM as well as the cytosol by severing F-actin and capping the barbed ends in the cytoplasm, establishing a novel mechanism that underlies the regulation of pollen tube growth. PMID:25804540

  2. Pattern formation in actin gels: A study in the mechanics of gels formed by the important cytoskeletal protein actin, especially as applied to cellular motility

    NASA Astrophysics Data System (ADS)

    Balter, Ariel

    We have studied pattern formation in actin gels to better understand how they function in biological systems, especially in the motility mechanism used by some pathogenic bacteria such as Listeria. By coating themselves with certain enzymes, these bacteria appropriate actin (a protein) from the surrounding host cell's cytoplasm and cause a network or "gel" of actin filaments to grow on their outer surface. As the resulting "comet tail" shaped protrusion grows, it pushes the bacterium away. In experiments, polystyrene beads coated with the same enzymes will also generate comet tails and swim in a very similar manner. However, these bead experiments have also generated anomalous results such as the formation of many comet tails. In some experiments, when two comet tails formed they systematically grew into regular, oppositely handed helices. The formation of any comet tails on a bead poses a physical conundrum. The bacterial enzyme coating is asymmetrical so the comet tail forms in a particular place. But the beads are symmetrical, so comet tails formation constitutes symmetry breaking and spontaneous pattern formation. We have modeled this process as a competition between elastic energy (which favors many tails) and chemical energy (which favors few tails). Our analytical model explains the factors that experimentally determine the number of tails, and numerical simulations confirm these predictions. To understand the helical tails, we did extensive data analysis involving image processing, statistical analysis and mathematical modeling of images of the helical tails. We identified some important features of how the twin tails form. For instance, the tail growth rate is independent of drag force, and bead rotation must accompany helical tail formation. We also created a physical model for helical growth. Numerical simulations of our model show that at very low Reynolds number, a cylindrical object growing under the conditions of an actin comet tail can spontaneously

  3. Bacterial Actins and Their Interactors.

    PubMed

    Gayathri, Pananghat

    2017-01-01

    Bacterial actins polymerize in the presence of nucleotide (preferably ATP), form a common arrangement of monomeric interfaces within a protofilament, and undergo ATP hydrolysis-dependent change in stability of the filament-all of which contribute to performing their respective functions. The relative stability of the filament in the ADP-bound form compared to that of ATP and the rate of addition of monomers at the two ends decide the filament dynamics. One of the major differences between eukaryotic actin and bacterial actins is the variety in protofilament arrangements and dynamics exhibited by the latter. The filament structure and the polymerization dynamics enable them to perform various functions such as shape determination in rod-shaped bacteria (MreB), cell division (FtsA), plasmid segregation (ParM family of actin-like proteins), and organelle positioning (MamK). Though the architecture and dynamics of a few representative filaments have been studied, information on the effect of interacting partners on bacterial actin filament dynamics is not very well known. The chapter reviews some of the structural and functional aspects of bacterial actins, with special focus on the effect that interacting partners exert on the dynamics of bacterial actins, and how these assist them to carry out the functions within the bacterial cell.

  4. Biogenesis of actin-like bacterial cytoskeletal filaments destined for positioning prokaryotic magnetic organelles.

    PubMed

    Pradel, Nathalie; Santini, Claire-Lise; Bernadac, Alain; Fukumori, Yoshihiro; Wu, Long-Fei

    2006-11-14

    Magnetosomes comprise a magnetic nanocrystal surrounded by a lipid bilayer membrane. These unique prokaryotic organelles align inside magnetotactic bacterial cells and serve as an intracellular compass allowing the bacteria to navigate along the geomagnetic field in aquatic environments. Cryoelectron tomography of Magnetospirillum strains has revealed that the magnetosome chain is surrounded by a network of filaments that may be composed of MamK given that the filaments are absent in the mamK mutant cells. The process of the MamK filament assembly is unknown. Here we prove the authenticity of the MamK filaments and show that MamK exhibits linear distribution inside Magnetospirillum sp. cells even in the area without magnetosomes. The mamK gene alone is sufficient to direct the synthesis of straight filaments in Escherichia coli, and one extremity of the MamK filaments is located at the cellular pole. By using dual fluorescent labeling of MamK, we found that MamK nucleates at multiple sites and assembles into mosaic filaments. Time-lapse experiments reveal that the assembly of the MamK filaments is a highly dynamic and kinetically asymmetrical process. MamK bundles might initiate the formation of a new filament or associate to one preexistent filament. Our results demonstrate the mechanism of biogenesis of prokaryotic cytoskeletal filaments that are structurally and functionally distinct from the known MreB and ParM filaments. In addition to positioning magnetosomes, other hypothetical functions of the MamK filaments in magnetotaxis might include anchoring magnetosomes and being involved in magnetic reception.

  5. Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium

    PubMed Central

    Kronlage, Cornelius; Schäfer-Herte, Marco; Böning, Daniel; Oberleithner, Hans; Fels, Johannes

    2015-01-01

    Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane. PMID:26287621

  6. A coat of filamentous actin prevents clustering of late-endosomal vacuoles in vivo.

    PubMed

    Drengk, Anja; Fritsch, Jürgen; Schmauch, Christian; Rühling, Harald; Maniak, Markus

    2003-10-14

    The endocytic pathway depends on the actin cytoskeleton. Actin contributes to internalization at the plasma membrane and to subsequent trafficking steps like propulsion through the cytoplasm, fusion of phagosomes with early endosomes, and transport from early to late endosomes. In vitro studies with mammalian endosomes and yeast vacuoles implicate actin in membrane fusion. Here, we investigate the function of the actin coat that surrounds late endosomes in Dictyostelium. Latrunculin treatment leads to aggregation of these endosomes into grape-like clusters and completely blocks progression of endocytic marker. In addition, the cells round up and stop moving. Because this drug treatment perturbs all actin assemblies in the cell simultaneously, we used a novel targeting approach to specifically study the function of the cytoskeleton in one subcellular location. To this end, we constructed a hybrid protein targeting cofilin, an actin depolymerizing protein, to late endosomes. As a consequence, the endosomal compartments lost their actin coats and aggregated, but these cells remained morphologically normal, and the kinetics of endocytic marker trafficking were unaltered. Therefore, the actin coat prevents the clustering of endosomes, which could be one safeguard mechanism precluding their docking and fusion.

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

    PubMed

    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.

  8. Light-Induced Movements of Chloroplasts and Nuclei Are Regulated in Both Cp-Actin-Filament-Dependent and -Independent Manners in Arabidopsis thaliana

    PubMed Central

    2016-01-01

    Light-induced chloroplast movement and attachment to the plasma membrane are dependent on actin filaments. In Arabidopsis thaliana, the short actin filaments on the chloroplast envelope, cp-actin filaments, are essential for chloroplast movement and positioning. Furthermore, cp-actin-filament-mediated chloroplast movement is necessary for the strong-light-induced nuclear avoidance response. The proteins CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for the generation and/or maintenance of cp-actin filaments in Arabidopsis. In land plants, CHUP1 and KAC family proteins play pivotal roles in the proper movement of chloroplasts and their attachment to the plasma membrane. Here, we report similar but distinct phenotypes in chloroplast and nuclear photorelocation movements between chup1 and kac1kac2 mutants. Measurement of chloroplast photorelocation movement indicated that kac1kac2, but not chup1, exhibited a clear strong-light-induced increase in leaf transmittance changes. The chloroplast movement in kac1kac2 depended on phototropin 2, CHUP1 and two other regulators for cp-actin filaments, PLASTID MOVEMENT IMPAIRED 1 and THRUMIN 1. Furthermore, kac1kac2 retained a weak but significant nuclear avoidance response although chup1 displayed a severe defect in the nuclear avoidance response. The kac1kac2chup1 triple mutant was completely defective in both chloroplast and nuclear avoidance responses. These results indicate that CHUP1 and the KACs function somewhat independently, but interdependently mediate both chloroplast and nuclear photorelocation movements. PMID:27310016

  9. Light-Induced Movements of Chloroplasts and Nuclei Are Regulated in Both Cp-Actin-Filament-Dependent and -Independent Manners in Arabidopsis thaliana.

    PubMed

    Suetsugu, Noriyuki; Higa, Takeshi; Gotoh, Eiji; Wada, Masamitsu

    2016-01-01

    Light-induced chloroplast movement and attachment to the plasma membrane are dependent on actin filaments. In Arabidopsis thaliana, the short actin filaments on the chloroplast envelope, cp-actin filaments, are essential for chloroplast movement and positioning. Furthermore, cp-actin-filament-mediated chloroplast movement is necessary for the strong-light-induced nuclear avoidance response. The proteins CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for the generation and/or maintenance of cp-actin filaments in Arabidopsis. In land plants, CHUP1 and KAC family proteins play pivotal roles in the proper movement of chloroplasts and their attachment to the plasma membrane. Here, we report similar but distinct phenotypes in chloroplast and nuclear photorelocation movements between chup1 and kac1kac2 mutants. Measurement of chloroplast photorelocation movement indicated that kac1kac2, but not chup1, exhibited a clear strong-light-induced increase in leaf transmittance changes. The chloroplast movement in kac1kac2 depended on phototropin 2, CHUP1 and two other regulators for cp-actin filaments, PLASTID MOVEMENT IMPAIRED 1 and THRUMIN 1. Furthermore, kac1kac2 retained a weak but significant nuclear avoidance response although chup1 displayed a severe defect in the nuclear avoidance response. The kac1kac2chup1 triple mutant was completely defective in both chloroplast and nuclear avoidance responses. These results indicate that CHUP1 and the KACs function somewhat independently, but interdependently mediate both chloroplast and nuclear photorelocation movements.

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

    PubMed Central

    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 min, which was required for completion of actin polymerization, two shapes of liposome were evident: dumbbell and disk. Elongation of the dumbbell-shaped liposomes was concomitant with actin polymerization. Polarization microscopy showed that actin filaments formed thick bundles in the liposomes and that these filaments lay contiguous to the periphery of the liposome. Localization of actin filaments in the liposomes was confirmed by observation of rhodamine phalloidin-conjugated actin filaments by fluorescence microscopy. Both dumbbell- and disk-shaped liposomes were rigid and kept their shapes as far as actin filaments were stabilized. In contrast, liposomes containing bovine serum albumin were fragile, and their shapes continually fluctuated from Brownian motion, indicating that the actin bundles served as mechanical support for the liposome shapes. Images PMID:1454846

  11. Entropic potential field formed for a linear-motor protein near a filament: Statistical-mechanical analyses using simple models.

    PubMed

    Amano, Ken-Ichi; Yoshidome, Takashi; Iwaki, Mitsuhiro; Suzuki, Makoto; Kinoshita, Masahiro

    2010-07-28

    We report a new progress in elucidating the mechanism of the unidirectional movement of a linear-motor protein (e.g., myosin) along a filament (e.g., F-actin). The basic concept emphasized here is that a potential field is entropically formed for the protein on the filament immersed in solvent due to the effect of the translational displacement of solvent molecules. The entropic potential field is strongly dependent on geometric features of the protein and the filament, their overall shapes as well as details of the polyatomic structures. The features and the corresponding field are judiciously adjusted by the binding of adenosine triphosphate (ATP) to the protein, hydrolysis of ATP into adenosine diphosphate (ADP)+Pi, and release of Pi and ADP. As the first step, we propose the following physical picture: The potential field formed along the filament for the protein without the binding of ATP or ADP+Pi to it is largely different from that for the protein with the binding, and the directed movement is realized by repeated switches from one of the fields to the other. To illustrate the picture, we analyze the spatial distribution of the entropic potential between a large solute and a large body using the three-dimensional integral equation theory. The solute is modeled as a large hard sphere. Two model filaments are considered as the body: model 1 is a set of one-dimensionally connected large hard spheres and model 2 is a double helical structure formed by two sets of connected large hard spheres. The solute and the filament are immersed in small hard spheres forming the solvent. The major findings are as follows. The solute is strongly confined within a narrow space in contact with the filament. Within the space there are locations with sharply deep local potential minima along the filament, and the distance between two adjacent locations is equal to the diameter of the large spheres constituting the filament. The potential minima form a ringlike domain in model 1

  12. Observation of Actin Filaments in Leydig Cells with a Contact-type Soft X-ray Microscope with Laser Plasma X-ray Source

    NASA Astrophysics Data System (ADS)

    Kado, Masataka; Ishino, Masahiko; Tamotsu, Satoshi; Yasuda, Keiko; Kishimoto, Maki; Nishikino, Masaharu; Kinjo, Yasuhito; Shinohara, Kunio

    Actin filaments in Leydig cells from mouse testes have been observed with a contact-type soft x-ray microscope with laser plasma x-ray source. The Leydig cells were fixed with paraformaldehyde, stained with Phalloidin, and observed with a confocal laser microscope prior to the observation with x-ray microscope. Obtained images by both of the confocal laser microscopy and the x-ray microscopy were directly compared and revealed that not only position of actin filaments but also the shapes can be identified each other. The actin filaments in the x-ray images were clearly recognized and their structures were obtained in more detail compared to those in the confocal laser microscope images.

  13. Eukaryotic cell locomotion depends on the propagation of self-organized reaction-diffusion waves and oscillations of actin filament assembly.

    PubMed

    Vicker, Michael G

    2002-04-15

    Actin filament (F-actin) assembly kinetics determines the locomotion and shape of crawling eukaryotic cells, but the nature of these kinetics and their determining reactions are unclear. Live BHK21 fibroblasts, mouse melanoma cells, and Dictyostelium amoebae, locomoting on glass and expressing Green Fluorescent Protein-actin fusion proteins, were examined by confocal microscopy. The cells demonstrated three-dimensional bands of F-actin, which propagated throughout the cytoplasm at rates usually ranging between 2 and 5 microm/min in each cell type and produced lamellipodia or pseudopodia at the cell boundary. F-actin's dynamic behavior and supramolecular spatial patterns resembled in detail self-organized chemical waves in dissipative, physico-chemical systems. On this basis, the present observations provide the first evidence of self-organized, and probably autocatalytic, chemical reaction-diffusion waves of reversible actin filament assembly in vertebrate cells and a comprehensive record of wave and locomotory dynamics in vegetative-stage Dictyostelium cells. The intensity and frequency of F-actin wavefronts determine locomotory cell projections and the rotating oscillatory waves, which structure the cell surface. F-actin assembly waves thus provide a fundamental, deterministic, and nonlinear mechanism of cell locomotion and shape, which complements mechanisms based exclusively on stochastic molecular reaction kinetics.

  14. Simulated ischaemia induces Ca2+-independent glutamatergic vesicle release through actin filament depolymerization in area CA1 of the hippocampus.

    PubMed

    Andrade, Adriana L; Rossi, David J

    2010-05-01

    Transient, non-catastrophic brain ischaemia can induce either a protected state against subsequent episodes of ischaemia (ischaemic preconditioning) or delayed, selective neuronal death. Altered glutamatergic signalling and altered Ca(2+) homeostasis have been implicated in both processes. Here we use simultaneous patch-clamp recording and Ca(2+) imaging to monitor early changes in glutamate release and cytoplasmic [Ca(2+)] ([Ca(2+)](c)) in an in vitro slice model of hippocampal ischaemia. In slices loaded with the Ca(2+)-sensitive dye Fura-2, ischaemia leads to an early increase in [Ca(2+)](c) that precedes the severe ischaemic depolarization (ID) associated with pan necrosis. The early increase in [Ca(2+)](c) is mediated by influx through the plasma membrane and release from internal stores, and parallels an early increase in vesicular glutamate release that manifests as a fourfold increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs). However, the increase in mEPSC frequency is not prevented by blocking the increase in [Ca(2+)](c), and the early rise in [Ca(2+)](c) is not affected by blocking ionotropic and metabotropic glutamate receptors. Thus, the increase in [Ca(2+)](c) and the increase in glutamate release are independent of each other. Stabilizing actin filaments with jaspamide or phalloidin prevented vesicle release induced by ischaemia. Our results identify several early cellular cascades triggered by ischaemia: Ca(2+) influx, Ca(2+) release from intracellular stores, actin filament depolymerization, and vesicular release of glutamate that depends on actin dynamics but not [Ca(2+)](c). All of these processes precede the catastrophic ID by several minutes, and thus represent potential target mechanisms to influence the outcome of an ischaemic episode.

  15. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

    PubMed Central

    Paredez, Alexander R.; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C.; Wang, Chung-Ju Rachel; Cande, W. Z.

    2011-01-01

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host. PMID:21444821

  16. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins.

    PubMed

    Paredez, Alexander R; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C; Wang, Chung-Ju Rachel; Cande, W Z

    2011-04-12

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.

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

    PubMed

    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

  18. PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front

    PubMed Central

    Nguyen, Trang Thi Thu; Park, Wei Sun; Park, Byung Ouk; Kim, Cha Yeon; Oh, Yohan; Kim, Jin Man; Choi, Hana; Kyung, Taeyoon; Kim, Cheol-Hee; Lee, Gabsang; Hahn, Klaus M.; Meyer, Tobias; Heo, Won Do

    2016-01-01

    Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration. PMID:27555588

  19. Nucleus-associated actin in Amoeba proteus.

    PubMed

    Berdieva, Mariia; Bogolyubov, Dmitry; Podlipaeva, Yuliya; Goodkov, Andrew

    2016-10-01

    The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.

  20. Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay.

    PubMed

    Debold, Edward P; Turner, Matthew A; Stout, Jordan C; Walcott, Sam

    2011-06-01

    Elevated levels of inorganic phosphate (P(i)) are believed to inhibit muscular force by reversing myosin's force-generating step. These same levels of P(i) can also affect muscle velocity, but the molecular basis underlying these effects remains unclear. We directly examined the effect of P(i) (30 mM) on skeletal muscle myosin's ability to translocate actin (V(actin)) in an in vitro motility assay. Manipulation of the pH enabled us to probe rebinding of P(i) to myosin's ADP-bound state, while changing the ATP concentration probed rebinding to the rigor state. Surprisingly, the addition of P(i) significantly increased V(actin) at both pH 6.8 and 6.5, causing a doubling of V(actin) at pH 6.5. To probe the mechanisms underlying this increase in speed, we repeated these experiments while varying the ATP concentration. At pH 7.4, the effects of P(i) were highly ATP dependent, with P(i) slowing V(actin) at low ATP (<500 μM), but with a minor increase at 2 mM ATP. The P(i)-induced slowing of V(actin), evident at low ATP (pH 7.4), was minimized at pH 6.8 and completely reversed at pH 6.5. These data were accurately fit with a simple detachment-limited kinetic model of motility that incorporated a P(i)-induced prolongation of the rigor state, which accounted for the slowing of V(actin) at low ATP, and a P(i)-induced detachment from a strongly bound post-power-stroke state, which accounted for the increase in V(actin) at high ATP. These findings suggest that P(i) differentially affects myosin function: enhancing velocity, if it rebinds to the ADP-bound state, while slowing velocity, if it binds to the rigor state.

  1. A 45,000-mol-wt protein from unfertilized sea urchin eggs severs actin filaments in a calcium-dependent manner and increases the steady-state concentration of nonfilamentous actin

    PubMed Central

    1984-01-01

    A 45,000-mol-wt protein has been purified from unfertilized sea urchin (Strongylocentrotus purpuratus) eggs. The isolation scheme includes DEAE cellulose ion-exchange chromatography, gel filtration, and hydroxylapatite chromatography. The homogeneity of the isolated protein is greater than 90% by SDS PAGE. The 45,000-mol-wt protein reduces the viscosity of actin filaments in a Ca2+-dependent manner. The free calcium concentration required for the activity of this protein is in the micromolar range. Electron microscopic studies reveal that the formation of short filaments parallels the decrease in viscosity. Energy transfer and sedimentation experiments indicate a net disassembly of actin filaments and an increase in the steady-state nonfilamentous actin concentration in the presence of Ca2+ ions and the 45,000-mol-wt protein. The increase in the steady-state nonfilamentous actin concentration is proportional to the amount of 45,000-mol-wt protein added. The actin molecules disassembled by the addition of the 45,000-mol-wt protein are capable of polymerization. PMID:6540784

  2. Morphology and viscoelasticity of actin networks formed with the mutually interacting crosslinkers: palladin and alpha-actinin.

