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Sample records for actin nucleation complex

  1. Structural Basis of Actin Filament Nucleation by Tandem W Domains

    PubMed Central

    Chen, Xiaorui; Ni, Fengyun; Tian, Xia; Kondrashkina, Elena; Wang, Qinghua; Ma, Jianpeng

    2013-01-01

    SUMMARY Spontaneous nucleation of actin is very inefficient in cells. To overcome this barrier, cells have evolved a set of actin filament nucleators to promote rapid nucleation and polymerization in response to specific stimuli. However, the molecular mechanism of actin nucleation remains poorly understood. This is hindered largely by the fact that actin nucleus, once formed, rapidly polymerizes into filament, thus making it impossible to capture stable multisubunit actin nucleus. Here, we report an effective double-mutant strategy to stabilize actin nucleus by preventing further polymerization. Employing this strategy, we solved the crystal structure of AMPPNP-actin in complex with the first two tandem W domains of Cordon-bleu (Cobl), a potent actin filament nucleator. Further sequence comparison and functional studies suggest that the nucleation mechanism of Cobl is probably shared by the p53 cofactor JMY, but not Spire. Moreover, the double-mutant strategy opens the way for atomic mechanistic study of actin nucleation and polymerization. PMID:23727244

  2. Identification of an ATP-controlled allosteric switch that controls actin filament nucleation by Arp2/3 complex

    PubMed Central

    Rodnick-Smith, Max; Liu, Su-Ling; Balzer, Connor J.; Luan, Qing; Nolen, Brad J.

    2016-01-01

    Nucleation of branched actin filaments by Arp2/3 complex is tightly regulated to control actin assembly in cells. Arp2/3 complex activation involves conformational changes brought about by ATP, Nucleation Promoting Factor (NPF) proteins, actin filaments and NPF-recruited actin monomers. To understand how these factors promote activation, we must first understand how the complex is held inactive in their absence. Here we demonstrate that the Arp3 C-terminal tail is a structural switch that prevents Arp2/3 complex from adopting an active conformation. The interaction between the tail and a hydrophobic groove in Arp3 blocks movement of Arp2 and Arp3 into an activated filament-like (short pitch) conformation. Our data indicate ATP binding destabilizes this interaction via an allosteric link between the Arp3 nucleotide cleft and the hydrophobic groove, thereby promoting the short-pitch conformation. Our results help explain how Arp2/3 complex is locked in an inactive state without activators and how autoinhibition is relieved. PMID:27417392

  3. Identification of an ATP-controlled allosteric switch that controls actin filament nucleation by Arp2/3 complex.

    PubMed

    Rodnick-Smith, Max; Liu, Su-Ling; Balzer, Connor J; Luan, Qing; Nolen, Brad J

    2016-01-01

    Nucleation of branched actin filaments by Arp2/3 complex is tightly regulated to control actin assembly in cells. Arp2/3 complex activation involves conformational changes brought about by ATP, Nucleation Promoting Factor (NPF) proteins, actin filaments and NPF-recruited actin monomers. To understand how these factors promote activation, we must first understand how the complex is held inactive in their absence. Here we demonstrate that the Arp3 C-terminal tail is a structural switch that prevents Arp2/3 complex from adopting an active conformation. The interaction between the tail and a hydrophobic groove in Arp3 blocks movement of Arp2 and Arp3 into an activated filament-like (short pitch) conformation. Our data indicate ATP binding destabilizes this interaction via an allosteric link between the Arp3 nucleotide cleft and the hydrophobic groove, thereby promoting the short-pitch conformation. Our results help explain how Arp2/3 complex is locked in an inactive state without activators and how autoinhibition is relieved. PMID:27417392

  4. A Nucleator Arms Race: Cellular Control of Actin Assembly

    PubMed Central

    Campellone, Kenneth G.; Welch, Matthew D.

    2010-01-01

    For more than a decade the Arp2/3 complex, a handful of nucleation-promoting factors, and formins were the only molecules known to directly nucleate actin filament formation de novo. However, the past several years have brought a surge in the discovery of mammalian proteins with roles in actin nucleation and dynamics. Newly recognized nucleation-promoting factors, such as WASH, WHAMM, and JMY stimulate Arp2/3 complex activity at distinct cellular locations. Formin nucleators with additional biochemical and cellular activities have also been uncovered. Finally, the Spire, Cordon-bleu, and Leiomodin nucleators have revealed new ways of overcoming the kinetic barriers to actin polymerization. PMID:20237478

  5. Membrane Targeting of the Spir·Formin Actin Nucleator Complex Requires a Sequential Handshake of Polar Interactions*

    PubMed Central

    Tittel, Janine; Welz, Tobias; Czogalla, Aleksander; Dietrich, Susanne; Samol-Wolf, Annette; Schulte, Markos; Schwille, Petra; Weidemann, Thomas; Kerkhoff, Eugen

    2015-01-01

    Spir and formin (FMN)-type actin nucleators initiate actin polymerization at vesicular membranes necessary for long range vesicular transport processes. Here we studied in detail the membrane binding properties and protein/protein interactions that govern the assembly of the membrane-associated Spir·FMN complex. Using biomimetic membrane models we show that binding of the C-terminal Spir-2 FYVE-type zinc finger involves both the presence of negatively charged lipids and hydrophobic contributions from the turret loop that intrudes the lipid bilayer. In solution, we uncovered a yet unknown intramolecular interaction between the Spir-2 FYVE-type domain and the N-terminal kinase non-catalytic C-lobe domain (KIND) that could not be detected in the membrane-bound state. Interestingly, we found that the intramolecular Spir-2 FYVE/KIND and the trans-regulatory Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions in living cells by fluorescence cross-correlation spectroscopy. The data corroborate a model according to which Spir-2 exists in two different states, a cytosolic monomeric conformation and a membrane-bound state in which the KIND domain is released and accessible for subsequent Fmn-2 recruitment. This sequence of interactions mechanistically couples membrane binding of Spir to the recruitment of FMN, a pivotal step for initiating actin nucleation at vesicular membranes. PMID:25564607

  6. 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.

  7. Regulation of actin nucleation and autophagosome formation.

    PubMed

    Coutts, Amanda S; La Thangue, Nicholas B

    2016-09-01

    Autophagy is a process of self-eating, whereby cytosolic constituents are enclosed by a double-membrane vesicle before delivery to the lysosome for degradation. This is an important process which allows for recycling of nutrients and cellular components and thus plays a critical role in normal cellular homeostasis as well as cell survival during stresses such as starvation or hypoxia. A large number of proteins regulate various stages of autophagy in a complex and still incompletely understood series of events. In this review, we will discuss recent studies which provide a growing body of evidence that actin dynamics and proteins that influence actin nucleation play an important role in the regulation of autophagosome formation and maturation. PMID:27147468

  8. Capping Protein Increases the Rate of Actin-based Motility by Promoting Filament Nucleation by the Arp2/3 Complex

    PubMed Central

    Akin, Orkun; Mullins, R. Dyche

    2008-01-01

    Summary Capping protein is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between capping protein and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex, and not by increasing the rate of filament elongation as previously suggested. One consequence of this coupling between capping and nucleation is that, while the rate of motility depends strongly on the concentration of capping protein and Arp2/3, the net rate of actin assembly is insensitive to changes in either factor. By reorganizing their architecture, dendritic actin networks harness the same assembly kinetics to drive different rates of motility. PMID:18510928

  9. Activation by Cdc42 and Pip2 of Wiskott-Aldrich Syndrome Protein (Wasp) Stimulates Actin Nucleation by Arp2/3 Complex

    PubMed Central

    Higgs, Henry N.; Pollard, Thomas D.

    2000-01-01

    We purified native WASp (Wiskott-Aldrich Syndrome protein) from bovine thymus and studied its ability to stimulate actin nucleation by Arp2/3 complex. WASp alone is inactive in the presence or absence of 0.5 μM GTP-Cdc42. Phosphatidylinositol 4,5 bisphosphate (PIP2) micelles allowed WASp to activate actin nucleation by Arp2/3 complex, and this was further enhanced twofold by GTP-Cdc42. Filaments nucleated by Arp2/3 complex and WASp in the presence of PIP2 and Cdc42 concentrated around lipid micelles and vesicles, providing that Cdc42 was GTP-bound and prenylated. Thus, the high concentration of WASp in neutrophils (9 μM) is dependent on interactions with both acidic lipids and GTP-Cdc42 to activate actin nucleation by Arp2/3 complex. The results also suggest that membrane binding increases the local concentrations of Cdc42 and WASp, favoring their interaction. PMID:10995437

  10. Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex.

    PubMed

    Rodnick-Smith, Max; Luan, Qing; Liu, Su-Ling; Nolen, Brad J

    2016-07-01

    The Arp2/3 (Actin-related proteins 2/3) complex is activated by WASP (Wiskott-Aldrich syndrome protein) family proteins to nucleate branched actin filaments that are important for cellular motility. WASP recruits actin monomers to the complex and stimulates movement of Arp2 and Arp3 into a "short-pitch" conformation that mimics the arrangement of actin subunits within filaments. The relative contribution of these functions in Arp2/3 complex activation and the mechanism by which WASP stimulates the conformational change have been unknown. We purified budding yeast Arp2/3 complex held in or near the short-pitch conformation by an engineered covalent cross-link to determine if the WASP-induced conformational change is sufficient for activity. Remarkably, cross-linked Arp2/3 complex bypasses the need for WASP in activation and is more active than WASP-activated Arp2/3 complex. These data indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation. During activation, WASP limits nucleation rates by releasing slowly from nascent branches. The cross-linked complex is inhibited by WASP's CA region, even though CA potently stimulates cross-linking, suggesting that slow WASP detachment masks the activating potential of the short-pitch conformational switch. We use structure-based mutations and WASP-Arp fusion chimeras to determine how WASP stimulates movement toward the short-pitch conformation. Our data indicate that WASP displaces the autoinhibitory Arp3 C-terminal tail from a hydrophobic groove at Arp3's barbed end to destabilize the inactive state, providing a mechanism by which WASP stimulates the short-pitch conformation and activates Arp2/3 complex. PMID:27325766

  11. Actin nucleation at the centrosome controls lymphocyte polarity.

    PubMed

    Obino, Dorian; Farina, Francesca; Malbec, Odile; Sáez, Pablo J; Maurin, Mathieu; Gaillard, Jérémie; Dingli, Florent; Loew, Damarys; Gautreau, Alexis; Yuseff, Maria-Isabel; Blanchoin, Laurent; Théry, Manuel; Lennon-Duménil, Ana-Maria

    2016-01-01

    Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome-regulated by the availability of the Arp2/3 complex-determines its capacity to polarize in response to external stimuli. PMID:26987298

  12. How capping protein enhances actin filament growth and nucleation on biomimetic beads

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe; Carlsson, Anders E.

    2015-12-01

    Capping protein (CP), which caps the growing ends of actin filaments, accelerates actin-based motility. Recent experiments on biomimetic beads have shown that CP also enhances the rate of actin filament nucleation. Proposed explanations for these phenomena include (i) the actin funneling hypothesis (AFH), in which the presence of CP increases the free-actin concentration, and (ii) the monomer gating model, in which CP binding to actin filament barbed ends makes more monomers available for filament nucleation. To establish how CP increases the rates of filament elongation and nucleation on biomimetic beads, we perform a quantitative modeling analysis of actin polymerization, using rate equations that include actin filament nucleation, polymerization and capping, as modified by monomer depletion near the surface of the bead. With one adjustable parameter, our simulation results match previously measured time courses of polymerized actin and filament number. The results support a version of the AFH where CP increases the local actin monomer concentration at the bead surface, but leaves the global free-actin concentration nearly constant. Because the rate of filament nucleation increases with the monomer concentration, the increased local monomer concentration enhances actin filament nucleation. We derive a closed-form formula for the characteristic CP concentration where the local free-actin concentration reaches half the bulk value, and find it to be comparable to the global Arp2/3 complex concentration. We also propose an experimental protocol for distinguishing branching nucleation of filaments from spontaneous nucleation.

  13. Actin nucleation at the centrosome controls lymphocyte polarity

    PubMed Central

    Obino, Dorian; Farina, Francesca; Malbec, Odile; Sáez, Pablo J.; Maurin, Mathieu; Gaillard, Jérémie; Dingli, Florent; Loew, Damarys; Gautreau, Alexis; Yuseff, Maria-Isabel; Blanchoin, Laurent; Théry, Manuel; Lennon-Duménil, Ana-Maria

    2016-01-01

    Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome—regulated by the availability of the Arp2/3 complex—determines its capacity to polarize in response to external stimuli. PMID:26987298

  14. Viruses that ride on the coat-tails of actin nucleation.

    PubMed

    Newsome, Timothy P; Marzook, N Bishara

    2015-10-01

    Actin nucleation drives a diversity of critical cellular processes and the motility of a select group of viral pathogens. Vaccinia virus and baculovirus, Autographa californica multiple nucleopolyhedrovirus, recruit and activate the cellular actin nucleator, the Arp2/3 complex, at the surface of virus particles thereby instigating highly localized actin nucleation. The extension of these filaments provides a mechanical force that bestows the ability to navigate the intracellular environment and promote their infectious cycles. This review outlines the viral and cellular proteins that initiate and regulate the signalling networks leading to viral modification of the actin cytoskeleton and summarizes recent insights into the role of actin-based virus transport. PMID:26459972

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

    SciTech Connect

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

    2012-12-09

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

  16. Dendritic Actin Filament Nucleation Causes Traveling Waves and Patches

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders E.

    2010-06-01

    The polymerization of actin via branching at a cell membrane containing nucleation-promoting factors 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 not involving myosin II. The effects of downregulation of key proteins on actin wave behavior are evaluated.

  17. Integration of linear and dendritic actin nucleation in Nck-induced actin comets

    PubMed Central

    Borinskaya, Sofya; Velle, Katrina B.; Campellone, Kenneth G.; Talman, Arthur; Alvarez, Diego; Agaisse, Hervé; Wu, Yi I.; Loew, Leslie M.; Mayer, Bruce J.

    2016-01-01

    The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails—dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, formin-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens. PMID:26609071

  18. Actin nucleation by WH2 domains at the autophagosome.

    PubMed

    Coutts, Amanda S; La Thangue, Nicholas B

    2015-01-01

    Autophagy is a catabolic process whereby cytosolic components and organelles are degraded to recycle key cellular materials. It is a constitutive process required for proper tissue homoeostasis but can be rapidly regulated by a variety of stimuli (for example, nutrient starvation and chemotherapeutic agents). JMY is a DNA damage-responsive p53 cofactor and actin nucleator important for cell survival and motility. Here we show that JMY regulates autophagy through its actin nucleation activity. JMY contains an LC3-interacting region, which is necessary to target JMY to the autophagosome where it enhances the autophagy maturation process. In autophagosomes, the integrity of the WH2 domains allows JMY to promote actin nucleation, which is required for efficient autophagosome formation. Thus our results establish a direct role for actin nucleation mediated by WH2 domain proteins that reside at the autophagosome. PMID:26223951

  19. Actin nucleation by WH2 domains at the autophagosome

    PubMed Central

    Coutts, Amanda S.; La Thangue, Nicholas B.

    2015-01-01

    Autophagy is a catabolic process whereby cytosolic components and organelles are degraded to recycle key cellular materials. It is a constitutive process required for proper tissue homoeostasis but can be rapidly regulated by a variety of stimuli (for example, nutrient starvation and chemotherapeutic agents). JMY is a DNA damage-responsive p53 cofactor and actin nucleator important for cell survival and motility. Here we show that JMY regulates autophagy through its actin nucleation activity. JMY contains an LC3-interacting region, which is necessary to target JMY to the autophagosome where it enhances the autophagy maturation process. In autophagosomes, the integrity of the WH2 domains allows JMY to promote actin nucleation, which is required for efficient autophagosome formation. Thus our results establish a direct role for actin nucleation mediated by WH2 domain proteins that reside at the autophagosome. PMID:26223951

  20. Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing.

    PubMed

    Miyoshi, Takushi; Tsuji, Takahiro; Higashida, Chiharu; Hertzog, Maud; Fujita, Akiko; Narumiya, Shuh; Scita, Giorgio; Watanabe, Naoki

    2006-12-18

    Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays. PMID:17178911

  1. Electrostatics control actin filament nucleation and elongation kinetics.

    PubMed

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

    2013-04-26

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

  2. 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

  3. Rab1 recruits WHAMM during membrane remodeling but limits actin nucleation

    PubMed Central

    Russo, Ashley J.; Mathiowetz, Alyssa J.; Hong, Steven; Welch, Matthew D.; Campellone, Kenneth G.

    2016-01-01

    Small G-proteins are key regulatory molecules that activate the actin nucleation machinery to drive cytoskeletal rearrangements during plasma membrane remodeling. However, the ability of small G-proteins to interact with nucleation factors on internal membranes to control trafficking processes has not been well characterized. Here we investigated roles for members of the Rho, Arf, and Rab G-protein families in regulating WASP homologue associated with actin, membranes, and microtubules (WHAMM), an activator of Arp2/3 complex–mediated actin nucleation. We found that Rab1 stimulated the formation and elongation of WHAMM-associated membrane tubules in cells. Active Rab1 recruited WHAMM to dynamic tubulovesicular structures in fibroblasts, and an active prenylated version of Rab1 bound directly to an N-terminal domain of WHAMM in vitro. In contrast to other G-protein–nucleation factor interactions, Rab1 binding inhibited WHAMM-mediated actin assembly. This ability of Rab1 to regulate WHAMM and the Arp2/3 complex represents a distinct strategy for membrane remodeling in which a Rab G-protein recruits the actin nucleation machinery but dampens its activity. PMID:26823012

  4. Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion

    PubMed Central

    Chazeau, Anaël; Mehidi, Amine; Nair, Deepak; Gautier, Jérémie J; Leduc, Cécile; Chamma, Ingrid; Kage, Frieda; Kechkar, Adel; Thoumine, Olivier; Rottner, Klemens; Choquet, Daniel; Gautreau, Alexis; Sibarita, Jean-Baptiste; Giannone, Grégory

    2014-01-01

    Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity. PMID:25293574

  5. The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway

    PubMed Central

    Michard, Céline; Sperandio, Daniel; Baïlo, Nathalie; Pizarro-Cerdá, Javier; LeClaire, Lawrence; Chadeau-Argaud, Elise; Pombo-Grégoire, Isabel; Hervet, Eva; Vianney, Anne; Gilbert, Christophe; Faure, Mathias; Cossart, Pascale

    2015-01-01

    ABSTRACT Legionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses. PMID:25944859

  6. Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes.

    PubMed

    Pylypenko, Olena; Welz, Tobias; Tittel, Janine; Kollmar, Martin; Chardon, Florian; Malherbe, Gilles; Weiss, Sabine; Michel, Carina Ida Luise; Samol-Wolf, Annette; Grasskamp, Andreas Till; Hume, Alistair; Goud, Bruno; Baron, Bruno; England, Patrick; Titus, Margaret A; Schwille, Petra; Weidemann, Thomas; Houdusse, Anne; Kerkhoff, Eugen

    2016-01-01

    There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes. PMID:27623148

  7. Two Pathways through Cdc42 Couple the N-Formyl Receptor to Actin Nucleation in Permeabilized Human Neutrophils

    PubMed Central

    Glogauer, M.; Hartwig, J.; Stossel, T.

    2000-01-01

    We developed a permeabilization method that retains coupling between N-formyl-methionyl-leucyl-phenylalanine tripeptide (FMLP) receptor stimulation, shape changes, and barbed-end actin nucleation in human neutrophils. Using GTP analogues, phosphoinositides, a phosphoinositide-binding peptide, constitutively active or inactive Rho GTPase mutants, and activating or inhibitory peptides derived from neural Wiskott-Aldrich syndrome family proteins (N-WASP), we identified signaling pathways leading from the FMLP receptor to actin nucleation that require Cdc42, but then diverge. One branch traverses the actin nucleation pathway involving N-WASP and the Arp2/3 complex, whereas the other operates through active Rac to promote actin nucleation. Both pathways depend on phosphoinositide expression. Since maximal inhibition of the Arp2/3 pathway leaves an N17Rac inhibitable alternate pathway intact, we conclude that this alternate involves phosphoinositide-mediated uncapping of actin filament barbed ends. PMID:10953003

  8. The Actin Nucleator Cobl Is Controlled by Calcium and Calmodulin

    PubMed Central

    Haag, Natja; Kessels, Michael M.; Qualmann, Britta

    2015-01-01

    Actin nucleation triggers the formation of new actin filaments and has the power to shape cells but requires tight control in order to bring about proper morphologies. The regulation of the members of the novel class of WASP Homology 2 (WH2) domain-based actin nucleators, however, thus far has largely remained elusive. Our study reveals signal cascades and mechanisms regulating Cordon-Bleu (Cobl). Cobl plays some, albeit not fully understood, role in early arborization of neurons and nucleates actin by a mechanism that requires a combination of all three of its actin monomer–binding WH2 domains. Our experiments reveal that Cobl is regulated by Ca2+ and multiple, direct associations of the Ca2+ sensor Calmodulin (CaM). Overexpression analyses and rescue experiments of Cobl loss-of-function phenotypes with Cobl mutants in primary neurons and in tissue slices demonstrated the importance of CaM binding for Cobl’s functions. Cobl-induced dendritic branch initiation was preceded by Ca2+ signals and coincided with local F-actin and CaM accumulations. CaM inhibitor studies showed that Cobl-mediated branching is strictly dependent on CaM activity. Mechanistic studies revealed that Ca2+/CaM modulates Cobl’s actin binding properties and furthermore promotes Cobl’s previously identified interactions with the membrane-shaping F-BAR protein syndapin I, which accumulated with Cobl at nascent dendritic protrusion sites. The findings of our study demonstrate a direct regulation of an actin nucleator by Ca2+/CaM and reveal that the Ca2+/CaM-controlled molecular mechanisms we discovered are crucial for Cobl’s cellular functions. By unveiling the means of Cobl regulation and the mechanisms, by which Ca2+/CaM signals directly converge on a cellular effector promoting actin filament formation, our work furthermore sheds light on how local Ca2+ signals steer and power branch initiation during early arborization of nerve cells—a key process in neuronal network formation. PMID

  9. Structure of a Bud6/Actin Complex Reveals a Novel WH2-like Actin Monomer Recruitment Motif.

    PubMed

    Park, Eunyoung; Graziano, Brian R; Zheng, Wei; Garabedian, Mikael; Goode, Bruce L; Eck, Michael J

    2015-08-01

    In budding yeast, the actin-binding protein Bud6 cooperates with formins Bni1 and Bnr1 to catalyze the assembly of actin filaments. The nucleation-enhancing activity of Bud6 requires both a "core" domain that binds to the formin and a "flank" domain that binds monomeric actin. Here, we describe the structure of the Bud6 flank domain in complex with actin. Two helices in Bud6(flank) interact with actin; one binds in a groove at the barbed end of the actin monomer in a manner closely resembling the helix of WH2 domains, a motif found in many actin nucleation factors. The second helix rises along the face of actin. Mutational analysis verifies the importance of these Bud6-actin contacts for nucleation-enhancing activity. The Bud6 binding site on actin overlaps with that of the formin FH2 domain and is also incompatible with inter-subunit contacts in F-actin, suggesting that Bud6 interacts only transiently with actin monomers during filament nucleation. PMID:26118535

  10. Chlamydia trachomatis Tarp cooperates with the Arp2/3 complex to increase the rate of actin polymerization

    PubMed Central

    Jiwani, Shahanawaz; Ohr, Ryan J.; Fischer, Elizabeth R.; Hackstadt, Ted; Alvarado, Stephenie; Romero, Adriana; Jewett, Travis J.