    PubMed

    Grooman, Brian; Fujiwara, Ikuko; Otey, Carol; Upadhyaya, Arpita

    2012-01-01

    Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not clearly understood. The ABP, palladin, is essential for the maintenance of cell morphology and the regulation of cell movement. Palladin coexists with α-actinin in stress fibers and focal adhesions and binds to both actin and α-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we characterized the micro-structure and mechanics of actin networks crosslinked with palladin and α-actinin. We first showed that palladin crosslinks actin filaments into bundled networks which are viscoelastic in nature. Our studies also showed that composite networks of α-actinin/palladin/actin behave very similar to pure palladin or pure [Formula: see text]-actinin networks. However, we found evidence that palladin and α-actinin synergistically modify network viscoelasticity. To our knowledge, this is the first quantitative characterization of the physical properties of actin networks crosslinked with two mutually interacting crosslinkers.

  3. Initial binding of Shiga toxin-producing Escherichia coli to host cells and subsequent induction of actin rearrangements depend on filamentous EspA-containing surface appendages.

    PubMed

    Ebel, F; Podzadel, T; Rohde, M; Kresse, A U; Krämer, S; Deibel, C; Guzmán, C A; Chakraborty, T

    1998-10-01

    Shiga toxin-producing Escherichia coli (STEC) induce so-called attaching and effacing lesions that enable the tight adherence of these pathogens to the gut epithelium. All of the genes necessary for this process are present in the locus of enterocyte effacement, which encodes a type III secretion system, the secreted Esp proteins and the surface protein intimin. In this study we sequenced the espA gene of STEC, generated and characterized a corresponding deletion mutant and raised EspA-specific monoclonal antibodies to analyse the functional role of this protein during infection. EspA was detected in often filament-like structures decorating all bacteria that had attached to HeLa cells. These appendages were especially prominent on bacteria that had not yet induced the formation of actin pedestals, indicating that they mediate the initial contact of STEC to their target cells. Consistently, a deletion of the espA gene completely abolished the capacity of such STEC mutants to bind to HeLa cells and to induce actin rearrangements. Surface appendages similar to those described in this study are also formed by Pseudomonas syringae and may represent a structural element common to many bacterial pathogens that deliver proteins into their target cells via a type III secretion system.

  4. A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

    PubMed Central

    Horsington, Jacquelyn; Lynn, Helena; Turnbull, Lynne; Cheng, Delfine; Braet, Filip; Diefenbach, Russell J.; Whitchurch, Cynthia B.; Karupiah, Guna; Newsome, Timothy P.

    2013-01-01

    Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36YdF virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36YdF infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36YdF extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5P189S mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin

  5. The Effects of Disease Models of Nuclear Actin Polymerization on the Nucleus

    PubMed Central

    Serebryannyy, Leonid A.; Yuen, Michaela; Parilla, Megan; Cooper, Sandra T.; de Lanerolle, Primal

    2016-01-01

    Actin plays a crucial role in regulating multiple processes within the nucleus, including transcription and chromatin organization. However, the polymerization state of nuclear actin remains controversial, and there is no evidence for persistent actin filaments in a normal interphase nucleus. Further, several disease pathologies are characterized by polymerization of nuclear actin into stable filaments or rods. These include filaments that stain with phalloidin, resulting from point mutations in skeletal α-actin, detected in the human skeletal disease intranuclear rod myopathy, and cofilin/actin rods that form in response to cellular stressors like heatshock. To further elucidate the effects of these pathological actin structures, we examined the nucleus in both cell culture models as well as isolated human tissues. We find these actin structures alter the distribution of both RNA polymerase II and chromatin. Our data suggest that nuclear actin filaments result in disruption of nuclear organization, which may contribute to the disease pathology. PMID:27774069

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

    SciTech Connect

    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.

  7. Polycation induced actin bundles.

    PubMed

    Muhlrad, Andras; Grintsevich, Elena E; Reisler, Emil

    2011-04-01

    Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4Å) MTS-1 (1,1-methanedyl bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.

  8. RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling Endosomes

    PubMed Central

    Wang, Yu; Liu, Ou; Zhang, Jing; Gleason, Adenrele; Yang, Zhenrong; Wang, Hui; Shi, Anbing; Grant, Barth D.

    2016-01-01

    EHBP-1 (Ehbp1) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction. Furthermore, our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin microfilaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. We propose a mechanism whereby RAB-10 promotes the ability of endosome-bound EHBP-1 to also bind to the actin cytoskeleton, thereby promoting endosomal tubulation. PMID:27272733

  9. WAVE2, N-WASP, and Mena facilitate cell invasion via phosphatidylinositol 3-kinase-dependent local accumulation of actin filaments.

    PubMed

    Takahashi, Kazuhide; Suzuki, Katsuo

    2011-11-01

    Cell migration is accomplished by the formation of cellular protrusions such as lamellipodia and filopodia. These protrusions result from actin filament (F-actin) rearrangement at the cell cortex by WASP/WAVE family proteins and Drosophila enabled (Ena)/vasodilator-stimulated factor proteins. However, the role of each of these actin cytoskeletal regulatory proteins in the regulation of three-dimensional cell invasion remains to be clarified. We found that platelet-derived growth factor (PDGF) induces invasion of MDA-MB-231 human breast cancer cells through invasion chamber membrane pores. This invasion was accompanied by intensive F-actin accumulation at the sites of cell infiltration. After PDGF stimulation, WAVE2, N-WASP, and a mammalian Ena (Mena) colocalized with F-actin at the sites of cell infiltration in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Depletion of WAVE2, N-WASP, or Mena by RNA interference (RNAi) abrogated both cell invasion and intensive F-actin accumulation at the invasion site. These results indicate that by mediating intensive F-actin accumulation at the sites of cell infiltration, WAVE2, N-WASP, and Mena are crucial for PI3K-dependent cell invasion induced by PDGF.

  10. Involvement of β- and γ-actin isoforms in actin cytoskeleton organization and migration abilities of bleb-forming human colon cancer cells

    PubMed Central

    Simiczyjew, Aleksandra; Mazur, Antonina Joanna; Dratkiewicz, Ewelina; Nowak, Dorota

    2017-01-01

    Amoeboid movement is characteristic for rounded cells, which do not form strong adhesion contacts with the ECM and use blebs as migratory protrusions. It is well known that actin is the main component of mature forms of these structures, but the exact role fulfilled by non-muscle actin isoforms β- and γ- in bleb formation and migration of these cells is still not fully understood. The aim of this study was to establish the role of β- and γ-actin in migration of bleb-forming cancer cells using isoform-specific antibodies and expression of fluorescently tagged actin isoforms. We observed, after staining with monoclonal antibodies, that both actins are present in these cells in the form of a cortical ring as well as in the area of blebs. Additionally, using simultaneous expression of differentially tagged β- and γ-actin in cells, we observed that the actin isoforms are present together in a single bleb. They were involved during bleb expansion as well as retraction. Also present in the area of these protrusions formed by both isoforms were the bleb markers–ezrin and myosin II. The overexpression of β- or γ-actin led to actin cytoskeletal rearrangement followed by the growth of migration and invasion abilities of examined human colon cancer cells, LS174T line. In summary these data prove that both actin isoforms have an impact on motility of bleb-forming cancer cells. Moreover, we conclude that monoclonal antibodies directed against actin isoforms in combination with the tagged actins are good tools to study their role in important biological processes. PMID:28333953

  11. Caenorhabditis elegans Kettin, a Large Immunoglobulin-like Repeat Protein, Binds to Filamentous Actin and Provides Mechanical Stability to the Contractile Apparatuses in Body Wall Muscle

    PubMed Central

    Ono, Kanako; Yu, Robinson; Mohri, Kurato

    2006-01-01

    Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegans muscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent non-Ig region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but it was enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with α-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction. PMID:16597697

  12. Characterization of the ATP-G-actin aggregates formed at low potassium chloride concentration.

    PubMed Central

    Grazi, E; Aleotti, A; Ferri, A

    1984-01-01

    The ATP-G-actin aggregates formed by incubation of ATP-G-actin in 7.5 mM-KCl were characterized by electron-microscopical observation, by high-pressure liquid chromatography and by the study of the 1,N6-etheno-ATP-ATP exchange reaction between the free and the actin-bound nucleotide. In 30 mM-KCl the initial rate of the reduced-viscosity increase is found to be directly related to the amount of the aggregates formed in the course of the preincubation in 7.5 mM-KCl. Images Fig. 1. PMID:6721856

  13. A new member of the Rho family, Rnd1, promotes disassembly of actin filament structures and loss of cell adhesion.

    PubMed

    Nobes, C D; Lauritzen, I; Mattei, M G; Paris, S; Hall, A; Chardin, P

    1998-04-06

    Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell-substrate adhesion leading to cell rounding (hence Rnd for "round"). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.

  14. C-terminal fragment of amebin promotes actin filament bundling, inhibits acto-myosin ATPase activity and is essential for amoeba migration.

    PubMed

    Jóźwiak, Jolanta; Rzhepetskyy, Yuriy; Sobczak, Magdalena; Kocik, Elżbieta; Skórzewski, Radosław; Kłopocka, Wanda; Rędowicz, Maria Jolanta

    2011-02-01

    Amebin [formerly termed as ApABP-FI; Sobczak et al. (2007) Biochem. Cell Biol. 85] is encoded in Amoeba proteus by two transcripts, 2672-nt and 1125-nt. A product of the shorter transcript (termed as C-amebin), comprising C-terminal 375 amino-acid-residue fragment of amebin, has been expressed and purified as the recombinant GST-fusion protein. GST-C-amebin bound both to monomeric and filamentous actin. The binding was Ca(2+)-independent and promoted filament bundling, as revealed with the transmission electron microscopy. GST-C-amebin significantly decreased MgATPase activity of rabbit skeletal muscle acto-S1. Removal with endoproteinase ArgC of a positively charged C-terminal region of GST-amebin containing KLASMWEQ sequence abolished actin-binding and bundling as well as the ATPase-inhibitory effect of C-amebin, indicating that this protein region was involved in the interaction with actin. Microinjection of amoebae with antibody against C-terminus of amebin significantly affected amoebae morphology, disturbed cell polarization and transport of cytoplasmic granules as well as blocked migration. These data indicate that amebin may be one of key regulators of the actin-cytoskeleton dynamics and actin-dependent motility in A. proteus.

  15. Localization by indirect immunofluorescence of tetrin, actin, and centrin to the oral apparatus and buccal cavity of the macrostomal form of Tetrahymena vorax.

    PubMed

    McLaughlin, Neil B; Buhse, Howard E

    2004-01-01

    We have taken advantage of the size of the macrostomal oral apparatus of Tetrahymena vorax to investigate the immunofluorescent localization of three cytoskeletal proteins--tetrin, actin, and centrin. Tetrin and actin antibodies co-localize to cross-connectives that anchor the membranelles. These antibodies also recognize the coarse filamentous reticulum, a filament associated with the undulating membrane. Actin-specific localization extends beyond the coarse filamentous reticulum-undulating membrane complex into a region called the specialized cytoplasm. A centrin antibody localizes to the fine filamentous reticulum which, along with microtubules of the oral ribs, circumscribes the cytostomal opening. Models of phagocytic contraction based on these data are presented.

  16. Role of Actin Filaments in Correlating Nuclear Shape and Cell Spreading

    PubMed Central

    Vishavkarma, Renu; Raghavan, Swetavalli; Kuyyamudi, Chandrashekar; Majumder, Abhijit; Dhawan, Jyotsna; Pullarkat, Pramod A.

    2014-01-01

    It is well known that substrate properties like stiffness and adhesivity influence stem cell morphology and differentiation. Recent experiments show that cell morphology influences nuclear geometry and hence gene expression profile. The mechanism by which surface properties regulate cell and nuclear properties is only beginning to be understood. Direct transmission of forces as well as chemical signalling are involved in this process. Here, we investigate the formal aspect by studying the correlation between cell spreading and nuclear deformation using Mesenchymal stem cells under a wide variety of conditions. It is observed that a robust quantitative relation holds between the cell and nuclear projected areas, irrespective of how the cell area is modified or when various cytoskeletal or nuclear components are perturbed. By studying the role of actin stress fibers in compressing the nucleus we propose that nuclear compression by stress fibers can lead to enhanced cell spreading due to an interplay between elastic and adhesion factors. The significance of myosin-II in regulating this process is also explored. We demonstrate this effect using a simple technique to apply external compressive loads on the nucleus. PMID:25251154

  17. Mammalian class I myosin, Myo1b, is monomeric and cross-links actin filaments as determined by hydrodynamic studies and electron microscopy.

    PubMed

    Stafford, Walter F; Walker, Matt L; Trinick, John A; Coluccio, Lynne M

    2005-01-01

    The class I myosin, Myo1b, is a calmodulin- and actin-associated molecular motor widely expressed in mammalian tissues. Analytical ultracentrifugation studies indicate that Myo1b purified from rat liver has a Stokes radius of 6.7 nm and a sedimentation coefficient, s(20,w), of 7.0 S with a predicted molar mass of 213 kg/mol. These results indicate that Myo1b is monomeric and consists primarily of a splice variant having five associated calmodulins. Molecular modeling based on the analytical ultracentrifugation studies are supported by electron microscopy studies that depict Myo1b as a single-headed, tadpole-shaped molecule with outer dimensions of 27.9 x 4.0 nm. Above a certain Myo1b/actin ratio, Myo1b bundles actin filaments presumably by virtue of a second actin-binding site. These studies provide new information regarding the oligomeric state and morphology of Myo1b and support a model in which Myo1b cross-links actin through a cryptic actin-binding site.

  18. Viscoelastic dynamics of actin filaments coupled to rotary F-ATPase: angular torque profile of the enzyme.

    PubMed Central

    Pänke, O; Cherepanov, D A; Gumbiowski, K; Engelbrecht, S; Junge, W

    2001-01-01

    ATP synthase (F(O)F(1)) operates as two rotary motor/generators coupled by a common shaft. Both portions, F(1) and F(O), are rotary steppers. Their symmetries are mismatched (C(3) versus C(10-14)). We used the curvature of fluorescent actin filaments, attached to the rotating c-ring, as a spring balance (flexural rigidity of 8. 10(-26) Nm(2)) to gauge the angular profile of the output torque at F(O) during ATP hydrolysis by F(1) (see theoretical companion article (. Biophys. J. 81:1234-1244.)). The large average output torque (50 +/- 6 pN. nm) proved the absence of any slip. Variations of the torque were small, and the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the threefold stepping and high activation barrier of the driving motor proper, the rather constant output torque implied a soft elastic power transmission between F(1) and F(O). It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate of the two counteracting and stepping motor/generators. PMID:11509339

  19. Disruption of actin filaments and suppression of pancreatic cancer cell viability and migration following treatment with polyisoprenylated cysteinyl amides

    PubMed Central

    Nkembo, Augustine T; Salako, Olufisayo; Poku, Rosemary A; Amissah, Felix; Ntantie, Elizabeth; Flores-Rozas, Hernan; Lamango, Nazarius S

    2016-01-01

    Pancreatic cancer is characterized by K-Ras mutations in over 90% of the cases. The mutations make the tumors aggressive and resistant to current therapies resulting in very poor prognoses. Valiant efforts to drug mutant K-Ras and related proteins for the treatment of cancers with Ras mutations have been elusive. The need thus persists for therapies to target and suppress the hyperactive K-Ras mutant proteins to normal levels of activity. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) of polyisoprenylated methylated protein methyl esterase (PMPMEase) were designed to disrupt polyisoprenylated protein metabolism and/or functions. The potential for PCAIs to serve as targeted anticancer agents for pancreatic cancer was evaluated in pancreatic ductal adenocarcinoma (PDAC) cell lines expressing mutant (MIAPaCa-2 and Panc-1) and wild type (BxPC-3) K-Ras proteins. The PCAIs inhibited MIAPaCa-2 and BxPC-3 cell viability and induced apoptosis with EC50 values as low as 1.9 µM. The PCAIs, at 0.5 µM, inhibited MIAPaCa-2 cell migration by 50%, inhibited colony formation and disrupted F-actin filament organization. The PCAIs blocked MIAPaCa-2 cell progression at the G0/G1 phase. These results reveal that the PCAIs disrupt pertinent biological processes that lead to pancreatic cancer progression and thus have the potential to act as targeted effective treatments for pancreatic cancer. PMID:27904769

  20. Septin 9_i2 is downregulated in tumors, impairs cancer cell migration and alters subnuclear actin filaments

    PubMed Central

    Verdier-Pinard, P.; Salaun, D.; Bouguenina, H.; Shimada, S.; Pophillat, M.; Audebert, S.; Agavnian, E.; Coslet, S.; Charafe-Jauffret, E.; Tachibana, T.; Badache, A.

    2017-01-01

    Functions of septin cytoskeletal polymers in tumorigenesis are still poorly defined. Their role in the regulation of cytokinesis and cell migration were proposed to contribute to cancer associated aneuploidy and metastasis. Overexpression of Septin 9 (Sept9) promotes migration of cancer cell lines. SEPT9 mRNA and protein expression is increased in breast tumors compared to normal and peritumoral tissues and amplification of SEPT9 gene was positively correlated with breast tumor progression. However, the existence of multiple isoforms of Sept9 is a confounding factor in the analysis of Sept9 functions. In the present study, we analyze the protein expression of Sept9_i2, an uncharacterized isoform, in breast cancer cell lines and tumors and describe its specific impact on cancer cell migration and Sept9 cytoskeletal distribution. Collectively, our results showed that, contrary to Sept9_i1, Sept9_i2 did not support cancer cell migration, and induced a loss of subnuclear actin filaments. These effects were dependent on Sept9_i2 specific N-terminal sequence. Sept9_i2 was strongly down-regulated in breast tumors compared to normal mammary tissues. Thus our data indicate that Sept9_i2 is a negative regulator of breast tumorigenesis. We propose that Sept9 tumorigenic properties depend on the balance between Sept9_i1 and Sept9_i2 expression levels. PMID:28338090

  1. Nuclear migration during karyogamy in rice zygotes is mediated by continuous convergence of actin meshwork toward the egg nucleus.

    PubMed

    Ohnishi, Yukinosuke; Okamoto, Takashi

    2017-03-01

    Fertilization is comprised of two sequential fusion processes; plasmogamy and karyogamy. Karyogamy completes with migration and fusion of the male and female nuclei in the fused cell. In animals, microtubules organized by the centrosome control female/male pronuclei migration. In contrast, the nuclear migration in fused gametes of angiosperms is controlled by actin filaments, but the mechanism that regulates actin filament-dependent nuclear migration is not clear. In this study, we prepared fused rice (Oryza sativa L.) gametes/zygotes using in vitro fertilization and observed the spatial and temporal movements of actin filaments and sperm nuclei. Our results show that actin filaments in egg cells form a meshwork structure surrounding the nuclei. Quantitative analysis of the actin meshwork dynamics suggests that actin meshwork converges toward the egg nucleus. In egg cells fused with sperm cells, actin filaments appeared to interact with a portion of the sperm nuclear membrane. The velocity of the actin filaments was positively correlated with the velocity of the sperm nucleus during karyogamy. These results suggest that sperm nuclear membrane and actin filaments physically interact with each other during karyogamy, and that the sperm nucleus migrates toward the egg nucleus through the convergence of the actin meshwork. Interestingly, actin filament velocity increased promptly after gamete fusion and was further elevated during nuclear fusion. In addition to the migration of gamete nuclei, convergence of actin meshwork may also be critical during early zygotic developments.

  2. Reversible stress softening of actin networks

    PubMed Central

    Chaudhuri, Ovijit; Parekh, Sapun H.; Fletcher, Daniel A.

    2011-01-01

    The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes1–3. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension4–8. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks. PMID:17230186

  3. What we talk about when we talk about nuclear actin

    PubMed Central

    Belin, Brittany J; Mullins, R Dyche

    2013-01-01

    In the cytoplasm, actin filaments form crosslinked networks that enable eukaryotic cells to transport cargo, change shape, and move. Actin is also present in the nucleus but, in this compartment, its functions are more cryptic and controversial. If we distill the substantial literature on nuclear actin down to its essentials, we find four, recurring, and more-or-less independent, claims: (1) crosslinked networks of conventional actin filaments span the nucleus and help maintain its structure and organize its contents; (2) assembly or contraction of filaments regulates specific nuclear events; (3) actin monomers moonlight as subunits of chromatin remodeling complexes, independent of their ability to form filaments; and (4) modified actin monomers or oligomers, structurally distinct from canonical, cytoskeletal filaments, mediate nuclear events by unknown mechanisms. We discuss the evidence underlying these claims and as well as their strengths and weaknesses. Next, we describe our recent work, in which we built probes specific for nuclear actin and used them to describe the form and distribution of actin in somatic cell nuclei. Finally, we discuss how different forms of nuclear actin may play different roles in different cell types and physiological contexts. PMID:23934079

  4. The Tobacco etch virus P3 protein forms mobile inclusions via the early secretory pathway and traffics along actin microfilaments.

    PubMed

    Cui, Xiaoyan; Wei, Taiyun; Chowda-Reddy, R V; Sun, Guangyu; Wang, Aiming

    2010-02-05

    Plant potyviruses encode two membrane proteins, 6K and P3. The 6K protein has been shown to induce virus replication vesicles. However, the function of P3 remains unclear. In this study, subcellular localization of the Tobacco etch virus (TEV) P3 protein was investigated in Nicotiana benthamiana leaf cells. The TEV P3 protein localized on the endoplasmic reticulum (ER) membrane and formed punctate inclusions in association with the Golgi apparatus. The trafficking of P3 to the Golgi was mediated by the early secretory pathway. The Golgi-associated punctate structures originated from the ER exit site (ERES). Deletion analyses identified P3 domains required for the retention of P3 at the Golgi. Moreover, the P3 punctate structure was found to traffic along the actin filaments and colocalize with the 6K-containing replication vesicles. Taken together, these data support previous suggestions that P3 may play dual roles in virus movement and replication.

  5. The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo

    PubMed Central

    Belmont, Lisa D.; Drubin, David G.

    1998-01-01

    Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables. PMID:9732289

  6. A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

    PubMed Central

    Bonello, Teresa T.; Janco, Miro; Hook, Jeff; Byun, Alex; Appaduray, Mark; Dedova, Irina; Hitchcock-DeGregori, Sarah; Hardeman, Edna C.; Stehn, Justine R.; Böcking, Till; Gunning, Peter W.

    2016-01-01

    The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development. PMID:26804624

  7. αT-Catenin Is a Constitutive Actin-binding α-Catenin That Directly Couples the Cadherin·Catenin Complex to Actin Filaments*

    PubMed Central

    Wickline, Emily D.; Dale, Ian W.; Merkel, Chelsea D.; Heier, Jonathon A.; Stolz, Donna B.

    2016-01-01

    α-Catenin is the primary link between the cadherin·catenin complex and the actin cytoskeleton. Mammalian αE-catenin is allosterically regulated: the monomer binds the β-catenin·cadherin complex, whereas the homodimer does not bind β-catenin but interacts with F-actin. As part of the cadherin·catenin complex, αE-catenin requires force to bind F-actin strongly. It is not known whether these properties are conserved across the mammalian α-catenin family. Here we show that αT (testes)-catenin, a protein unique to amniotes that is expressed predominantly in the heart, is a constitutive actin-binding α-catenin. We demonstrate that αT-catenin is primarily a monomer in solution and that αT-catenin monomer binds F-actin in cosedimentation assays as strongly as αE-catenin homodimer. The β-catenin·αT-catenin heterocomplex also binds F-actin with high affinity unlike the β-catenin·αE-catenin complex, indicating that αT-catenin can directly link the cadherin·catenin complex to the actin cytoskeleton. Finally, we show that a mutation in αT-catenin linked to arrhythmogenic right ventricular cardiomyopathy, V94D, promotes homodimerization, blocks β-catenin binding, and in cardiomyocytes disrupts localization at cell-cell contacts. Together, our data demonstrate that αT-catenin is a constitutively active actin-binding protein that can physically couple the cadherin·catenin complex to F-actin in the absence of tension. We speculate that these properties are optimized to meet the demands of cardiomyocyte adhesion. PMID:27231342

  8. The JCMT Gould Belt Survey: properties of star-forming filaments in Orion A North

    NASA Astrophysics Data System (ADS)

    Salji, C. J.; Richer, J. S.; Buckle, J. V.; Francesco, J. Di; Hatchell, J.; Hogerheijde, M.; Johnstone, D.; Kirk, H.; Ward-Thompson, D.; JCMT GBS Consortium

    2015-05-01

    We develop and apply a Hessian-based filament detection algorithm to submillimetre continuum observations of Orion A North. The resultant filament radial density profiles are fitted with beam-convolved line-of-sight Plummer-profiles using Markov chain Monte Carlo techniques. The posterior distribution of the radial decay parameter demonstrates that the majority of filaments exhibit p = 1.5-3, with a mode at p = 2.2, suggesting deviation from the Ostriker p = 4 isothermal, equilibrium, self-gravitating cylinder. The spatial distribution of young stellar objects relative to the high column density filaments is investigated, yielding a lower limit on the star-forming age of the integral-shaped filament ˜1.4 Myr. Additionally, inferred lifetimes of filaments are examined which suggest long-term filament accretion, varying rates of star formation, or both. Theoretical filament stability measures are determined with the aid of HARP C18O J = 3-2 observations and indicate that the majority of filaments are gravitationally subcritical, despite the presence of young protostars. The results from this investigation are consistent with the one-dimensional accretion flow filament model recently observed in numerical simulations.

  9. Actin dynamics and cofilin-actin rods in Alzheimer disease

    PubMed Central

    Bamburg, James R.; Bernstein, Barbara W.

    2017-01-01

    Cytoskeletal abnormalities and synaptic loss, typical of both familial and sporadic Alzheimer disease (AD), are induced by diverse stresses such as neuroinflammation, oxidative stress, and energetic stress, each of which may be initiated or enhanced by proinflammatory cytokines or amyloid-β (Aβ) peptides. Extracellular Aβ-containing plaques and intracellular phospho-tau-containing neurofibrillary tangles are postmortem pathologies required to confirm AD and have been the focus of most studies. However, AD brain, but not normal brain, also have increased levels of cytoplasmic rod-shaped bundles of filaments composed of ADF/cofilin-actin in a 1:1 complex (rods). Cofilin, the major ADF/cofilin isoform in mammalian neurons, severs actin filaments at low cofilin/actin ratios and stabilizes filaments at high cofilin/actin ratios. It binds cooperatively to ADP-actin subunits in F-actin. Cofilin is activated by dephosphorylation and may be oxidized in stressed neurons to form disulfide-linked dimers, required for bundling cofilin-actin filaments into stable rods. Rods form within neurites causing synaptic dysfunction by sequestering cofilin, disrupting normal actin dynamics, blocking transport, and exacerbating mitochondrial membrane potential loss. Aβ and proinflammatory cytokines induce rods through a cellular prion protein-dependent activation of NADPH oxidase and production of reactive oxygen species. Here we review recent advances in our understanding of cofilin biochemistry, rod formation, and the development of cognitive deficits. We will then discuss rod formation as a molecular pathway for synapse loss that may be common between all three prominent current AD hypotheses, thus making rods an attractive therapeutic target. PMID:26873625

  10. Structural polymorphism of the actin-espin system: a prototypical system of filaments and linkers in stereocilia.

    PubMed

    Purdy, Kirstin R; Bartles, James R; Wong, Gerard C L

    2007-02-02

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  11. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin R.; Bartles, James R.; Wong, Gerard C. L.

    2007-02-01

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system’s phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  12. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    SciTech Connect

    Purdy, Kirstin R.; Wong, Gerard C. L.; Bartles, James R.

    2007-02-02

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  13. Actin, microvilli, and the fertilization cone of sea urchin eggs

    PubMed Central

    1980-01-01

    Sea urchin eggs and oocytes at the germinal vesicle stage were fixed at various times after insemination, and thin sections were examined. Actin filaments can first be found in the cortical cytoplasm 1 min after insemination, and by 2 min enormous numbers of filaments are present. At these early stages, the filaments are only occasionally organized into bundles, but one end of many filaments contacts the plasma membrane. By 3 min, and even more dramatically by 5 min after insemination, the filaments become progressively more often found in bundles that lie parallel to the long axis of the microvilli and the fertilization cones. By 7 min, the bundles of filaments in the cone are maximally pronounced, with virtually all the filaments lying parallel to one another. Decoration of the filaments with subfragment 1 of myosin shows that, in both the microvilli and the cones, the filaments are unidirectionally polarized with the arrowheads pointing towards the cell center. The efflux of H+ from the eggs was measured as a function of time after insemination. The rapid phase of H+ efflux occurs at the same time as actin polymerization. From these results it appears that the formation of bundles of actin filaments in microvilli and in cones is a two-step process, involving actin polymerization to form filaments, randomly oriented but in most cases having one end in contact with the plasma membrane, followed by the zippering together of the filaments by macromolecular bridges. PMID:6893988

  14. 12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells

    SciTech Connect

    Shiba, Yoshiki; Sasaki, Yasuto; Kanno, Yoshinobu )

    1988-10-01

    The effect of a tumor-promoting phorbol ester on the binding of fibronectin-coated beads to 3T3-L1 cells was studied to clarify the relationship between the binding of fibronectin to the cells, cell adhesion, and the organization of actin filaments. Interference-reflection microscopy revealed focal contacts of 3T3-L1 cells with the substratum. Stress fibers observed after rhodomine-phalloidin staining were well-developed in the cells. Treatment of the cells for 20 min with 12-O-tetradecanoylphorbol-13-acetate (TPA), but not with phorbol, disrupted focal contacts and caused a reorganization of stress fibers to generate actin ribbons. Treatment of the cells with TPA enhanced the binding of beads coated with human plasma fibronectin to the cells, as observed after incubation for 6 h with the beads. The TPA-induced increase in the percentage of cells with bound beads was dependent on the duration of treatment with TPA and on the concentration of TPA. Treatment of the cells with TPA also enhanced proliferation of cells in a dose-dependent manner. Furthermore, treatment of the cells with phorbol did not enhance the binding of beads coated with fibronectin. These results suggest that TPA specifically enhances the binding of fibronectin-coated beads to 3T3-L1 cells, and that TPA-induced binding of the beads may be related to disruption of focal contacts and reorganization of actin filaments.

  15. A structural study of F-actin - filamin networks

    NASA Astrophysics Data System (ADS)

    Ahrens-Braunstein, Ashley; Nguyen, Lam; Hirst, Linda

    2010-03-01

    The cell's ability to move and contract is attributed to the semi-flexible filamentous protein, F -actin, one of the three filaments in the cytoskeleton. Actin bundling can be formed by a cross-linking actin binding protein (ABP) filamin. By examining filamin's cross-linking abilities at different concentrations and molar ratios, we can study the flexibility, structure and multiple network formations created when cross-linking F-actin with this protein. We have studied the phase diagram of this protein system using fluorescence microscopy, analyzing the network structures observed in the context of a coarse grained molecular dynamics simulation carried out by our group.

  16. The Human Arp2/3 Complex Is Composed of Evolutionarily Conserved Subunits and Is Localized to Cellular Regions of Dynamic Actin Filament Assembly

    PubMed Central

    Welch, Matthew D.; DePace, Angela H.; Verma, Suzie; Iwamatsu, Akihiro; Mitchison, Timothy J.

    1997-01-01

    The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (Arp complex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat–containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion. PMID:9230079

  17. The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly.

    PubMed

    Welch, M D; DePace, A H; Verma, S; Iwamatsu, A; Mitchison, T J

    1997-07-28

    The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells. The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex). We have determined the predicted amino acid sequence of all seven subunits. Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution. Human Arp2 and Arp3 are very similar to family members from other species. p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex. p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families. We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution. Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails. They were not detected in cellular bundles of actin filaments. Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

  18. The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies.

    PubMed

    van Hemert, Martijn J; Lamers, Gerda E M; Klein, Dionne C G; Oosterkamp, Tjerk H; Steensma, H Yde; van Heusden, G Paul H

    2002-04-16

    The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of approximately 10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells.

  19. The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies

    PubMed Central

    van Hemert, Martijn J.; Lamers, Gerda E. M.; Klein, Dionne C. G.; Oosterkamp, Tjerk H.; Steensma, H. Yde; van Heusden, G. Paul H.

    2002-01-01

    The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of ≈10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells. PMID:11929974

  20. Vacuolated Beggiatoa-like filaments from different hypersaline environments form a novel genus.

    PubMed

    Hinck, Susanne; Mussmann, Marc; Salman, Verena; Neu, Thomas R; Lenk, Sabine; Beer, Dirk de; Jonkers, Henk M

    2011-12-01

    In this study, members of a specific group of thin (6-14 µm filament diameter), vacuolated Beggiatoa-like filaments from six different hypersaline microbial mats were morphologically and phylogenetically characterized. Therefore, enrichment cultures were established, filaments were stained with fluorochromes to show intracellular structures and 16S rRNA genes were sequenced. Morphological characteristics of Beggiatoa-like filaments, in particular the presence of intracellular vacuoles, and the distribution of nucleic acids were visualized. In the intracellular vacuole nitrate reached concentrations of up to 650 mM. Fifteen of the retrieved 16S rRNA gene sequences formed a monophyletic cluster and were phylogenetically closely related (≥ 94.4% sequence identity). Sequences of known filamentous sulfide-oxidizing genera Beggiatoa and Thioploca that comprise non-vacuolated and vacuolated filaments from diverse habitats clearly delineated from this cluster. The novel monophyletic cluster was furthermore divided into two sub-clusters: one contained sequences originating from Guerrero Negro (Mexico) microbial mats and the other comprised sequences from five distinct Spanish hypersaline microbial mats from Ibiza, Formentera and Lake Chiprana. Our data suggest that Beggiatoa-like filaments from hypersaline environments displaying a thin filament diameter contain nitrate-storing vacuoles and are phylogenetically separate from known Beggiatoa. Therefore, we propose a novel genus for these organisms, which we suggest to name 'Candidatus Allobeggiatoa'.

  1. Microstructure dependent filament forming kinetics in HfO2 programmable metallization cells

    NASA Astrophysics Data System (ADS)

    Clarke, Heidi; Brown, Timothy; Hu, Jianjun; Ganguli, Raj; Reed, Amber; Voevodin, Andrey; Shamberger, Patrick J.

    2016-10-01

    Variability remains the principal concern for commercialization of HfO2 based resistance switching devices. Here, we investigate the role of thermal processing conditions on internal structure of atomic layer deposited HfO2 thin films, and the impact of that structure on filament forming kinetics of p+ Si/HfO2/Cu and TiN/HfO2/Cu devices. Regardless of bias polarity or electrode metal, filament formation times are at least one order of magnitude shorter in polycrystalline than in amorphous films, which we attribute to the presence of fast ion migration along grain boundaries. Within polycrystalline films, filament formation times are correlated with degree of crystalline orientation. Inter-device variability in forming time is roughly equivalent across HfO2 film processing conditions. The kinetics of filament forming are shown to be highly dependent on HfO2 microstructure, with possible implications for the inter-device variability of subsequent switching cycles.

  2. Actin organization in chick embryo fibroblasts after influenza virus infection. I. Isolation and characterization of actin from chick embryo cells.

    PubMed

    Krizanová, O; Závodská, E; Solariková, L; Ciampor, F; Kocisková, D

    1984-05-01

    Comparison of two starting materials for actin purification has shown that preparation of actin from aceton-dried cytoskeleton was more effective than from native chick embryos (CE). The isolated actin formed a single band of Mr = 42-43000 in SDS-PAGE; less purified samples revealed additional faint bands. G form of actin (non-polymerized) inhibited the activity of DNase I, electron microscopy showed actin filaments and bundles formed upon its polymerization. The freshly purified homogeneous actin has not lost its DNase I-inhibiting activity when incubated for 60 min at 35 degrees or 45 degrees C. Older or less purified actin samples kept under similar conditions showed 18-25% decrease of their DNase I-inhibiting activity and a loss of their polymerization ability. Digestion with trypsin caused a decrease of DNase I-inhibiting activity of fresh as well as for older actin samples.

  3. A “Mechanistic” Explanation of the Multiple Helical Forms Adopted by Bacterial Flagellar Filaments

    PubMed Central

    Calladine, C.R.; Luisi, B.F.; Pratap, J.V.

    2013-01-01

    The corkscrew-like flagellar filaments emerging from the surface of bacteria such as Salmonella typhimurium propel the cells toward nutrient and away from repellents. This kind of motility depends upon the ability of the flagellar filaments to adopt a range of distinct helical forms. A filament is typically constructed from ~ 30,000 identical flagellin molecules, which self-assemble into a tubular structure containing 11 near-longitudinal protofilaments. A “mechanical” model, in which the flagellin building block has the capacity to switch between two principal interfacial states, predicts that the filament can assemble into a “canonical” family of 12 distinct helical forms, each having unique curvature and twist: these include two “extreme” straight forms having left- and right-handed twists, respectively, and 10 intermediate helical forms. Measured shapes of the filaments correspond well with predictions of the model. This report is concerned with two unanswered questions. First, what properties of the flagellin determine which of the 12 discrete forms is preferred? Second, how does the interfacial “switch” work, at a molecular level? Our proposed solution of these problems is based mainly on a detailed examination of differences between the available electron cryo-microscopy structures of the straight L and R filaments, respectively. PMID:23274110

  4. UNC-45/CRO1/She4p (UCS) Protein Forms Elongated Dimer and Joins Two Myosin Heads Near Their Actin Binding Region

    SciTech Connect

    H Shi; G Blobel

    2011-12-31

    UNC-45/CRO1/She4p (UCS) proteins have variously been proposed to affect the folding, stability, and ATPase activity of myosins. They are the only proteins known to interact directly with the motor domain. To gain more insight into UCS function, we determined the atomic structure of the yeast UCS protein, She4p, at 2.9 {angstrom} resolution. We found that 16 helical repeats are organized into an L-shaped superhelix with an amphipathic N-terminal helix dangling off the short arm of the L-shaped molecule. In the crystal, She4p forms a 193-{angstrom}-long, zigzag-shaped dimer through three distinct and evolutionary conserved interfaces. We have identified She4p's C-terminal region as a ligand for a 27-residue-long epitope on the myosin motor domain. Remarkably, this region consists of two adjacent, but distinct, binding epitopes localized at the nucleotide-responsive cleft between the nucleotide- and actin-filament-binding sites. One epitope is situated inside the cleft, the other outside the cleft. After ATP hydrolysis and Pi ejection, the cleft narrows at its base from 20 to 12 {angstrom} thereby occluding the inside the cleft epitope, while leaving the adjacent, outside the cleft binding epitope accessible to UCS binding. Hence, one cycle of higher and lower binding affinity would accompany one ATP hydrolysis cycle and a single step in the walk on an actin filament rope. We propose that a UCS dimer links two myosins at their motor domains and thereby functions as one of the determinants for step size of myosin on actin filaments.

  5. Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

    PubMed Central

    1994-01-01

    Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin. PMID:8163553

  6. Dendritic Actin Nucleation Causes Traveling Waves and Patches

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2010-03-01

    Reversible polymerization of the intracellular protein actin into semiflexible filaments is crucial for cell motion and environmental sensing. Recent studies have shown that polymerized actin can spontaneously form traveling waves and/or moving patches. I investigate possible mechanisms for such phenomena by numerically simulating the ``dendritic nucleation'' model of actin network growth. The simulations treat the growth of an actin network on a flat portion of a cell membrane, using a stochastic-growth method which calculates an explicit three-dimensional network structure. The calculations treat processes including filament growth, capping, branching, severing, and Brownian motion. The dynamics of membrane proteins stimulating actin polymerization are also included: they diffuse in the membrane, and detach/deactivate in the presence of polymerized actin. The simulations show three types of polymerized-actin behavior: 1) traveling waves, 2) coherently moving patches, and 3) random fluctuations with occasional moving patches. Wave formation is favored at low free-actin concentrations by a long reattachment time for the membrane proteins, and by weakness of the attractive interaction between filaments and the membrane. Raising the free-actin concentration results in a randomly varying distribution of polymerized actin. Lowering the free-actin concentration below the optimal value for waves causes the waves to break up into patches which, however, move coherently. Effects of similar magnitude are predicted when other intracellular protein concentrations are varied. Diffusion of the membrane proteins slows the waves, and, if fast enough, stops them completely, resulting in the formation of a static spot.

  7. F-actin dismantling through a Redox-driven synergy between Mical and cofilin

    PubMed Central

    Grintsevich, Elena E.; Yesilyurt, Hunkar Gizem; Rich, Shannon K.; Hung, Ruei-Jiun; Terman, Jonathan R.; Reisler, Emil

    2016-01-01

    Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF/cofilins/twinstar, sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. Here we uncover a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. We find that Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared to either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodeling, axon guidance, and Semaphorin/Plexin repulsion. Mical and cofilin, therefore, form a Redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties. PMID:27454820

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

    PubMed

    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.

  9. Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements.

    PubMed

    Buxa, Stefanie V; Degola, Francesca; Polizzotto, Rachele; De Marco, Federica; Loschi, Alberto; Kogel, Karl-Heinz; di Toppi, Luigi Sanità; van Bel, Aart J E; Musetti, Rita

    2015-01-01

    Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by 'Candidatus Phytoplasma solani,' the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Western blot analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes.