    2012-01-01

    Actin polymerization is required for Chlamydia trachomatis entry into nonphagocytic host cells. Host and chlamydial actin nucleators are essential for internalization of chlamydiae by eukaryotic cells. The host cell Arp2/3 complex and the chlamydial translocated actin recruiting phosphoprotein (Tarp) are both required for entry. Tarp and the Arp2/3 complex exhibit unique actin polymerization kinetics individually, but the molecular details of how these two actin nucleators cooperate to promote bacterial entry is not understood. In this study we provide biochemical evidence that the two actin nucleators act synergistically by co-opting the unique attributes of each to enhance the dynamics of actin filament formation. This process is independent of Tarp phosphorylation. We further demonstrate that Tarp colocalization with actin filaments is independent of the Tarp phosphorylation domain. The results are consistent with a model in which chlamydial and host cell actin nucleators cooperate to increase the rate of actin filament formation. PMID:22465117

  11. CASK and protein 4.1 support F-actin nucleation on neurexins.

    PubMed

    Biederer, T; Sudhof, T C

    2001-12-21

    Rearrangements of the actin cytoskeleton are involved in a variety of cellular processes from locomotion of cells to morphological alterations of the cell surface. One important question is how local interactions of cells with the extracellular space are translated into alterations of their membrane organization. To address this problem, we studied CASK, a member of the membrane-associated guanylate kinase homologues family of adaptor proteins. CASK has been shown to bind the erythrocyte isoform of protein 4.1, a class of proteins that promote formation of actin/spectrin microfilaments. In neurons, CASK also interacts via its PDZ domain with the cytosolic C termini of neurexins, neuron-specific cell-surface proteins. We now show that CASK binds a brain-enriched isoform of protein 4.1, and nucleates local assembly of actin/spectrin filaments. These interactions can be reconstituted on the cytosolic tail of neurexins. Furthermore, CASK can be recovered with actin filaments prepared from rat brain extracts, and neurexins are recruited together with CASK and protein 4.1 into these actin filaments. Thus, analogous to the PDZ-domain protein p55 and glycophorin C at the erythrocyte membrane, a similar complex comprising CASK and neurexins exists in neurons. Our data suggest that intercellular junctions formed by neurexins, such as junctions initiated by beta-neurexins with neuroligins, are at least partially coupled to the actin cytoskeleton via an interaction with CASK and protein 4.1. PMID:11604393

  12. Dimeric WH2 repeats of VopF sequester actin monomers into non-nucleating linear string conformations: An X-ray scattering study.

    PubMed

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

    2015-05-01

    VopF and VopL are highly similar virulence-factors of Vibrio cholerae and Vibrio parahaemolyticus respectively that disrupt the host's actin cytoskeleton, using a unique organization in dimerized WH2 repeats. Association of dimerized WH2 domains with the barbed face of actin confers multifunctional activities to VopF in vitro, including G-actin sequestration and filament nucleation, barbed end tracking and uncapping. Here, small angle X-ray scattering (SAXS) measurements of complexes of VopF with actin and structural modeling reveal that VopF stabilizes linear actin-strings that differ from canonical actin filament architectures but represent non-polymerizable sequestered forms of actin. The results exclude that VopL binds the pointed end of actin filaments in the template filament nucleation mechanism derived from crystallographic studies. PMID:25818509

  13. 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

  14. Genome-Wide siRNA Screen Identifies Complementary Signaling Pathways Involved in Listeria Infection and Reveals Different Actin Nucleation Mechanisms during Listeria Cell Invasion and Actin Comet Tail Formation

    PubMed Central

    Kühbacher, Andreas; Emmenlauer, Mario; Rämo, Pauli; Kafai, Natasha; Dehio, Christoph

    2015-01-01

    ABSTRACT Listeria monocytogenes enters nonphagocytic cells by a receptor-mediated mechanism that is dependent on a clathrin-based molecular machinery and actin rearrangements. Bacterial intra- and intercellular movements are also actin dependent and rely on the actin nucleating Arp2/3 complex, which is activated by host-derived nucleation-promoting factors downstream of the cell receptor Met during entry and by the bacterial nucleation-promoting factor ActA during comet tail formation. By genome-wide small interfering RNA (siRNA) screening for host factors involved in bacterial infection, we identified diverse cellular signaling networks and protein complexes that support or limit these processes. In addition, we could precise previously described molecular pathways involved in Listeria invasion. In particular our results show that the requirements for actin nucleators during Listeria entry and actin comet tail formation are different. Knockdown of several actin nucleators, including SPIRE2, reduced bacterial invasion while not affecting the generation of comet tails. Most interestingly, we observed that in contrast to our expectations, not all of the seven subunits of the Arp2/3 complex are required for Listeria entry into cells or actin tail formation and that the subunit requirements for each of these processes differ, highlighting a previously unsuspected versatility in Arp2/3 complex composition and function. PMID:25991686

  15. Arp2/3 complex-dependent actin networks constrain myosin II function in driving retrograde actin flow.

    PubMed

    Yang, Qing; Zhang, Xiao-Feng; Pollard, Thomas D; Forscher, Paul

    2012-06-25

    The Arp2/3 complex nucleates actin filaments to generate networks at the leading edge of motile cells. Nonmuscle myosin II produces contractile forces involved in driving actin network translocation. We inhibited the Arp2/3 complex and/or myosin II with small molecules to investigate their respective functions in neuronal growth cone actin dynamics. Inhibition of the Arp2/3 complex with CK666 reduced barbed end actin assembly site density at the leading edge, disrupted actin veils, and resulted in veil retraction. Strikingly, retrograde actin flow rates increased with Arp2/3 complex inhibition; however, when myosin II activity was blocked, Arp2/3 complex inhibition now resulted in slowing of retrograde actin flow and veils no longer retracted. Retrograde flow rate increases induced by Arp2/3 complex inhibition were independent of Rho kinase activity. These results provide evidence that, although the Arp2/3 complex and myosin II are spatially segregated, actin networks assembled by the Arp2/3 complex can restrict myosin II-dependent contractility with consequent effects on growth cone motility. PMID:22711700

  16. Electrostatic Interactions Between the Bni1p Formin FH2 Domain and Actin Influence Actin Filament Nucleation

    PubMed Central

    Baker, Joseph L.; Courtemanche, Naomi; Parton, Daniel L.; McCullagh, Martin; Pollard, Thomas D.; Voth, Gregory A.

    2014-01-01

    SUMMARY Formins catalyze nucleation and growth of actin filaments. Here we study the structure and interactions of actin with the FH2 domain of budding yeast formin Bni1p. We built an all-atom model of the formin dimer on an Oda actin filament 7-mer and studied structural relaxation and inter-protein interactions by molecular dynamics simulations. These simulations produced a refined model for the FH2 dimer associated with the barbed end of the filament and revealed electrostatic interactions between the formin knob and actin target-binding cleft. Mutations of two formin residues contributing to these interactions (R1423N, K1467L or both) reduced the interaction energies between the proteins, and in coarse-grained simulations the formin lost more inter-protein contacts with an actin dimer than with an actin 7-mer. Biochemical experiments confirmed a strong influence of these mutations on Bni1p-mediated actin filament nucleation, but not elongation, suggesting that different interactions contribute to these two functions of formins. PMID:25482541

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

    SciTech Connect

    Daniel Szymanski

    2012-10-23

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

  18. Tobacco Arp3 is localized to actin-nucleating sites in vivo

    PubMed Central

    Maisch, Jan; Fišerová, Jindřiška; Fischer, Lukáš; Nick, Peter

    2009-01-01

    The polarity of actin is a central determinant of intracellular transport in plant cells. To visualize actin polarity in living plant cells, the tobacco homologue of the actin-related protein 3 (ARP3) was cloned and a fusion with the red fluorescent protein (RFP) was generated. Upon transient expression of these fusions in the tobacco cell line BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2), punctate structures were observed near the nuclear envelope and in the cortical plasma. These dots could be shown to decorate actin filaments by expressing RFP–ARP3 in a marker line, where actin was tagged by GFP (green fluorescent protein)–FABD (fimbrin actin-binding domain 2). When actin filaments were disrupted by latrunculin B or by prolonged cold treatment, and subsequently allowed to recover, the actin filaments reformed from the RFP–ARP3 structures, that therefore represented actin nucleation sites. The intracellular distribution of these sites was followed during the formation of pluricellular files, and it was observed that the density of RFP–ARP3 increased in the apex of the polarized, terminal cells of a file, whereas it was equally distributed in the central cells of a file. These findings are interpreted in terms of position-dependent differences of actin organization. PMID:19129161

  19. Mechanism of actin filament nucleation by the bacterial effector VopL

    SciTech Connect

    Yu, Bingke; Cheng, Hui-Chun; Brautigam, Chad A.; Tomchick, Diana R.; Rosen, Michael K.

    2012-05-02

    Vibrio parahaemolyticus protein L (VopL) is an actin nucleation factor that induces stress fibers when injected into eukaryotic host cells. VopL contains three N-terminal Wiskott-Aldrich homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). We describe crystallographic and biochemical analyses of filament nucleation by VopL. The WH2 element of VopL does not nucleate on its own and requires the VCD for activity. The VCD forms a U-shaped dimer in the crystal, stabilized by a terminal coiled coil. Dimerization of the WH2 motifs contributes strongly to nucleation activity, as do contacts of the VCD to actin. Our data lead to a model in which VopL stabilizes primarily lateral (short-pitch) contacts between actin monomers to create the base of a two-stranded filament. Stabilization of lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.

  20. A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast

    PubMed Central

    Dudin, Omaya; Bendezú, Felipe O.; Groux, Raphael; Laroche, Thierry; Seitz, Arne

    2015-01-01

    Cell–cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure—the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion. PMID:25825517

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

    PubMed Central

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

    2015-01-01

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

  2. Formin 1 Regulates Ectoplasmic Specialization in the Rat Testis Through Its Actin Nucleation and Bundling Activity.

    PubMed

    Li, Nan; Mruk, Dolores D; Wong, Chris K C; Han, Daishu; Lee, Will M; Cheng, C Yan

    2015-08-01

    During spermatogenesis, developing spermatids and preleptotene spermatocytes are transported across the adluminal compartment and the blood-testis barrier (BTB), respectively, so that spermatids line up near the luminal edge to prepare for spermiation, whereas preleptotene spermatocytes enter the adluminal compartment to differentiate into late spermatocytes to prepare for meiosis I/II. These cellular events involve actin microfilament reorganization at the testis-specific, actin-rich Sertoli-spermatid and Sertoli-Sertoli cell junction called apical and basal ectoplasmic specialization (ES). Formin 1, an actin nucleation protein known to promote actin microfilament elongation and bundling, was expressed at the apical ES but limited to stage VII of the epithelial cycle, whereas its expression at the basal ES/BTB stretched from stage III to stage VI, diminished in stage VII, and was undetectable in stage VIII tubules. Using an in vitro model of studying Sertoli cell BTB function by RNA interference and biochemical assays to monitor actin bundling and polymerization activity, a knockdown of formin 1 in Sertoli cells by approximately 70% impeded the tight junction-permeability function. This disruptive effect on the tight junction barrier was mediated by a loss of actin microfilament bundling and actin polymerization capability mediated by changes in the localization of branched actin-inducing protein Arp3 (actin-related protein 3), and actin bundling proteins Eps8 (epidermal growth factor receptor pathway substrate 8) and palladin, thereby disrupting cell adhesion. Formin 1 knockdown in vivo was found to impede spermatid adhesion, transport, and polarity, causing defects in spermiation in which elongated spermatids remained embedded into the epithelium in stage IX tubules, mediated by changes in the spatiotemporal expression of Arp3, Eps8, and palladin. In summary, formin 1 is a regulator of ES dynamics. PMID:25901598

  3. 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

  4. Crystal structure of a nuclear actin ternary complex.

    PubMed

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

    2016-08-01

    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

  5. Plasma membrane-associated SCAR complex subunits promote cortical F-actin accumulation and normal growth characteristics in Arabidopsis roots

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ARP2/3 complex, a highly conserved nucleator of F-actin polymerization, and its activator, the SCAR complex, have been shown to play important roles in leaf epidermal cell morphogenesis in Arabidopsis. However, the intracellular site(s) and function(s) of SCAR complex and ARP2/3 complex-depende...

  6. An Arp2/3 nucleated F-actin shell fragments nuclear membranes at nuclear envelope breakdown in starfish oocytes.

    PubMed

    Mori, Masashi; Somogyi, Kálmán; Kondo, Hiroshi; Monnier, Nilah; Falk, Henning J; Machado, Pedro; Bathe, Mark; Nédélec, François; Lénárt, Péter

    2014-06-16

    Animal cells disassemble and reassemble their nuclear envelopes (NEs) upon each division. Nuclear envelope breakdown (NEBD) serves as a major regulatory mechanism by which mixing of cytoplasmic and nuclear compartments drives the complete reorganization of cellular architecture, committing the cell for division. Breakdown is initiated by phosphorylation-driven partial disassembly of the nuclear pore complexes (NPCs), increasing their permeability but leaving the overall NE structure intact. Subsequently, the NE is rapidly broken into membrane fragments, defining the transition from prophase to prometaphase and resulting in complete mixing of cyto- and nucleoplasm. However, the mechanism underlying this rapid NE fragmentation remains largely unknown. Here, we show that NE fragmentation during NEBD in starfish oocytes is driven by an Arp2/3 complex-nucleated F-actin "shell" that transiently polymerizes on the inner surface of the NE. Blocking the formation of this F-actin shell prevents membrane fragmentation and delays entry of large cytoplasmic molecules into the nucleus. We observe spike-like protrusions extending from the F-actin shell that appear to "pierce" the NE during the fragmentation process. Finally, we show that NE fragmentation is essential for successful reproduction, because blocking this process in meiosis leads to formation of aneuploid eggs. PMID:24909322

  7. The WAVE Regulatory Complex Links Diverse Receptors to the Actin Cytoskeleton

    PubMed Central

    Chen, Baoyu; Chen, Zhucheng; Brinkmann, Klaus; Pak, Chi W.; Liao, Yuxing; Shi, Shuoyong; Henry, Lisa; Grishin, Nick V.; Bogdan, Sven; Rosen, Michael K.

    2014-01-01

    SUMMARY The WAVE regulatory complex (WRC) controls actin cytoskeletal dynamics throughout the cell by stimulating the actin nucleating activity of the Arp2/3 complex at distinct membrane sites. However, the factors that recruit the WRC to specific locations remain poorly understood. Here we have identified a large family of potential WRC ligands, consisting of ~120 diverse membrane proteins including protocadherins, ROBOs, netrin receptors, Neuroligins, GPCRs and channels. Structural, biochemical and cellular studies reveal that a novel sequence motif that defines these ligands binds to a highly conserved interaction surface of the WRC formed by the Sra and Abi subunits. Mutating this binding surface in flies resulted in defects in actin cytoskeletal organization and egg morphology during oogenesis, leading to female sterility. Our findings directly link diverse membrane proteins to the WRC and actin cytoskeleton, and have broad physiological and pathological ramifications in metazoans. PMID:24439376

  8. Ligand-induced activation of a formin–NPF pair leads to collaborative actin nucleation

    PubMed Central

    Graziano, Brian R.; Jonasson, Erin M.; Pullen, Jessica G.; Gould, Christopher J.

    2013-01-01

    Formins associate with other nucleators and nucleation-promoting factors (NPFs) to stimulate collaborative actin assembly, but the mechanisms regulating these interactions have been unclear. Yeast Bud6 has an established role as an NPF for the formin Bni1, but whether it also directly regulates the formin Bnr1 has remained enigmatic. In this paper, we analyzed NPF-impaired alleles of bud6 in a bni1Δ background and found that Bud6 stimulated Bnr1 activity in vivo. Furthermore, Bud6 bound directly to Bnr1, but its NPF effects were masked by a short regulatory sequence, suggesting that additional factors may be required for activation. We isolated a novel in vivo binding partner of Bud6, Yor304c-a/Bil1, which colocalized with Bud6 and functioned in the Bnr1 pathway for actin assembly. Purified Bil1 bound to the regulatory sequence in Bud6 and triggered NPF effects on Bnr1. These observations define a new mode of formin regulation, which has important implications for understanding NPF-nucleator pairs in diverse systems. PMID:23671312

  9. Neutrophils establish rapid and robust WAVE complex polarity in an actin-dependent fashion

    PubMed Central

    Millius, Arthur; Dandekar, Sheel N.; Houk, Andrew R.; Weiner, Orion D.

    2009-01-01

    Asymmetric intracellular signals enable cells to migrate in response to external cues. The multiprotein WAVE (SCAR/WASF) complex activates the actin-nucleating Arp2/3 complex [1-4] and localizes to propagating “waves”, which direct actin assembly during neutrophil migration [5, 6]. Here, we observe similar WAVE complex dynamics in other mammalian cells and analyze WAVE complex dynamics during the establishment of neutrophil polarity. Earlier models proposed that either spatially-biased generation [7] or selection of protrusions [8] enables chemotaxis. These models require existing morphological polarity to control protrusions. Similar spatially-biased generation and selection of WAVE complex recruitment occur in morphologically unpolarized neutrophils during the development of their first protrusions. Additionally, several mechanisms limit WAVE complex recruitment during polarization and movement: intrinsic cues restrict WAVE complex distribution during the establishment of polarity, and asymmetric intracellular signals constrain WAVE complex distribution in morphologically polarized cells. External gradients can overcome both intrinsic biases and control WAVE complex localization. Following latrunculin-mediated inhibition of actin polymerization, addition and removal of agonist gradients globally recruits and releases the WAVE complex from the membrane. Under these conditions the WAVE complex no longer polarizes, despite the presence of strong external gradients. Thus, actin polymer and the WAVE complex reciprocally interact during polarization. PMID:19200726

  10. A complex gene superfamily encodes actin in petunia.

    PubMed Central

    Baird, W V; Meagher, R B

    1987-01-01

    We have shown by several independent criteria that actin is encoded by a very large and complex superfamily of genes in Petunia. Several cDNA and genomic probes encoding actins from diverse organisms (Dictyostelium, Drosophila, chicken and soybean) hybridize to hundreds of restriction fragments in the petunia genome. Actin-hybridizing sequences were isolated from a petunia genomic library at a rate of at least 200 per genome equivalent. Twenty randomly selected actin-hybridizing clones were characterized in more detail. DNA sequence data from four representative and highly divergent clones, PAc2, PAc3, PAc4 and PAc7, demonstrate that these actin-like sequences are related to functional actin genes. Intron positions typical of other known plant actin genes are conserved in these clones. Four of six clones analyzed (PAc1, PAc2, PAc3, PAc4) hybridize to leaf mRNA of the same size (1.7 kb) as that reported for other plant actin mRNAs and to a slightly smaller mRNA species (1.5 kb). Five distinct subfamilies of actin-related genes were characterized which varied in size from a few members to several dozen members. It is clear from our data that other actin gene subfamilies must also exist within the genome. Possible mechanisms of actin gene amplification and genome turnover are discussed. Images Fig. 1. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3428258

  11. Structure of the F-actin-tropomyosin complex.

    PubMed

    von der Ecken, Julian; Müller, Mirco; Lehman, William; Manstein, Dietmar J; Penczek, Pawel A; Raunser, Stefan

    2015-03-01

    Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg(2+) and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted

  12. Multilayer defects nucleated by substrate pits: a comparison of actinic inspection and non-actinic inspection techniques

    SciTech Connect

    Barty, A; Goldberg, K; Kearney, P; Rekawa, S; LaFontaine, B; Wood, O; Taylor, J S; Han, H

    2006-10-02

    The production of defect-free mask blanks remains a key challenge for EUV lithography. Mask-blank inspection tools must be able to accurately detect all critical defects while simultaneously having the minimum possible false-positive detection rate. We have recently observed and here report the identification of bump-type buried substrate defects, that were below the detection limit of a non-actinic (i.e. non-EUV) in inspection tool. Presently, the occurrence inspection of pit-type defects, their printability, and their detectability with actinic techniques and non-actinic commercial tools, has become a significant concern. We believe that the most successful strategy for the development of effective non-actinic mask inspection tools will involve the careful cross-correlation with actinic inspection and lithographic printing. In this way, the true efficacy of prototype inspection tools now under development can be studied quantitatively against relevant benchmarks. To this end we have developed a dual-mode actinic mask inspection system capable of scanning mask blanks for defects (with simultaneous EUV bright-field and dark-field detection) and imaging those same defects with a zoneplate microscope that matches or exceeds the resolution of EUV steppers.

  13. CDK5RAP2 stimulates microtubule nucleation by the gamma-tubulin ring complex.

    PubMed

    Choi, Yuk-Kwan; Liu, Pengfei; Sze, Siu Kwan; Dai, Chao; Qi, Robert Z

    2010-12-13

    CDK5RAP2 is a human microcephaly protein that contains a γ-tubulin complex (γ-TuC)-binding domain conserved in Drosophila melanogaster centrosomin and Schizosaccharomyces pombe Mto1p and Pcp1p, which are γ-TuC-tethering proteins. In this study, we show that this domain within CDK5RAP2 associates with the γ-tubulin ring complex (γ-TuRC) to stimulate its microtubule-nucleating activity and is therefore referred to as the γ-TuRC-mediated nucleation activator (γ-TuNA). γ-TuNA but not its γ-TuC-binding-deficient mutant stimulates microtubule nucleation by purified γ-TuRC in vitro and induces extensive, γ-TuRC-dependent nucleation of microtubules in a microtubule regrowth assay. γ-TuRC bound to γ-TuNA contains NME7, FAM128A/B, and actin in addition to γ-tubulin and GCP2-6. RNA interference-mediated depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, although γ-TuRC assembly is unaffected. Collectively, these results suggest that the γ-TuNA found in CDK5RAP2 has regulatory functions in γ-TuRC-mediated microtubule nucleation. PMID:21135143

  14. WASH complex regulates Arp2/3 complex for actin-based polar body extrusion in mouse oocytes

    PubMed Central

    Wang, Fei; Zhang, Liang; Zhang, Guang-Li; Wang, Zhen-Bo; Cui, Xiang-Shun; Kim, Nam-Hyung; Sun, Shao-Chen

    2014-01-01

    Prior to their fertilization, oocytes undergo asymmetric division, which is regulated by actin filaments. Recently, WASH complex were identified as actin nucleation promoting factors (NPF) that activated Arp2/3 complex. However, the roles of WASH complex remain uncertain, particularly for oocyte polarization and asymmetric division. Here, we examined the functions of two important subunits of a WASH complex, WASH1 and Strumpellin, during mouse oocyte meiosis. Depleting WASH1 or disrupting Strumpellin activity by WASH1 morpholino (MO) injection or Strumpellin antibody injection decreased polar body extrusion and caused oocyte symmetric division, and this may have been due to spindle formation and migration defects. Time lapse microscopy showed that actin filaments distribution and relative amount at the membrane and in the cytoplasm of oocytes was significantly decreased after disrupting WASH complex. In addition, Arp2/3 complex expression was reduced after WASH1 depletion. Thus, our data indicated that WASH complex regulated Arp2/3 complex and were required for cytokinesis and following polar body extrusion during mouse oocyte meiotic maturation. PMID:24998208

  15. A Rickettsia WASP-like protein activates the Arp2/3 complex and mediates actin-based motility.

    PubMed

    Jeng, Robert L; Goley, Erin D; D'Alessio, Joseph A; Chaga, Oleg Y; Svitkina, Tatyana M; Borisy, Gary G; Heinzen, Robert A; Welch, Matthew D

    2004-08-01

    Spotted fever group Rickettsia are obligate intracellular pathogens that exploit the host cell actin cytoskeleton to promote motility and cell-to-cell spread. Although other pathogens such as Listeria monocytogenes use an Arp2/3 complex-dependent nucleation mechanism to generate comet tails consisting of Y-branched filament arrays, Rickettsia polymerize tails consisting of unbranched filaments by a previously unknown mechanism. We identified genes in several Rickettsia species encoding proteins (termed RickA) with similarity to the WASP family of Arp2/3-complex activators. Rickettsia rickettsii RickA activated both the nucleation and Y-branching activities of the Arp2/3 complex like other WASP-family proteins, and was sufficient to direct the motility of microscopic beads in cell extracts. Actin tails generated by RickA-coated beads consisted of Y-branched filament networks. These data suggest that Rickettsia use an Arp2/3 complex-dependent actin-nucleation mechanism similar to that of other pathogens. We propose that additional Rickettsia or host factors reorganize the Y-branched networks into parallel arrays in a manner similar to a recently proposed model of filopodia formation. PMID:15236643

  16. Arp2/3 complex ATP hydrolysis promotes lamellipodial actin network disassembly but is dispensable for assembly

    PubMed Central

    Ingerman, Elena; Hsiao, Jennifer Ying

    2013-01-01

    We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits expanded and delayed disassembly of lamellipodial actin networks and the effect of mutant subunits was additive. Arp2 and Arp3 ATP hydrolysis mutants remained in lamellipodial networks longer and traveled greater distances from the plasma membrane, even in networks still containing wild-type Arp2/3 complex. In vitro, wild-type and ATP hydrolysis mutant Arp2/3 complexes each nucleated actin and built similar dendritic networks. However, networks constructed with Arp2/3 hydrolysis-defective mutants were more resistant to disassembly by cofilin. Our results indicate that ATP hydrolysis on both Arp2 and Arp3 contributes to dissociation of the complex from the actin network but is not strictly necessary for lamellipodial network disassembly. PMID:23439681

  17. Broadening the Spectrum of Actin-Based Protrusive Activity Mediated by Arp2/3 Complex-Facilitated Polymerization: Motility of Cytoplasmic Ridges and Tubular Projections

    PubMed Central

    Henson, John H.; Gianakas, Anastasia D.; Henson, Lauren H.; Lakin, Christina L.; Voss, Meagen K.; Bewersdorf, Joerg; Oldenbourg, Rudolf; Morris, Robert L.