  10. CARMIL is a potent capping protein antagonist: identification of a conserved CARMIL domain that inhibits the activity of capping protein and uncaps capped actin filaments.

    PubMed

    Uruno, Takehito; Remmert, Kirsten; Hammer, John A

    2006-04-14

    Acanthamoeba CARMIL was previously shown to co-purify with capping protein (CP) and to bind pure CP. Here we show that this interaction inhibits the barbed end-capping activity of CP. Even more strikingly, this interaction drives the uncapping of actin filaments previously capped with CP. These activities are CP-specific; CARMIL does not inhibit the capping activities of either gelsolin or CapG and does not uncap gelsolin-capped filaments. Although full-length (FL) CARMIL (residues 1-1121) possesses both anti-CP activities, C-terminal fragments like glutathione S-transferase (GST)-P (940-1121) that contain the CARMIL CP binding site are at least 10 times more active. We localized the full activities of GST-P to its C-terminal 51 residues (1071-1121). This sequence contains a stretch of 25 residues that is highly conserved in CARMIL proteins from protozoa, flies, worms, and vertebrates (CARMIL Homology domain 3; CAH3). Point mutations showed that the majority of the most highly conserved residues within CAH3 are critical for the anti-CP activity of GST-AP (862-1121). Finally, we found that GST-AP binds CP approximately 20-fold more tightly than does FL-CARMIL. This observation together with the elevated activities of C-terminal fragments relative to FL-CARMIL suggests that FL-CARMIL might exist primarily in an autoinhibited state. Consistent with this idea, proteolytic cleavage of FL-CARMIL with thrombin generated an approximately 14-kDa C-terminal fragment that expresses full anti-CP activities. We propose that, after some type of physiological activation event, FL-CARMIL could function in vivo as a potent CP antagonist. Given the pivotal role that CP plays in determining the global actin phenotype of cells, our results suggest that CARMIL may play an important role in the physiological regulation of actin assembly.

  11. On the nature of star-forming filaments - II. Subfilaments and velocities

    NASA Astrophysics Data System (ADS)

    Smith, Rowan J.; Glover, Simon C. O.; Klessen, Ralf S.; Fuller, Gary A.

    2016-02-01

    We show that hydrodynamic turbulent cloud simulations naturally produce large filaments made up of a network of smaller and coherent subfilaments. Such simulations resemble observations of filaments and fibres in nearby molecular clouds. The subfilaments are dynamical features formed at the stagnation points of the turbulent velocity field where shocks dissipate the turbulent energy. They are a ubiquitous feature of the simulated clouds, which appear from the beginning of the simulation and are not formed by gradual fragmentation of larger filaments. Most of the subfilaments are gravitationally subcritical and do not fragment into cores, however, there is also a significant fraction of supercritical subfilaments which break up into star-forming cores. The subfilaments are coherent along their length, and the residual velocities along their spine show that they are subsonically contracting without any ordered rotation on scales of ˜0.1 pc. Accretion flows along the subfilaments can feed material into star-forming cores embedded within the network. The overall mass in subfilaments and the number of subfilaments increases as the cloud evolves. We propose that the formation of filaments and subfilaments is a natural consequence of the turbulent cascade in the complex multiphase interstellar medium. Subfilaments are formed by the high wavenumber, small-scale modes in the turbulent velocity field. These are then stretched by local shear motions and gathered together by a combination of low wavenumber modes and gravitational contraction on larger scales, and by doing so build up the extended filaments observed in column density maps.

  12. Resemblance of actin-binding protein/actin gels to covalently crosslinked networks

    NASA Astrophysics Data System (ADS)

    Janmey, Paul A.; Hvidt, Søren; Lamb, Jennifer; Stossel, Thomas P.

    1990-05-01

    THE maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex1,2. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel sol' transformations result from the rearrangement of cortical actin-rich networks3. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, α-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments4: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels Theologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.

  13. Differential Effects of Caldesmon on the Intermediate Conformational States of Polymerizing Actin*

    PubMed Central

    Huang, Renjian; Grabarek, Zenon; Wang, Chih-Lueh Albert

    2010-01-01

    The actin-binding protein caldesmon (CaD) reversibly inhibits smooth muscle contraction. In non-muscle cells, a shorter CaD isoform co-exists with microfilaments in the stress fibers at the quiescent state, but the phosphorylated CaD is found at the leading edge of migrating cells where dynamic actin filament remodeling occurs. We have studied the effect of a C-terminal fragment of CaD (H32K) on the kinetics of the in vitro actin polymerization by monitoring the fluorescence of pyrene-labeled actin. Addition of H32K or its phosphorylated form either attenuated or accelerated the pyrene emission enhancement, depending on whether it was added at the early or the late phase of actin polymerization. However, the CaD fragment had no effect on the yield of sedimentable actin, nor did it affect the actin ATPase activity. Our findings can be explained by a model in which nascent actin filaments undergo a maturation process that involves at least two intermediate conformational states. If present at early stages of actin polymerization, CaD stabilizes one of the intermediate states and blocks the subsequent filament maturation. Addition of CaD at a later phase accelerates F-actin formation. The fact that CaD is capable of inhibiting actin filament maturation provides a novel function for CaD and suggests an active role in the dynamic reorganization of the actin cytoskeleton. PMID:19889635

  14. Dynamic actin cycling through mitochondrial subpopulations locally regulates the fission–fusion balance within mitochondrial networks

    PubMed Central

    Moore, Andrew S.; Wong, Yvette C.; Simpson, Cory L.; Holzbaur, Erika L. F.

    2016-01-01

    Mitochondria form interconnected networks that dynamically remodel in response to cellular needs. Using live-cell imaging, we investigate the role of the actin cytoskeleton in regulating mitochondrial fission and fusion. We identify cycling of actin filaments onto and off of subsets of cellular mitochondria. The association of actin filaments with mitochondrial subpopulations is transient; actin quickly disassembles, then reassembles around a distinct subpopulation, efficiently cycling through all cellular mitochondria within 14 min. The focal assembly of actin induces local, Drp1-dependent fragmentation of the mitochondrial network. On actin disassembly, fragmented mitochondria undergo rapid fusion, leading to regional recovery of the tubular mitochondrial network. Cycling requires dynamic actin polymerization and is blocked by inhibitors of both Arp2/3 and formins. We propose that cyclic assembly of actin onto mitochondria modulates the fission/fusion balance, promotes network remodelling and content mixing, and thus may serve as an essential mechanism regulating mitochondrial network homeostasis. PMID:27686185

  15. X-ray scattering study of actin polymerization nuclei assembled by tandem W domains

    SciTech Connect

    Rebowski, Grzegorz; Boczkowska, Malgorzata; Hayes, David B.; Guo, Liang; Irving, Thomas C.; Dominguez, Roberto

    2008-08-27

    The initiation of actin polymerization in cells requires actin filament nucleators. With the exception of formins, known filament nucleators use the Wiskott-Aldrich syndrome protein (WASP) homology 2 (WH2 or W) domain for interaction with actin. A common architecture, found in Spire, Cobl, VopL, and VopF, consists of tandem W domains that tie together three to four actin monomers to form a polymerization nucleus. Uncontrollable polymerization has prevented the structural investigation of such nuclei. We have engineered stable nuclei consisting of an actin dimer and a trimer stabilized by tandem W domain hybrid constructs and studied their structures in solution by x-ray scattering. We show that Spire-like tandem W domains stabilize a polymerization nucleus by lining up actin subunits along the long-pitch helix of the actin filament. Intersubunit contacts in the polymerization nucleus, thought to involve the DNase I-binding loop of actin, coexist with the binding of the W domain in the cleft between actin subdomains 1 and 3. The successful stabilization of filament-like multiactin assemblies opens the way to the crystallographic investigation of intersubunit contacts in the actin filament.

  16. Form-Finding Model Shows How Cytoskeleton Network Stiffness Is Realized

    PubMed Central

    Gong, Jinghai; Zhang, Daxu; Tseng, Yiider; Li, Baolong; Wirtz, Denis; Schafer, Benjamin William

    2013-01-01

    In eukaryotic cells the actin-cytoskeletal network provides stiffness and the driving force that contributes to changes in cell shape and cell motility, but the elastic behavior of this network is not well understood. In this paper a two dimensional form-finding model is proposed to investigate the elasticity of the actin filament network. Utilizing an initially random array of actin filaments and actin-cross-linking proteins the form-finding model iterates until the random array is brought into a stable equilibrium configuration. With some care given to actin filament density and length, distance between host sites for cross-linkers, and overall domain size the resulting configurations from the form-finding model are found to be topologically similar to cytoskeletal networks in real cells. The resulting network may then be mechanically exercised to explore how the actin filaments deform and align under load and the sensitivity of the network’s stiffness to actin filament density, length, etc. Results of the model are consistent with the experimental literature, e.g. actin filaments tend to re-orient in the direction of stretching; and the filament relative density, filament length, and actin-cross-linking protein’s relative density, control the actin-network stiffness. The model provides a ready means of extension to more complicated domains and a three-dimensional form-finding model is under development as well as models studying the formation of actin bundles. PMID:24146992

  17. RefilinB (FAM101B) targets FilaminA to organize perinuclear actin networks and regulates nuclear shape

    PubMed Central

    Gay, Olivia; Gilquin, Benoît; Nakamura, Fumihiko; Jenkins, Zandra A.; McCartney, Rosannah; Krakow, Deborah; Deshiere, Alexandre; Assard, Nicole; Hartwig, John H.; Robertson, Stephen P.; Baudier, Jacques

    2011-01-01

    The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actin-binding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial–mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actin-based network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations. PMID:21709252

  18. Mechanics model for actin-based motility

    NASA Astrophysics Data System (ADS)

    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.

  19. Mechanics model for actin-based motility.

    PubMed

    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.

  20. Crystal structure of a nuclear actin ternary complex.

    PubMed

    Cao, Tingting; Sun, Lingfei; Jiang, Yuxiang; Huang, Shanjin; Wang, Jiawei; Chen, Zhucheng

    2016-08-09

    Actin polymerizes and forms filamentous structures (F-actin) in the cytoplasm of eukaryotic cells. It also exists in the nucleus and regulates various nucleic acid transactions, particularly through its incorporation into multiple chromatin-remodeling complexes. However, the specific structure of actin and the mechanisms that regulate its polymeric nature inside the nucleus remain unknown. Here, we report the crystal structure of nuclear actin (N-actin) complexed with actin-related protein 4 (Arp4) and the helicase-SANT-associated (HSA) domain of the chromatin remodeler Swr1. The inner face and barbed end of N-actin are sequestered by interactions with Arp4 and the HSA domain, respectively, which prevents N-actin from polymerization and binding to many actin regulators. The two major domains of N-actin are more twisted than those of globular actin (G-actin), and its nucleotide-binding pocket is occluded, freeing N-actin from binding to and regulation by ATP. These findings revealed the salient structural features of N-actin that distinguish it from its cytoplasmic counterpart and provide a rational basis for its functions and regulation inside the nucleus.

  1. Crystal structure of a nuclear actin ternary complex

    PubMed Central

    Cao, Tingting; Sun, Lingfei; Jiang, Yuxiang; Huang, Shanjin; Wang, Jiawei; Chen, Zhucheng

    2016-01-01

    Actin polymerizes and forms filamentous structures (F-actin) in the cytoplasm of eukaryotic cells. It also exists in the nucleus and regulates various nucleic acid transactions, particularly through its incorporation into multiple chromatin-remodeling complexes. However, the specific structure of actin and the mechanisms that regulate its polymeric nature inside the nucleus remain unknown. Here, we report the crystal structure of nuclear actin (N-actin) complexed with actin-related protein 4 (Arp4) and the helicase-SANT–associated (HSA) domain of the chromatin remodeler Swr1. The inner face and barbed end of N-actin are sequestered by interactions with Arp4 and the HSA domain, respectively, which prevents N-actin from polymerization and binding to many actin regulators. The two major domains of N-actin are more twisted than those of globular actin (G-actin), and its nucleotide-binding pocket is occluded, freeing N-actin from binding to and regulation by ATP. These findings revealed the salient structural features of N-actin that distinguish it from its cytoplasmic counterpart and provide a rational basis for its functions and regulation inside the nucleus. PMID:27457955

  2. Mechanical force-induced polymerization and depolymerization of F-actin at water/solid interfaces

    NASA Astrophysics Data System (ADS)

    Zhang, Xueqiang; Hu, Xiuyuan; Lei, Haozhi; Hu, Jun; Zhang, Yi

    2016-03-01

    Actin molecules are among the three main cytoskeleton proteins of cells and undergo rapid cycling to regulate critical processes such as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. Although extensive studies have been carried out on the dynamics as well as biological functions of actin polymerization and depolymerization both in vivo and in vitro, the molecular mechanisms by which cells sense and respond to mechanical signals are not fully understood. In particular, little attention has been paid to the effect of a physical force that is exerted directly on the actin cytoskeleton. In this paper, we have explored how the mechanical force affects the actin polymerization and depolymerization behaviors at water/solid interfaces using an atomic force microscope (AFM) operated in liquid. By raster scanning an AFM probe on a substrate surface with a certain load, it was found that actin monomers could polymerize into filaments without the help of actin related proteins (ARPs). Further study indicated that actin monomers were inclined to form filaments only under a small scanning load. The polymerized actin filaments would be depolymerized when the mechanical force was stronger. A possible mechanism has been suggested to explain the mechanical force induced actin polymerization.Actin molecules are among the three main cytoskeleton proteins of cells and undergo rapid cycling to regulate critical processes such as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. Although extensive studies have been carried out on the dynamics as well as biological functions of actin polymerization and depolymerization both in vivo and in vitro, the molecular mechanisms by which cells sense and respond to mechanical signals are not fully understood. In particular, little attention has been paid to the effect of a physical force that is exerted directly on the actin cytoskeleton. In this paper, we have explored how the mechanical force affects the actin

  3. Experimental observation of a traveling plasma grating formed by two crossing filaments in gases

    SciTech Connect

    Durand, Magali; Liu Yi; Forestier, Benjamin; Houard, Aurelien; Mysyrowicz, Andre

    2011-03-21

    The spatial motion and effective duration of a traveling plasma grating formed by two interfering femtosecond laser filaments in gases is characterized by its Doppler effect imparted on a probe pulse. The shift velocity determined experimentally agrees with the theoretical calculations.

  4. Cell envelope components influencing filament length in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

    PubMed

    Burnat, Mireia; Schleiff, Enrico; Flores, Enrique

    2014-12-01

    Heterocyst-forming cyanobacteria grow as chains of cells (known as trichomes or filaments) that can be hundreds of cells long. The filament consists of individual cells surrounded by a cytoplasmic membrane and peptidoglycan layers. The cells, however, share a continuous outer membrane, and septal proteins, such as SepJ, are important for cell-cell contact and filament formation. Here, we addressed a possible role of cell envelope components in filamentation, the process of producing and maintaining filaments, in the model cyanobacterium Anabaena sp. strain PCC 7120. We studied filament length and the response of the filaments to mechanical fragmentation in a number of strains with mutations in genes encoding cell envelope components. Previously published peptidoglycan- and outer membrane-related gene mutants and strains with mutations in two genes (all5045 and alr0718) encoding class B penicillin-binding proteins isolated in this work were used. Our results show that filament length is affected in most cell envelope mutants, but the filaments of alr5045 and alr2270 gene mutants were particularly fragmented. All5045 is a dd-transpeptidase involved in peptidoglycan elongation during cell growth, and Alr2270 is an enzyme involved in the biosynthesis of lipid A, a key component of lipopolysaccharide. These results indicate that both components of the cell envelope, the murein sacculus and the outer membrane, influence filamentation. As deduced from the filament fragmentation phenotypes of their mutants, however, none of these elements is as important for filamentation as the septal protein SepJ.

  5. Structural Evidence for Actin-like Filaments in Toxoplasma gondii Using High-Resolution Low-Voltage Field Emission Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Schatten, Heide; Sibley, L. David; Ris, Hans

    2003-08-01

    The protozoan parasite Toxoplasma gondii is representative of a large group of parasites within the phylum Apicomplexa, which share a highly unusual motility system that is crucial for locomotion and active host cell invasion. Despite the importance of motility in the pathology of these unicellular organisms, the motor mechanisms for locomotion remain uncertain, largely because only limited data exist about composition and organization of the cytoskeleton. By using cytoskeleton stabilizing protocols on membrane-extracted parasites and novel imaging with high-resolution low-voltage field emission scanning electron microscopy (LVFESEM), we were able to visualize for the first time a network of actin-sized filaments just below the cell membrane. A complex cytoskeletal network remained after removing the actin-sized fibers with cytochalasin D, revealing longitudinally arranged, subpellicular microtubules and intermediate-sized fibers of 10 nm, which, in stereo images, are seen both above and below the microtubules. These approaches open new possibilities to characterize more fully the largely unexplored and unconventional cytoskeletal motility complex in apicomplexan parasites.

  6. Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding

    PubMed Central

    Arden, Susan D.; Tumbarello, David A.; Butt, Tariq; Kendrick-Jones, John; Buss, Folma

    2016-01-01

    Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo. As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability. PMID:27474411

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

    SciTech Connect

    Juvvadi, Praveen Rao; Belina, Detti; Soderblom, Erik J.; Moseley, M. Arthur; Steinbach, William J.

    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.

  8. Three-color FRET expands the ability to quantify the interactions of several proteins involved in actin filament nucleation

    NASA Astrophysics Data System (ADS)

    Wallrabe, Horst; Sun, Yuansheng; Fang, Xiaolan; Periasamy, Ammasi; Bloom, George

    2012-03-01

    With traditional 2-color Förster Resonance Energy Transfer (FRET) microscopy, valuable quantitative analyses can be conducted. Correlations of donor (D), acceptor (A) and their ratios (D:A) with energy transfer efficiency (E%) or distance (r) allows measurement of changes between control and experimental samples; also, clustered vs. random assembly of cellular components can be differentiated. Essentially, only the above three parameters D, A and D:A vs. E% are the basis for these deductions. 3-color FRET uses the same basic parameters, but exponentially expands the opportunities to quantify interrelationships among 3 cellular components. We investigated a number of questions based on the results of a triple combination (F1-F2-F3) of TFPNWASP/ Venus-IQGAP1/mCherry-Actin - all involved in the nucleation of actin - to apply the extensive analysis assay possible with 3-color FRET. How do changing N-WASP or IQGAP1 fluorescence levels affect actin fluorescence? What is the effect on E% of NWASP-actin by IQGAP1 or E% of IQGAP1-actin by N-WASP? These and other questions are explored in the context of all proteins of interest being in FRET distance vs. any two in the absence of the third. 4 cases are compared based on bleed-through corrected FRET: (1) all 3 interact, (2) only F1- F3 and F2-F3 [not F1-F2], (3) only F1-F2 and F2-F3 interact [not F1-F3], (4) only F1-F2 and F1-F3 interact [not F2-F3]. Other than describing the methodology in detail, several biologically relevant results are presented showing how E% (i.e. distance), fluorescence levels and ratios are affected in each of the cases. These correlations can only be observed in a 3-fluorophore combination. 3-color FRET will greatly expand the investigative range of quantitative analysis for the life-science researcher.

  9. Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the Parasite Toxoplasma gondiiD⃞

    PubMed Central

    Sahoo, Nivedita; Beatty, Wandy; Heuser, John; Sept, David; Sibley, L. David

    2006-01-01

    Toxoplasma is a protozoan parasite in the phylum Apicomplexa, which contains a number of medically important parasites that rely on a highly unusual form of motility termed gliding to actively penetrate their host cells. Parasite actin filaments regulate gliding motility, yet paradoxically filamentous actin is rarely detected in these parasites. To investigate the kinetics of this unusual parasite actin, we expressed TgACT1 in baculovirus and purified it to homogeneity. Biochemical analysis showed that Toxoplasma actin (TgACT1) rapidly polymerized into filaments at a critical concentration that was 3-4-fold lower than conventional actins, yet it failed to copolymerize with mammalian actin. Electron microscopic analysis revealed that TgACT1 filaments were 10 times shorter and less stable than rabbit actin. Phylogenetic comparison of actins revealed a limited number of apicomplexan-specific residues that likely govern the unusual behavior of parasite actin. Molecular modeling identified several key alterations that affect interactions between monomers and that are predicted to destabilize filaments. Our findings suggest that conserved molecular differences in parasite actin favor rapid cycles of assembly and disassembly that govern the unusual form of gliding motility utilized by apicomplexans. PMID:16319175

  10. Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics.