    2014-01-01

    Arp2/3 complex-facilitated actin polymerization plays an essential role in a variety of cellular functions including motility, adherence, endocytosis and trafficking. In the present study we employ the sea urchin coelomocyte experimental model system to test the hypotheses that Arp2/3 complex-nucleated actin assembly mediates the motility of two unusual cellular protrusions; the cytoplasmic ridges present during coelomocyte spreading, and inducible, tubular-shaped, and neurite-like projections. Our investigations couple pharmacological manipulation employing inhibitors of actin polymerization and the Arp2/3 complex with a wide array of imaging methods including digitally enhanced phase contrast, DIC and polarization light microscopy of live cells; conventional, confocal and super-resolution light microscopy of fluorescently labeled cells; and scanning and transmission electron microscopy. Taken together, the results of this study indicate that Arp2/3 complex-facilitated actin polymerization underlies the motility of coelomocyte cytoplasmic ridges and tubular projections, that these processes are related to each other, and that they have been preliminarily identified in other cell types. The results also highlight the broad spectrum of actin-based protrusive activities dependent on the Arp2/3 complex and provide additional insights into the pervasive nature of this ubiquitous actin nucleator. Furthermore we provide the first evidence of a possible mechanistic difference between the impacts of the small molecule drugs BDM and CK666 on the Arp2/3 complex. PMID:25111797

  18. Signalling to actin assembly via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3 complex.

    PubMed Central

    Millard, Thomas H; Sharp, Stewart J; Machesky, Laura M

    2004-01-01

    The assembly of a branched network of actin filaments provides the mechanical propulsion that drives a range of dynamic cellular processes, including cell motility. The Arp2/3 complex is a crucial component of such filament networks. Arp2/3 nucleates new actin filaments while bound to existing filaments, thus creating a branched network. In recent years, a number of proteins that activate the filament nucleation activity of Arp2/3 have been identified, most notably the WASP (Wiskott-Aldrich syndrome protein) family. WASP-family proteins activate the Arp2/3 complex, and consequently stimulate actin assembly, in response to extracellular signals. Structural studies have provided a significant refinement in our understanding of the molecular detail of how the Arp2/3 complex nucleates actin filaments. There has also been much progress towards an understanding of the complicated signalling processes that regulate WASP-family proteins. In addition, the use of gene disruption in a number of organisms has led to new insights into the specific functions of individual WASP-family members. The present review will discuss the Arp2/3 complex and its regulators, in particular the WASP-family proteins. Emphasis will be placed on recent developments in the field that have furthered our understanding of actin dynamics and cell motility. PMID:15040784

  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. Insights into the Influence of Nucleotides on Actin Family Proteins from Seven Structures of Arp2/3 Complex

    SciTech Connect

    Nolen,B.; Pollard, T.

    2007-01-01

    ATP is required for nucleation of actin filament branches by Arp2/3 complex, but the influence of ATP binding and hydrolysis are poorly understood. We determined crystal structures of bovine Arp2/3 complex cocrystalized with various bound adenine nucleotides and cations. Nucleotide binding favors closure of the nucleotide binding cleft of Arp3, but no large scale conformational changes in the complex. Thus, ATP binding does not directly activate Arp2/3 complex, but is part of a network of interactions that contribute to nucleation. We compared nucleotide-induced conformational changes of residues lining the cleft in Arp3 and actin structures to construct a movie depicting the proposed ATPase cycle for the actin family. Chemical crosslinking stabilized subdomain 1 of Arp2, revealing new electron density for 69 residues in this subdomain. Steric clashes with Arp3 appear to be responsible for intrinsic disorder of subdomains 1 and 2 of Arp2 in inactive Arp2/3 complex.

  1. Capping of the barbed ends of actin filaments by a high-affinity profilin-actin complex.

    PubMed

    DiNubile, M J; Huang, S

    1997-01-01

    Profilin, a ubiquitous 12 to 15-kDa protein, serves many functions, including sequestering monomeric actin, accelerating nucleotide exchange on actin monomers, decreasing the critical concentration of the barbed end of actin filaments, and promoting actin polymerization when barbed ends are free. Most previous studies have focused on profilin itself rather than its complex with actin. A high-affinity profilin-actin complex (here called profilactin) can be isolated from a poly-(L)-proline (PLP) column by sequential elution with 3 M and 7 M urea. Profilactin inhibited the elongation rate of pyrenyl-G-actin from filament seeds in a concentration- and time-dependent manner. Much greater inhibition of elongation was observed with spectrin-F-actin than gelsolin-F-actin seeds, suggesting that the major effect of profilactin was due to capping the barbed ends of actin filaments. Its dissociation constant for binding to filament ends was 0.3 microM; the on- and off-rate constants were estimated to be 1.7 x 10(3) M-1 s-1 and 4.5 x 10(-4) s-1, respectively. Purified profilin (obtained by repetitive applications to a PLP column and assessed by silver-stained polyacylamide gels) did not slow the elongation rate of pyrenyl-G-actin from filament seeds. Capping protein could not be detected by Western blotting in the profilactin preparation, but low concentrations of gelsolin did contaminate our preparation. However, prolonged incubation with either calcium or EGTA did not affect capping activity, implying that contaminating gelsolin-actin complexes were not primarily responsible for the observed capping activity. Reapplication of the profilactin preparation to PLP-coupled Sepharose removed both profilin and actin and concurrently eliminated its capping activity. Profilactin that was reapplied to uncoupled Sepharose retained its capping activity. Phosphatidylinositol-4,5-bisphosphate (PIP2) was the most potent phosphoinositol in reducing the capping activity of profilactin

  2. Arp2/3 complex-deficient mouse fibroblasts are viable and have normal leading-edge actin structure and function

    PubMed Central

    Di Nardo, Alessia; Cicchetti, Gregor; Falet, Hervé; Hartwig, John H.; Stossel, Thomas P.; Kwiatkowski, David J.

    2005-01-01

    RNA interference silencing of up to 90% of Arp3 protein expression, a major subunit of the Arp2/3 complex, proportionately decreases the intracellular motility of Listeria monocytogenes and actin nucleation activity ascribable to the Arp2/3 complex in mouse embryonic fibroblasts. However, the Arp2/3-deficient cells exhibit unimpaired lamellipodial actin network structure, translational locomotion, spreading, actin assembly, and ruffling responses. In addition, Arp3-silenced cells expressing neural Wiskott-Aldrich syndrome protein-derived peptides that inhibit Arp2/3 complex function in wild-type cells retained normal PDGF-induced ruffling. The Arp2/3 complex can be dispensable for leading-edge actin remodeling. PMID:16254049

  3. Immersion freezing of ice nucleation active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Wex, H.; Šantl-Temkiv, T.; Voigtländer, J.; Niedermeier, D.; Stratmann, F.

    2013-06-01

    Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS), the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between -5 °C to -38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA) bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about -6 °C to about -10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei) which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a) the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b) the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice nucleation are attached

  4. Immersion freezing of ice nucleating active protein complexes

    NASA Astrophysics Data System (ADS)

    Hartmann, S.; Augustin, S.; Clauss, T.; Voigtländer, J.; Niedermeier, D.; Wex, H.; Stratmann, F.

    2012-08-01

    Biological particles, e.g. bacteria and their Ice Nucleating Active (INA) protein complexes, might play an important role for the ice formation in atmospheric mixed-phase clouds. Therefore, the immersion freezing behavior of INA protein complexes generated from a SnomaxTM solution/suspension was investigated as function of temperature in a range of -5 °C to -38 °C at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). The immersion freezing of droplets containing small numbers of INA protein complexes occurs in a temperature range of -7 °C and -10 °C. The experiments performed in the lower temperature range, where all droplets freeze which contain at least one INA protein complex, are used to determine the average number of INA protein complexes present, assuming that the INA protein complexes are Poisson distributed over the droplet ensemble. Knowing the average number of INA protein complexes, the heterogeneous ice nucleation rate and rate coefficient of a single INA protein complex is determined by using the newly-developed CHESS model (stoCHastic model of idEntical poiSSon distributed ice nuclei). Therefore, we assume the ice nucleation process to be of stochastic nature, and a parameterization of the INA protein complex's nucleation rate. Analyzing the results of immersion freezing experiments from literature (SnomaxTM and Pseudomonas syringae bacteria), to results gained in this study, demonstrates that first, a similar temperature dependence of the heterogeneous ice nucleation rate for a single INA protein complex was found in all experiments, second, the shift of the ice fraction curves to higher temperatures can be explained consistently by a higher average number of INA protein complexes being present in the droplet ensemble, and finally the heterogeneous ice nucleation rate of one single INA protein complex might be also applicable for intact Pseudomonas syringae bacteria cells. The results obtained in this study allow a new perspective on the

  5. Atomic structure of the actin: DNase I complex

    NASA Astrophysics Data System (ADS)

    Kabsch, Wolfgang; Mannherz, Hans Georg; Suck, Dietrich; Pai, Emil F.; Holmes, Kenneth C.

    1990-09-01

    The atomic models of the complex between rabbit skeletal muscle actin and bovine pancreatic deoxyribonuclease I both in the ATP and ADP forms have been determined byo X-ray analysis at an effective resolution of 2.8 Å and 3 Å, respectively. The two structures are very similar. The actin molecule consists of two domains which can be further subdivided into two subdomains. ADP or ATP is located in the cleft between the domains with a calcium ion bound to the β- or β- and γ-phosphates, respectively. The motif of a five-stranded (3 sheet consisting of a (3 meander and a right handed βαβ unit appears in each domain suggesting that gene duplication might have occurred. These sheets have the same topology as that found in hexokinase.

  6. Actin related protein complex subunit 1b controls sperm release, barrier integrity and cell division during adult rat spermatogenesis.

    PubMed

    Kumar, Anita; Dumasia, Kushaan; Deshpande, Sharvari; Gaonkar, Reshma; Balasinor, N H

    2016-08-01

    Actin remodeling is a vital process for signaling, movement and survival in all cells. In the testes, extensive actin reorganization occurs at spermatid-Sertoli cell junctions during sperm release (spermiation) and at inter Sertoli cell junctions during restructuring of the blood testis barrier (BTB). During spermiation, tubulobulbar complexes (TBCs), rich in branched actin networks, ensure recycling of spermatid-Sertoli cell junctional molecules. Similar recycling occurs during BTB restructuring around the same time as spermiation occurs. Actin related protein 2/3 complex is an essential actin nucleation and branching protein. One of its subunits, Arpc1b, was earlier found to be down-regulated in an estrogen-induced rat model of spermiation failure. Also, Arpc1b was found to be estrogen responsive through estrogen receptor beta in seminiferous tubule culture. Here, knockdown of Arpc1b by siRNA in adult rat testis led to defects in spermiation caused by failure in TBC formation. Knockdown also compromised BTB integrity and caused polarity defects of mature spermatids. Apart from these effects pertaining to Sertoli cells, Arpc1b reduction perturbed ability of germ cells to enter G2/M phase thus hindering cell division. In summary, Arpc1b, an estrogen responsive gene, is a regulator of spermiation, mature spermatid polarity, BTB integrity and cell division during adult spermatogenesis. PMID:27113856

  7. Assaying microtubule nucleation by the γ-tubulin ring complex.

    PubMed

    Choi, Yuk-Kwan; Qi, Robert Z

    2014-01-01

    Microtubule organization by microtubule-organizing centers such as the centrosome requires γ-tubulin, which exists in the γ-tubulin ring complex (γTuRC) that nucleates microtubules. The γTuRC is a ring-shaped, macromolecular complex whose core components are γ-tubulin and the γ-tubulin complex proteins. Despite the recent identification of additional γTuRC components, the molecular composition and regulatory properties of the complex remain poorly understood. The ability to purify the γTuRC at a large scale for characterization may hold a key to understanding the mechanism by which the γTuRC nucleates microtubules. In this chapter, we describe methods to isolate the γTuRC from human cell cultures and to perform assays on the purified γTuRC. PMID:24630104

  8. Stability of actin-lysozyme complexes formed in cystic fibrosis disease.

    PubMed

    Mohammadinejad, Sarah; Ghamkhari, Behnoush; Abdolmaleki, Sarah

    2016-08-21

    Finding the conditions for destabilizing actin-lysozyme complexes is of biomedical importance in preventing infections in cystic fibrosis. In this manuscript, the effects of different charge-mutants of lysozyme and salt concentration on the stability of actin-lysozyme complexes are studied using Langevin dynamics simulation. A coarse-grained model of F-actin is used in which both its twist and bending rigidities are considered. We observe that the attraction between F-actins is stronger in the presence of wild-type lysozymes relative to the mutated lysozymes of lower charges. By calculating the potential of mean force between F-actins, we conclude that the stability of actin-lysozyme complexes is decreased by reducing the charge of lysozyme mutants. The distributions of different lysozyme charge-mutants show that wild-type (+9e) lysozymes are mostly accumulated in the center of triangles formed by three adjacent F-actins, while lysozyme mutants of charges +7e and +5e occupy the bridging regions between F-actins. Low-charge mutants of lysozyme (+3e) distribute uniformly around F-actins. A rough estimate of the electrostatic energy for these different distributions proves that the distribution in which lysozymes reside in the center of triangles leads to more stable complexes. Also our results in the presence of a salt suggest that at physiological salt concentration of airway, F-actin complexes are not formed by charge-reduced mutants of lysozyme. The findings are interesting because if we can design charge-reduced lysozyme mutants with considerable antibacterial activity, they are not sequestered inside F-actin aggregates and can play their role as antibacterial agents against airway infection. PMID:27436705

  9. MICAL-Family Proteins: Complex Regulators of the Actin Cytoskeleton

    PubMed Central

    Giridharan, Sai Srinivas Panapakkam

    2014-01-01

    Abstract Significance: The molecules interacting with CasL (MICAL) family members participate in a multitude of activities, including axonal growth cone repulsion, membrane trafficking, apoptosis, and bristle development in flies. An interesting feature of MICAL proteins is the presence of an N-terminal flavo-mono-oxygenase domain. This mono-oxygenase domain generates redox potential with which MICALs can either oxidize proteins or produce reactive oxygen species (ROS). Actin is one such protein that is affected by MICAL function, leading to dramatic cytoskeletal rearrangements. This review describes the MICAL-family members, and discusses their mechanisms of actin-binding and regulation of actin cytoskeleton organization. Recent Advances: Recent studies show that MICALs directly induce oxidation of actin molecules, leading to actin depolymerization. ROS production by MICALs also causes oxidation of collapsin response mediator protein-2, a microtubule assembly promoter, which subsequently undergoes phosphorylation. Critical Issues: MICAL proteins oxidize proteins through two mechanisms: either directly by oxidizing methionine residues or indirectly via the production of ROS. It remains unclear whether MICAL proteins employ both mechanisms or whether the activity of MICAL-family proteins might vary with different substrates. Future Directions: The identification of additional substrates oxidized by MICAL will shed new light on MICAL protein function. Additional directions include expanding studies toward the MICAL-like homologs that lack flavin adenine dinucleotide domains and oxidation activity. Antioxid. Redox Signal. 20, 2059–2073. PMID:23834433

  10. 25 Years of Tension over Actin Binding to the Cadherin Cell Adhesion Complex: The Devil is in the Details.

    PubMed

    Nelson, W James; Weis, William I

    2016-07-01

    Over the past 25 years, there has been a conceptual (re)evolution in understanding how the cadherin cell adhesion complex, which contains F-actin-binding proteins, binds to the actin cytoskeleton. There is now good synergy between structural, biochemical, and cell biological results that the cadherin-catenin complex binds to F-actin under force. PMID:27166091

  11. Structure and Stability of Self-Assembled Actin-Lysozyme Complexes in Salty Water

    SciTech Connect

    Sanders, Lori K.; Guaqueta, Camilo; Lee, Jae-Wook; Slimmer, Scott C.; Luijten, Erik; Angelini, Thomas E.; Wong, Gerard C.L.

    2005-09-02

    Interactions between actin, an anionic polyelectrolyte, and lysozyme, a cationic globular protein, have been examined using a combination of synchrotron small-angle x-ray scattering and molecular dynamics simulations. Lysozyme initially bridges pairs of actin filaments, which relax into hexagonally coordinated columnar complexes comprised of actin held together by incommensurate one-dimensional close-packed arrays of lysozyme macroions. These complexes are found to be stable even in the presence of significant concentrations of monovalent salt, which is quantitatively explained from a redistribution of salt between the condensed and the aqueous phases.

  12. A Role for Myosin-I in Actin Assembly through Interactions with Vrp1p, Bee1p, and the Arp2/3 Complex

    PubMed Central

    Evangelista, Marie; Klebl, Bert M.; Tong, Amy H.Y.; Webb, Bradley A.; Leeuw, Thomas; Leberer, Ekkehard; Whiteway, Malcolm; Thomas, David Y.; Boone, Charles

    2000-01-01

    Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly. PMID:10648568

  13. 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

  14. Spatial control of actin polymerization during neutrophil chemotaxis

    PubMed Central

    Weiner, Orion D.; Servant, Guy; Welch, Matthew D.; Mitchison, Timothy J.; Sedat, John W.; Bourne, Henry R.

    2010-01-01

    Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients. PMID:10559877

  15. Regulation of the actin cytoskeleton by the Ndel1-Tara complex is critical for cell migration

    PubMed Central

    Hong, Ji-Ho; Kwak, Yongdo; Woo, Youngsik; Park, Cana; Lee, Seol-Ae; Lee, Haeryun; Park, Sung Jin; Suh, Yeongjun; Suh, Bo Kyoung; Goo, Bon Seong; Mun, Dong Jin; Sanada, Kamon; Nguyen, Minh Dang; Park, Sang Ki

    2016-01-01

    Nuclear distribution element-like 1 (Ndel1) plays pivotal roles in diverse biological processes and is implicated in the pathogenesis of multiple neurodevelopmental disorders. Ndel1 function by regulating microtubules and intermediate filaments; however, its functional link with the actin cytoskeleton is largely unknown. Here, we show that Ndel1 interacts with TRIO-associated repeat on actin (Tara), an actin-bundling protein, to regulate cell movement. In vitro wound healing and Boyden chamber assays revealed that Ndel1- or Tara-deficient cells were defective in cell migration. Moreover, Tara overexpression induced the accumulation of Ndel1 at the cell periphery and resulted in prominent co-localization with F-actin. This redistribution of Ndel1 was abolished by deletion of the Ndel1-interacting domain of Tara, suggesting that the altered peripheral localization of Ndel1 requires a physical interaction with Tara. Furthermore, co-expression of Ndel1 and Tara in SH-SY5Y cells caused a synergistic increase in F-actin levels and filopodia formation, suggesting that Tara facilitates cell movement by sequestering Ndel1 at peripheral structures to regulate actin remodeling. Thus, we demonstrated that Ndel1 interacts with Tara to regulate cell movement. These findings reveal a novel role of the Ndel1-Tara complex in actin reorganization during cell movement. PMID:27546710

  16. Regulation of the actin cytoskeleton by the Ndel1-Tara complex is critical for cell migration.

    PubMed

    Hong, Ji-Ho; Kwak, Yongdo; Woo, Youngsik; Park, Cana; Lee, Seol-Ae; Lee, Haeryun; Park, Sung Jin; Suh, Yeongjun; Suh, Bo Kyoung; Goo, Bon Seong; Mun, Dong Jin; Sanada, Kamon; Nguyen, Minh Dang; Park, Sang Ki

    2016-01-01

    Nuclear distribution element-like 1 (Ndel1) plays pivotal roles in diverse biological processes and is implicated in the pathogenesis of multiple neurodevelopmental disorders. Ndel1 function by regulating microtubules and intermediate filaments; however, its functional link with the actin cytoskeleton is largely unknown. Here, we show that Ndel1 interacts with TRIO-associated repeat on actin (Tara), an actin-bundling protein, to regulate cell movement. In vitro wound healing and Boyden chamber assays revealed that Ndel1- or Tara-deficient cells were defective in cell migration. Moreover, Tara overexpression induced the accumulation of Ndel1 at the cell periphery and resulted in prominent co-localization with F-actin. This redistribution of Ndel1 was abolished by deletion of the Ndel1-interacting domain of Tara, suggesting that the altered peripheral localization of Ndel1 requires a physical interaction with Tara. Furthermore, co-expression of Ndel1 and Tara in SH-SY5Y cells caused a synergistic increase in F-actin levels and filopodia formation, suggesting that Tara facilitates cell movement by sequestering Ndel1 at peripheral structures to regulate actin remodeling. Thus, we demonstrated that Ndel1 interacts with Tara to regulate cell movement. These findings reveal a novel role of the Ndel1-Tara complex in actin reorganization during cell movement. PMID:27546710

  17. Neuronal Actin Dynamics, Spine Density and Neuronal Dendritic Complexity Are Regulated by CAP2.

    PubMed

    Kumar, Atul; Paeger, Lars; Kosmas, Kosmas; Kloppenburg, Peter; Noegel, Angelika A; Peche, Vivek S

    2016-01-01

    Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2(gt/gt) mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2(gt/gt) with increased number of excitatory synapses. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2(gt/gt) neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin Ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics. PMID:27507934

  18. TOR complex 2-Ypk1 signaling regulates actin polarization via reactive oxygen species.

    PubMed

    Niles, Brad J; Powers, Ted

    2014-12-01

    The evolutionarily conserved mTOR complex 2 (mTORC2) signaling pathway is an important regulator of actin cytoskeletal architecture and, as such, is a candidate target for preventing cancer cell motility and invasion. Remarkably, the precise mechanism(s) by which mTORC2 regulates the actin cytoskeleton have remained elusive. Here we show that in budding yeast, TORC2 and its downstream kinase Ypk1 regulate actin polarization by controlling reactive oxygen species (ROS) accumulation. Specifically, we find that TORC2-Ypk1 regulates actin polarization both by vacuole-related ROS, controlled by the phospholipid flippase kinase Fpk1 and sphingolipids, and by mitochondria-mediated ROS, controlled by the PKA subunit Tpk3. In addition, we find that the protein kinase C (Pkc1)/MAPK cascade, a well-established regulator of actin, acts downstream of Ypk1 to regulate ROS, in part by promoting degradation of the oxidative stress responsive repressor, cyclin C. Furthermore, we show that Ypk1 regulates Pkc1 activity through proper localization of Rom2 at the plasma membrane, which is also dependent on Fpk1 and sphingolipids. Together these findings demonstrate important links between TORC2/Ypk1 signaling, Fpk1, sphingolipids, Pkc1, and ROS as regulators of actin and suggest that ROS may play an important role in mTORC2-dependent dysregulation of the actin cytoskeleton in cancer cells. PMID:25253719

  19. Neuronal Actin Dynamics, Spine Density and Neuronal Dendritic Complexity Are Regulated by CAP2

    PubMed Central

    Kumar, Atul; Paeger, Lars; Kosmas, Kosmas; Kloppenburg, Peter; Noegel, Angelika A.; Peche, Vivek S.

    2016-01-01

    Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2gt/gt mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2gt/gt with increased number of excitatory synapses. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2gt/gt neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin Ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics. PMID:27507934

  20. 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.

  1. Structural and Biochemical Studies of Actin in Complex with Synthetic Macrolide Tail Analogues

    DOE PAGESBeta

    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 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

  2. Actin-dependent propulsion of endosomes and lysosomes byrecruitment of n-wasp

    SciTech Connect

    Taunton J; Rowning BA; Coughlin ML; Wu M; Moon RT; Mitchison TJ; Larabell CA

    2000-02-07

    We examined the spatial and temporal control of actin assembly in living Xenopus eggs. Within minutes of egg activation,dynamic actin-rich comet tails appeared on a subset of cytoplasmic vesicles that were enriched in protein kinase C (PKC), causing the vesicles to move through the cytoplasm. Actin comet tail formation in vivo was stimulated by the PKC activator phorbol myristate acetate (PMA),and this process could be reconstituted in a cell-free system. We used this system to define the characteristics that distinguish vesicles associated with actin comet tails from other vesicles in the extract. We found that the protein, N-WASP, was recruited to the surface of every vesicle associated with an actin comet tail, suggesting that vesicle movement results from actin assembly nucleated by the Arp2/3 complex, the immediate downstream target of N-WASP, The motile vesicles accumulated the dye acridine orange, a marker for endosomes and lysosomes. Furthermore, vesicles associated with actin comet tails had the morphological features of multivesicular endosomes as revealed by electron microscopy. Endosomes and lysosomes from mammalian cells preferentially nucleated actin assembly and moved in the Xenopus egg extract system. These results define endosomes and lysosomes as recruitment sites for the actin nucleation machinery and demonstrate that actin assembly contributes to organelle movement. Conversely, by nucleating actin assembly, intracellular membranes may contribute to the dynamic organization of the actin cytoskeleton.

  3. Activity of a gelsolin-like actin modulator in rat skeletal muscle under protein catabolic conditions.

    PubMed Central

    D'Haese, J; Rutschmann, M; Dahlmann, B; Hinssen, H

    1987-01-01

    A gelsolin-like actin-modulating protein was isolated from rat skeletal muscle and characterized with respect to its interaction with actin. The protein, with a molecular mass of approx. 85 kDa, forms a stoichiometric complex with two actin molecules and is activated by micromolar concentrations of Ca2+. It effectively severs actin filaments and promotes nucleation of actin polymerization. The activity of this protein is detectable already in crude extracts by its capability to reduce the steady state viscosity of actin. Actin-modulating activities were determined in muscle extracts of rats kept under protein catabolic conditions, i.e. as generated by corticosterone treatment and starvation. In both cases we found a marked increase of modulator activity. The possibility is discussed that the increased activity of actin modulator indicates a fragmentation of actin filaments prior to the proteolytic degradation of actin. Images Fig. 2. PMID:3435453

  4. Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis

    PubMed Central

    Borek, Weronika E.; Groocock, Lynda M.; Samejima, Itaru; Zou, Juan; de Lima Alves, Flavia; Rappsilber, Juri; Sawin, Kenneth E.

    2015-01-01

    Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation ‘switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation. PMID:26243668

  5. Reconstitution of Actin-based Motility by Vasodilator-stimulated Phosphoprotein (VASP) Depends on the Recruitment of F-actin Seeds from the Solution Produced by Cofilin*

    PubMed Central

    Siton, Orit; Bernheim-Groswasser, Anne

    2014-01-01

    Vasodilator-stimulated phosphoprotein (VASP) is active in many filopodium-based and cytoskeleton reorganization processes. It is not fully understood how VASP directly functions in actin-based motility and how regulatory proteins affect its function. Here, we combine bead motility assay and single filament experiments. In the presence of a bundling component, actin bundles that grow from the surface of WT-VASP-coated beads induced movement of the beads. VASP promotes actin-based movement alone, in the absence of other actin nucleators. We propose that at physiological salt conditions VASP nucleation activity is too weak to promote motility and bundle formation. Rather, VASP recruits F-actin seeds from the solution and promotes their elongation. Cofilin has a crucial role in the nucleation of these F-actin seeds, notably under conditions of unfavorable spontaneous actin nucleation. We explored the role of multiple VASP variants. We found that the VASP-F-actin binding domain is required for the recruitment of F-actin seeds from the solution. We also found that the interaction of profilin-actin complexes with the VASP-proline-rich domain and the binding of the VASP-F-actin binding domain to the side of growing filaments is critical for transforming actin polymerization into motion. At the single filament level, profilin mediates both filament elongation rate and VASP anti-capping activity. Binding of profilin-actin complexes increases the polymerization efficiency by VASP but decreases its efficiency as an anti-capper; binding of free profilin creates the opposite effect. Finally, we found that an additional component such as methylcellulose or fascin is required for actin bundle formation and motility mediated by VASP. PMID:25246528

  6. Isoform diversity in the Arp2/3 complex determines actin filament dynamics.

    PubMed

    Abella, Jasmine V G; Galloni, Chiara; Pernier, Julien; Barry, David J; Kjær, Svend; Carlier, Marie-France; Way, Michael

    2016-01-01

    The Arp2/3 complex consists of seven evolutionarily conserved subunits (Arp2, Arp3 and ARPC1-5) and plays an essential role in generating branched actin filament networks during many different cellular processes. In mammals, however, the ARPC1 and ARPC5 subunits are each encoded by two isoforms that are 67% identical. This raises the possibility that Arp2/3 complexes with different properties may exist.  We found that Arp2/3 complexes containing ARPC1B and ARPC5L are significantly better at promoting actin assembly than those with ARPC1A and ARPC5, both in cells and in vitro. Branched actin networks induced by complexes containing ARPC1B or ARPC5L are also disassembled ∼2-fold slower than those formed by their counterparts. This difference reflects the ability of cortactin to stabilize ARPC1B- and ARPC5L- but not ARPC1A- and ARPC5-containing complexes against coronin-mediated disassembly. Our observations demonstrate that the Arp2/3 complex in higher eukaryotes is actually a family of complexes with different properties. PMID:26655834

  7. Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenic Escherichia coli Tir effector.

    PubMed

    Nieto-Pelegrin, Elvira; Kenny, Brendan; Martinez-Quiles, Narcisa

    2014-01-01

    Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area. PMID:25482634

  8. The Association of the Arabidopsis Actin-Related Protein2/3 Complex with Cell Membranes Is Linked to Its Assembly Status But Not Its Activation1[W][OA

    PubMed Central

    Kotchoni, Simeon O.; Zakharova, Taya; Mallery, Eileen L.; Le, Jie; El-Assal, Salah El-Din; Szymanski, Daniel B.

    2009-01-01

    In growing plant cells, the combined activities of the cytoskeleton, endomembrane, and cell wall biosynthetic systems organize the cytoplasm and define the architecture and growth properties of the cell. These biosynthetic machineries efficiently synthesize, deliver, and recycle the raw materials that support cell expansion. The precise roles of the actin cytoskeleton in these processes are unclear. Certainly, bundles of actin filaments position organelles and are a substrate for long-distance intracellular transport, but the functional linkages between dynamic actin filament arrays and the cell growth machinery are poorly understood. The Arabidopsis (Arabidopsis thaliana) “distorted group” mutants have defined protein complexes that appear to generate and convert small GTPase signals into an Actin-Related Protein2/3 (ARP2/3)-dependent actin filament nucleation response. However, direct biochemical knowledge about Arabidopsis ARP2/3 and its cellular distribution is lacking. In this paper, we provide biochemical evidence for a plant ARP2/3. The plant complex utilizes a conserved assembly mechanism. ARPC4 is the most critical core subunit that controls the assembly and steady-state levels of the complex. ARP2/3 in other systems is believed to be mostly a soluble complex that is locally recruited and activated. Unexpectedly, we find that Arabidopsis ARP2/3 interacts strongly with cell membranes. Membrane binding is linked to complex assembly status and not to the extent to which it is activated. Mutant analyses implicate ARP2 as an important subunit for membrane association. PMID:19801398

  9. Actin kinetics shapes cortical network structure and mechanics

    PubMed Central

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

    2016-01-01

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

  10. Stabilization of actin bundles by a dynamin 1/cortactin ring complex is necessary for growth cone filopodia.

    PubMed

    Yamada, Hiroshi; Abe, Tadashi; Satoh, Ayano; Okazaki, Nana; Tago, Shota; Kobayashi, Kinue; Yoshida, Yumi; Oda, Yoshiya; Watanabe, Masami; Tomizawa, Kazuhito; Matsui, Hideki; Takei, Kohji

    2013-03-01

    Dynamin GTPase, a key molecule in endocytosis, mechanically severs the invaginated membrane upon GTP hydrolysis. Dynamin functions also in regulating actin cytoskeleton, but the mechanisms are yet to be defined. Here we show that dynamin 1, a neuronal isoform of dynamin, and cortactin form ring complexes, which twine around F-actin bundles and stabilize them. By negative-staining EM, dynamin 1-cortactin complexes appeared as "open" or "closed" rings depending on guanine nucleotide conditions. By pyrene actin assembly assay, dynamin 1 stimulated actin assembly in mouse brain cytosol. In vitro incubation of F-actin with both dynamin 1 and cortactin led to the formation of long and thick actin bundles, on which dynamin 1 and cortactin were periodically colocalized in puncta. A depolymerization assay revealed that dynamin 1 and cortactin increased the stability of actin bundles, most prominently in the presence of GTP. In rat cortical neurons and human neuroblastoma cell line, SH-SY5Y, both dynamin 1 and cortactin localized on actin filaments and the bundles at growth cone filopodia as revealed by immunoelectron microscopy. In SH-SY5Y cell, acute inhibition of dynamin 1 by application of dynamin inhibitor led to growth cone collapse. Cortactin knockdown also reduced growth cone filopodia. Together, our results strongly suggest that dynamin 1 and cortactin ring complex mechanically stabilizes F-actin bundles in growth cone filopodia. Thus, the GTPase-dependent mechanochemical enzyme property of dynamin is commonly used both in endocytosis and regulation of F-actin bundles by a dynamin 1-cortactin complex. PMID:23467367

  11. Three-dimensional structure of the complex of actin and DNase I at 4.5 A resolution.

    PubMed Central

    Kabsch, W; Mannherz, H G; Suck, D

    1985-01-01

    The shape of an actin subunit has been derived from an improved 6 A map of the complex of rabbit skeletal muscle actin and bovine pancreatic DNase I obtained by X-ray crystallographic methods. The three-dimensional structure of DNase I determined independently at 2.5 A resolution was compared with the DNase I electron density in the actin:DNase map. The two structures are very similar at 6 A resolution thus leading to an unambiguous identification of actin as well as DNase I electron density. Furthermore the correct hand of the actin structure is determined from the DNase I atomic structure. The resolution of the actin structure was extended to 4.5 A by using a single heavy-atom derivative and the knowledge of the atomic coordinates of DNase I. The dimensions of an actin subunit are 67 A X 40 A X 37 A. It consists of a small and a large domain, the small domain containing the N terminus. Actin is an alpha,beta-protein with a beta-pleated sheet in each domain. These sheets are surrounded by several alpha-helices, comprising at least 40% of the structure. The phosphate peak of the adenine nucleotide is located between the two domains. The complex of actin and DNase I as found in solution (i.e., the actin:DNase I contacts which do not depend on crystal packing) was deduced from a comparison of monoclinic with orthorhombic crystals. Residues 44-46, 51, 52, 60-62 of DNase I are close to a loop region in the small domain of actin. At a distance of approximately 15 A there is a second contact in the large domain in which Glu13 of DNase I is involved. A possible binding region for myosin is discussed. Images Fig. 1. Fig. 2. Fig. 3. PMID:4065103

  12. Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments

    PubMed Central

    Thiam, Hawa-Racine; Vargas, Pablo; Carpi, Nicolas; Crespo, Carolina Lage; Raab, Matthew; Terriac, Emmanuel; King, Megan C.; Jacobelli, Jordan; Alberts, Arthur S.; Stradal, Theresia; Lennon-Dumenil, Ana-Maria; Piel, Matthieu

    2016-01-01

    Cell migration has two opposite faces: although necessary for physiological processes such as immune responses, it can also have detrimental effects by enabling metastatic cells to invade new organs. In vivo, migration occurs in complex environments and often requires a high cellular deformability, a property limited by the cell nucleus. Here we show that dendritic cells, the sentinels of the immune system, possess a mechanism to pass through micrometric constrictions. This mechanism is based on a rapid Arp2/3-dependent actin nucleation around the nucleus that disrupts the nuclear lamina, the main structure limiting nuclear deformability. The cells' requirement for Arp2/3 to pass through constrictions can be relieved when nuclear stiffness is decreased by suppressing lamin A/C expression. We propose a new role for Arp2/3 in three-dimensional cell migration, allowing fast-moving cells such as leukocytes to rapidly and efficiently migrate through narrow gaps, a process probably important for their function. PMID:26975831

  13. Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments.

    PubMed

    Thiam, Hawa-Racine; Vargas, Pablo; Carpi, Nicolas; Crespo, Carolina Lage; Raab, Matthew; Terriac, Emmanuel; King, Megan C; Jacobelli, Jordan; Alberts, Arthur S; Stradal, Theresia; Lennon-Dumenil, Ana-Maria; Piel, Matthieu

    2016-01-01

    Cell migration has two opposite faces: although necessary for physiological processes such as immune responses, it can also have detrimental effects by enabling metastatic cells to invade new organs. In vivo, migration occurs in complex environments and often requires a high cellular deformability, a property limited by the cell nucleus. Here we show that dendritic cells, the sentinels of the immune system, possess a mechanism to pass through micrometric constrictions. This mechanism is based on a rapid Arp2/3-dependent actin nucleation around the nucleus that disrupts the nuclear lamina, the main structure limiting nuclear deformability. The cells' requirement for Arp2/3 to pass through constrictions can be relieved when nuclear stiffness is decreased by suppressing lamin A/C expression. We propose a new role for Arp2/3 in three-dimensional cell migration, allowing fast-moving cells such as leukocytes to rapidly and efficiently migrate through narrow gaps, a process probably important for their function. PMID:26975831

  14. Nitrosative modifications of the Ca2+ release complex and actin underlie arthritis-induced muscle weakness

    PubMed Central

    Yamada, Takashi; Fedotovskaya, Olga; Cheng, Arthur J; Cornachione, Anabelle S; Minozzo, Fabio C; Aulin, Cecilia; Fridén, Cecilia; Turesson, Carl; Andersson, Daniel C; Glenmark, Birgitta; Lundberg, Ingrid E; Rassier, Dilson E; Westerblad, Håkan; Lanner, Johanna T

    2015-01-01

    Objective Skeletal muscle weakness is a prominent clinical feature in patients with rheumatoid arthritis (RA), but the underlying mechanism(s) is unknown. Here we investigate the mechanisms behind arthritis-induced skeletal muscle weakness with special focus on the role of nitrosative stress on intracellular Ca2+ handling and specific force production. Methods Nitric oxide synthase (NOS) expression, degree of nitrosative stress and composition of the major intracellular Ca2+ release channel (ryanodine receptor 1, RyR1) complex were measured in muscle. Changes in cytosolic free Ca2+ concentration ([Ca2+]i) and force production were assessed in single-muscle fibres and isolated myofibrils using atomic force cantilevers. Results The total neuronal NOS (nNOS) levels were increased in muscles both from collagen-induced arthritis (CIA) mice and patients with RA. The nNOS associated with RyR1 was increased and accompanied by increased [Ca2+]i during contractions of muscles from CIA mice. A marker of peroxynitrite-derived nitrosative stress (3-nitrotyrosine, 3-NT) was increased on the RyR1 complex and on actin of muscles from CIA mice. Despite increased [Ca2+]i, individual CIA muscle fibres were weaker than in healthy controls, that is, force per cross-sectional area was decreased. Furthermore, force and kinetics were impaired in CIA myofibrils, hence actin and myosin showed decreased ability to interact, which could be a result of increased 3-NT content on actin. Conclusions Arthritis-induced muscle weakness is linked to nitrosative modifications of the RyR1 protein complex and actin, which are driven by increased nNOS associated with RyR1 and progressively increasing Ca2+ activation. PMID:24854355

  15. The centrosome is an actin-organizing center

    PubMed Central

    Farina, Francesca; Gaillard, Jérémie; Guérin, Christophe; Couté, Yohann; Sillibourne, James; Blanchoin, Laurent; Théry, Manuel

    2016-01-01

    Microtubules and actin filaments are the two main cytoskeleton networks supporting intracellular architecture and cell polarity. The centrosome nucleates and anchors microtubules and is therefore considered to be the main microtubule-organizing center. However, recurring, yet unexplained, observations have pointed towards a connection between the centrosome and actin filaments. Here we have used isolated centrosomes to demonstrate that the centrosome can directly promote actin filament assembly. A cloud of centrosome-associated actin filaments could be identified in living cells as well. Actin-filament nucleation at the centrosome was mediated by the nucleation promoting factor WASH in combination with the Arp2/3 complex. Pericentriolar material 1 (PCM1) appeared to modulate the centrosomal actin network by regulating Arp2/3 complex and WASH recruitment to the centrosome. Hence our results reveal an additional facet of the centrosome as an intracellular organizer and provide mechanistic insights into how the centrosome can function as an actin filament-organizing center. PMID:26655833

  16. Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

    SciTech Connect

    Baek, Kyuwon; Liu, Xiong; Ferron, Francois; Shu, Shi; Korn, Edward D.; Dominguez, Roberto

    2008-08-27

    On starvation, Dictyostelium cells aggregate to form multicellular fruiting bodies containing spores that germinate when transferred to nutrient-rich medium. This developmental cycle correlates with the extent of actin phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells but high in viable mature spores. Here we describe high-resolution crystal structures of pY53-actin and unphosphorylated actin in complexes with gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate group on Tyr-53 makes hydrogen-bonding interactions with residues of the DNase I-binding loop (D-loop) of actin, resulting in a more stable conformation of the D-loop than in the unphosphorylated structures. A more rigidly folded D-loop may explain some of the previously described properties of pY53-actin, including its increased critical concentration for polymerization, reduced rates of nucleation and pointed end elongation, and weak affinity for DNase I. We show here that phosphorylation of Tyr-53 inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide exchange on actin. The structure of profilin-Dictyostelium-actin is strikingly similar to previously determined structures of profilin-{beta}-actin and profilin-{alpha}-actin. By comparing this representative set of profilin-actin structures with other structures of actin, we highlight the effects of profilin on the actin conformation. In the profilin-actin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4.7 deg. rotation of the two major domains of actin relative to each other. As a result, the nucleotide cleft becomes moderately more open in the profilin-actin complex, probably explaining the stimulation of nucleotide exchange on actin by profilin.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. TACC3 protein regulates microtubule nucleation by affecting γ-tubulin ring complexes.

    PubMed

    Singh, Puja; Thomas, Geethu Emily; Gireesh, Koyikulangara K; Manna, Tapas K

    2014-11-14

    Centrosome-mediated microtubule nucleation is essential for spindle assembly during mitosis. Although γ-tubulin complexes have primarily been implicated in the nucleation process, details of the underlying mechanisms remain poorly understood. Here, we demonstrated that a member of the human transforming acidic coiled-coil (TACC) protein family, TACC3, plays a critical role in microtubule nucleation at the centrosome. In mitotic cells, TACC3 knockdown substantially affected the assembly of microtubules in the astral region and impaired microtubule nucleation at the centrosomes. The TACC3 depletion-induced mitotic phenotype was rescued by expression of the TACC3 C terminus predominantly consisting of the TACC domain, suggesting that the TACC domain plays an important role in microtubule assembly. Consistently, experiments with the recombinant TACC domain of TACC3 demonstrated that this domain possesses intrinsic microtubule nucleating activity. Co-immunoprecipitation and sedimentation experiments revealed that TACC3 mediates interactions with proteins of both the γ-tubulin ring complex (γ-TuRC) and the γ-tubulin small complex (γ-TuSC). Interestingly, TACC3 depletion resulted in reduced levels of γ-TuRC and increased levels of γ-TuSC, indicating that the assembly of γ-TuRC from γ-TuSC requires TACC3. Detailed analyses suggested that TACC3 facilitates the association of γ-TuSC-specific proteins with the proteins known to be involved in the assembly of γ-TuRC. Consistent with such a role for TACC3, the suppression of TACC3 disrupted localization of γ-TuRC proteins to the centrosome. Our findings reveal that TACC3 is involved in the regulation of microtubule nucleation at the centrosome and functions in the stabilization of the γ-tubulin ring complex assembly. PMID:25246530

  20. Two actin-related proteins are shared functional components of the chromatin-remodeling complexes RSC and SWI/SNF.

    PubMed

    Cairns, B R; Erdjument-Bromage, H; Tempst, P; Winston, F; Kornberg, R D

    1998-11-01

    The yeast Saccharomyces cerevisiae contains two related chromatin-remodeling complexes, RSC and SWI/SNF, which are shown to share the actin-related proteins Arp7 and Arp9. Depending on the genetic background tested, arp7 delta and arp9 delta mutants are either inviable or show greatly impaired growth and Swi-/Snf- mutant phenotypes. Unlike swi/snf mutants, viable arp7 delta or arp9 delta mutants have an Spt- phenotype, suggesting that RSC affects transcription. Temperature-sensitive mutations in ARP7 and ARP9 were isolated, and the amino acid changes support the structural relationship of Arp7 and Arp9 to actin. However, site-directed mutations predicted to impair ATP binding or hydrolysis did not detectably affect Arp7 or Arp9 function. Our results suggest that actin-related proteins perform important roles in chromatin-remodeling complexes by virtue of structural rather than enzymatic similarities to actin. PMID:9844636

  1. 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

  2. Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly.