    PubMed

    Speranza, Luisa; Giuliano, Teresa; Volpicelli, Floriana; De Stefano, M Egle; Lombardi, Loredana; Chambery, Angela; Lacivita, Enza; Leopoldo, Marcello; Bellenchi, Gian C; di Porzio, Umberto; Crispino, Marianna; Perrone-Capano, Carla

    2015-01-01

    Recent studies have indicated that the serotonin receptor subtype 7 (5-HT7R) plays a crucial role in shaping neuronal morphology during embryonic and early postnatal life. Here we show that pharmacological stimulation of 5-HT7R using a highly selective agonist, LP-211, enhances neurite outgrowth in neuronal primary cultures from the cortex, hippocampus and striatal complex of embryonic mouse brain, through multiple signal transduction pathways. All these signaling systems, involving mTOR, the Rho GTPase Cdc42, Cdk5, and ERK, are known to converge on the reorganization of cytoskeletal proteins that subserve neurite outgrowth. Indeed, our data indicate that neurite elongation stimulated by 5-HT7R is modulated by drugs affecting actin polymerization. In addition, we show, by 2D Western blot analyses, that treatment of neuronal cultures with LP-211 alters the expression profile of cofilin, an actin binding protein involved in microfilaments dynamics. Furthermore, by using microfluidic chambers that physically separate axons from the soma and dendrites, we demonstrate that agonist-dependent activation of 5-HT7R stimulates axonal elongation. Our results identify for the first time several signal transduction pathways, activated by stimulation of 5-HT7R, that converge to promote cytoskeleton reorganization and consequent modulation of axonal elongation. Therefore, the activation of 5-HT7R might represent one of the key elements regulating CNS connectivity and plasticity during development.

  11. Role of Active Contraction and Tropomodulins in Regulating Actin Filament Length and Sarcomere Structure in Developing Zebrafish Skeletal Muscle

    PubMed Central

    Mazelet, Lise; Parker, Matthew O.; Li, Mei; Arner, Anders; Ashworth, Rachel

    2016-01-01

    Whilst it is recognized that contraction plays an important part in maintaining the structure and function of mature skeletal muscle, its role during development remains undefined. In this study the role of movement in skeletal muscle maturation was investigated in intact zebrafish embryos using a combination of genetic and pharmacological approaches. An immotile mutant line (cacnb1ts25) which lacks functional voltage-gated calcium channels (dihydropyridine receptors) in the muscle and pharmacological immobilization of embryos with a reversible anesthetic (Tricaine), allowed the study of paralysis (in mutants and anesthetized fish) and recovery of movement (reversal of anesthetic treatment). The effect of paralysis in early embryos (aged between 17 and 24 hours post-fertilization, hpf) on skeletal muscle structure at both myofibrillar and myofilament level was determined using both immunostaining with confocal microscopy and small angle X-ray diffraction. The consequences of paralysis and subsequent recovery on the localization of the actin capping proteins Tropomodulin 1 & 4 (Tmod) in fish aged from 17 hpf until 42 hpf was also assessed. The functional consequences of early paralysis were investigated by examining the mechanical properties of the larval muscle. The length-force relationship, active and passive tension, was measured in immotile, recovered and control skeletal muscle at 5 and 7 day post-fertilization (dpf). Recovery of muscle function was also assessed by examining swimming patterns in recovered and control fish. Inhibition of the initial embryonic movements (up to 24 hpf) resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralyzed fish by 42 hpf. In conclusion, myofibril organization is regulated by a dual mechanism involving movement-dependent and movement-independent processes. The initial contractile event itself drives the localization of Tmod1 to its sarcomeric position

  12. Actinous enigma or enigmatic actin

    PubMed Central

    Povarova, Olga I; Uversky, Vladimir N; Kuznetsova, Irina M; Turoverov, Konstantin K

    2014-01-01

    Being the most abundant protein of the eukaryotic cell, actin continues to keep its secrets for more than 60 years. Everything about this protein, its structure, functions, and folding, is mysteriously counterintuitive, and this review represents an attempt to solve some of the riddles and conundrums commonly found in the field of actin research. In fact, actin is a promiscuous binder with a wide spectrum of biological activities. It can exist in at least three structural forms, globular, fibrillar, and inactive (G-, F-, and I-actin, respectively). G-actin represents a thermodynamically instable, quasi-stationary state, which is formed in vivo as a result of the energy-intensive, complex posttranslational folding events controlled and driven by cellular folding machinery. The G-actin structure is dependent on the ATP and Mg2+ binding (which in vitro is typically substituted by Ca2+) and protein is easily converted to the I-actin by the removal of metal ions and by action of various denaturing agents (pH, temperature, and chemical denaturants). I-actin cannot be converted back to the G-form. Foldable and “natively folded” forms of actin are always involved in interactions either with the specific protein partners, such as Hsp70 chaperone, prefoldin, and the CCT chaperonin during the actin folding in vivo or with Mg2+ and ATP as it takes place in the G-form. We emphasize that the solutions for the mysteries of actin multifunctionality, multistructurality, and trapped unfolding can be found in the quasi-stationary nature of this enigmatic protein, which clearly possesses many features attributed to both globular and intrinsically disordered proteins.

  13. Residual stress analysis in forming process of filament wound thick-walled CFRP pipes

    SciTech Connect

    Kondo, Toshimi; Sekine, Hideki; Nakano, Kunio

    1995-11-01

    Residual stress analysis for the cracking phenomenon of filament would thick-walled CFRP pipes, which frequently occurs in the forming process of curing and thermal cycling through the course of the wet filament winding, was made from both the experimental and theoretical points of view. A simple analytical model to study the cracking in the CFRP pipes was proposed. The pipes are multilayered and reinforced in the axial and circumferential directions alternatively by carbon fibers. Taking account of the anisotropy of mechanical and thermal properties including the shrinkage strain, which depend considerably on the temperature, the residual stresses in the CFRP pipes were elucidated in the forming process, particularly, in cooling of the cure process.

  14. Arf1 and Arf6 Promote Ventral Actin Structures formed by acute Activation of Protein Kinase C and Src

    PubMed Central

    Caviston, Juliane P.; Cohen, Lee Ann; Donaldson, Julie G.

    2016-01-01

    Arf proteins regulate membrane traffic and organelle structure. Although Arf6 is known to initiate actin-based changes in cell surface architecture, Arf1 may also function at the plasma membrane. Here we show that acute activation of protein kinase C (PKC) induced by the phorbol ester PMA led to the formation of motile actin structures on the ventral surface of Beas-2b cells, a lung bronchial epithelial cell line. Ventral actin structures also formed in PMA-treated HeLa cells that had elevated levels of Arf activation. For both cell types, formation of the ventral actin structures was enhanced by expression of active forms of either Arf1 or Arf6, and by the expression of guanine nucleotide exchange factors that activate these Arfs. By contrast, formation of these structures was blocked by inhibitors of PKC and Src, and required phosphatidylinositol 4, 5-bisphosphate, Rac, Arf6 and Arf1. Furthermore, expression of ASAP1, an Arf1 GTPase activating protein (GAP) was more effective at inhibiting the ventral actin structures than was ACAP1, an Arf6 GAP. This study adds to the expanding role for Arf1 in the periphery and identifies a requirement for Arf1, a “Golgi Arf”, in the reorganization of the cortical actin cytoskeleton on ventral surfaces, against the substratum. PMID:24916416

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Mitotic cells form actin-based bridges with adjacent cells to provide intercellular communication during rounding.

    PubMed

    Fykerud, Tone A; Knudsen, Lars M; Totland, Max Z; Sørensen, Vigdis; Dahal-Koirala, Shiva; Lothe, Ragnhild A; Brech, Andreas; Leithe, Edward

    2016-11-01

    In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed "mitotic nanotubes," were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding.

  17. Mitotic cells form actin-based bridges with adjacent cells to provide intercellular communication during rounding

    PubMed Central

    Fykerud, Tone A.; Knudsen, Lars M.; Totland, Max Z.; Dahal-Koirala, Shiva; Lothe, Ragnhild A.; Brech, Andreas; Leithe, Edward

    2016-01-01

    ABSTRACT In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed “mitotic nanotubes,” were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding. PMID:27625181

  18. New mechanism of γ-H2AX generation: Surfactant-induced actin disruption causes deoxyribonuclease I translocation to the nucleus and forms DNA double-strand breaks.

    PubMed

    Zhao, Xiaoxu; Yang, Gang; Toyooka, Tatsushi; Ibuki, Yuko

    2015-12-01

    We previously showed that nonionic surfactants, nonylphenol polyethoxylates (NPEOs), induced phosphorylation of histone H2AX, forming γ-H2AX. In this study, we analyzed the mechanism of γ-H2AX generation by an NPEO with 15 ethylene oxide units (NPEO(15)). In MCF-7 breast carcinoma cells, NPEO(15) treatment induced γ-H2AX in a dose-dependent manner. EDTA and ZnCl2, two inhibitors of deoxyribonuclease I (DNase I), inhibited both the γ-H2AX and DNA double-strand breaks induced by NPEO(15). NPEO(15) disrupted filamentous actin and released free DNase I as detected by cell fractionation analysis. Based on immunofluorescence staining of DNase I and monitoring DNase I-GFP localization, DNase I was translocated from the cytosol to the nucleus of cells after treatment with NPEO(15). This translocation did not occur with the common DNA damage inducers ultraviolet B irradiation and hydrogen peroxide. Other surfactants, Tween 20, Triton X-100 and Nonidet P-40, also generated γ-H2AX. These results show that γ-H2AX induction by surfactants including NPEOs, occurs via a new mechanism involving release of free DNase I with actin disruption. This mechanism is distinct from the process of γ-H2AX generation caused by direct chemically induced DNA damage.

  19. Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis.

    PubMed

    Stossel, T P; Hartwig, J H

    1976-03-01

    Actin and myosin of rabbit pulmonary macrophages are influenced by two other proteins. A protein cofactor is required for the actin activation of macrophage myosin Mg2 ATPase activity, and a high molecular weight actin-binding protein aggregates actin filaments (Stossel T.P., and J.H. Hartwig. 1975. J. Biol. Chem. 250:5706-5711)9 When warmed in 0.34 M sucrose solution containing Mg2-ATP and dithiothreitol, these four proteins interact cooperatively. Acin-binding protein in the presence of actin causes the actin to form a gel, which liquifies when cooled. The myosin contracts the gel into an aggregate, and the rate of aggregation is accelerated by the cofactor. Therefore, we believe that these four proteins also effec the temperature-dependent gelation and aggregation of crude sucrose extracts pulmonary macrophages containing Mg2-ATP and dithiothreitol. The gelled extracts are composed of tangled filaments. Relative to homogenates of resting macrophages, the distribution of actin-binding protein in homogenates of phagocytizing macrophages is altered such that 2-6 times more actin-binding protein is soluble. Sucrose extracts of phagocytizing macrophages gel more rapidly than extracts of resting macrophages. Phagocytosis by pulmonary macrophages involves the formation of peripheral pseudopods containing filaments. The findings suggest that the actin-binding protein initiates a cooperative interaction of contractile proteins to generate cytoplasmic gelation, and that phagocytosis influences the behavior of the actin-binding protein.

  20. Microstructure of the multiple-filamentation zone formed by femtosecond laser radiation in a solid dielectric

    SciTech Connect

    Geints, Yu E; Zemlyanov, A A; Kabanov, A M; Petrov, A V; Golik, S S

    2016-02-28

    The regularities of multiple filamentation of gigawatt femtosecond laser pulses in a solid dielectric (optical glass) have been considered. The fine spatial structure of the plasma region that is formed under glass photoionisation and accompanies the formation of light filaments is analysed experimentally and by means of numerical simulation. The dependence of the number, position, and extension of individual 'generations' of plasma channels on the laser pulse energy has been investigated for the first time. It is found that the distribution of the number of plasma channels over the length of a dielectric sample has a maximum, the position of which correlates well with the position of the nonlinear focus of the light beam as a whole; at the same time, the average channel length decreases with increasing pulse power, whereas the number of successive channel 'generations', on the contrary, increases. (interaction of laser radiation with matter. laser plasma)

  1. Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments.

    PubMed

    Schevzov, Galina; Kee, Anthony J; Wang, Bin; Sequeira, Vanessa B; Hook, Jeff; Coombes, Jason D; Lucas, Christine A; Stehn, Justine R; Musgrove, Elizabeth A; Cretu, Alexandra; Assoian, Richard; Fath, Thomas; Hanoch, Tamar; Seger, Rony; Pleines, Irina; Kile, Benjamin T; Hardeman, Edna C; Gunning, Peter W

    2015-07-01

    ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor-stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells.

  2. Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments

    PubMed Central

    Schevzov, Galina; Kee, Anthony J.; Wang, Bin; Sequeira, Vanessa B.; Hook, Jeff; Coombes, Jason D.; Lucas, Christine A.; Stehn, Justine R.; Musgrove, Elizabeth A.; Cretu, Alexandra; Assoian, Richard; Fath, Thomas; Hanoch, Tamar; Seger, Rony; Pleines, Irina; Kile, Benjamin T.; Hardeman, Edna C.; Gunning, Peter W.

    2015-01-01

    ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor–stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells. PMID:25971798

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

    PubMed

    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.

  4. Spontaneous actin dynamics in contractile rings

    NASA Astrophysics Data System (ADS)

    Kruse, Karsten; Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Riveline, Daniel

    Networks of polymerizing actin filaments are known to be capable to self-organize into a variety of structures. For example, spontaneous actin polymerization waves have been observed in living cells in a number of circumstances, notably, in crawling neutrophils and slime molds. During later stages of cell division, they can also spontaneously form a contractile ring that will eventually cleave the cell into two daughter cells. We present a framework for describing networks of polymerizing actin filaments, where assembly is regulated by various proteins. It can also include the effects of molecular motors. We show that the molecular processes driven by these proteins can generate various structures that have been observed in contractile rings of fission yeast and mammalian cells. We discuss a possible functional role of each of these patterns. The work was supported by Agence Nationale de la Recherche, France, (ANR-10-LABX-0030-INRT) and by Deutsche Forschungsgemeinschaft through SFB1027.

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

    PubMed

    Yokota, Etsuo; Tominaga, Motoki; Mabuchi, Issei; Tsuji, Yasunori; Staiger, Christopher J; Oiwa, Kazuhiro; Shimmen, Teruo

    2005-10-01

    From germinating pollen of lily, two types of villins, P-115-ABP and P-135-ABP, have been identified biochemically. Ca(2+)-CaM-dependent actin-filament binding and bundling activities have been demonstrated for both villins previously. Here, we examined the effects of lily villins on the polymerization and depolymerization of actin. P-115-ABP and P-135-ABP present in a crude protein extract prepared from germinating pollen bound to a DNase I affinity column in a Ca(2+)-dependent manner. Purified P-135-ABP reduced the lag period that precedes actin filament polymerization from monomers in the presence of either Ca(2+) or Ca(2+)-CaM. These results indicated that P-135-ABP can form a complex with G-actin in the presence of Ca(2+) and this complex acts as a nucleus for polymerization of actin filaments. However, the nucleation activity of P-135-ABP is probably not relevant in vivo because the assembly of G-actin saturated with profilin, a situation that mimics conditions found in pollen, was not accelerated in the presence of P-135-ABP. P-135-ABP also enhanced the depolymerization of actin filaments during dilution-mediated disassembly. Growth from filament barbed ends in the presence of Ca(2+)-CaM was also prevented, consistent with filament capping activity. These results suggested that lily villin is involved not only in the arrangement of actin filaments into bundles in the basal and shank region of the pollen tube, but also in regulating and modulating actin dynamics through its capping and depolymerization (or fragmentation) activities in the apical region of the pollen tube, where there is a relatively high concentration of Ca(2+).

  6. Endospore-forming filamentous bacteria symbiotic in termites: ultrastructure and growth in culture of Arthromitus

    NASA Technical Reports Server (NTRS)

    Margulis, L.; Olendzenski, L.; Afzelius, B. A.

    1990-01-01

    Many morphologically distinguishable filamentous spore-forming bacteria symbiotic in the paunch (hypertrophied hindguts) of wood-eating insects have been seen since Arthromitus was first described and named as a plant by Leidy in 1850. Previous descriptions were inadequate for acceptance of the group in modern bacteriological literature. Twenty-two distinguishable arthromitids in nine different arthropod hosts are recorded on the basis of microscopic studies. Five are named, including two whose ultrastructure are detailed: Arthromitus chasei sp. nov. that lives in the damp wood-eating termite Zootermopsis angusticollis (from the west coast of North America) and Arthromitus reticulitermitidis sp. nov. from the subterranean west coast termite Reticulitermes tibialis. A pterotermiditis from the desert termite Pterotermitidis occidentis; A. zootermopsidis, also from Z. angusticollis; and A. cristatus (Leidy, 1881) from Reticulitermes flavipes of eastern North America are also named here. Characterized by trichomes that show a morphogenetic sequence from no spores through immature spores to mature spores with spore filaments, Arthromitus symbionts can be identified as members of the genus by light microscopy and habitat. Electron microscopy reveals their remarkable complexity. They attach by spore filaments to various objects including the host gut wall; their maturation extends distally toward the termite lumen. By surface sterilization of the termite, maceration of the paunch, exposure to boiling temperatures and plating on soft acetate agar, the heat resistant nature of the spores and facultatively aerobic nature of Arthromitus sp. (from Zootermopsis) was demonstrated.

  7. Modelling the chemistry of star-forming filaments - I. H2 and CO chemistry

    NASA Astrophysics Data System (ADS)

    Seifried, D.; Walch, S.

    2016-06-01

    We present simulations of star-forming filaments incorporating on of the largest chemical network used to date on-the-fly in a 3D-magnetohydrodynamic (MHD) simulation. The network contains 37 chemical species and about 300 selected reaction rates. For this, we use the newly developed package KROME (Grassi et al.). We combine the KROME package with an algorithm which allows us to calculate the column density and attenuation of the interstellar radiation field necessary to properly model heating and ionization rates. Our results demonstrate the feasibility of using such a complex chemical network in 3D-MHD simulations on modern supercomputers. We perform simulations with different strengths of the interstellar radiation field and the cosmic ray ionization rate. We find that, towards the centre of the filaments, there is gradual conversion of hydrogen from H to H2 as well as of C+ over C to CO. Moreover, we find a decrease of the dust temperature towards the centre of the filaments in agreement with recent HERSCHEL observations.

  8. Deafness and espin-actin self-organization in stereocilia

    NASA Astrophysics Data System (ADS)

    Wong, Gerard C. L.

    2009-03-01

    Espins are F-actin-bundling proteins associated with large parallel actin bundles found in hair cell stereocilia in the ear, as well as brush border microvilli and Sertoli cell junctions. We examine actin bundle structures formed by different wild-type espin isoforms, fragments, and naturally-occurring human espin mutants linked to deafness and/or vestibular dysfunction. The espin-actin bundle structure consisted of a hexagonal arrangement of parallel actin filaments in a non-native twist state. We delineate the structural consequences caused by mutations in espin's actin-bundling module. For espin mutation with a severely damaged actin-bundling module, which are implicated in deafness in mice and humans, oriented nematic-like actin filament structures, which strongly impinges on bundle mechanical stiffness. Finally, we examine what makes espin different, via a comparative study of bundles formed by espin and those formed by fascin, a prototypical bundling protein found in functionally different regions of the cell, such as filopodia.

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

    PubMed

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R; Bryce, Nicole S; Whan, Renee M; Hardeman, Edna C; Fath, Thomas; Schevzov, Galina; Gunning, Peter W

    2015-01-01

    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.

  10. Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

    PubMed Central

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R.; Bryce, Nicole S.; Whan, Renee M.; Hardeman, Edna C.

    2015-01-01

    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. PMID:25978408

  11. Structural and biochemical studies of actin in complex with synthetic macrolide tail analogues.

    PubMed

    Pereira, Jose H; Petchprayoon, Chutima; Hoepker, Alexander C; Moriarty, Nigel W; Fink, Sarah J; Cecere, Giuseppe; Paterson, Ian; Adams, Paul D; Marriott, Gerard

    2014-10-01

    The actin filament-binding and filament-severing activities of the aplyronine, kabiramide, and reidispongiolide families of marine macrolides are located within the hydrophobic tail region of the molecule. Two synthetic tail analogues of aplyronine C (SF-01 and GC-04) are shown to bind to G-actin with dissociation constants of (285±33) and (132±13) nM, respectively. The crystal structures of actin complexes with GC-04, SF-01, and kabiramide C reveal a conserved mode of tail binding within the cleft that forms between subdomains (SD) 1 and 3. Our studies support the view that filament severing is brought about by specific binding of the tail region to the SD1/SD3 cleft on the upper protomer, which displaces loop-D from the lower protomer on the same half-filament. With previous studies showing that the GC-04 analogue can sever actin filaments, it is argued that the shorter complex lifetime of tail analogues with F-actin would make them more effective at severing filaments compared with plasma gelsolin. Structure-based analyses are used to suggest more reactive or targetable forms of GC-04 and SF-01, which may serve to boost the capacity of the serum actin scavenging system, to generate antibody conjugates against tumor cell antigens, and to decrease sputum viscosity in children with cystic fibrosis.