    PubMed

    Lee, Il-Hyung; Kai, Hiroyuki; Carlson, Lars-Anders; Groves, Jay T; Hurley, James H

    2015-12-29

    The endosomal sorting complexes required for transport (ESCRT) machinery functions in HIV-1 budding, cytokinesis, multivesicular body biogenesis, and other pathways, in the course of which it interacts with concave membrane necks and bud rims. To test the role of membrane shape in regulating ESCRT assembly, we nanofabricated templates for invaginated supported lipid bilayers. The assembly of the core ESCRT-III subunit CHMP4B/Snf7 is preferentially nucleated in the resulting 100-nm-deep membrane concavities. ESCRT-II and CHMP6 accelerate CHMP4B assembly by increasing the concentration of nucleation seeds. Superresolution imaging was used to visualize CHMP4B/Snf7 concentration in a negatively curved annulus at the rim of the invagination. Although Snf7 assemblies nucleate slowly on flat membranes, outward growth onto the flat membrane is efficiently nucleated at invaginations. The nucleation behavior provides a biophysical explanation for the timing of ESCRT-III recruitment and membrane scission in HIV-1 budding. PMID:26668364

  3. 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.

  4. Actomyosin-dependent formation of the mechanosensitive talin-vinculin complex reinforces actin anchoring

    NASA Astrophysics Data System (ADS)

    Ciobanasu, Corina; Faivre, Bruno; Le Clainche, Christophe

    2014-01-01

    The force generated by the actomyosin cytoskeleton controls focal adhesion dynamics during cell migration. This process is thought to involve the mechanical unfolding of talin to expose cryptic vinculin-binding sites. However, the ability of the actomyosin cytoskeleton to directly control the formation of a talin-vinculin complex and the resulting activity of the complex are not known. Here we develop a microscopy assay with pure proteins in which the self-assembly of actomyosin cables controls the association of vinculin to a talin-micropatterned surface in a reversible manner. Quantifications indicate that talin refolding is limited by vinculin dissociation and modulated by the actomyosin network stability. Finally, we show that the activation of vinculin by stretched talin induces a positive feedback that reinforces the actin-talin-vinculin association. This in vitro reconstitution reveals the mechanism by which a key molecular switch senses and controls the connection between adhesion complexes and the actomyosin cytoskeleton.

  5. Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling.

    PubMed

    McGarvey, Jennifer C; Xiao, Kunhong; Bowman, Shanna L; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W Bruce; Ardura, Juan A; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A; Friedman, Peter A

    2016-05-20

    The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor. PMID:27008860

  6. 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. PMID:26240174

  7. An actin-dependent annexin complex mediates plasma membrane repair in muscle.

    PubMed

    Demonbreun, Alexis R; Quattrocelli, Mattia; Barefield, David Y; Allen, Madison V; Swanson, Kaitlin E; McNally, Elizabeth M

    2016-06-20

    Disruption of the plasma membrane often accompanies cellular injury, and in muscle, plasma membrane resealing is essential for efficient recovery from injury. Muscle contraction, especially of lengthened muscle, disrupts the sarcolemma. To define the molecular machinery that directs repair, we applied laser wounding to live mammalian myofibers and assessed translocation of fluorescently tagged proteins using high-resolution microscopy. Within seconds of membrane disruption, annexins A1, A2, A5, and A6 formed a tight repair "cap." Actin was recruited to the site of damage, and annexin A6 cap formation was both actin dependent and Ca(2+) regulated. Repair proteins, including dysferlin, EHD1, EHD2, MG53, and BIN1, localized adjacent to the repair cap in a "shoulder" region enriched with phosphatidlyserine. Dye influx into muscle fibers lacking both dysferlin and the related protein myoferlin was substantially greater than control or individual null muscle fibers, underscoring the importance of shoulder-localized proteins. These data define the cap and shoulder as subdomains within the repair complex accumulating distinct and nonoverlapping components. PMID:27298325

  8. A network of spectrin and plectin surrounds the actin cuffs of apical tubulobulbar complexes in the rat.

    PubMed

    Aristaeus de Asis, Marc; Pires, Manuel; Lyon, Kevin; Vogl, A Wayne

    2013-07-01

    Tubulobulbar complexes (TBCs) are actin-related endocytic structures that internalize intercellular junctions in the seminiferous epithelium. The structures consist of elongate tubular projections of the attached plasma membranes of two adjacent cells that project into Sertoli cells. This double membrane core is cuffed by a dentritic actin network and is capped at its end by a clathrin-coated pit. Here we explore the possibility that elements of the spectrin cytoskeleton are associated with clusters of tubulobulbar complexes that develop at adhesion junctions between late spermatids and Sertoli cells at the apex of the epithelium, and extend what is known about the distribution of plectin at the sites. Cryo-sections of perfusion-fixed testes and apical processes of Sertoli cells mechanically dissociated from perfusion-fixed testes were probed for spectrin, EPB41, and actin and analyzed using conventional fluorescence microscopy and confocal microscopy. Data sets from confocal microscopy were analyzed further in three-dimensional reconstructions using computer software. Additional apical Sertoli cell processes were probed for plectin and analyzed using conventional fluorescence microscopy. Antibodies generated against elements of the spectrin cytoskeleton react with material around and between the actin cuffs of tubulobulbar complexes, but appear excluded from the actin cuffs themselves. A similar staining pattern occurs with a probe for plectin. Immunoelectron microscopy confirmed the staining patterns observed by fluourescence microscopy. Based on our results, we suggest that a network of spectrin and plectin forms a scaffold around tubulobulbar complexes that may provide support for the actin network that cuffs each complex and also link adjacent complexes together. PMID:24381803

  9. A network of spectrin and plectin surrounds the actin cuffs of apical tubulobulbar complexes in the rat

    PubMed Central

    Aristaeus de Asis, Marc; Pires, Manuel; Lyon, Kevin; Vogl, A Wayne

    2013-01-01

    Tubulobulbar complexes (TBCs) are actin-related endocytic structures that internalize intercellular junctions in the seminiferous epithelium. The structures consist of elongate tubular projections of the attached plasma membranes of two adjacent cells that project into Sertoli cells. This double membrane core is cuffed by a dentritic actin network and is capped at its end by a clathrin-coated pit. Here we explore the possibility that elements of the spectrin cytoskeleton are associated with clusters of tubulobulbar complexes that develop at adhesion junctions between late spermatids and Sertoli cells at the apex of the epithelium, and extend what is known about the distribution of plectin at the sites. Cryo-sections of perfusion-fixed testes and apical processes of Sertoli cells mechanically dissociated from perfusion-fixed testes were probed for spectrin, EPB41, and actin and analyzed using conventional fluorescence microscopy and confocal microscopy. Data sets from confocal microscopy were analyzed further in three-dimensional reconstructions using computer software. Additional apical Sertoli cell processes were probed for plectin and analyzed using conventional fluorescence microscopy. Antibodies generated against elements of the spectrin cytoskeleton react with material around and between the actin cuffs of tubulobulbar complexes, but appear excluded from the actin cuffs themselves. A similar staining pattern occurs with a probe for plectin. Immunoelectron microscopy confirmed the staining patterns observed by fluourescence microscopy. Based on our results, we suggest that a network of spectrin and plectin forms a scaffold around tubulobulbar complexes that may provide support for the actin network that cuffs each complex and also link adjacent complexes together. PMID:24381803

  10. Circulating Complexes of the Vitamin D Binding Protein with G-Actin Induce Lung Inflammation by Targeting Endothelial Cells

    PubMed Central

    Ge, Lingyin; Trujillo, Glenda; Miller, Edmund J.; Kew, Richard R.

    2013-01-01

    This study investigated the actin scavenger function of the vitamin D binding protein (DBP) in vivo using DBP null (−/−) mice. Intravenous injection of G-actin into wild-type (DBP+/+) and DBP−/− mice showed that contrary to expectations, DBP+/+ mice developed more severe acute lung inflammation. Inflammation was restricted to the lung and pathological changes were clearly evident at 1.5 and 4 hours post-injection but were largely resolved by 24 hours. Histology of DBP+/+ lungs revealed noticeably more vascular leakage, hemorrhage and thickening of the alveolar wall. Flow cytometry analysis of whole lung homogenates showed significantly increased neutrophil infiltration into DBP+/+ mouse lungs at 1.5 and 4 hours. Increased amounts of protein and leukocytes were also noted in bronchoalveolar lavage fluid from DBP+/+ mice 4 hours after actin injection. In vitro, purified DBP-actin complexes did not activate complement or neutrophils but induced injury and death of cultured human lung microvascular endothelial cells (HLMVEC) and human umbilical vein endothelial cells (HUVEC). Cells treated with DBP-actin showed a significant reduction in viability at 4 hours, this effect was reversible if cells were cultured in fresh media for another 24 hours. However, a 24-hour treatment with DBP-actin complexes showed a significant increase in cell death (95% for HLMVEC, 45% for HUVEC). The mechanism of endothelial cell death was via both caspase-3 dependent (HUVEC) and independent (HLMVEC) pathways. These results demonstrate that elevated levels and/or prolonged exposure to DBP-actin complexes may induce endothelial cell injury and death, particularly in the lung microvasculature. PMID:24268110

  11. A complex of ZO-1 and the BAR-domain protein TOCA-1 regulates actin assembly at the tight junction

    PubMed Central

    Van Itallie, Christina M.; Tietgens, Amber Jean; Krystofiak, Evan; Kachar, Bechara; Anderson, James M.

    2015-01-01

    Assembly and sealing of the tight junction barrier are critically dependent on the perijunctional actin cytoskeleton, yet little is known about physical and functional links between barrier-forming proteins and actin. Here we identify a novel functional complex of the junction scaffolding protein ZO-1 and the F-BAR–domain protein TOCA-1. Using MDCK epithelial cells, we show that an alternative splice of TOCA-1 adds a PDZ-binding motif, which binds ZO-1, targeting TOCA-1 to barrier contacts. This isoform of TOCA-1 recruits the actin nucleation–promoting factor N-WASP to tight junctions. CRISPR-Cas9–mediated knockout of TOCA-1 results in increased paracellular flux and delayed recovery in a calcium switch assay. Knockout of TOCA-1 does not alter FRAP kinetics of GFP ZO-1 or occludin, but longer term (12 h) time-lapse microscopy reveals strikingly decreased tight junction membrane contact dynamics in knockout cells compared with controls. Reexpression of TOCA-1 with, but not without, the PDZ-binding motif rescues both altered flux and membrane contact dynamics. Ultrastructural analysis shows actin accumulation at the adherens junction in TOCA-1–knockout cells but unaltered freeze-fracture fibril morphology. Identification of the ZO-1/TOCA-1 complex provides novel insights into the underappreciated dependence of the barrier on the dynamic nature of cell-to-cell contacts and perijunctional actin. PMID:26063734

  12. EFC/F-BAR proteins and the N-WASP–WIP complex induce membrane curvature-dependent actin polymerization

    PubMed Central

    Takano, Kazunari; Toyooka, Kiminori; Suetsugu, Shiro

    2008-01-01

    Extended Fer-CIP4 homology (EFC)/FCH-BAR (F-BAR) domains generate and bind to tubular membrane structures of defined diameters that are involved in the formation and fission of endocytotic vesicles. Formin-binding protein 17 (FBP17) and Toca-1 contain EFC/F-BAR domains and bind to neural Wiskott–Aldrich syndrome protein (N-WASP), which links phosphatidylinositol (4,5)-bisphosphate (PIP2) and the Rho family GTPase Cdc42 to the Arp2/3 complex. The N-WASP–WASP-interacting protein (WIP) complex, a predominant form of N-WASP in cells, is known to be activated by Toca-1 and Cdc42. Here, we show that N-WASP–WIP complex-mediated actin polymerization is activated by phosphatidylserine-containing membranes depending on membrane curvature in the presence of Toca-1 or FBP17 and in the absence of Cdc42 and PIP2. Cdc42 further promoted the activation of actin polymerization by N-WASP–WIP. Toca-1 or FBP17 recruited N-WASP–WIP to the membrane. Conserved acidic residues near the SH3 domain of Toca-1 and FBP17 positioned the N-WASP–WIP to be spatially close to the membrane for activation of actin polymerization. Therefore, curvature-dependent actin polymerization is stimulated by spatially appropriate interactions of EFC/F-BAR proteins and the N-WASP–WIP complex with the membrane. PMID:18923421

  13. Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba

    PubMed Central

    Mullins, R. Dyche; Stafford, Walter F.; Pollard, Thomas D.

    1997-01-01

    The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actinrelated proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of ∼13 × 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 μM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actinrich regions of Acanthamoeba. PMID:9015304

  14. 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.

  15. Schip1 Is a Novel Podocyte Foot Process Protein that Mediates Actin Cytoskeleton Rearrangements and Forms a Complex with Nherf2 and Ezrin

    PubMed Central

    Perisic, Ljubica; Rodriguez, Patricia Q.; Hultenby, Kjell; Sun, Ying; Lal, Mark; Betsholtz, Christer; Uhlén, Mathias; Wernerson, Annika; Hedin, Ulf; Pikkarainen, Timo; Tryggvason, Karl; Patrakka, Jaakko

    2015-01-01

    Background Podocyte foot process effacement accompanied by actin cytoskeleton rearrangements is a cardinal feature of many progressive human proteinuric diseases. Results By microarray profiling of mouse glomerulus, SCHIP1 emerged as one of the most highly enriched transcripts. We detected Schip1 protein in the kidney glomerulus, specifically in podocytes foot processes. Functionally, Schip1 inactivation in zebrafish by morpholino knock-down results in foot process disorganization and podocyte loss leading to proteinuria. In cultured podocytes Schip1 localizes to cortical actin-rich regions of lamellipodia, where it forms a complex with Nherf2 and ezrin, proteins known to participate in actin remodeling stimulated by PDGFβ signaling. Mechanistically, overexpression of Schip1 in vitro causes accumulation of cortical F-actin with dissolution of transversal stress fibers and promotes cell migration in response to PDGF-BB stimulation. Upon actin disassembly by latrunculin A treatment, Schip1 remains associated with the residual F-actin-containing structures, suggesting a functional connection with actin cytoskeleton possibly via its interaction partners. A similar assay with cytochalasin D points to stabilization of cortical actin cytoskeleton in Schip1 overexpressing cells by attenuation of actin depolymerisation. Conclusions Schip1 is a novel glomerular protein predominantly expressed in podocytes, necessary for the zebrafish pronephros development and function. Schip1 associates with the cortical actin cytoskeleton network and modulates its dynamics in response to PDGF signaling via interaction with the Nherf2/ezrin complex. Its implication in proteinuric diseases remains to be further investigated. PMID:25807495

  16. Cortical actin filament organization in developing and functioning stomatal complexes of Zea mays and Triticum turgidum.

    PubMed

    Panteris, Emmanuel; Galatis, Basil; Quader, Hartmut; Apostolakos, Panagiotis

    2007-07-01

    Cortical actin filament (AF) organization was studied in detail in developing stomatal complexes of the grasses Zea mays and Triticum turgidum. AF arrays during the whole stomatal complex development are dynamic, partly following the pattern of cortical microtubule (MT) organization. They also exhibit particular patterns of organization, spatially and temporarily restricted. Among AF arrays, the radial ones that underlie young guard cell (GC) periclinal walls, those that line the bulbous GC ends and the AF ring at the junction between subsidiary cells (SCs) and GCs are described here for the first time. Although many similarities in cortical AF organization exist among the stomatal cells of both plants studied, considerable differences have also been observed between them. Our data reveal that the expanding areas of stomatal cell walls are lined by distinct cortical AF aggregations that probably protect the plasmalemma against mechanical stresses. Experimental AF disruption does not seem to affect detectably stomatal cell morphogenesis. Moreover, the structural and experimental data of this study revealed that, in contrast to the elliptical stomata, in the dumbbell-shaped ones the AFs and MTs seem not to be involved in the mechanism of opening and closing of the stomatal pore. PMID:17443701

  17. Ca2+-calmodulin regulates fesselin-induced actin polymerization.

    PubMed

    Schroeter, Mechthild; Chalovich, Joseph M

    2004-11-01

    Fesselin is a proline-rich actin-binding protein that was isolated from avian smooth muscle. Fesselin bundles actin and accelerates actin polymerization by facilitating nucleation. We now show that this polymerization of actin can be regulated by Ca(2+)-calmodulin. Fesselin was shown to bind to immobilized calmodulin in the presence of Ca(2+). The fesselin-calmodulin interaction was confirmed by a Ca(2+)-dependent increase in 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) fluorescence upon addition of fesselin to MIANS-labeled wheat germ calmodulin. The affinity was estimated to be approximately 10(9) M(-1). The affinity of Ca(2+)-calmodulin to the fesselin F-actin complex was approximately 10(8) M(-1). Calmodulin binding to fesselin appeared to be functionally significant. In the presence of fesselin and calmodulin, the polymerization of actin was Ca(2+)-dependent. Ca(2+)-free calmodulin either had no effect or enhanced the ability of fesselin to accelerate actin polymerization. Ca(2+)-calmodulin not only reversed the stimulatory effect of fesselin but reduced the rate of polymerization below that observed in the absence of fesselin. While Ca(2+)-calmodulin had a large effect on the interaction of fesselin with G-actin, the effect on F-actin was small. Neither the binding of fesselin to F-actin nor the subsequent bundling of F-actin was greatly affected by Ca(2+)-calmodulin. Fesselin may function as an actin-polymerizing factor that is regulated by Ca(2+) levels. PMID:15504050

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

    PubMed Central

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

    2015-01-01

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

  19. Bacterial actin: architecture of the ParMRC plasmid DNA partitioning complex.

    PubMed

    Salje, Jeanne; Löwe, Jan

    2008-08-20

    The R1 plasmid employs ATP-driven polymerisation of the actin-like protein ParM to move newly replicated DNA to opposite poles of a bacterial cell. This process is essential for ensuring accurate segregation of the low-copy number plasmid and is the best characterised example of DNA partitioning in prokaryotes. In vivo, ParM only forms long filaments when capped at both ends by attachment to a centromere-like region parC, through a small DNA-binding protein ParR. Here, we present biochemical and electron microscopy data leading to a model for the mechanism by which ParR-parC complexes bind and stabilise elongating ParM filaments. We propose that the open ring formed by oligomeric ParR dimers with parC DNA wrapped around acts as a rigid clamp, which holds the end of elongating ParM filaments while allowing entry of new ATP-bound monomers. We propose a processive mechanism by which cycles of ATP hydrolysis in polymerising ParM drives movement of ParR-bound parC DNA. Importantly, our model predicts that each pair of plasmids will be driven apart in the cell by just a single double helical ParM filament. PMID:18650930

  20. Actinic Keratosis

    MedlinePlus

    ... rashes clinical tools newsletter | contact Share | Actinic Keratosis (Solar Keratosis) Information for adults A A A Actinic ... the touch. Overview Actinic keratoses, also known as solar keratoses, are small rough or scaly areas of ...

  1. Three-dimensional reconstruction of a co-complex of F-actin with antibody Fab fragments to actin's NH2 terminus.

    PubMed Central

    Orlova, A; Yu, X; Egelman, E H

    1994-01-01

    We have decorated F-actin with Fab fragments of antibodies to actin residues 1-7. These antibody fragments do not strongly affect the rigor binding of myosin S-1 to actin, but do affect the binding of S-1 to actin in the presence of nucleotide (DasGupta, G., and E. Reisler, 1989. J. Mol. Biol. 207:833-836; 1991. Biochemistry. 30:9961-9966; 1992. Biochemistry. 31:1836-1841). Although the binding constant is rather low, we estimate that we have achieved about 85% occupancy of the actin sites. Three-dimensional reconstructions from electron micrographs of both negatively stained and frozen-hydrated filaments show that the Fab fragment is bound at the location of the NH2 terminus in the model of Holmes et al. (Holmes, K.C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature. 347:37-44) for F-actin, excluding very different orientations of the actin subunit in the filament. Most of the mass of the antibody is not visualized, which is due to the large mobility of the NH2 terminus in F-actin, differences in binding angle within the polyclonal antibody population, or a combination of both of these possibilities. Images FIGURE 1 FIGURE 5 FIGURE 7 FIGURE 10 PMID:8161679

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

    PubMed

    Block, Jennifer; Breitsprecher, Dennis; Kühn, Sonja; Winterhoff, Moritz; Kage, Frieda; Geffers, Robert; Duwe, Patrick; Rohn, Jennifer L; Baum, Buzz; Brakebusch, Cord; Geyer, Matthias; Stradal, Theresia E B; Faix, Jan; Rottner, Klemens

    2012-06-01

    Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia. PMID:22608513

  3. Actin polymerization is stimulated by actin cross-linking protein palladin.

    PubMed

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G; Orlova, Albina; Egelman, Edward H; Beck, Moriah R

    2016-02-15

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the co-ordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. In the present study, 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 cross-linking 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 globular or monomeric actin (G-actin), akin to metal ions, either through charge neutralization or through conformational changes. PMID:26607837

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

    PubMed Central

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

    2015-01-01

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

  5. Profilin-1 serves as a gatekeeper for actin assembly by Arp2/3-dependent and –independent pathways

    PubMed Central

    Rotty, Jeremy D.; Wu, Congying; Haynes, Elizabeth M.; Suarez, Cristian; Winkelman, Jonathan D.; Johnson, Heath E.; Haugh, Jason M.; Kovar, David R.; Bear, James E.

    2015-01-01

    SUMMARY Cells contain multiple F-actin assembly pathways including the Arp2/3 complex, formins, and Ena/VASP, which have largely been analyzed separately. They collectively generate the bulk of F-actin from a common pool of G-actin; however, the interplay/competition between these pathways remains poorly understood. Using fibroblast lines derived from an Arpc2 conditional knockout mouse, we established matched-pair cells with and without the Arp2/3 complex. Arpc2−/− cells lack lamellipodia and migrate slower than WT cells, but have F-actin levels indistinguishable from controls. Actin assembly in Arpc2−/− cells was resistant to cytochalasin-D and was highly dependent on profilin-1 and Ena/VASP, but not formins. Profilin-1 depletion in WT cells increased F-actin and Arp2/3 complex in lamellipodia. Conversely, addition of exogenous profilin-1 inhibited Arp2/3 complex actin nucleation in vitro and in vivo. These observations suggest that antagonism of the Arp2/3 complex by profilin-1 in cells maintains actin homeostasis by balancing Arp2/3 complex-dependent and independent actin assembly pathways. PMID:25543281

  6. Complex relationship between TCTP, microtubules and actin microfilaments regulates cell shape in normal and cancer cells

    PubMed Central

    Bazile, Franck; Pascal, Aude; Arnal, Isabelle; Le Clainche, Christophe; Chesnel, Franck; Kubiak, Jacek Z.