  12. Structural and Biochemical Studies of Actin in Complex with Synthetic Macrolide Tail Analogues

    SciTech Connect

    Pereira, Jose H.; Petchprayoon, Chutima; Hoepker, Alexander C.; Moriarty, Nigel W.; Fink, Sarah J.; Cecere, Giuseppe; Paterson, Ian; Adams, Paul D.; Marriott, Gerard

    2014-07-22

    The actin filament-binding and filament-severing activities of the aplyronine, kabiramide, and reidispongiolide families of marine macrolides are located within the hydrophobic tail region of the molecule. Two synthetic tail analogues of aplyronine C (SF-01 and GC-04) are shown to bind to G-actin with dissociation constants of (285±33) and (132±13) nM, respectively. The crystal structures of actin complexes with GC-04, SF-01, and kabiramide C reveal a conserved mode of tail binding within the cleft that forms between subdomains (SD) 1 and 3. Our studies support the view that filament severing is brought about by specific binding of the tail region to the SD1/SD3 cleft on the upper protomer, which displaces loop-D from the lower protomer on the same half-filament. With previous studies showing that the GC-04 analogue can sever actin filaments, it is argued that the shorter complex lifetime of tail analogues with F-actin would make them more effective at severing filaments compared with plasma gelsolin. In conclusion, structure-based analyses are used to suggest more reactive or targetable forms of GC-04 and SF-01, which may serve to boost the capacity of the serum actin scavenging system, to generate antibody conjugates against tumor cell antigens, and to decrease sputum viscosity in children with cystic fibrosis.

  13. Structural and Biochemical Studies of Actin in Complex with Synthetic Macrolide Tail Analogues

    DOE PAGES

    Pereira, Jose H.; Petchprayoon, Chutima; Hoepker, Alexander C.; ...

    2014-07-22

    The actin filament-binding and filament-severing activities of the aplyronine, kabiramide, and reidispongiolide families of marine macrolides are located within the hydrophobic tail region of the molecule. Two synthetic tail analogues of aplyronine C (SF-01 and GC-04) are shown to bind to G-actin with dissociation constants of (285±33) and (132±13) nM, respectively. The crystal structures of actin complexes with GC-04, SF-01, and kabiramide C reveal a conserved mode of tail binding within the cleft that forms between subdomains (SD) 1 and 3. Our studies support the view that filament severing is brought about by specific binding of the tail region tomore » the SD1/SD3 cleft on the upper protomer, which displaces loop-D from the lower protomer on the same half-filament. With previous studies showing that the GC-04 analogue can sever actin filaments, it is argued that the shorter complex lifetime of tail analogues with F-actin would make them more effective at severing filaments compared with plasma gelsolin. In conclusion, structure-based analyses are used to suggest more reactive or targetable forms of GC-04 and SF-01, which may serve to boost the capacity of the serum actin scavenging system, to generate antibody conjugates against tumor cell antigens, and to decrease sputum viscosity in children with cystic fibrosis.« less

  14. Amplification of actin polymerization forces

    PubMed Central

    Dmitrieff, Serge; Nédélec, François

    2016-01-01

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments. PMID:27002174

  15. Amplification of actin polymerization forces.

    PubMed

    Dmitrieff, Serge; Nédélec, François

    2016-03-28

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments.

  16. Axonal actin in action: Imaging actin dynamics in neurons.

    PubMed

    Ladt, Kelsey; Ganguly, Archan; Roy, Subhojit

    2016-01-01

    Actin is a highly conserved, key cytoskeletal protein involved in numerous structural and functional roles. In neurons, actin has been intensively investigated in axon terminals-growth cones-and dendritic spines, but details about actin structure and dynamics in axon shafts have remained obscure for decades. A major barrier in the field has been imaging actin. Actin exists as soluble monomers (G-actin) as well as actin filaments (F-actin), and labeling actin with conventional fluorescent probes like GFP/RFP typically leads to a diffuse haze that makes it difficult to discern kinetic behaviors. In a recent publication, we used F-actin selective probes to visualize actin dynamics in axons, resolving striking actin behaviors that have not been described before. However, using these probes to visualize actin dynamics is challenging as they can cause bundling of actin filaments; thus, experimental parameters need to be strictly optimized. Here we describe some practical methodological details related to using these probes for visualizing F-actin dynamics in axons.

  17. Filamentous structures in skeletal muscle: anchors for the subsarcolemmal space.

    PubMed

    Khairani, Astrid Feinisa; Tajika, Yuki; Takahashi, Maiko; Ueno, Hitoshi; Murakami, Tohru; Soenggono, Arifin; Yorifuji, Hiroshi

    2015-03-01

    In skeletal muscle fibers, intermediate filaments and actin filaments provide structural support to the myofibrils and the sarcolemma. For many years, it was poorly understood from ultrastructural observations that how these filamentous structures were kept anchored. The present study was conducted to determine the architecture of filamentous anchoring structures in the subsarcolemmal space and the intermyofibrils. The diaphragms (Dp) of adult wild type and mdx mice (mdx is a model for Duchenne muscular dystrophy) were subjected to tension applied perpendicular to the long axis of the muscle fibers, with or without treatment with 1% Triton X-100 or 0.03% saponin. These experiments were conducted to confirm the presence and integrity of the filamentous anchoring structures. Transmission electron microscopy revealed that these structures provide firm transverse connections between the sarcolemma and peripheral myofibrils. Most of the filamentous structures appeared to be inserted into subsarcolemmal densities, forming anchoring connections between the sarcolemma and peripheral myofibrils. In some cases, actin filaments were found to run longitudinally in the subsarcolemmal space to connect to the sarcolemma or in some cases to connect to the intermyofibrils as elongated thin filaments. These filamentous anchoring structures were less common in the mdx Dp. Our data suggest that the transverse and longitudinal filamentous structures form an anchoring system in the subsarcolemmal space and the intermyofibrils.

  18. Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible.

    PubMed

    Yu, X; Egelman, E H

    1992-09-05

    We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli. The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6.1 subunits per turn of a 76 A pitch helix, and appears to be inactive with respect to all RecA activity. The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6.2 subunits per turn of a 94 A pitch helix. Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state. The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds. A three-dimensional reconstruction of the inactive state shows the same general features as the 83 A pitch filament present in the RecA crystal. This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament.

  19. The septin AspB in Aspergillus nidulans forms bars and filaments and plays roles in growth emergence and conidiation.

    PubMed

    Hernández-Rodríguez, Yainitza; Hastings, Susan; Momany, Michelle

    2012-03-01

    In yeast, septins form rings at the mother-bud neck and function as diffusion barriers. In animals, septins form filaments that can colocalize with other cytoskeletal elements. In the filamentous fungus Aspergillus nidulans there are five septin genes, aspA (an ortholog of Saccharomyces cerevisiae CDC11), aspB (an ortholog of S. cerevisiae CDC3), aspC (an ortholog of S. cerevisiae CDC12), aspD (an ortholog of S. cerevisiae CDC10), and aspE (found only in filamentous fungi). The aspB gene was previously reported to be the most highly expressed Aspergillus nidulans septin and to be essential. Using improved gene targeting techniques, we found that deletion of aspB is not lethal but results in delayed septation, increased emergence of germ tubes and branches, and greatly reduced conidiation. We also found that AspB-green fluorescent protein (GFP) localizes as rings and collars at septa, branches, and emerging layers of the conidiophore and as bars and filaments in conidia and hyphae. Bars are found in dormant and isotropically expanding conidia and in subapical nongrowing regions of hyphae and display fast movements. Filaments form as the germ tube emerges, localize to hyphal and branch tips, and display slower movements. All visible AspB-GFP structures are retained in ΔaspD and lost in ΔaspA and ΔaspC strains. Interestingly, in the ΔaspE mutant, AspB-GFP rings, bars, and filaments are visible in early growth, but AspB-GFP rods and filaments disappear after septum formation. AspE orthologs are only found in filamentous fungi, suggesting that this class of septins might be required for stability of septin bars and filaments in highly polar cells.

  20. Sedimentary Biosignatures of Social Organization in Cone-Forming Filamentous Bacteria

    NASA Astrophysics Data System (ADS)

    Tice, M. M.; Gong, J.; Zeng, Z.; Sneed, J.; Wehner, M.; Sparks, D. W.

    2013-12-01

    Conical mats consisting of centimeter-scale steep-sided cones growing above flat basal films form some of the most distinctive fossil microbial communities in the geologic record. Cones have been hypothesized to form by the initially random motion of filamentous bacteria into small tangled clumps followed by the phototactic motion of the same bacteria up resulting slopes. More recent models of cone development suggest that they form in response to growth in stagnant fluids where diffusion limits exchange of nutrients and wastes with the environment. Determining the biological and environmental factors that promote cone formation will be important for interpreting the geological record of fossil mats and stromatolites, on Earth and potentially on Mars. Here we report the results of new experiments demonstrating complex social organization of cone-forming communities and a novel biosignature of the growth of such communities on sandy sediments, as well as detection of that biosignature in 3.2 Ga fossil mats of the Moodies Group (Barberton greenstone belt, South Africa). In order to investigate the processes involved in cone formation, we grew cultures of a filamentous cyanobacterium originally isolated from tufted cones in Yellowstone National Park, Montana, U.S.A. (Leptolyngbya sp. Y-WT-2000 Cl 1). During early mat development, filaments coat sand grain surfaces and aggregate into ~100-μm-long tufts, or mutually aligned bundles of filaments. Tufts are highly motile, bridging sand grains and merging to form larger tufts. After 10-14 days of growth, tufts aggregate during the early morning into centers composed of many tufts that wave vertically and along the sand surface. Centers move across the sediment surface during the middle of the day and merge along bridging tufts. These bridges transmit force to the underlying sediment and are capable of rolling sand grains. At this stage, mats are composed of small mobile centers that disperse along streams of co

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

    PubMed Central

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

    2016-01-01

    Objective: 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. Methods: 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. Results: The migration toward the cathode was significantly higher in the cells treated with the 200-μA monophasic pulsed microcurrent than in the controls (P < .01) without any change in cell viability; treatment with 300-μA monophasic pulsed 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 < .05). Conclusion: The results show that 200 μA is an effective monophasic pulsed microcurrent intensity to promote migration toward the cathode, and this intensity could regulate polarization of migration-related intracellular factors in human dermal fibroblasts. PMID:26819649

  2. 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

    NASA Technical Reports Server (NTRS)

    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.

  3. Inhibitory effect of a surfactant on pure cultures of a filamentous and a floc forming micro-organism.

    PubMed

    Caravelli, A; Giannuzzi, L; Zaritzky, N

    2007-02-01

    Activated sludge is the most widely used biological process for wastewater treatment. Inorganic and organic compounds are removed by a biotic community in the aeration basin. Problems of these systems are loss of settleability and poor sludge compaction due to excessive growth of filamentous micro-organisms. The filamentous bulking can be controlled by the addition of chemical agents. Strong oxidants, such as chlorine, are utilized to eliminate filamentous bacteria; however, these substances also tend to attack floc-forming bacteria and to cause process breakdown. Besides, chlorine may become hazardous owing to the formation of chemical products as chloramines. Surfactant addition constitutes an interesting alternative for the control of filamentous bulking. In this work the effect of a surfactant Triton X-100 (octylphenol ethoxylate), on the respiratory activity (RA) of pure cultures of a filamentous (Sphaerotilus natans) and a floc-former microorganism (Acinetobacter anitratus) was evaluated. In the concentration range tested (60-220 mg l(-1)), the surfactant was observed to exhibit high RA specific inhibition of the filamentous micro-organism with no significant effect on the floc-forming bacteria. Light microscopy observations showed that the surfactant induced cell lysis, leaving only empty sheaths in the case of filamentous micro-organisms. A kinetic equation to predict the microbial RA fraction of a S. natans pure culture as a function of surfactant concentration and contact time was proposed. The effect of Triton X-100 on the inactivation of pure cultures of both micro-organisms was compared to that of chlorine. Triton X-100 results were adequate to eliminate filamentous bacteria emerging as an alternative for filamentous bulking treatment.

  4. A-type Lamins Form Distinct Filamentous Networks with Differential Nuclear Pore Complex Associations.

    PubMed

    Xie, Wei; Chojnowski, Alexandre; Boudier, Thomas; Lim, John S Y; Ahmed, Sohail; Ser, Zheng; Stewart, Colin; Burke, Brian

    2016-10-10

    The nuclear lamina is a universal feature of metazoan nuclear envelopes (NEs) [1]. In mammalian cells, it appears as a 10-30 nm filamentous layer at the nuclear face of the inner nuclear membrane (INM) and is composed primarily of A- and B-type lamins, members of the intermediate filament family [2]. While providing structural integrity to the NE, the lamina also represents an important signaling and regulatory platform [3]. Two A-type lamin isoforms, lamins A and C (LaA and LaC), are expressed in most adult human cells. Encoded by a single gene, these proteins are largely identical, diverging only in their C-terminal tail domains. By contrast with that of LaC, the unique LaA tail undergoes extensive processing, including farnesylation and endo-proteolysis [4, 5]. However, functional differences between LaA and LaC are still unclear. Compounding this uncertainty, the structure of the lamina remains ill defined. In this study, we used BioID, an in vivo proximity-labeling method to identify differential interactors of A-type lamins [6]. One of these, Tpr, a nuclear pore complex (NPC) protein, is highlighted by its selective association with LaC. By employing superresolution microscopy, we demonstrate that this Tpr association is mirrored in enhanced interaction of LaC with NPCs. Further superresolution studies visualizing both endogenous A- and B-type lamins have allowed us to construct a nanometer-scale model of the mammalian nuclear lamina. Our data indicate that different A- and B-type lamin species assemble into separate filament networks that together form an extended composite structure at the nuclear periphery providing attachment sites for NPCs, thereby regulating their distribution.

  5. Feedback Interactions of Polymerized Actin with the Cell Membrane: Waves, Pulses, and Oscillations

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    Polymerized filaments of the protein actin have crucial functions in cell migration, and in bending the cell membrane to drive endocytosis or the formation of protrusions. The nucleation and polymerization of actin filaments are controlled by upstream agents in the cell membrane, including nucleation-promoting factors (NPFs) that activate the Arp2/3 complex to form new branches on pre-existing filaments. But polymerized actin (F-actin) also feeds back on the assembly of NPFs. We explore the effects of the resulting feedback loop of F-actin and NPFs on two phenomena: actin pulses that drive endocytosis in yeast, and actin waves traveling along the membrane of several cell types. In our model of endocytosis in yeast, the actin network is grown explicitly in three dimensions, exerts a negative feedback interaction on localized patch of NPFs in the membrane, and bends the membrane by exerting a distribution of forces. This model explains observed actin and NPF pulse dynamics, and the effects of several interventions including i) NPF mutations, ii) inhibition of actin polymerization, and iii) deletion of a protein that allows F-actin to bend the cell membrane. The model predicts that mutation of the active region of an NPF will enhance the accumulation of that NPF, and we confirm this prediction by quantitative fluorescence microscopy. For actin waves, we treat a similar model, with NPFs distributed over a larger region of the cell membrane. This model naturally generates actin waves, and predicts a transition from wave behavior to spatially localized oscillations when NPFs are confined to a small region. We also predict a transition from waves to static polarization as the negative-feedback coupling between F-actin and the NPFs is reduced. Supported by NIGMS Grant R01 GM107667.

  6. Microtubules as Platforms for Assaying Actin Polymerization In Vivo

    PubMed Central

    Oelkers, J. Margit; Vinzenz, Marlene; Nemethova, Maria; Jacob, Sonja; Lai, Frank P. L.; Block, Jennifer; Szczodrak, Malgorzata; Kerkhoff, Eugen; Backert, Steffen; Schlüter, Kai; Stradal, Theresia E. B.; Small, J. Victor

    2011-01-01

    The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process. PMID:21603613

  7. Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

    PubMed Central

    Provost, P; Doucet, J; Stock, A; Gerisch, G; Samuelsson, B; Rådmark, O

    2001-01-01

    Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction. PMID:11583571

  8. Actin-binding proteins implicated in the formation of the punctate actin foci stimulated by the self-incompatibility response in Papaver.

    PubMed

    Poulter, Natalie S; Staiger, Christopher J; Rappoport, Joshua Z; Franklin-Tong, Vernonica E

    2010-03-01

    The actin cytoskeleton is a key target for signaling networks and plays a central role in translating signals into cellular responses in eukaryotic cells. Self-incompatibility (SI) is an important mechanism responsible for preventing self-fertilization. The SI system of Papaver rhoeas pollen involves a Ca(2+)-dependent signaling network, including massive actin depolymerization as one of the earliest cellular responses, followed by the formation of large actin foci. However, no analysis of these structures, which appear to be aggregates of filamentous (F-)actin based on phalloidin staining, has been carried out to date. Here, we characterize and quantify the formation of F-actin foci in incompatible Papaver pollen tubes over time. The F-actin foci increase in size over time, and we provide evidence that their formation requires actin polymerization. Once formed, these SI-induced structures are unusually stable, being resistant to treatments with latrunculin B. Furthermore, their formation is associated with changes in the intracellular localization of two actin-binding proteins, cyclase-associated protein and actin-depolymerizing factor. Two other regulators of actin dynamics, profilin and fimbrin, do not associate with the F-actin foci. This study provides, to our knowledge, the first insights into the actin-binding proteins and mechanisms involved in the formation of these intriguing structures, which appear to be actively formed during the SI response.

  9. Holding back the microfilament--structural insights into actin and the actin-monomer-binding proteins of apicomplexan parasites.

    PubMed

    Olshina, Maya A; Wong, Wilson; Baum, Jake

    2012-05-01

    Parasites from the phylum Apicomplexa are responsible for several major diseases of man, including malaria and toxoplasmosis. These highly motile protozoa use a conserved actomyosin-based mode of movement to power tissue traversal and host cell invasion. The mode termed as 'gliding motility' relies on the dynamic turnover of actin, whose polymerisation state is controlled by a markedly limited number of identifiable regulators when compared with other eukaryotic cells. Recent studies of apicomplexan actin regulator structure-in particular those of the core triad of monomer-binding proteins, actin-depolymerising factor/cofilin, cyclase-associated protein/Srv2, and profilin-have provided new insights into possible mechanisms of actin regulation in parasite cells, highlighting divergent structural features and functions to regulators from other cellular systems. Furthermore, the unusual nature of apicomplexan actin itself is increasingly coming into the spotlight. Here, we review recent advances in understanding of the structure and function of actin and its regulators in apicomplexan parasites. In particular we explore the paradox between there being an abundance of unpolymerised actin, its having a seemingly increased potential to form filaments relative to vertebrate actin, and the apparent lack of visible, stable filaments in parasite cells.

  10. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  11. The Role of Magnetic Fields in High-Mass Star-Forming Filaments

    NASA Astrophysics Data System (ADS)

    Stephens, Ian

    Filaments are ubiquitous in the star formation process. Planck has revealed that magnetic fields are perpendicular to the densest filaments, which are the birthplace of high-mass stars, suggesting that fields help funnel gas into the filaments. However, the resolved field morphologies and strengths in the dense filaments are unknown. We propose HAWC+ 53 and 214 um polarimetric observations toward two filaments, the Snake (G11.1) and G18.6, to unveil the field morphology. Such observations will probe the filament field morphology at the subarcminute scale over the largest spatial extent to date: 25 and 9 pc respectively. We expect to have over 400 independent beams worth of detections. From the field morphology, we will test the hub-filament theory and investigate how the magnetic field strength and morphology changes with evolution and size-scale.

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

    PubMed

    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.