    2009-01-01

    Translationally Controlled Tumor-associated Protein (TCTP) is a ubiquitous and highly conserved protein implicated in cancers. Here we demonstrate that interactions of TCTP with microtubules (MTs) are functionally important but indirect, and we reveal novel interaction of TCTP with the actin cytoskeleton. Firstly, immunofluorescence in Xenopus XL2 cells revealed cytoplasmic fibers stained with TCTP but not with tubulin antibodies, as well as MT-free of TCTP. Furthermore, TCTP localized to a subset of actin-rich fibers in migrating cells. Secondly XlTCTP did not affect in vitro assembly/disassembly of MTs, and lacked MT binding affinity both in pull-down assays and in cell-free extracts. Although TCTP also failed to bind to purified F-actin, it associated with microfilaments in cell-free extracts. Thirdly, TCTP concentrated in mitotic spindle did not colocalize with MTs, and was easily dissociated from these structures except at the poles. Finally, RNAi knockdown of TCTP in XL2 and HeLa cells provoked drastic, MT-dependent, shape change. These data show that although TCTP interacts with MTs it does not behave as classic MT Associated Protein (MAP). Our evidence for an association of TCTP with F-actin structures, and for an involvement in cell shape regulation, implicates this protein in integrating cytoskeletal interations both in interphase and mitosis providing a new avenue to fully understand the role of TCTP. PMID:19168579

  7. Capping complex formation at the slow-growing end of the actin filament.

    PubMed

    Kostyukova, A S

    2008-12-01

    Actin filaments are polar; their barbed (fast-growing) and pointed (slow-growing) ends differ in structure and dynamic properties. The slow-growing end is regulated by tropomodulins, a family of capping proteins that require tropomyosins for optimal function. There are four tropomodulin isoforms; their distributions vary depending on tissue type and change during development. The C-terminal half of tropomodulin contains one compact domain represented by alternating alpha-helices and beta-structures. The tropomyosin-independent actin-capping site is located at the C-terminus. The N-terminal half has no regular structure; however, it contains a tropomyosin-dependent actin-capping site and two tropomyosin-binding sites. One tropomodulin molecule can bind two tropomyosin molecules. Effectiveness of tropomodulin binding to tropomyosin depends on the tropomyosin isoform. Regulation of tropomodulin binding at the pointed end as well as capping effectiveness in the presence of specific tropomyosins may affect formation of local cytoskeleton and dynamics of actin filaments in cells. PMID:19216712

  8. Membrane Tension Acts Through PLD2 and mTORC2 to Limit Actin Network Assembly During Neutrophil Migration

    PubMed Central

    Diz-Muñoz, Alba; Thurley, Kevin; Chintamen, Sana; Altschuler, Steven J.; Fletcher, Daniel A.; Weiner, Orion D.

    2016-01-01

    For efficient polarity and migration, cells need to regulate the magnitude and spatial distribution of actin assembly. This process is coordinated by reciprocal interactions between the actin cytoskeleton and mechanical forces. Actin polymerization-based protrusion increases tension in the plasma membrane, which in turn acts as a long-range inhibitor of actin assembly. These interactions form a negative feedback circuit that limits the magnitude of membrane tension in neutrophils and prevents expansion of the existing front and the formation of secondary fronts. It has been suggested that the plasma membrane directly inhibits actin assembly by serving as a physical barrier that opposes protrusion. Here we show that efficient control of actin polymerization-based protrusion requires an additional mechanosensory feedback cascade that indirectly links membrane tension with actin assembly. Specifically, elevated membrane tension acts through phospholipase D2 (PLD2) and the mammalian target of rapamycin complex 2 (mTORC2) to limit actin nucleation. In the absence of this pathway, neutrophils exhibit larger leading edges, higher membrane tension, and profoundly defective chemotaxis. Mathematical modeling suggests roles for both the direct (mechanical) and indirect (biochemical via PLD2 and mTORC2) feedback loops in organizing cell polarity and motility—the indirect loop is better suited to enable competition between fronts, whereas the direct loop helps spatially organize actin nucleation for efficient leading edge formation and cell movement. This circuit is essential for polarity, motility, and the control of membrane tension. PMID:27280401

  9. Membrane Tension Acts Through PLD2 and mTORC2 to Limit Actin Network Assembly During Neutrophil Migration.

    PubMed

    Diz-Muñoz, Alba; Thurley, Kevin; Chintamen, Sana; Altschuler, Steven J; Wu, Lani F; Fletcher, Daniel A; Weiner, Orion D

    2016-06-01

    For efficient polarity and migration, cells need to regulate the magnitude and spatial distribution of actin assembly. This process is coordinated by reciprocal interactions between the actin cytoskeleton and mechanical forces. Actin polymerization-based protrusion increases tension in the plasma membrane, which in turn acts as a long-range inhibitor of actin assembly. These interactions form a negative feedback circuit that limits the magnitude of membrane tension in neutrophils and prevents expansion of the existing front and the formation of secondary fronts. It has been suggested that the plasma membrane directly inhibits actin assembly by serving as a physical barrier that opposes protrusion. Here we show that efficient control of actin polymerization-based protrusion requires an additional mechanosensory feedback cascade that indirectly links membrane tension with actin assembly. Specifically, elevated membrane tension acts through phospholipase D2 (PLD2) and the mammalian target of rapamycin complex 2 (mTORC2) to limit actin nucleation. In the absence of this pathway, neutrophils exhibit larger leading edges, higher membrane tension, and profoundly defective chemotaxis. Mathematical modeling suggests roles for both the direct (mechanical) and indirect (biochemical via PLD2 and mTORC2) feedback loops in organizing cell polarity and motility-the indirect loop is better suited to enable competition between fronts, whereas the direct loop helps spatially organize actin nucleation for efficient leading edge formation and cell movement. This circuit is essential for polarity, motility, and the control of membrane tension. PMID:27280401

  10. Synthetic polyamines: new compounds specific to actin dynamics for mammalian cell and fission yeast

    PubMed Central

    Riveline, Daniel; Thiagarajan, Raghavan; Lehn, Jean-Marie; Carlier, Marie-France

    2014-01-01

    Actin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes like adhesion, immune synapse, cortex dynamics or cytokinesis are achieved by combining acto-myosin contractility and actin assembly in a complex and not fully understood manner. New chemical compounds are therefore needed to disentangle acto-myosin and actin dynamics. We have found that synthetic, cell permeant, short polyamines are promising new actin regulators in this context. They generate growth and stabilization of lamellipodia within minutes by slowing down the actin assembly/disassembly cycle and facilitating nucleation. We now report that these polyamines also slow down cytokinetic ring closure in fission yeast. This shows that these synthetic compounds are active also in yeasts, and these experiments specifically highlight that actin depolymerization is involved in the ring closure. Thus, synthetic polyamines appear to be potentially powerful agents in a quantitative approach to the role of actin in complex processes in cell biology, developmental biology and potentially cancer research. PMID:25664996

  11. Synthetic polyamines: new compounds specific to actin dynamics for mammalian cell and fission yeast.

    PubMed

    Riveline, Daniel; Thiagarajan, Raghavan; Lehn, Jean-Marie; Carlier, Marie-France

    2014-01-01

    Actin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes like adhesion, immune synapse, cortex dynamics or cytokinesis are achieved by combining acto-myosin contractility and actin assembly in a complex and not fully understood manner. New chemical compounds are therefore needed to disentangle acto-myosin and actin dynamics. We have found that synthetic, cell permeant, short polyamines are promising new actin regulators in this context. They generate growth and stabilization of lamellipodia within minutes by slowing down the actin assembly/disassembly cycle and facilitating nucleation. We now report that these polyamines also slow down cytokinetic ring closure in fission yeast. This shows that these synthetic compounds are active also in yeasts, and these experiments specifically highlight that actin depolymerization is involved in the ring closure. Thus, synthetic polyamines appear to be potentially powerful agents in a quantitative approach to the role of actin in complex processes in cell biology, developmental biology and potentially cancer research. PMID:25664996

  12. Ion-association complexes unite classical and non-classical theories for the biomimetic nucleation of calcium phosphate.

    PubMed

    Habraken, Wouter J E M; Tao, Jinhui; Brylka, Laura J; Friedrich, Heiner; Bertinetti, Luca; Schenk, Anna S; Verch, Andreas; Dmitrovic, Vladimir; Bomans, Paul H H; Frederik, Peter M; Laven, Jozua; van der Schoot, Paul; Aichmayer, Barbara; de With, Gijsbertus; DeYoreo, James J; Sommerdijk, Nico A J M

    2013-01-01

    Despite its importance in many industrial, geological and biological processes, the mechanism of crystallization from supersaturated solutions remains a matter of debate. Recent discoveries show that in many solution systems nanometre-sized structural units are already present before nucleation. Still little is known about the structure and role of these so-called pre-nucleation clusters. Here we present a combination of in situ investigations, which show that for the crystallization of calcium phosphate these nanometre-sized units are in fact calcium triphosphate complexes. Under conditions in which apatite forms from an amorphous calcium phosphate precursor, these complexes aggregate and take up an extra calcium ion to form amorphous calcium phosphate, which is a fractal of Ca(2)(HPO(4))(3)(2-) clusters. The calcium triphosphate complex also forms the basis of the crystal structure of octacalcium phosphate and apatite. Finally, we demonstrate how the existence of these complexes lowers the energy barrier to nucleation and unites classical and non-classical nucleation theories. PMID:23422675

  13. Force Feedback Controls Motor Activity and Mechanical Properties of Self-Assembling Branched Actin Networks.

    PubMed

    Bieling, Peter; Li, Tai-De; Weichsel, Julian; McGorty, Ryan; Jreij, Pamela; Huang, Bo; Fletcher, Daniel A; Mullins, R Dyche

    2016-01-14

    Branched actin networks--created by the Arp2/3 complex, capping protein, and a nucleation promoting factor--generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency. Microscopically, increased density reflects increased filament number and altered geometry but no change in average length. Macroscopically, increased density enhances network stiffness and resistance to mechanical failure beyond those of isotropic actin networks. These effects endow branched actin networks with memory of their mechanical history that shapes their material properties and motor activity. This work reveals intrinsic force feedback mechanisms by which mechanical resistance makes self-assembling actin networks stiffer, stronger, and more powerful. PMID:26771487

  14. Filament assembly by Spire: key residues and concerted actin binding.

    PubMed

    Rasson, Amy S; Bois, Justin S; Pham, Duy Stephen L; Yoo, Haneul; Quinlan, Margot E

    2015-02-27

    The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers. PMID:25234086

  15. Filament Assembly by Spire: Key Residues and Concerted Actin Binding

    PubMed Central

    Rasson, Amy S.; Bois, Justin S.; Pham, Duy Stephen L.; Yoo, Haneul; Quinlan, Margot E.

    2014-01-01

    The most recently identified class of actin nucleators, WASp Homology domain 2 (WH2) – nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or Sc), plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of Sc in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within Sc that are critical for its activity. Using this information we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that Sc binds actin filaments, in addition to monomers. PMID:25234086

  16. Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes

    SciTech Connect

    Wang, Yanli; Juranek, Stefan; Li, Haitao; Sheng, Gang; Wardle, Greg S.; Tuschl, Thomas; Patel, Dinshaw J.

    2009-10-21

    The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5'-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg{sup 2+}-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3'-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures.

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

    Summary Burkholderia pseudomallei and B. mallei are bacterial pathogens that cause melioidosis and glanders, while their close relative B. thailandensis is nonpathogenic. All use the trimeric autotransporter BimA to facilitate actin-based motility, host cell fusion and dissemination. Here, we show that BimA orthologs mimic different host actin-polymerizing proteins. B. thailandensis BimA activates the host Arp2/3 complex. In contrast, B. pseudomallei and B. mallei BimA mimic host Ena/VASP actin polymerases in their ability to nucleate, elongate and bundle filaments by associating with barbed ends, as well as in their use of WH2 motifs and oligomerization for activity. Mechanistic differences among BimA orthologs resulted in distinct actin filament organization and motility parameters, which affected the efficiency of cell fusion during infection. Our results identify bacterial Ena/VASP mimics and reveal that pathogens imitate the full spectrum of host actin-polymerizing pathways, suggesting that mimicry of different polymerization mechanisms influences key parameters of infection. PMID:25860613

  19. Structural and Biochemical Characterization of Two Binding Sites for Nucleation-promoting Factor WASp-VCA on Arp2/3 Complex

    SciTech Connect

    S Ti; C Jurgenson; B Nolen; T Pollard

    2011-12-31

    Actin-related protein (Arp) 2/3 complex mediates the formation of actin filament branches during endocytosis and at the leading edge of motile cells. The pathway of branch formation is ambiguous owing to uncertainty regarding the stoichiometry and location of VCA binding sites on Arp2/3 complex. Isothermal titration calorimetry showed that the CA motif from the C terminus of fission yeast WASP (Wsp1p) bound to fission yeast and bovine Arp2/3 complex with a stoichiometry of 2 to 1 and very different affinities for the two sites (K{sub d}s of 0.13 and 1.6 {micro}M for fission yeast Arp2/3 complex). Equilibrium binding, kinetic, and cross-linking experiments showed that (i) CA at high-affinity site 1 inhibited Arp2/3 complex binding to actin filaments, (ii) low-affinity site 2 had a higher affinity for CA when Arp2/3 complex was bound to actin filaments, and (iii) Arp2/3 complex had a much higher affinity for free CA than VCA cross-linked to an actin monomer. Crystal structures showed the C terminus of CA bound to the low-affinity site 2 on Arp3 of bovine Arp2/3 complex. The C helix is likely to bind to the barbed end groove of Arp3 in a position for VCA to deliver the first actin subunit to the daughter filament.

  20. Single-molecule visualization of a formin-capping protein ‘decision complex' at the actin filament barbed end

    PubMed Central

    Bombardier, Jeffrey P.; Eskin, Julian A.; Jaiswal, Richa; Corrêa, Ivan R.; Xu, Ming-Qun; Goode, Bruce L.; Gelles, Jeff

    2015-01-01

    Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived ‘decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells. PMID:26566078

  1. Myosins, Actin and Autophagy.

    PubMed

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

    2016-08-01

    Myosin motor proteins working together with the actin cytoskeleton drive a wide range of cellular processes. In this review, we focus on their roles in autophagy - the pathway the cell uses to ensure homeostasis by targeting pathogens, misfolded proteins and damaged organelles for degradation. The actin cytoskeleton regulated by a host of nucleating, anchoring and stabilizing proteins provides the filament network for the delivery of essential membrane vesicles from different cellular compartments to the autophagosome. Actin networks have also been implicated in structurally supporting the expanding phagophore, moving autophagosomes and enabling efficient fusion with the lysosome. Only a few myosins have so far been shown to play a role in autophagy. Non-muscle myosin IIA functions in the early stages delivering membrane for the initial formation of the autophagosome, whereas myosin IC and myosin VI are involved in the final stages providing specific membranes for autophagosome maturation and its fusion with the lysosome. PMID:27146966

  2. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar) ... Some actinic keratoses become squamous cell skin cancer . Have your health care provider look at all skin growths as soon as you find them. Your provider will ...

  3. Rapid non-equilibrium turnover fluidizes entangled F-actin solutions

    NASA Astrophysics Data System (ADS)

    McCall, Patrick M.; Kovar, David R.; Gardel, Margaret L.

    The actin cytoskeleton of living cells is a semiflexible polymer network which regulates cell division, motility, and morphogenesis by controlling cell shape. These complex shape-changing processes require both mechanical deformation and remodeling of the actin cytoskeleton. Molecular motors generate internal forces to drive deformation, while cytoskeletal remodeling is regulated by non-equilibrium polymer turnover. Although the mechanical properties of equilibrium actin filament (F-actin) networks are well-described by theories of semiflexible polymers, these theories do not incorporate the effects of non-equilibrium turnover. To address this experimentally, we developed a model system in which both the turnover rate and the length distribution of purified F-actin can be tuned independently at steady-state through the combined action of actin regulatory proteins. Specifically we tune the concentrations of cofilin, profilin, and formin to regulate F-actin severing, recycling, and nucleation, respectively. We find that the actin turnover rate can be tuned by cofilin up to 25-fold (31 +/- 2 subunits/sec/filament). Surprisingly, changes in turnover rate have no effect on the steady-state F-actin length distribution, which is instead set by formin concentration. Passive microrheology measurements show that increased turnover leads to striking fluidization in both entangled and crosslinked networks. Non-equilibrium turnover thus enables modulation of network mechanics, which impacts force transmission and material deformation.

  4. Actin-based propulsion of functionalized hard versus fluid spherical objects

    NASA Astrophysics Data System (ADS)

    Delatour, Vincent; Shekhar, Shashank; Reymann, Anne-Cécile; Didry, Dominique; Diêp Lê, Kim Hô; Romet-Lemonne, Guillaume; Helfer, Emmanuèle; Carlier, Marie-France

    2008-02-01

    The directed polymerization of a branched actin network against a functionalized surface drives cell protrusions and organelle propulsion in living cells. Solid microspheres or giant unilamellar vesicles, functionalized with neural Wiskott Aldrich syndrome protein (N-WASP), initiate the formation of a branched actin array using actin-related protein 2/3 (Arp2/3) complex, when placed in a motility assay reconstituted with pure proteins. These systems are useful biomimetic models of actin-based propulsion that allow to address how the interplay between the physical properties of the functionalized surface and the dynamics of the actin cytoskeleton determines motile behavior. Both solid beads and deformable vesicles display either continuous or saltatory propulsive motions, which are analyzed comparatively; we show that the deformability of liposomes and the mobility of N-WASP at the lipid surface affect the dynamic and structural parameters of the actin meshwork. Our results indicate that beads and vesicles use different mechanisms to translate insertional polymerization of actin at their surface into directed movement: stress relaxation within the actin gel prevents the accumulation of filaments at the front of moving beads, while segregation of nucleators reduces actin polymerization at the front of moving vesicles.

  5. Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy

    PubMed Central

    2013-01-01

    Background In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. Results We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. Conclusions Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities. PMID:23937664

  6. Arabidopsis CROOKED encodes for the smallest subunit of the ARP2/3 complex and controls cell shape by region specific fine F-actin formation.

    PubMed

    Mathur, Jaideep; Mathur, Neeta; Kirik, Victor; Kernebeck, Birgit; Srinivas, Bhylahalli Purushottam; Hülskamp, Martin

    2003-07-01

    The generation of a specific cell shape requires differential growth, whereby specific regions of the cell expand more relative to others. The Arabidopsis crooked mutant exhibits aberrant cell shapes that develop because of mis-directed expansion, especially during a rapid growth phase. GFP-aided visualization of the F-actin cytoskeleton and the behavior of subcellular organelles in different cell-types in crooked and wild-type Arabidopsis revealed that localized expansion is promoted in cellular regions with fine F-actin arrays but is restricted in areas that maintain dense F-actin. This suggested that a spatiotemporal distinction between fine versus dense F-actin in a growing cell could determine the final shape of the cell. CROOKED was molecularly identified as the plant homolog of ARPC5, the smallest sub-unit of the ARP2/3 complex that in other organisms is renowned for its role in creating dendritic arrays of fine F-actin. Rescue of crooked phenotype by the human ortholog provides the first molecular evidence for the presence and functional conservation of the complex in higher plants. Our cell-biological and molecular characterization of CROOKED suggests a general actin-based mechanism for regulating differential growth and generating cell shape diversity. PMID:12783786

  7. Formin' actin in the nucleus.

    PubMed

    Baarlink, Christian; Grosse, Robert

    2014-01-01

    Many if not most proteins can, under certain conditions, change cellular compartments, such as, for example, shuttling from the cytoplasm to the nucleus. Thus, many proteins may exert functions in various and very different subcellular locations, depending on the signaling context. A large amount of actin regulatory proteins has been detected in the mammalian cell nucleus, although their potential roles are much debated and are just beginning to emerge. Recently, members of the formin family of actin nucleators were also reported to dynamically localize to the nuclear environment. Here we discuss our findings that specific diaphanous-related formins can promote nuclear actin assembly in a signal-dependent manner. PMID:24637338

  8. Linker of nucleoskeleton and cytoskeleton (LINC) complex-mediated actin-dependent nuclear positioning orients centrosomes in migrating myoblasts

    PubMed Central

    Chang, Wakam; Antoku, Susumu; Östlund, Cecilia; Worman, Howard J; Gundersen, Gregg G

    2015-01-01

    Myoblast migration is essential for muscle development and repair; however, the factors that contribute to the polarity of migrating myoblasts are relatively unknown. We find that randomly migrating C2C12 myoblasts orient their centrosomes in the direction of migration. Using wounded monolayers, we further show that centrosome orientation is stimulated by the serum factor lysophosphatidic acid (LPA) and involves the rearward movement of the nucleus while the centrosome is maintained at the cell centroid. The rate of nuclear movement correlated with that of actin retrograde flow and both cytochalasin D and blebbistatin prevented nuclear movement and centrosome orientation. Actin-dependent rearward nuclear movement in fibroblasts is mediated by assembly of nuclear membrane nesprin-2G and SUN2 LINC complexes into transmembrane actin-associated nuclear (TAN) lines anchored by A-type lamins and emerin. In C2C12 myoblasts, depletion of nesprin-2G, SUN2 or lamin A/C prevented nuclear movement and endogenous nesprin-2G and a chimeric GFP-mini-nesprin-2G formed TAN lines during nuclear movement. Depleting nesprin-2G strongly interfered with directed cell migration and reduced the efficiency of myoblast fusion into multinucleated myotubes. Our results show that nuclear movement contributes to centrosome orientation and polarity for efficient migration and fusion of myoblasts. Given that mutations in the genes encoding A-type lamins, nesprin-2 and SUN2 cause Emery-Dreifuss muscular dystrophy and related myopathies, our results have implications for understanding the mechanism of disease pathogenesis. PMID:25587885

  9. Genetically encoded photoswitching of actin assembly through the Cdc42-WASP-Arp2/3 complex pathway

    PubMed Central

    Leung, Daisy W.; Otomo, Chinatsu; Chory, Joanne; Rosen, Michael K.

    2008-01-01

    General methods to engineer genetically encoded, reversible, light-mediated control over protein function would be useful in many areas of biomedical research and technology. We describe a system that yields such photo-control over actin assembly. We fused the Rho family GTPase Cdc42 in its GDP-bound form to the photosensory domain of phytochrome B (PhyB) and fused the Cdc42 effector, the Wiskott-Aldrich Syndrome Protein (WASP), to the light-dependent PhyB-binding domain of phytochrome interacting factor 3 (Pif3). Upon red light illumination, the fusion proteins bind each other, activating WASP, and consequently stimulating actin assembly by the WASP target, the Arp2/3 complex. Binding and WASP activation are reversed by far-red illumination. Our approach, in which the biochemical specificity of the nucleotide switch in Cdc42 is overridden by the light-dependent PhyB-Pif3 interaction, should be generally applicable to other GTPase-effector pairs. PMID:18728185

  10. The prototypical 4.1R-10-kDa domain and the 4.1g-10-kDa paralog mediate fodrin-actin complex formation.

    PubMed

    Kontrogianni-Konstantopoulos, A; Frye, C S; Benz, E J; Huang, S C

    2001-06-01

    A complex family of 4.1R isoforms has been identified in non-erythroid tissues. In this study we characterized the exonic composition of brain 4.1R-10-kDa or spectrin/actin binding (SAB) domain and identified the minimal sequences required to stimulate fodrin/F-actin association. Adult rat brain expresses predominantly 4.1R mRNAs that carry an extended SAB, consisting of the alternative exons 14/15/16 and part of the constitutive exon 17. Exon 16 along with sequences carried by exon 17 is necessary and sufficient to induce formation of fodrin-actin-4.1R ternary complexes. The ability of the respective SAB domains of 4.1 homologs to sediment fodrin/actin was also investigated. 4.1G-SAB stimulates association of fodrin/actin, although with an approximately 2-fold reduced efficiency compared with 4.1R-10-kDa, whereas 4.1N and 4.1B do not. Sequencing of the corresponding domains revealed that 4.1G-SAB carries a cassette that shares significant homology with 4.1R exon 16, whereas the respective sequence is divergent in 4.1N and absent from brain 4.1B. An approximately 150-kDa 4.1R and an approximately 160-kDa 4.1G isoforms are present in PC12 lysates that occur in vivo in a supramolecular complex with fodrin and F-actin. Moreover, proteins 4.1R and 4.1G are distributed underneath the plasma membrane in PC12 cells. Collectively, these observations suggest that brain 4.1R and 4.1G may modulate the membrane mechanical properties of neuronal cells by promoting fodrin/actin association. PMID:11274145

  11. Comprehensive analysis of motions in molecular dynamics trajectories of the actin capping protein and its inhibitor complexes.

    PubMed

    Koike, Ryotaro; Takeda, Shuichi; Maéda, Yuichiro; Ota, Motonori

    2016-07-01

    The actin capping protein (CP) binds to actin filaments to block further elongation. The capping activity is inhibited by proteins V-1 and CARMIL interacting with CP via steric and allosteric mechanisms, respectively. The crystal structures of free CP, CP/V-1, and CP/CARMIL complexes suggest that the binding of CARMIL alters the flexibility of CP rather than the overall structure of CP, and this is an allosteric inhibition mechanism. Here, we performed molecular dynamics (MD) simulations of CP in the free form, and in complex with CARMIL or V-1. The resulting trajectories were analyzed exhaustively using Motion Tree, which identifies various rigid-body motions ranging from small local motions to large domain motions. After enumerating all the motions, CP flexibilities with different ligands were characterized by a list of frequencies for 20 dominant rigid-body motions, some of which were not identified in previous studies. The comparative analysis highlights the influence of the binding of the CARMIL peptide to CP flexibility. In free CP and the CP/V-1 complex, domain motions around a large crevice between the N-stalk and the CP-S domain occur frequently. The CARMIL peptide binds the crevice and suppresses the motions effectively. In addition, the binding of the CARMIL peptide enhances and alters local motions around the pocket that participates in V-1 binding. These newly identified motions are likely to suppress the binding of V-1 to CP. The observed changes in CP motion provide insights that describe the mechanism of allosteric regulation by CARMIL through modulating CP flexibility. Proteins 2016; 84:948-956. © 2016 Wiley Periodicals, Inc. PMID:27028786

  12. GMFβ controls branched actin content and lamellipodial retraction in fibroblasts

    PubMed Central

    Haynes, Elizabeth M.; Asokan, Sreeja B.; King, Samantha J.; Johnson, Heath E.; Haugh, Jason M.

    2015-01-01

    The lamellipodium is an important structure for cell migration containing branched actin nucleated via the Arp2/3 complex. The formation of branched actin is relatively well studied, but less is known about its disassembly and how this influences migration. GMF is implicated in both Arp2/3 debranching and inhibition of Arp2/3 activation. Modulation of GMFβ, a ubiquitous GMF isoform, by depletion or overexpression resulted in changes in lamellipodial dynamics, branched actin content, and migration. Acute pharmacological inhibition of Arp2/3 by CK-666, coupled to quantitative live-cell imaging of the complex, showed that depletion of GMFβ decreased the rate of branched actin disassembly. These data, along with mutagenesis studies, suggest that debranching (not inhibition of Arp2/3 activation) is a primary activity of GMFβ in vivo. Furthermore, depletion or overexpression of GMFβ disrupted the ability of cells to directionally migrate to a gradient of fibronectin (haptotaxis). These data suggest that debranching by GMFβ plays an important role in branched actin regulation, lamellipodial dynamics, and directional migration. PMID:26101216

  13. Calcium influx through CRAC channels controls actin organization and dynamics at the immune synapse

    PubMed Central

    Hartzell, Catherine A; Jankowska, Katarzyna I; Burkhardt, Janis K; Lewis, Richard S

    2016-01-01

    T cell receptor (TCR) engagement opens Ca2+ release-activated Ca2+ (CRAC) channels and triggers formation of an immune synapse between T cells and antigen-presenting cells. At the synapse, actin reorganizes into a concentric lamellipod and lamella with retrograde actin flow that helps regulate the intensity and duration of TCR signaling. We find that Ca2+ influx is required to drive actin organization and dynamics at the synapse. Calcium acts by promoting actin depolymerization and localizing actin polymerization and the actin nucleation promotion factor WAVE2 to the periphery of the lamellipod while suppressing polymerization elsewhere. Ca2+-dependent retrograde actin flow corrals ER tubule extensions and STIM1/Orai1 complexes to the synapse center, creating a self-organizing process for CRAC channel localization. Our results demonstrate a new role for Ca2+ as a critical regulator of actin organization and dynamics at the synapse, and reveal potential feedback loops through which Ca2+ influx may modulate TCR signaling. DOI: http://dx.doi.org/10.7554/eLife.14850.001 PMID:27440222

  14. Calcium influx through CRAC channels controls actin organization and dynamics at the immune synapse.

    PubMed

    Hartzell, Catherine A; Jankowska, Katarzyna I; Burkhardt, Janis K; Lewis, Richard S

    2016-01-01

    T cell receptor (TCR) engagement opens Ca(2+) release-activated Ca(2+) (CRAC) channels and triggers formation of an immune synapse between T cells and antigen-presenting cells. At the synapse, actin reorganizes into a concentric lamellipod and lamella with retrograde actin flow that helps regulate the intensity and duration of TCR signaling. We find that Ca(2+) influx is required to drive actin organization and dynamics at the synapse. Calcium acts by promoting actin depolymerization and localizing actin polymerization and the actin nucleation promotion factor WAVE2 to the periphery of the lamellipod while suppressing polymerization elsewhere. Ca(2+)-dependent retrograde actin flow corrals ER tubule extensions and STIM1/Orai1 complexes to the synapse center, creating a self-organizing process for CRAC channel localization. Our results demonstrate a new role for Ca(2+) as a critical regulator of actin organization and dynamics at the synapse, and reveal potential feedback loops through which Ca(2+) influx may modulate TCR signaling. PMID:27440222

  15. Structure and Biochemical Properties of Fission Yeast Arp2/3 Complex Lacking the Arp2 Subunit

    SciTech Connect

    Nolen, B.; Pollard, T

    2008-01-01

    Arp2/3 (actin-related protein 2/3) complex is a seven-subunit complex that nucleates branched actin filaments in response to cellular signals. Nucleation-promoting factors such as WASp/Scar family proteins activate the complex by facilitating the activating conformational change and recruiting the first actin monomer for the daughter branch. Here we address the role of the Arp2 subunit in the function of Arp2/3 complex by isolating a version of the complex lacking Arp2 (Arp2? Arp2/3 complex) from fission yeast. An x-ray crystal structure of the ?Arp2 Arp2/3 complex showed that the rest of the complex is unperturbed by the loss of Arp2. However, the Arp2? Arp2/3 complex was inactive in actin nucleation assays, indicating that Arp2 is essential to form a branch. A fluorescence anisotropy assay showed that Arp2 does not contribute to the affinity of the complex for Wsp1-VCA, a Schizosaccharomyces pombe nucleation-promoting factor protein. Fluorescence resonance energy transfer experiments showed that the loss of Arp2 does not prevent VCA from recruiting an actin monomer to the complex. Truncation of the N terminus of ARPC5, the smallest subunit in the complex, increased the yield of Arp2? Arp2/3 complex during purification but did not compromise nucleation activity of the full Arp2/3 complex.

  16. Quantifying actin wave modulation on periodic topography

    NASA Astrophysics Data System (ADS)

    Guven, Can; Driscoll, Meghan; Sun, Xiaoyu; Parker, Joshua; Fourkas, John; Carlsson, Anders; Losert, Wolfgang

    2014-03-01

    Actin is the essential builder of the cell cytoskeleton, whose dynamics are responsible for generating the necessary forces for the formation of protrusions. By exposing amoeboid cells to periodic topographical cues, we show that actin can be directionally guided via inducing preferential polymerization waves. To quantify the dynamics of these actin waves and their interaction with the substrate, we modify a technique from computer vision called ``optical flow.'' We obtain vectors that represent the apparent actin flow and cluster these vectors to obtain patches of newly polymerized actin, which represent actin waves. Using this technique, we compare experimental results, including speed distribution of waves and distance from the wave centroid to the closest ridge, with actin polymerization simulations. We hypothesize the modulation of the activity of nucleation promotion factors on ridges (elevated regions of the surface) as a potential mechanism for the wave-substrate coupling. Funded by NIH grant R01GM085574.

  17. Collapsin Response Mediator Protein-1 Regulates Arp2/3-dependent Actin Assembly.

    PubMed

    Yu-Kemp, Hui-Chia; Brieher, William M

    2016-01-01

    Listeria monocytogenes is a bacterial parasite that uses host proteins to assemble an Arp2/3-dependent actin comet tail to power its movement through the host cell. Initiation of comet tail assembly is more efficient in cytosol than it is under defined conditions, indicating that unknown factors contribute to the reaction. We therefore fractionated cytosol and identified CRMP-1 as a factor that facilitates Arp2/3-dependent Listeria actin cloud formation in the presence of Arp2/3 and actin alone. It also scored as an important factor for Listeria actin comet tail formation in brain cytosol. CRMP-1 does not nucleate actin assembly on its own, nor does it directly activate the Arp2/3 complex. Rather, CRMP-1 scored as an auxiliary factor that promoted the ability of Listeria ActA protein to activate the Arp2/3 complex to trigger actin assembly. CRMP-1 is one member of a family of five related proteins that modulate cell motility in response to extracellular signals. Our results demonstrate an important role for CRMP-1 in Listeria actin comet tail formation and open the possibility that CRMP-1 controls cell motility by modulating Arp2/3 activation. PMID:26598519

  18. An Elmo–Dock complex locally controls Rho GTPases and actin remodeling during cadherin-mediated adhesion

    PubMed Central

    Collins, Caitlin

    2014-01-01

    Cell–cell contact formation is a dynamic process requiring the coordination of cadherin-based cell–cell adhesion and integrin-based cell migration. A genome-wide RNA interference screen for proteins required specifically for cadherin-dependent cell–cell adhesion identified an Elmo–Dock complex. This was unexpected as Elmo–Dock complexes act downstream of integrin signaling as Rac guanine-nucleotide exchange factors. In this paper, we show that Elmo2 recruits Dock1 to initial cell–cell contacts in Madin–Darby canine kidney cells. At cell–cell contacts, both Elmo2 and Dock1 are essential for the rapid recruitment and spreading of E-cadherin, actin reorganization, localized Rac and Rho GTPase activities, and the development of strong cell–cell adhesion. Upon completion of cell–cell adhesion, Elmo2 and Dock1 no longer localize to cell–cell contacts and are not required subsequently for the maintenance of cell–cell adhesion. These studies show that Elmo–Dock complexes are involved in both integrin- and cadherin-based adhesions, which may help to coordinate the transition of cells from migration to strong cell–cell adhesion. PMID:25452388

  19. Investigation of Water Nucleation on Designed Single Crystalline Oxide Surfaces - a Step Towards Understanding the Complex Behavior of Atmospheric Aerosols

    NASA Astrophysics Data System (ADS)

    Du, Y.; Wang, B.; Lyubinetsky, I.; Laskin, A.; Kulkarni, G.; Knopf, D. A.; Guenther, A. B.

    2014-12-01

    The interaction between water molecules and solid surfaces during water/ice nucleation is of extreme importance in physical, biological, geological, and environmental research. Aerosols in the atmosphere, including inorganic mineral dust particles and organic compounds from biogenic and anthropogenic sources, are recognized to be effective ice nuclei (IN) that lead to the formation of ice crystals. These ice crystals play important roles in climate through their interactions with solar and terrestrial radiation. However, a detailed understanding is hampered by the fact that the aerosols vary in size, chemical composition, morphology, crystal orientation, and local defects. In EMSL, some of those challenges can be addressed by utilizing state-of-the-art synthesis and characterization capabilities. By using molecular beam epitaxy (MBE), we are able to synthesis materials, such as TiO2 and Fe2O3, that are commonly found in mineral dusts in their single crystalline thin film form with controlled surface termination. Fundamental studies on these designed surfaces allow us to vary some of the variables independently so that a concrete cause and effect relationship can be established. In this study, we grow epitaxial rutile TiO2(110) and anatase TiO2(001) films and investigate water adsorption and water nucleation processes on these surfaces by ultra-high vacuum scanning tunneling microscopy (STM) and environmental scanning electron microscopy (E-SEM). The surface reaction dynamics revealed can be of critical importance in understanding the water/ice nucleation process on complex aerosols.

  20. Platelet actin nodules are podosome-like structures dependent on Wiskott-Aldrich syndrome protein and ARP2/3 complex.

    PubMed

    Poulter, Natalie S; Pollitt, Alice Y; Davies, Amy; Malinova, Dessislava; Nash, Gerard B; Hannon, Mike J; Pikramenou, Zoe; Rappoport, Joshua Z; Hartwig, John H; Owen, Dylan M; Thrasher, Adrian J; Watson, Stephen P; Thomas, Steven G

    2015-01-01

    The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott-Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp(-/-) mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet-platelet interaction and their absence contributes to the bleeding diathesis of Wiskott-Aldrich syndrome. PMID:26028144

  1. Association of hepatitis C virus replication complexes with microtubules and actin filaments is dependent on the interaction of NS3 and NS5A.

    PubMed

    Lai, Chao-Kuen; Jeng, King-Song; Machida, Keigo; Lai, Michael M C

    2008-09-01

    The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A. PMID:18562541

  2. Platelet actin nodules are podosome-like structures dependent on Wiskott–Aldrich syndrome protein and ARP2/3 complex

    PubMed Central

    Poulter, Natalie S.; Pollitt, Alice Y.; Davies, Amy; Malinova, Dessislava; Nash, Gerard B.; Hannon, Mike J.; Pikramenou, Zoe; Rappoport, Joshua Z.; Hartwig, John H.; Owen, Dylan M.; Thrasher, Adrian J.; Watson, Stephen P.; Thomas, Steven G.

    2015-01-01

    The actin nodule is a novel F-actin structure present in platelets during early spreading. However, only limited detail is known regarding nodule organization and function. Here we use electron microscopy, SIM and dSTORM super-resolution, and live-cell TIRF microscopy to characterize the structural organization and signalling pathways associated with nodule formation. Nodules are composed of up to four actin-rich structures linked together by actin bundles. They are enriched in the adhesion-related proteins talin and vinculin, have a central core of tyrosine phosphorylated proteins and are depleted of integrins at the plasma membrane. Nodule formation is dependent on Wiskott–Aldrich syndrome protein (WASp) and the ARP2/3 complex. WASp−/− mouse blood displays impaired platelet aggregate formation at arteriolar shear rates. We propose actin nodules are platelet podosome-related structures required for platelet–platelet interaction and their absence contributes to the bleeding diathesis of Wiskott–Aldrich syndrome. PMID:26028144

  3. Cortactin promotes exosome secretion by controlling branched actin dynamics.

    PubMed

    Sinha, Seema; Hoshino, Daisuke; Hong, Nan Hyung; Kirkbride, Kellye C; Grega-Larson, Nathan E; Seiki, Motoharu; Tyska, Matthew J; Weaver, Alissa M

    2016-07-18

    Exosomes are extracellular vesicles that influence cellular behavior and enhance cancer aggressiveness by carrying bioactive molecules. The mechanisms that regulate exosome secretion are poorly understood. Here, we show that the actin cytoskeletal regulatory protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in cancer cells leads to a respective decrease or increase in exosome secretion, without altering exosome cargo content. Live-cell imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular late endosomes (MVEs). Regulation of exosome secretion by cortactin requires binding to the branched actin nucleating Arp2/3 complex and to actin filaments. Furthermore, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin MVE docking sites and exosome secretion. Functionally, the addition of purified exosomes to cortactin-knockdown cells rescued defects of those cells in serum-independent growth and invasion. These data suggest a model in which cortactin promotes exosome secretion by stabilizing cortical actin-rich MVE docking sites. PMID:27402952

  4. Actinic keratosis

    MedlinePlus

    ... example, if you work outdoors) Had many severe sunburns early in life Are older Symptoms Actinic keratosis ... and tanning salons. Other things to know about sun exposure: Sun exposure is stronger in or near surfaces ...

  5. Actinic Cheilitis

    MedlinePlus

    ... is a precancerous condition related to cumulative lifetime sun exposure. The lower lip is most often affected. Individuals ... Wearing barrier clothing (eg, wide-brimmed hats) and sunscreen-containing lip balms can aid in preventing actinic ...

  6. Molecular Architecture of Synaptic Actin Cytoskeleton in Hippocampal Neurons Reveals a Mechanism of Dendritic Spine Morphogenesis

    PubMed Central

    Korobova, Farida

    2010-01-01

    Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility. PMID:19889835

  7. Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization.

    PubMed

    Johansen, Jesper; Alfaro, Gabriel; Beh, Christopher T

    2016-08-01

    Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott-Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization. PMID:27526190

  8. Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization

    PubMed Central

    Johansen, Jesper; Alfaro, Gabriel; Beh, Christopher T.

    2016-01-01

    Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott—Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization. PMID:27526190

  9. Regimes of wave type patterning driven by refractory actin feedback: transition from static polarization to dynamic wave behaviour.

    PubMed

    Holmes, W R; Carlsson, A E; Edelstein-Keshet, L

    2012-08-01

    Patterns of waves, patches, and peaks of actin are observed experimentally in many living cells. Models of this phenomenon have been based on the interplay between filamentous actin (F-actin) and its nucleation promoting factors (NPFs) that activate the Arp2/3 complex. Here we present an alternative biologically-motivated model for F-actin-NPF interaction based on properties of GTPases acting as NPFs. GTPases (such as Cdc42, Rac) are known to promote actin nucleation, and to have active membrane-bound and inactive cytosolic forms. The model is a natural extension of a previous mathematical mini-model of small GTPases that generates static cell polarization. Like other modellers, we assume that F-actin negative feedback shapes the observed patterns by suppressing the trailing edge of NPF-generated wave-fronts, hence localizing the activity spatially. We find that our NPF-actin model generates a rich set of behaviours, spanning a transition from static polarization to single pulses, reflecting waves, wave trains, and oscillations localized at the cell edge. The model is developed with simplicity in mind to investigate the interaction between nucleation promoting factor kinetics and negative feedback. It explains distinct types of pattern initiation mechanisms, and identifies parameter regimes corresponding to distinct behaviours. We show that weak actin feedback yields static patterning, moderate feedback yields dynamical behaviour such as travelling waves, and strong feedback can lead to wave trains or total suppression of patterning. We use a recently introduced nonlinear bifurcation analysis to explore the parameter space of this model and predict its behaviour with simulations validating those results. PMID:22785332

  10. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization

    PubMed Central

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2016-01-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis. PMID:25664724

  11. Dynamic Interplay of Smooth Muscle α-Actin Gene-Regulatory Proteins Reflects the Biological Complexity of Myofibroblast Differentiation

    PubMed Central

    Strauch, Arthur Roger; Hariharan, Seethalakshmi

    2013-01-01

    Myofibroblasts (MFBs) are smooth muscle-like cells that provide contractile force required for tissue repair during wound healing. The leading agonist for MFB differentiation is transforming growth factor β1 (TGFβ1) that induces transcription of genes encoding smooth muscle α-actin (SMαA) and interstitial collagen that are markers for MFB differentiation. TGFβ1 augments activation of Smad transcription factors, pro-survival Akt kinase, and p38 MAP kinase as well as Wingless/int (Wnt) developmental signaling. These actions conspire to activate β-catenin needed for expression of cyclin D, laminin, fibronectin, and metalloproteinases that aid in repairing epithelial cells and their associated basement membranes. Importantly, β-catenin also provides a feed-forward stimulus that amplifies local TGFβ1 autocrine/paracrine signaling causing transition of mesenchymal stromal cells, pericytes, and epithelial cells into contractile MFBs. Complex, mutually interactive mechanisms have evolved that permit several mammalian cell types to activate the SMαA promoter and undergo MFB differentiation. These molecular controls will be reviewed with an emphasis on the dynamic interplay between serum response factor, TGFβ1-activated Smads, Wnt-activated β-catenin, p38/calcium-activated NFAT protein, and the RNA-binding proteins, Purα, Purβ, and YB-1, in governing transcriptional and translational control of the SMαA gene in injury-activated MFBs. PMID:24832798

  12. Saccharomyces cerevisiae Kelch Proteins and Bud14 Protein Form a Stable 520-kDa Formin Regulatory Complex That Controls Actin Cable Assembly and Cell Morphogenesis*

    PubMed Central

    Gould, Christopher J.; Chesarone-Cataldo, Melissa; Alioto, Salvatore L.; Salin, Bénédicte; Sagot, Isabelle; Goode, Bruce L.

    2014-01-01

    Formins perform essential roles in actin assembly and organization in vivo, but they also require tight regulation of their activities to produce properly functioning actin structures. Saccharomyces cerevisiae Bud14 is one member of an emerging class of formin regulators that target the FH2 domain to inhibit actin polymerization, but little is known about how these regulators are themselves controlled in vivo. Kelch proteins are critical for cell polarity and morphogenesis in a wide range of organisms, but their mechanistic roles in these processes are still largely undefined. Here, we report that S. cerevisiae Kelch proteins, Kel1 and Kel2, associate with Bud14 in cell extracts to form a stable 520-kDa complex with an apparent stoichiometry of 2:2:1 Bud14/Kel1/Kel2. Using pairwise combinations of GFP- and red fluorescent protein-tagged proteins, we show that Kel1, Kel2, and Bud14 interdependently co-localize at polarity sites. By analyzing single, double, and triple mutants, we show that Kel1 and Kel2 function in the same pathway as Bud14 in regulating Bnr1-mediated actin cable formation. Loss of any component of the complex results in long, bent, and hyper-stable actin cables, accompanied by defects in secretory vesicle traffic during polarized growth and septum formation during cytokinesis. These observations directly link S. cerevisiae Kelch proteins to the control of formin activity, and together with previous observations made for S. pombe homologues tea1p and tea3p, they have broad implications for understanding Kelch function in other systems. PMID:24828508

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

    PubMed

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

    2012-06-01

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

  14. Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.

    PubMed Central

    Prendergast, G C; Davide, J P; deSolms, S J; Giuliani, E A; Graham, S L; Gibbs, J B; Oliff, A; Kohl, N E

    1994-01-01

    A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin. Images PMID:8196657

  15. Profilin connects actin assembly with microtubule dynamics.

    PubMed

    Nejedla, Michaela; Sadi, Sara; Sulimenko, Vadym; de Almeida, Francisca Nunes; Blom, Hans; Draber, Pavel; Aspenström, Pontus; Karlsson, Roger

    2016-08-01

    Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro-tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element. PMID:27307590

  16. Actinic reticuloid

    SciTech Connect

    Marx, J.L.; Vale, M.; Dermer, P.; Ragaz, A.; Michaelides, P.; Gladstein, A.H.

    1982-09-01

    A 58-year-old man has his condition diagnosed as actinic reticuloid on the basis of clinical and histologic findings and phototesting data. He had clinical features resembling mycosis fungoides in light-exposed areas. Histologic findings disclosed a bandlike infiltrate with atypical mononuclear cells in the dermis and scattered atypical cells in the epidermis. Electron microscopy disclosed mononuclear cells with bizarre, convoluted nuclei, resembling cerebriform cells of Lutzner. Phototesting disclosed a diminished minimal erythemal threshold to UV-B and UV-A. Microscopic changes resembling actinic reticuloid were reproduced in this patient 24 and 72 hours after exposure to 15 minimal erythemal doses of UV-B.