  13. Structural and viscoelastic properties of actin networks formed by espin or pathologically relevant espin mutants.

    PubMed

    Lieleg, Oliver; Schmoller, Kurt M; Purdy Drew, Kirstin R; Claessens, Mireille M A E; Semmrich, Christine; Zheng, Lili; Bartles, James R; Bausch, Andreas R

    2009-11-09

    The structural organization of the cytoskeleton determines its viscoelastic response which is crucial for the correct functionality of living cells. Both the mechanical response and microstructure of the cytoskeleton are regulated on a microscopic level by the local activation of different actin binding and/or bundling proteins (ABPs). Misregulations in the expression of these ABPs or mutations in their sequence can entail severe cellular dysfunctions and diseases. Here, we study the structural and viscoelastic properties of reconstituted actin networks cross-linked by the ABP espin and compare the obtained network properties to those of other bundled actin networks. Moreover, we quantify the impact of pathologically relevant espin mutations on the viscoelastic properties of these cytoskeletal networks.

  14. 3D actin network centerline extraction with multiple active contours.

    PubMed

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2014-02-01

    Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and actin cables. 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 propose 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 Total Internal Reflection Fluorescence Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy. Quantitative evaluation of the method using synthetic images shows that for images with SNR above 5.0, the average vertex error measured by the distance between our result and ground truth is 1 voxel, and the average Hausdorff distance is below 10 voxels.

  15. Arabidopsis Villins Promote Actin Turnover at Pollen Tube Tips and Facilitate the Construction of Actin Collars[W

    PubMed Central

    Qu, Xiaolu; Zhang, Hua; Xie, Yurong; Wang, Juan; Chen, Naizhi; Huang, Shanjin

    2013-01-01

    Apical actin filaments are crucial for pollen tube tip growth. However, the specific dynamic changes and regulatory mechanisms associated with actin filaments in the apical region remain largely unknown. Here, we have investigated the quantitative dynamic parameters that underlie actin filament growth and disappearance in the apical regions of pollen tubes and identified villin as the major player that drives rapid turnover of actin filaments in this region. Downregulation of Arabidopsis thaliana VILLIN2 (VLN2) and VLN5 led to accumulation of actin filaments at the pollen tube apex. Careful analysis of single filament dynamics showed that the severing frequency significantly decreased, and the lifetime significantly increased in vln2 vln5 pollen tubes. These results indicate that villin-mediated severing is critical for turnover and departure of actin filaments originating in the apical region. Consequently, the construction of actin collars was affected in vln2 vln5 pollen tubes. In addition to the decrease in severing frequency, actin filaments also became wavy and buckled in the apical cytoplasm of vln2 vln5 pollen tubes. These results suggest that villin confers rigidity upon actin filaments. Furthermore, an observed decrease in skewness of actin filaments in the subapical region of vln2 vln5 pollen tubes suggests that villin-mediated bundling activity may also play a role in the construction of actin collars. Thus, our data suggest that villins promote actin turnover at pollen tube tips and facilitate the construction of actin collars. PMID:23715472

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

    PubMed

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

    2014-08-05

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

  17. F-actin distribution and function during sexual development in Eimeria maxima.

    PubMed

    Frölich, Sonja; Wallach, Michael

    2015-06-01

    To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.

  18. SWAP70 Organizes the Actin Cytoskeleton and Is Essential for Phagocytosis.

    PubMed

    Baranov, Maksim V; Revelo, Natalia H; Dingjan, Ilse; Maraspini, Riccardo; Ter Beest, Martin; Honigmann, Alf; van den Bogaart, Geert

    2016-11-01

    Actin plays a critical role during the early stages of pathogenic microbe internalization by immune cells. In this study, we identified a key mechanism of actin filament tethering and stabilization to the surface of phagosomes in human dendritic cells. We found that the actin-binding protein SWAP70 is specifically recruited to nascent phagosomes by binding to the lipid phosphatidylinositol (3,4)-bisphosphate. Multi-color super-resolution stimulated emission depletion (STED) microscopy revealed that the actin cage surrounding early phagosomes is formed by multiple concentric rings containing SWAP70. SWAP70 colocalized with and stimulated activation of RAC1, a known activator of actin polymerization, on phagosomes. Genetic ablation of SWAP70 impaired actin polymerization around phagosomes and resulted in a phagocytic defect. These data show a key role for SWAP70 as a scaffold for tethering the peripheral actin cage to phagosomes.

  19. The actin cytoskeleton in myofibroblast differentiation: Ultrastructure defining form and driving function

    PubMed Central

    Sandbo, Nathan; Dulin, Nickolai

    2011-01-01

    Myofibroblasts are modified fibroblasts, characterized by the presence of a well-developed contractile apparatus, and the formation of robust actin stress fibers. These mechanically active cells are thought to orchestrate extracellular matrix remodeling during normal wound healing in response to tissue injury, and in aberrant tissue remodeling found in fibrosing disorders. This review surveys the understanding of the role of actin stress fibers in myofibroblast biology. From its original description as a defining ultrastructural and morphologic feature, to well-accepted observations demonstrating its participation in contraction, focal adhesion maturation, and extracellular matrix reorganization, and finally to more recent observations demonstrating its role in transducing mechanical force into biochemical signals, transcriptional control of genes involved in locomotion, contraction, and matrix reorganization, and the localized regulation of mRNA translation. This breadth of functionality of the actin stress fiber serves to reinforce and amplify its mechanical function, via induced expression of proteins that themselves augment contraction, focal adhesion formation, and matrix remodeling. In composite, the functions of the actin cytoskeleton are most often aligned, allowing for the integration and amplification of signals promoting both myofibroblast differentiation and matrix remodeling during fibrogenesis. PMID:21925115

  20. Molecular Studies of Filamentous and Biofilm-Forming Hyperthermophilic Communities in Yellowstone National Park

    NASA Astrophysics Data System (ADS)

    Summons, R. E.; Meyer-Dombard, D. R.; Bradley, A. S.; Dibbell, A. K.; Fredricks, H. F.; Hinrichs, K.; Jahnke, L. L.; Shock, E.; Amend, J. P.

    2005-12-01

    The Aquificales, the most deeply-branching order of Bacteria in the phylogenetic tree of life, comprises eight recognized thermophilic genera, including Aquifex, Hydrogenobacter, and Thermocrinis. The common metabolism for these Bacteria, when grown in culture, is the oxidation of hydrogen with molecular oxygen (Knallgas reaction). Aquificales have been identified by molecular techniques (16S rRNA gene surveys, fluorescent in situ hybridization) in Yellowstone National Park (YNP), sea vent chimneys and fluids, and many other terrestrial and marine locations. In situ, Aquificales can reside as biofilms on vent sinters but they also commonly form filamentous communities, otherwise known as pink streamers, which attach to solid substrates. Initial 16S rRNA gene surveys conducted on streamer communities from Octopus Spring YNP indicated that these were low diversity ecosystems dominated by a few phylotypes including Thermocrinis sp., Thermotoga sp. and one other bacterial clade (Reysenbach et al 1994). Archaea were notable for their absence. In one of the first geobiological studies of pink streamers and vent biofilms in Yellowstone National Park, Jahnke and coworkers (2001) used classical lipidological techniques to compare Aquificales cultures with environmental samples to show that YNP pink filaments were more phylogenetically diverse and physiologically more complex than the early genomic studies indicated. The presence of archaeol, the range and structures of other lipids and a wide dispersion in the carbon isotopic signatures of biomass and individual lipids (-15 to -27%) showed that Archaea were present in pink filament communities and that there was, at least, one additional bacterial group besides the dominant Aquificales component. New molecular studies that comprise analyses of 16S rRNA genes and total lipid extracts by liquid chromatography and mass spectrometry and chemical degradation with gas chromatography and mass spectrometry now show that Crenarchaea

  1. Role of Actin Polymerization in Cell Locomotion: Molecules and Models

    PubMed Central

    Bearer, E. L.

    2015-01-01

    Actin filaments forming at the anterior margin of a migrating cell are essential for the formation of filopodia, lamellipodia, and pseudopodia, the “feet” that the cell extends before it. These structures in turn are required for cell locomotion. Yet the molecular nature of the “nucleator” that seeds the polymerization of actin at the leading edge is unknown. Recent advances, including video microscopy of actin dynamics, discovery of proteins unique to the leading edge such as ponticulin, the Mab 2E4 antigen, and ABP 120, and novel experimental models of actin polymerization such as the actin-based movements of intracellular parasites, promise to shed light on this problem in the near future. PMID:8323743

  2. Actin-associated Proteins in the Pathogenesis of Podocyte Injury.

    PubMed

    He, Fang-Fang; Chen, Shan; Su, Hua; Meng, Xian-Fang; Zhang, Chun

    2013-11-01

    Podocytes have a complex cellular architecture with interdigitating processes maintained by a precise organization of actin filaments. The actin-based foot processes of podocytes and the interposed slit diaphragm form the final barrier to proteinuria. The function of podocytes is largely based on the maintenance of the normal foot process structure with actin cytoskeleton. Cytoskeletal dynamics play important roles during normal podocyte development, in maintenance of the healthy glomerular filtration barrier, and in the pathogenesis of glomerular diseases. In this review, we focused on recent findings on the mechanisms of organization and reorganization of these actin-related molecules in the pathogenesis of podocyte injury and potential therapeutics targeting the regulation of actin cytoskeleton in podocytopathies.

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

    PubMed

    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.

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

    PubMed Central

    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

  5. Polyelectrolyte properties of filamentous biopolymers and their consequences in biological fluids.

    PubMed

    Janmey, Paul A; Slochower, David R; Wang, Yu-Hsiu; Wen, Qi; Cēbers, Andrejs

    2014-03-14

    Anionic polyelectrolyte filaments are common in biological cells. DNA, RNA, the cytoskeletal filaments F-actin, microtubules, and intermediate filaments, and polysaccharides such as hyaluronan that form the pericellular matrix all have large net negative charge densities distributed over their surfaces. Several filamentous viruses with diameters and stiffnesses similar to those of cytoskeletal polymers also have similar negative charge densities. Extracellular protein filaments such collagen, fibrin and elastin, in contrast, have notably smaller charge densities and do not behave as highly charged polyelectrolytes in solution. This review summarizes data that demonstrate generic counterion-mediated effects on four structurally unrelated biopolymers of similar charge density: F-actin, vimentin, Pf1 virus, and DNA, and explores the possible biological and pathophysiological consequences of the polyelectrolyte properties of biological filaments.

  6. Mechanical detection of a long-range actin network emanating from a biomimetic cortex.

    PubMed

    Bussonnier, Matthias; Carvalho, Kevin; Lemière, Joël; Joanny, Jean-François; Sykes, Cécile; Betz, Timo

    2014-08-19

    Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the "actin cloud" and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.

  7. Elucidating Key Motifs Required for Arp2/3-Dependent and Independent Actin Nucleation by Las17/WASP

    PubMed Central

    Urbanek, Agnieszka N.; Smaczynska-de Rooij, Iwona I.

    2016-01-01

    Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. Arp2/3 is a well-characterised protein complex that can promote nucleation of new filaments, though its activity requires additional nucleation promotion factors (NPFs). The best recognized of these factors are the WASP family of proteins that contain binding motifs for both monomeric actin and for Arp2/3. Previously we demonstrated that the yeast WASP homologue, Las17, in addition to activating Arp2/3 can also nucleate actin filaments de novo, independently of Arp2/3. This activity is dependent on its polyproline rich region. Through biochemical and in vivo analysis we have now identified key motifs within the polyproline region that are required for nucleation and elongation of actin filaments, and have addressed the role of the WH2 domain in the context of actin nucleation without Arp2/3. We have also demonstrated that full length Las17 is able to bind liposomes giving rise to the possibility of direct linkage of nascent actin filaments to specific membrane sites to which Las17 has been recruited. Overall, we propose that Las17 functions as the key initiator of de novo actin filament formation at endocytic sites by nucleating, elongating and tethering nascent filaments which then serve as a platform for Arp2/3 recruitment and function. PMID:27637067

  8. Effects of polymerization and nucleotide identity on the conformational dynamics of the bacterial actin homolog MreB.

    PubMed

    Colavin, Alexandre; Hsin, Jen; Huang, Kerwyn Casey

    2014-03-04

    The assembly of protein filaments drives many cellular processes, from nucleoid segregation, growth, and division in single cells to muscle contraction in animals. In eukaryotes, shape and motility are regulated through cycles of polymerization and depolymerization of actin cytoskeletal networks. In bacteria, the actin homolog MreB forms filaments that coordinate the cell-wall synthesis machinery to regulate rod-shaped growth and contribute to cellular stiffness through unknown mechanisms. Like actin, MreB is an ATPase and requires ATP to polymerize, and polymerization promotes nucleotide hydrolysis. However, it is unclear whether other similarities exist between MreB and actin because the two proteins share low sequence identity and have distinct cellular roles. Here, we use all-atom molecular dynamics simulations to reveal surprising parallels between MreB and actin structural dynamics. We observe that MreB exhibits actin-like polymerization-dependent structural changes, wherein polymerization induces flattening of MreB subunits, which restructures the nucleotide-binding pocket to favor hydrolysis. MreB filaments exhibited nucleotide-dependent intersubunit bending, with hydrolyzed polymers favoring a straighter conformation. We use steered simulations to demonstrate a coupling between intersubunit bending and the degree of flattening of each subunit, suggesting cooperative bending along a filament. Taken together, our results provide molecular-scale insight into the diversity of structural states of MreB and the relationships among polymerization, hydrolysis, and filament properties, which may be applicable to other members of the broad actin family.

  9. Actin depolymerizing factors ADF7 and ADF10 play distinct roles during pollen development and pollen tube growth.

    PubMed

    Daher, Firas Bou; Geitmann, Anja

    2012-07-01

    An important player in actin remodeling is the actin depolymerizing factor (ADF) which increases actin filament treadmilling rates. Previously, we had prepared fluorescent protein fusions of two Arabidopsis pollen specific ADFs, ADF7 and ADF10. These had enabled us to determine the temporal expression patterns and subcellular localization of these proteins during male gametophyte development. Here we generated stable transformants containing both chimeric genes allowing for simultaneous imaging and direct comparison. One of the striking differences between the two proteins was the localization profile in the growing pollen tube apex. Whereas ADF10 was associated with the filamentous actin array forming the subapical actin fringe, ADF7 was present in the same cytoplasmic region, but in diffuse form. This suggests that ADF7 is involved in the high actin turnover that is likely to occur in the fringe by continuously and efficiently depolymerizing filamentous actin and supplying monomeric actin to the advancing end of the fringe. The possibility to visualize both of these pollen-specific ADFs simultaneously opens avenues for future research into the regulatory function of actin binding proteins in pollen.

  10. A dynamin-actin interaction is required for vesicle scission during endocytosis in yeast.

    PubMed

    Palmer, Sarah E; Smaczynska-de Rooij, Iwona I; Marklew, Christopher J; Allwood, Ellen G; Mishra, Ritu; Johnson, Simeon; Goldberg, Martin W; Ayscough, Kathryn R

    2015-03-30

    Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that--like wild-type Vps1--it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission.

  11. Motion of a helical vortex filament in superfluid 4He under the extrinsic form of the local induction approximation

    NASA Astrophysics Data System (ADS)

    Van Gorder, Robert A.

    2013-08-01

    Very recently, Shivamoggi ["Vortex motion in superfluid 4He: Reformulation in the extrinsic vortex-filament coordinate space," Phys. Rev. B 84, 012506 (2011)], 10.1103/PhysRevB.84.012506 studied the extrinsic form of the local induction approximation (LIA) for the motion of a Kelvin wave on a vortex filament in superfluid 4He, and obtained some results in a cubic approximation. Presently, we study the motion of helical vortex filaments in superfluid 4He under the exact fully nonlinear LIA considered in potential form by Van Gorder ["Fully nonlinear local induction equation describing the motion of a vortex filament in superfluid 4He," J. Fluid Mech. 707, 585 (2012)], 10.1017/jfm.2012.308 and obtained from the Biot-Savart law through the equations of Hall and Vinen ["The rotation of liquid helium II. I. Experiments on the propagation of second sound in uniformly rotating helium II," Proc. R. Soc. London, Ser. A 238, 204 (1956)], 10.1098/rspa.1956.0214 including superfluid friction terms. Nonlinear dispersion relations governing the helical Kelvin wave on such a vortex filament are derived in exact form, from which we may exactly calculate the phase and group velocity of the Kelvin wave. With this, we classify the motion of a helical Kelvin wave on a vortex filament under the LIA. The dispersion relations and results, which follow are exact in nature, in contrast to most results in the literature, which are usually numerical approximations. As such, our results accurately capture the qualitative behavior of the Kelvin waves under the LIA. Extensions to other frameworks are discussed.

  12. Actin Mechanics and Fragmentation*

    PubMed Central

    De La Cruz, Enrique M.; Gardel, Margaret L.

    2015-01-01

    Cell physiological processes require the regulation and coordination of both mechanical and dynamical properties of the actin cytoskeleton. Here we review recent advances in understanding the mechanical properties and stability of actin filaments and how these properties are manifested at larger (network) length scales. We discuss how forces can influence local biochemical interactions, resulting in the formation of mechanically sensitive dynamic steady states. Understanding the regulation of such force-activated chemistries and dynamic steady states reflects an important challenge for future work that will provide valuable insights as to how the actin cytoskeleton engenders mechanoresponsiveness of living cells. PMID:25957404

  13. Direct tests of muscle cross-bridge theories: predictions of a Brownian dumbbell model for position-dependent cross-bridge lifetimes and step sizes with an optically trapped actin filament.

    PubMed Central

    Smith, D A

    1998-01-01

    Force and displacement events from a single myosin molecule interacting with an actin filament suspended between optically trapped beads (Finer, J. T., R. M. Simmons, and J. A. Spudich. 1994. Nature. 368:113-119) can be interpreted in terms of a generalized cross-bridge model that includes the effects of Brownian forces on the beads. Steady-state distributions of force and displacement can be obtained directly from a generalized Smoluchowski equation for Brownian motion of the actin-bead "dumbbell," and time series from Monte Carlo simulations of the corresponding Langevin equation. When the frequency spectrum of Brownian motion extends beyond cross-bridge transition rates, the inverse mean lifetimes of force/displacement pulses are given by cross-bridge rate constants averaged over a Boltzmann distribution of Brownian noise. These averaged rate constants reflect the strain-dependence of the rate constants for the stationary filament, most faithfully at high trap stiffness. Hence, measurements of the lifetimes and displacements of single events as a function of the resting position of the dumbbell can provide a direct test of different cross-bridge theories of muscle contraction. Quantitative demonstrations are given for Huxley models with 1) faster binding or 2) slower dissociation at positive cross-bridge strain. Predictions for other models can be inferred from the averaging procedure. PMID:9826619

  14. Myosin IIIB uses an actin-binding motif in its espin-1 cargo to reach the tips of actin protrusions.

    PubMed

    Merritt, Raymond C; Manor, Uri; Salles, Felipe T; Grati, M'hamed; Dose, Andrea C; Unrath, William C; Quintero, Omar A; Yengo, Christopher M; Kachar, Bechara

    2012-02-21

    Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip localization is lost when we remove the ESPN1 C terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip localization reveals a novel mechanism for the cell to regulate myosin tip localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations.

  15. Actin-binding proteins sensitively mediate F-actin bundle stiffness

    NASA Astrophysics Data System (ADS)

    Claessens, Mireille M. A. E.; Bathe, Mark; Frey, Erwin; Bausch, Andreas R.

    2006-09-01

    Bundles of filamentous actin (F-actin) form primary structural components of a broad range of cytoskeletal processes including filopodia, sensory hair cell bristles and microvilli. Actin-binding proteins (ABPs) allow the cell to tailor the dimensions and mechanical properties of the bundles to suit specific biological functions. Therefore, it is important to obtain quantitative knowledge on the effect of ABPs on the mechanical properties of F-actin bundles. Here we measure the bending stiffness of F-actin bundles crosslinked by three ABPs that are ubiquitous in eukaryotes. We observe distinct regimes of bundle bending stiffness that differ by orders of magnitude depending on ABP type, concentration and bundle size. The behaviour observed experimentally is reproduced quantitatively by a molecular-based mechanical model in which ABP shearing competes with F-actin extension/compression. Our results shed new light on the biomechanical function of ABPs and demonstrate how single-molecule properties determine mesoscopic behaviour. The bending mechanics of F-actin fibre bundles are general and have implications for cytoskeletal mechanics and for the rational design of functional materials.

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

    PubMed Central

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

    2016-01-01

    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and the trans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H+-translocating ATPase (V-ATPase), whose V1 domain subunits B and C bind actin. We have generated a GFP-tagged subunit B2 construct (GFP-B2) that is incorporated into the V1 domain, which in turn is coupled to the V0 sector. 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-V0 domains, which entails subunit B2 translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunits B2 and C1 and actin were detected. In addition, Golgi membrane lipid order disruption by d-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0 domains of V-ATPase through the binding of microfilaments to subunits B and C and 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. PMID:26872971

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

    PubMed

    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.

  18. Controllable Terahertz Radiation from a Linear-Dipole Array Formed by a Two-Color Laser Filament in Air

    NASA Astrophysics Data System (ADS)

    Zhang, Zhelin; Chen, Yanping; Chen, Min; Zhang, Zhen; Yu, Jin; Sheng, Zhengming; Zhang, Jie

    2016-12-01

    We demonstrate effective control on the carrier-envelope phase and angular distribution as well as the peak intensity of a nearly single-cycle terahertz pulse emitted from a laser filament formed by two-color, the fundamental and the corresponding second harmonics, femtosecond laser pulses propagating in air. Experimentally, such control has been performed by varying the filament length and the initial phase difference between the two-color laser components. A linear-dipole-array model, including the descriptions of both the generation (via laser field ionization) and propagation of the emitted terahertz pulse, is proposed to present a quantitative interpretation of the observations. Our results contribute to the understanding of terahertz generation in a femtosecond laser filament and suggest a practical way to control the electric field of a terahertz pulse for potential applications.

  19. In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins.

    PubMed

    Zimmermann, Dennis; Morganthaler, Alisha N; Kovar, David R; Suarez, Cristian

    2016-01-01

    Characterizing the biochemical and biophysical properties of purified proteins is critical to understand the underlying molecular mechanisms that facilitate complicated cellular processes such as cytokinesis. Here we outline in vitro assays to investigate the effects of cytokinesis actin-binding proteins on actin filament dynamics and organization. We describe (1) multicolor single-molecule TIRF microscopy actin assembly assays, (2) "bulk" pyrene actin assembly/disassembly assays, and (3) "bulk" sedimentation actin filament binding and bundling assays.

  20. EXPANDED VERY LARGE ARRAY OBSERVATIONS OF THE BARNARD 5 STAR-FORMING CORE: EMBEDDED FILAMENTS REVEALED

    SciTech Connect

    Pineda, Jaime E.; Longmore, Steven; Goodman, Alyssa A.; Arce, Hector G.; Caselli, Paola; Corder, Stuartt

    2011-09-20

    We present {approx}6.'5 x 8' Expanded Very Large Array (EVLA) mosaic observations of the NH{sub 3} (1,1) emission in the Barnard 5 region in Perseus, with an angular resolution of 6''. This map covers the coherent region, where the dense gas presents subsonic non-thermal motions (as seen from single dish observations with the Green Bank Telescope, GBT). The combined EVLA and GBT observations reveal, for the first time, a striking filamentary structure (20'' wide or 5000 AU at the distance of Perseus) in this low-mass star-forming region. The integrated intensity profile of this structure is consistent with models of an isothermal filament in hydrostatic equilibrium. The observed separation between the B5-IRS1 young stellar object (YSO), in the central region of the core, and the northern starless condensation matches the Jeans length of the dense gas. This suggests that the dense gas in the coherent region is fragmenting. The observed region displays a narrow velocity dispersion, where most of the gas shows evidence for subsonic turbulence and where little spatial variations are present. It is only close to the YSO where an increase in the velocity dispersion is found, but still displaying subsonic non-thermal motions.

  1. Melghiribacillus thermohalophilus gen. nov., sp. nov., a novel filamentous, endospore-forming, thermophilic and halophilic bacterium.

    PubMed

    Addou, Nariman Ammara; Schumann, Peter; Spröer, Cathrin; Ben Hania, Wajdi; Hacene, Hocine; Fauque, Guy; Cayol, Jean-Luc; Fardeau, Marie-Laure

    2015-04-01

    A novel filamentous, endospore-forming, thermophilic and moderately halophilic bacterium designated strain Nari2A(T) was isolated from soil collected from an Algerian salt lake, Chott Melghir. The novel isolate was Gram-staining-positive, aerobic, catalase-negative and oxidase-positive. Optimum growth occurred at 50-55 °C, 7-10% (w/v) NaCl and pH 7-8. The strain exhibited 95.4, 95.4 and 95.2% 16S rRNA gene sequence similarity to Thalassobacillus devorans G19.1(T), Sediminibacillus halophilus EN8d(T) and Virgibacillus kekensis YIM-kkny16(T), respectively. The major menaquinone was MK-7. The polar lipid profile consisted of phosphatidylglycerol, diphosphatidylglycerol, three unknown phosphoglycolipids and two unknown phospholipids. The predominant cellular fatty acids were iso-C(15 : 0) and iso-C(17 : 0). The DNA G+C content was 41.9 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain Nari2A(T) is considered to represent a novel species of a new genus in the family Bacillaceae , order Bacillales , for which the name Melghiribacillus thermohalophilus gen. nov., sp. nov. is proposed. The type strain of Melghiribacillus thermohalophilus is Nari2A(T) ( = DSM 25894(T) = CCUG 62543(T)).

  2. Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase

    PubMed Central

    1995-01-01

    Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti- phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas

  3. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development

    PubMed Central

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-01-01

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated. PMID:27385345

  4. Actin Skeletons at the Membrane as Liquid Crystal Elastomers

    NASA Astrophysics Data System (ADS)

    Discher, Dennis; Dalhaimer, Paul; Levine, Alex; Lubensky, Tom

    2002-03-01

    Actin filaments crosslinked by proteins such as spectrin form plasma membrane networks in a number of cell-types, including the red blood cell and the outer hair cell of the inner ear. Actin filaments are stiff compared to spectrin and can be considered hard rods. We statistically simulate network phase behavior at finite temperature by Monte Carlo methods, and explore the effects of spectrin and actin length as well as isotropic and shear stresses. Relative lengths required for a zero pressure nematic phase are determined, for exmaple, and indicate structural requirements for obtaining a 2D anisotropic elastomer. Emerging studies of network elasticity examine the anisotropic state and begin to probe the relevance of hyper-soft modes to hearing.

  5. F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex

    PubMed Central

    Murrell, Michael P.; Gardel, Margaret L.

    2012-01-01

    Here we develop a minimal model of the cell actomyosin cortex by forming a quasi-2D cross-linked filamentous actin (F-actin) network adhered to a model cell membrane and contracted by myosin thick filaments. Myosin motors generate both compressive and tensile stresses on F-actin and consequently induce large bending fluctuations, which reduces their effective persistence length to <1 μm. Over a large range of conditions, we show the extent of network contraction corresponds exactly to the extent of individual F-actin shortening via buckling. This demonstrates an essential role of buckling in breaking the symmetry between tensile and compressive stresses to facilitate mesoscale network contraction of up to 80% strain. Portions of buckled F-actin with a radius of curvature ∼300 nm are prone to severing and thus compressive stresses mechanically coordinate contractility with F-actin severing, the initial step of F-actin turnover. Finally, the F-actin curvature acquired by myosin-induced stresses can be further constrained by adhesion of the network to a membrane, accelerating filament severing but inhibiting the long-range transmission of the stresses necessary for network contractility. Thus, the extent of membrane adhesion can regulate the coupling between network contraction and F-actin severing. These data demonstrate the essential role of the nonlinear response of F-actin to compressive stresses in potentiating both myosin-mediated contractility and filament severing. This may serve as a general mechanism to mechanically coordinate contractility and cortical dynamics across diverse actomyosin assemblies in smooth muscle and nonmuscle cells. PMID:23213249

  6. Spatial control of the actin cytoskeleton in Drosophila epithelial cells.

    PubMed

    Baum, B; Perrimon, N

    2001-10-01

    The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Here we combine genetics and cell biology to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. We find that profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, CAP-a Drosophila homologue of Adenylyl Cyclase Associated Proteins-functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. We therefore propose that CAP, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila.

  7. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

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

    SciTech Connect

    Kumakura, Michiko; Kawaguchi, Atsushi Nagata, Kyosuke

    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.

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

    SciTech Connect

    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.

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

    PubMed Central

    Liu, Yao; Zhu, Wenhan; Tan, Yunhao; Nakayasu, Ernesto S.; Staiger, Christopher J.

    2017-01-01

    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. PMID:28129393

  11. Changes in morphology of actin filaments and expression of alkaline phosphatase at 3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds.

    PubMed

    Goncharenko, A V; Malyuchenko, N V; Moisenovich, A M; Kotlyarova, M S; Arkhipova, A Yu; Kon'kov, A S; Agapov, I I; Molochkov, A V; Moisenovich, M M; Kirpichnikov, M P

    2016-09-01

    3D cultivation of MG-63 osteoblast-like cells on mineralized fibroin scaffolds leads to an increase in the expression of alkaline phosphatase, an early marker of bone formation. Increased expression is associated with the actin cytoskeleton reorganization under the influence of 3D cultivation and osteogenic calcium phosphate component of the microcarrier.

  12. The Interplay between Viscoelastic and Thermodynamic Properties Determines the Birefringence of F-Actin Gels

    PubMed Central

    Helfer, Emmanuèle; Panine, Pierre; Carlier, Marie-France; Davidson, Patrick

    2005-01-01

    F-actin gels of increasing concentrations (25–300 μM) display in vitro a progressive onset of birefringence due to orientational ordering of actin filaments. At F-actin concentrations <100 μM, this birefringence can be erased and restored at will by sonication and gentle flow, respectively. Hence, the orientational ordering does not result from a thermodynamic transition to a nematic phase but instead is due to mechanical stresses stored in the gels. In contrast, at F-actin concentrations ≥100 μM, gels display spontaneous birefringence recovery, at rest, which is the sign of true nematic ordering, in good agreement with statistical physics models of the isotropic/nematic transition. Well-aligned samples of F-actin gels could be produced and their small-angle x-ray scattering patterns are quite anisotropic. These patterns show no sign of filament positional short-range order and could be modeled by averaging the form factor with the Maier-Saupe nematic distribution function. The derived nematic order parameter S of the gels ranged from S = 0.7 at 300 μM to S = 0.4 at 25 μM. Both birefringence and small-angle x-ray scattering data indicate that, even in absence of cross-linking proteins, spontaneous cooperative alignment of actin filaments may arise in motile regions of living cells where F-actin concentrations can reach values of a few 100 μM. PMID:15863487

  13. Solid friction between soft filaments.

    PubMed

    Ward, Andrew; Hilitski, Feodor; Schwenger, Walter; Welch, David; Lau, A W C; Vitelli, Vincenzo; Mahadevan, L; Dogic, Zvonimir

    2015-06-01

    Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments' overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes's drag, can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. Our findings demonstrate how altering a filament's elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials.

  14. Disease-associated mutant alpha-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity.

    PubMed

    Weins, Astrid; Schlondorff, Johannes S; Nakamura, Fumihiko; Denker, Bradley M; Hartwig, John H; Stossel, Thomas P; Pollak, Martin R

    2007-10-09

    Alpha-actinin-4 is a widely expressed protein that employs an actin-binding site with two calponin homology domains to crosslink actin filaments (F-actin) in a Ca(2+)-sensitive manner in vitro. An inherited, late-onset form of kidney failure is caused by point mutations in the alpha-actinin-4 actin-binding domain. Here we show that alpha-actinin-4/F-actin aggregates, observed in vivo in podocytes of humans and mice with disease, likely form as a direct result of the increased actin-binding affinity of the protein. We document that exposure of a buried actin-binding site 1 in mutant alpha-actinin-4 causes an increase in its actin-binding affinity, abolishes its Ca(2+) regulation in vitro, and diverts its normal localization from actin stress fibers and focal adhesions in vivo. Inactivation of this buried actin-binding site returns the affinity of the mutant to that of the WT protein and abolishes aggregate formation in cells. In vitro, actin filaments crosslinked by the mutant alpha-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT alpha-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of alpha-actinin with F-actin in podocytes in vivo. We propose that a conformational change with full exposure of actin-binding site 1 could function as a switch mechanism to regulate the actin-binding affinity of alpha-actinin and possibly other calponin homology domain proteins under physiological conditions.

  15. Nanosecond electric pulses trigger actin responses in plant cells

    SciTech Connect

    Berghoefer, Thomas; Eing, Christian; Flickinger, Bianca; Hohenberger, Petra; Wegner, Lars H.; Frey, Wolfgang; Nick, Peter

    2009-09-25

    We have analyzed the cellular effects of nanosecond pulsed electrical fields on plant cells using fluorescently tagged marker lines in the tobacco cell line BY-2 and confocal laser scanning microscopy. We observe a disintegration of the cytoskeleton in the cell cortex, followed by contraction of actin filaments towards the nucleus, and disintegration of the nuclear envelope. These responses are accompanied by irreversible permeabilization of the plasma membrane manifest as uptake of Trypan Blue. By pretreatment with the actin-stabilizing drug phalloidin, the detachment of transvacuolar actin from the cell periphery can be suppressed, and this treatment can also suppress the irreversible perforation of the plasma membrane. We discuss these findings in terms of a model, where nanosecond pulsed electric fields trigger actin responses that are key events in the plant-specific form of programmed cell death.

  16. Cardiac myosin-binding protein C decorates F-actin: Implications for cardiac function

    PubMed Central

    Whitten, Andrew E.; Jeffries, Cy M.; Harris, Samantha P.; Trewhella, Jill

    2008-01-01

    Cardiac myosin-binding protein C (cMyBP-C) is an accessory protein of striated muscle sarcomeres that is vital for maintaining regular heart function. Its 4 N-terminal regulatory domains, C0-C1-m-C2 (C0C2), influence actin and myosin interactions, the basic contractile proteins of muscle. Using neutron contrast variation data, we have determined that C0C2 forms a repeating assembly with filamentous actin, where the C0 and C1 domains of C0C2 attach near the DNase I-binding loop and subdomain 1 of adjacent actin monomers. Direct interactions between the N terminus of cMyBP-C and actin thereby provide a mechanism to modulate the contractile cycle by affecting the regulatory state of the thin filament and its ability to interact with myosin. PMID:19011110

  17. An ecto-protein tyrosine phosphatase of Entamoeba histolytica induces cellular detachment by disruption of actin filaments in HeLa cells.

    PubMed

    Anaya-Ruiz, M; Pérez-Santos, J L M; Talamás-Rohana, P

    2003-07-01

    Actin cytoskeleton disruption in host cells has been demonstrated for PTPases from pathogenic microorganisms. In this work, we analysed whether the secreted acid phosphatase from Entamoeba histolytica has phosphotyrosine phosphatase activity and the possibility that this activity may participate in damaging host cells. The secreted acid phosphatase of E. histolytica, which catalyses p-nitrophenyl phosphate hydrolysis at acid pH values, was found to have phosphotyrosine phosphatase activity. The enzymatic properties of phosphotyrosine phosphatase and acid phosphatase were virtually identical and included: Km values of 10 x 10(-4) M, no requirement for divalent cations, and sensitivity to molybdate, vanadate, and tungstate. The phosphotyrosyl phosphatase activity caused significant levels of cell rounding and detachment correlating with disruption of the actin stress fibres in HeLa cells. Thus, our data suggest that secreted phosphotyrosine phosphatase could play a cytotoxic role during amoebic infection.

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

    PubMed Central

    Tang, Haosu; Bidone, Tamara C.

    2015-01-01

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

  19. Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

    PubMed Central

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G.; Orlova, Albina; Egelman, Edward H.; Beck, Moriah R.

    2016-01-01

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. Here we show that the actin binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro crosslinking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of G-actin, akin to metal ions, either through charge neutralization or conformational changes. PMID:26607837

  20. Structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum.

    PubMed

    Kim, Min-Kyu; Kim, Ji-Hye; Kim, Ji-Sun; Kang, Sa-Ouk

    2015-09-01

    The crystal structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum was solved by Ca(2+)/S-SAD phasing and refined at 1.89 Å resolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Its in vitro F-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216-244 in the C-domain form part of the strongest actin-binding sites (193-254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216-244 fit into a pocket between actin subdomains -1 and -2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions.

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

    PubMed

    Hwang, Wonmuk; Eryilmaz, Esma

    2014-07-11

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

  2. Kinetic Signature of Fractal-like Filament Networks Formed by Orientational Linear Epitaxy

    NASA Astrophysics Data System (ADS)

    Hwang, Wonmuk; Eryilmaz, Esma

    2014-07-01

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

  3. Actin dynamics: old friends with new stories.

    PubMed

    Staiger, Christopher J; Blanchoin, Laurent

    2006-12-01

    Actin dynamics, or the rapid turnover of actin filaments, play a central role in numerous cellular processes. A large and diverse cast of characters, accessory proteins known as actin-binding proteins, modulate actin dynamics. They do this by binding to the monomer pool, interacting with the side and ends of filaments, creating breaks along a filament, and generating new filaments de novo. Recent biochemical and single-filament imaging analyses of several conserved classes of plant actin-binding proteins reveal unusual and unexpected properties. Examples that are highlighted in this review include: an abundant monomer-binding protein that catalyzes nucleotide exchange; a barbed-end capping protein that is dissociated from filament ends by the signaling lipid, phosphatidic acid; a villin-like bundling protein that lacks all Ca(2+)-regulated activities; and a formin family member that is non-processive and is sufficient to generate actin filament bundles. These and other stories motivate a careful description of the properties of plant proteins in vitro as a prelude to greater insight into the molecular mechanism(s) underlying the regulation of actin dynamics in vivo.

  4. Formation and ingression of division furrow can progress under the inhibitory condition of actin polymerization in ciliate Tetrahymena pyriformis.

    PubMed

    Shimizu, Yuhta; Kushida, Yasuharu; Kiriyama, Shuhei; Nakano, Kentaro; Numata, Osamu

    2013-12-01

    In eukaryotic cells that multiply by binary fission, the interaction of actin filaments with myosin II in the contractile ring is widely recognized to generate force for membrane ingression into the cleavage furrow; however, the expression of myosin II is restricted in animals, yeast, fungi, and amoeba (collectively, unikonts). No corresponding motor protein capable of forming mini-filaments that could exert sufficient tension to cleave the cell body is found in bikonts, consisting of planta, algae, and most protozoa; however, cells in some bikont lineages multiply by binary fission, as do animal cells. Of these, the ciliate Tetrahymena is known to form an actin ring beneath the division furrow in cytokinesis. Here, we investigated the role of filamentous actin in the cytokinesis of Tetrahymena pyriformis by treating synchronized dividing cells with an actin-inhibiting drug, Latrunculin-A. Video microscopic observation of live cells undergoing cytokinesis was performed, and contrary to expectation, we found that initiation of furrow ingression and its progress are not suppressed under the inhibitory condition of actin polymerization in Tetrahymena cells. We suggest that an actin filament-independent mechanism of binary fission may have been acquired during the evolution in this organism.

  5. Spectroscopic Study of Star-forming Galaxies in Filaments and the Field at z~0.5: Evidence for Environmental Dependence of Electron Density

    NASA Astrophysics Data System (ADS)

    Darvish, Behnam; Mobasher, Bahram; Sobral, David; Hemmati, Shoubaneh; Nayyeri, Hooshang; Shivaei, Irene

    2016-01-01

    We study the physical properties of a spectroscopic sample of 28 star-forming galaxies in a large filamentary structure in the COSMOS field at z~0.53, with spectroscopic data taken with the Keck/DEIMOS spectrograph, and compare them with a control sample of 30 field galaxies. We spectroscopically confirm the presence of a large galaxy filament (~ 8 Mpc), along which five confirmed X-ray groups exist. We show that within the uncertainties, the ionization parameter, equivalent width (EW), EW versus specific star-formation rate (sSFR) relation, EW versus stellar mass relation, line-of-sight velocity dispersion, dynamical mass, and stellar-to-dynamical mass ratio are similar for filament and field star-forming galaxies. However, we show that on average, filament star-forming galaxies are more metal-enriched (~ 0.1-0.15 dex), possibly due to the inflow of the already enriched intrafilamentary gas into filament galaxies. Moreover, we show that electron densities are significantly lower (a factor of ~17) in filament star-forming systems compared to those in the field, possibly because of a longer star-formation timescale for filament star-forming galaxies. Our results highlight the potential pre-processing role of galaxy filaments and intermediate-density environments on the evolution of galaxies, which has been highly underestimated.

  6. Spectroscopic Study of Star-forming Galaxies in Filaments and the Field at z ~ 0.5: Evidence for Environmental Dependence of Electron Density

    NASA Astrophysics Data System (ADS)

    Darvish, Behnam; Mobasher, Bahram; Sobral, David; Hemmati, Shoubaneh; Nayyeri, Hooshang; Shivaei, Irene

    2015-12-01

    We study the p