  17. Mesoscopic model of actin-based propulsion.

    PubMed

    Zhu, Jie; Mogilner, Alex

    2012-01-01

    Two theoretical models dominate current understanding of actin-based propulsion: microscopic polymerization ratchet model predicts that growing and writhing actin filaments generate forces and movements, while macroscopic elastic propulsion model suggests that deformation and stress of growing actin gel are responsible for the propulsion. We examine both experimentally and computationally the 2D movement of ellipsoidal beads propelled by actin tails and show that neither of the two models can explain the observed bistability of the orientation of the beads. To explain the data, we develop a 2D hybrid mesoscopic model by reconciling these two models such that individual actin filaments undergoing nucleation, elongation, attachment, detachment and capping are embedded into the boundary of a node-spring viscoelastic network representing the macroscopic actin gel. Stochastic simulations of this 'in silico' actin network show that the combined effects of the macroscopic elastic deformation and microscopic ratchets can explain the observed bistable orientation of the actin-propelled ellipsoidal beads. To test the theory further, we analyze observed distribution of the curvatures of the trajectories and show that the hybrid model's predictions fit the data. Finally, we demonstrate that the model can explain both concave-up and concave-down force-velocity relations for growing actin networks depending on the characteristic time scale and network recoil. To summarize, we propose that both microscopic polymerization ratchets and macroscopic stresses of the deformable actin network are responsible for the force and movement generation. PMID:23133366

  18. Steady-state nuclear actin levels are determined by export competent actin pool.

    PubMed

    Skarp, Kari-Pekka; Huet, Guillaume; Vartiainen, Maria K

    2013-10-01

    A number of studies in the last decade have irrevocably promoted actin into a fully fledged member of the nuclear compartment, where it, among other crucial tasks, facilitates transcription and chromatin remodeling. Changes in nuclear actin levels have been linked to different cellular processes: decreased nuclear actin to quiescence and increased nuclear actin to differentiation. Importin 9 and exportin 6 transport factors are responsible for the continuous nucleocytoplasmic shuttling of actin, but the mechanisms, which result in modulated actin levels, have not been characterized. We find that in cells growing under normal growth conditions, the levels of nuclear actin vary considerably from cell to cell. To understand the basis for this, we have extensively quantified several cellular parameters while at the same time recording the import and export rates of green fluorescent protein (GFP)-tagged actin. Surprisingly, our dataset shows that the ratio of nuclear to cytoplasmic fluorescence intensity, but not nuclear shape, size, cytoplasm size, or their ratio, correlates negatively with both import and export rate of actin. This suggests that high-nuclear actin content is maintained by both diminished import and export. The high nuclear actin containing cells still show high mobility of actin, but it is not export competent, suggesting increased binding of actin to nuclear complexes. Creation of such export incompetent actin pool would ensure enough actin is retained in the nucleus and make it available for the various nuclear functions described for actin. PMID:23749625

  19. Ldb1-nucleated transcription complexes function as primary mediators of global erythroid gene activation.

    PubMed

    Li, LiQi; Freudenberg, Johannes; Cui, Kairong; Dale, Ryan; Song, Sang-Hyun; Dean, Ann; Zhao, Keji; Jothi, Raja; Love, Paul E

    2013-05-30

    Erythropoiesis is dependent on the lineage-specific transcription factors Gata1, Tal1, and Klf1. Several erythroid genes have been shown to require all 3 factors for their expression, suggesting that they function synergistically; however, there is little direct evidence for widespread cooperation. Gata1 and Tal1 can assemble within higher-order protein complexes (Ldb1 complexes) that include the adapter molecules Lmo2 and Ldb1. Ldb1 proteins are capable of coassociation, and long-range Ldb1-mediated oligomerization of enhancer- and promoter-bound Ldb1 complexes has been shown to be required for β-globin gene expression. In this study, we generated a genomewide map of Ldb1 complex binding sites that revealed widespread binding at erythroid genes and at known erythroid enhancer elements. Ldb1 complex binding sites frequently colocalized with Klf1 binding sites and with consensus binding motifs for other erythroid transcription factors. Transcriptomic analysis demonstrated a strong correlation between Ldb1 complex binding and Ldb1 dependency for gene expression and identified a large cohort of genes coregulated by Ldb1 complexes and Klf1. Together, these results provide a foundation for defining the mechanism and scope of Ldb1 complex activity during erythropoiesis. PMID:23610375

  20. Complex Nucleation Process of Large North Chile Earthquakes, Implications for Early Warning Systems

    NASA Astrophysics Data System (ADS)

    Ruiz, S.; Meneses, G.; Sobiesiak, M.; Madariaga, R. I.

    2014-12-01

    We studied the nucleation process of Northern Chile events that included the large earthquakes of Tocopilla 2007 Mw 7.8 and Iquique 2014 Mw 8.1, as well as the background seismicity recorded from 2011 to 2013 by the ILN temporary network and the IPOC and CSN permanent networks. We built our catalogue of 393 events starting from the CSN catalogue, which has a completeness of magnitude Mw > 3.0 in Northern Chile. We re-located and computed moment magnitude for each event. We also computed Early Warning (EW) parameters - Pd, Pv, τc and IV2 - for each event including 13 earthquakes of Mw>6.0 that occurred between 2007-2012. We also included part of the seismicity from March-April 2014 period. We find that Pd, Pv and IV2 are good estimators of magnitude for interplate thrust and intraplate intermediate depth events with Mw between 4.0 and 6.0. However, the larger magnitude events show a saturation of the EW parameters. The Tocopilla 2007 and Iquique 2014 earthquake sequences were studied in detail. Almost all events with Mw>6.0 present precursory signals so that the largest amplitudes occur several seconds after the first P wave arrival. The recent Mw 8.1 Iquique 2014 earthquake was preceded by low amplitude P waves for 20 s before the main asperity was broken. The magnitude estimation can improve if we consider longer P wave windows in the estimation of EW parameters. There was, however, a practical limit during the Iquique earthquake because the first S waves arrived before the arrival of the P waves from the main rupture. The 4 s P-wave Pd parameter estimated Mw 7.1 for the Mw 8.1 Iquique 2014 earthquake and Mw 7.5 for the Mw 7.8 Tocopilla 2007 earthquake.

  1. 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

  2. Role of G protein signaling in the formation of the fibrin(ogen)-integrin αIIbβ3-actin cytoskeleton complex in platelets.

    PubMed

    Budnik, Ivan; Shenkman, Boris; Savion, Naphtali

    2016-09-01

    Effective platelet function requires formation of a physical link between fibrin(ogen), integrin αIIbβ3, and cytoplasmic actin filaments. We investigated the role of the Gαq, Gαi, and Gα12/13 families of heterotrimeric GTP-binding proteins (G proteins) in the assembly of a ligand-αIIbβ3-actin cytoskeleton complex. Selective and combined activation of the G proteins was achieved by using combinations of various platelet agonists and inhibitors. Formation and stability of fibrinogen-αIIbβ3 interaction were evaluated by the extent of platelet aggregation and the rate of eptifibatide-induced platelet disaggregation; association of αIIbβ3 with the cytoskeleton was analyzed by western blot. Formation of the fibrin-αIIbβ3-actin cytoskeleton complex was evaluated by rotational thromboelastometry assay in which clot formation was induced by the mixture of reptilase and factor XIIIa. We demonstrated that involvement of heterotrimeric G proteins in the formation of the ligand-αIIbβ3-cytoskeleton complex depends on whether fibrinogen or fibrin serves as the integrin ligand. Formation of the fibrinogen-αIIbβ3-cytoskeleton complex requires combined activation of at least two G protein pathways while the maximal αIIbβ3-cytoskeleton association and the strongest αIIbβ3-fibrinogen binding supporting irreversible platelet aggregation require combined activation of all three-Gαq, Gαi, and Gα12/13-G protein families. In contrast, formation of the fibrin-αIIbβ3-cytoskeleton complex mediating clot retraction is critically dependent on the activation of the Gαi family, especially on the activation of Gαz. PMID:27026498

  3. De novo actin polymerization is required for model Hirano body formation in Dictyostelium

    PubMed Central

    Dong, Yun; Shahid-Salles, Sonbol; Sherling, Dan; Fechheimer, Nathan; Iyer, Nathan; Wells, Lance; Fechheimer, Marcus

    2016-01-01

    ABSTRACT Hirano bodies are eosinophilic, actin-rich inclusions found in autopsied brains in numerous neurodegenerative diseases. The mechanism of Hirano body formation is unknown. Mass spectrometry analysis was performed to identify proteins from partially purified model Hirano bodies from Dictyostelium. This analysis identified proteins primarily belonging to ribosomes, proteasomes, mitochondria and cytoskeleton. Profilin, Arp/2/3 and WASH identified by mass spectrometry were found to colocalise with model Hirano bodies. Due to their roles in actin regulation, we selected these proteins for further investigation. Inhibition of the Arp2/3 complex by CK666 prevented formation of model Hirano bodies. Since Arp2/3 activation occurs via the WASH or WAVE complex, we next investigated how these proteins affect Hirano body formation. Whereas model Hirano bodies could form in WASH-deficient cells, they failed to form in cells lacking HSPC300, a member of the WAVE complex. We identified other proteins required for Hirano body formation that include profilin and VASP, an actin nucleation factor. In the case of VASP, both its G- and F-actin binding domains were required for model Hirano body formation. Collectively, our results indicate that de novo actin polymerization is required to form model Hirano bodies. PMID:27215322

  4. De novo actin polymerization is required for model Hirano body formation in Dictyostelium.

    PubMed

    Dong, Yun; Shahid-Salles, Sonbol; Sherling, Dan; Fechheimer, Nathan; Iyer, Nathan; Wells, Lance; Fechheimer, Marcus; Furukawa, Ruth

    2016-01-01

    Hirano bodies are eosinophilic, actin-rich inclusions found in autopsied brains in numerous neurodegenerative diseases. The mechanism of Hirano body formation is unknown. Mass spectrometry analysis was performed to identify proteins from partially purified model Hirano bodies from Dictyostelium This analysis identified proteins primarily belonging to ribosomes, proteasomes, mitochondria and cytoskeleton. Profilin, Arp/2/3 and WASH identified by mass spectrometry were found to colocalise with model Hirano bodies. Due to their roles in actin regulation, we selected these proteins for further investigation. Inhibition of the Arp2/3 complex by CK666 prevented formation of model Hirano bodies. Since Arp2/3 activation occurs via the WASH or WAVE complex, we next investigated how these proteins affect Hirano body formation. Whereas model Hirano bodies could form in WASH-deficient cells, they failed to form in cells lacking HSPC300, a member of the WAVE complex. We identified other proteins required for Hirano body formation that include profilin and VASP, an actin nucleation factor. In the case of VASP, both its G- and F-actin binding domains were required for model Hirano body formation. Collectively, our results indicate that de novo actin polymerization is required to form model Hirano bodies. PMID:27215322

  5. Major Histocompatibility Complex Class II Inhibits Fas Antigen-Mediated Gastric Mucosal Cell Apoptosis through Actin-Dependent Inhibition of Receptor Aggregation

    PubMed Central

    Stoicov, Calin; Cai, Xun; Li, Hanchen; Klucevsek, Kristine; Carlson, Jane; Saffari, Reza; Houghton, JeanMarie

    2005-01-01

    Escape from normal apoptotic controls is thought to be essential for the development of cancer. During Helicobacter pylori infection, the leading cause of gastric cancer, activation of the Fas antigen (Fas Ag) apoptotic pathway is responsible for early atrophy and tissue loss. As disease progresses, metaplastic and dysplastic glands arise which express Fas Ag but are resistant to apoptosis and are believed to be the precursor cells for adenocarcinoma. In this report, we show that one mechanism of acquired Fas resistance is inhibition of receptor aggregation via a major histocompatibility complex class II (MHCII)-mediated, actin-dependent mechanism. For these studies we used the well-described C57BL/6 mouse model of Helicobacter pylori and Helicobacter felis infection. Under normal conditions, Fas Ag is expressed at low levels, and MHCII expression on gastric mucosal cells is negligible. With infection and inflammation, both receptors are upregulated, and 6.1% of gastric mucosal cells express MHCII in combination with Fas Ag. Using the rat gastric mucosal cell line RGM-1 transfected with murine Fas Ag and MHCIIαβ chains, we demonstrate that MHCII prevents Fas receptor aggregation and inhibits Fas-mediated signaling through its effects on the actin cytoskeleton. Depolymerization of actin with cytochalasin D allows receptors to aggregate and restores Fas sensitivity. These findings offer one mechanism by which gastric mucosal cells acquire Fas resistance. PMID:16177302

  6. A Pan1/End3/Sla1 complex links Arp2/3-mediated actin assembly to sites of clathrin-mediated endocytosis

    PubMed Central

    Sun, Yidi; Leong, Nicole T.; Wong, Tiffany; Drubin, David G.

    2015-01-01

    More than 60 highly conserved proteins appear sequentially at sites of clathrin-mediated endocytosis in yeast and mammals. The yeast Eps15-related proteins Pan1 and End3 and the CIN85-related protein Sla1 are known to interact with each other in vitro, and they all appear after endocytic-site initiation but before endocytic actin assembly, which facilitates membrane invagination/scission. Here we used live-cell imaging in parallel with genetics and biochemistry to explore comprehensively the dynamic interactions and functions of Pan1, End3, and Sla1. Our results indicate that Pan1 and End3 associate in a stable manner and appear at endocytic sites before Sla1. The End3 C-terminus is necessary and sufficient for its cortical localization via interaction with Pan1, whereas the End3 N-terminus plays a crucial role in Sla1 recruitment. We systematically examined the dynamic behaviors of endocytic proteins in cells in which Pan1 and End3 were simultaneously eliminated, using the auxin-inducible degron system. The results lead us to propose that endocytic-site initiation and actin assembly are separable processes linked by a Pan1/End3/Sla1 complex. Finally, our study provides mechanistic insights into how Pan1 and End3 function with Sla1 to coordinate cargo capture with actin assembly. PMID:26337384

  7. Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics.

    PubMed

    Roybal, Kole T; Buck, Taráz E; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J; Ambler, Rachel; Tunbridge, Helen M; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F

    2016-01-01

    Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems. PMID:27095595

  8. Diffusion, capture and recycling of SCAR/WAVE and Arp2/3 complexes observed in cells by single-molecule imaging.

    PubMed

    Millius, Arthur; Watanabe, Naoki; Weiner, Orion D

    2012-03-01

    The SCAR/WAVE complex drives lamellipodium formation by enhancing actin nucleation by the Arp2/3 complex. Phosphoinositides and Rac activate the SCAR/WAVE complex, but how SCAR/WAVE and Arp2/3 complexes converge at sites of nucleation is unknown. We analyzed the single-molecule dynamics of WAVE2 and p40 (subunits of the SCAR/WAVE and Arp2/3 complexes, respectively) in XTC cells. We observed lateral diffusion of both proteins and captured the transition of p40 from diffusion to network incorporation. These results suggest that a diffusive 2D search facilitates binding of the Arp2/3 complex to actin filaments necessary for nucleation. After nucleation, the Arp2/3 complex integrates into the actin network and undergoes retrograde flow, which results in its broad distribution throughout the lamellipodium. By contrast, the SCAR/WAVE complex is more restricted to the cell periphery. However, with single-molecule imaging, we also observed WAVE2 molecules undergoing retrograde motion. WAVE2 and p40 have nearly identical speeds, lifetimes and sites of network incorporation. Inhibition of actin retrograde flow does not prevent WAVE2 association and disassociation with the membrane but does inhibit WAVE2 removal from the actin cortex. Our results suggest that membrane binding and diffusion expedites the recruitment of nucleation factors to a nucleation site independent of actin assembly, but after network incorporation, ongoing actin polymerization facilitates recycling of SCAR/WAVE and Arp2/3 complexes. PMID:22349699

  9. A stable transcription factor complex nucleated by oligomeric AML1–ETO controls leukaemogenesis

    SciTech Connect

    Sun, Xiao-Jian; Wang, Zhanxin; Wang, Lan; Jiang, Yanwen; Kost, Nils; Soong, T. David; Chen, Wei-Yi; Tang, Zhanyun; Nakadai, Tomoyoshi; Elemento, Olivier; Fischle, Wolfgang; Melnick, Ari; Patel, Dinshaw J.; Nimer, Stephen D.; Roeder, Robert G.

    2013-06-30

    Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression. AML1–ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia, is a transcription factor implicated in both gene repression and activation. AML1–ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis, making it important to identify co-regulatory factors that ‘read’ the NHR2 oligomerization and contribute to leukaemogenesis. Here we show that, in human leukaemic cells, AML1–ETO resides in and functions through a stable AML1–ETO-containing transcription factor complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1–ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2–N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1–ETO-induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1–ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukaemia.

  10. Polycystin-1 regulates actin cytoskeleton organization and directional cell migration through a novel PC1-Pacsin 2-N-Wasp complex.

    PubMed

    Yao, Gang; Su, Xuefeng; Nguyen, Vy; Roberts, Kristina; Li, Xiaogang; Takakura, Ayumi; Plomann, Markus; Zhou, Jing

    2014-05-15

    How epithelial cells form a tubule with defined length and lumen diameter remains a fundamental question in cell and developmental biology. Loss of control of tubule lumen size in multiple organs including the kidney, liver and pancreas features polycystic kidney disease (PKD). To gain insights into autosomal dominant polycystic kidney disease, we performed yeast two-hybrid screens using the C-terminus of polycystin-1 (PC1) as bait. Here, we report that PC1 interacts with Pacsin 2, a cytoplasmic phosphoprotein that has been implicated in cytoskeletal organization, vesicle trafficking and more recently in cell intercalation during gastrulation. PC1 binds to a 107-residue fragment containing the α3 helix of the F-BAR domain of Pacsin 2 via a coiled-coil domain in its C-tail. PC1 and Pacsin 2 co-localize on the lamellipodia of migrating kidney epithelial cells. PC1 and Pacsin 2-deficient kidney epithelial cells migrate at a slower speed with reduced directional persistency. We further demonstrate that PC1, Pacsin 2 and N-Wasp are in the same protein complex, and both PC1 and Pacsin 2 are required for N-Wasp/Arp2/3-dependent actin remodeling. We propose that PC1 modulates actin cytoskeleton rearrangements and directional cell migration through the Pacsin 2/N-Wasp/Arp2/3 complex, which consequently contributes to the establishment and maintenance of the sophisticated tubular architecture. Disruption of this complex contributes to cyst formation in PKD. PMID:24385601

  11. Heterogeneous nucleation or homogeneous nucleation?

    NASA Astrophysics Data System (ADS)

    Liu, X. Y.

    2000-06-01

    The generic heterogeneous effect of foreign particles on three dimensional nucleation was examined both theoretically and experimentally. It shows that the nucleation observed under normal conditions includes a sequence of progressive heterogeneous processes, characterized by different interfacial correlation function f(m,x)s. At low supersaturations, nucleation will be controlled by the process with a small interfacial correlation function f(m,x), which results from a strong interaction and good structural match between the foreign bodies and the crystallizing phase. At high supersaturations, nucleation on foreign particles having a weak interaction and poor structural match with the crystallizing phase (f(m,x)→1) will govern the kinetics. This frequently leads to the false identification of homogeneous nucleation. Genuine homogeneous nucleation, which is the up-limit of heterogeneous nucleation, may not be easily achievable under gravity. In order to check these results, the prediction is confronted with nucleation experiments of some organic and inorganic crystals. The results are in excellent agreement with the theory.

  12. D-AKAP2:PKA RII:PDZK1 ternary complex structure: insights from the nucleation of a polyvalent scaffold.

    PubMed

    Sarma, Ganapathy N; Moody, Issa S; Ilouz, Ronit; Phan, Ryan H; Sankaran, Banumathi; Hall, Randy A; Taylor, Susan S

    2015-01-01

    A-kinase anchoring proteins (AKAPs) regulate cAMP-dependent protein kinase (PKA) signaling in space and time. Dual-specific AKAP2 (D-AKAP2/AKAP10) binds with high affinity to both RI and RII regulatory subunits of PKA and is anchored to transporters through PDZ domain proteins. Here, we describe a structure of D-AKAP2 in complex with two interacting partners and the exact mechanism by which a segment that on its own is disordered presents an α-helix to PKA and a β-strand to PDZK1. These two motifs nucleate a polyvalent scaffold and show how PKA signaling is linked to the regulation of transporters. Formation of the D-AKAP2: PKA binary complex is an important first step for high affinity interaction with PDZK1, and the structure reveals important clues toward understanding this phenomenon. In contrast to many other AKAPs, D-AKAP2 does not interact directly with the membrane protein. Instead, the interaction is facilitated by the C-terminus of D-AKAP2, which contains two binding motifs-the D-AKAP2AKB and the PDZ motif-that are joined by a short linker and only become ordered upon binding to their respective partner signaling proteins. The D-AKAP2AKB binds to the D/D domain of the R-subunit and the C-terminal PDZ motif binds to a PDZ domain (from PDZK1) that serves as a bridging protein to the transporter. This structure also provides insights into the fundamental question of why D-AKAP2 would exhibit a differential mode of binding to the two PKA isoforms. PMID:25348485

  13. CLIC5 Stabilizes Membrane-Actin Filament Linkages at the Base of Hair Cell Stereocilia in a Molecular Complex with Radixin, Taperin, and Myosin VI

    PubMed Central

    Salles, Felipe T.; Andrade, Leonardo R.; Tanda, Soichi; Grati, M’hamed; Plona, Kathleen L.; Gagnon, Leona H.; Johnson, Kenneth R.; Kachar, Bechara; Berryman, Mark A.

    2015-01-01

    Chloride intracellular channel 5 protein (CLIC5) was originally isolated from microvilli in complex with actin binding proteins including ezrin, a member of the Ezrin-Radixin-Moesin (ERM) family of membrane-cytoskeletal linkers. CLIC5 concentrates at the base of hair cell stereocilia and is required for normal hearing and balance in mice, but its functional significance is poorly understood. This study investigated the role of CLIC5 in postnatal development and maintenance of hair bundles. Confocal and scanning electron microscopy of CLIC5-deficient jitterbug (jbg) mice revealed progressive fusion of stereocilia as early as postnatal day 10. Radixin (RDX), protein tyrosine phosphatase receptor Q (PTPRQ), and taperin (TPRN), deafness-associated proteins that also concentrate at the base of stereocilia, were mislocalized in fused stereocilia of jbg mice. TPRQ and RDX were dispersed even prior to stereocilia fusion. Biochemical assays showed interaction of CLIC5 with ERM proteins, TPRN, and possibly myosin VI (MYO6). In addition, CLIC5 and RDX failed to localize normally in fused stereocilia of MYO6 mutant mice. Based on these findings, we propose a model in which these proteins work together as a complex to stabilize linkages between the plasma membrane and subjacent actin cytoskeleton at the base of stereocilia. PMID:24285636

  14. CLIC5 stabilizes membrane-actin filament linkages at the base of hair cell stereocilia in a molecular complex with radixin, taperin, and myosin VI.

    PubMed

    Salles, Felipe T; Andrade, Leonardo R; Tanda, Soichi; Grati, M'hamed; Plona, Kathleen L; Gagnon, Leona H; Johnson, Kenneth R; Kachar, Bechara; Berryman, Mark A

    2014-01-01

    Chloride intracellular channel 5 protein (CLIC5) was originally isolated from microvilli in complex with actin binding proteins including ezrin, a member of the Ezrin-Radixin-Moesin (ERM) family of membrane-cytoskeletal linkers. CLIC5 concentrates at the base of hair cell stereocilia and is required for normal hearing and balance in mice, but its functional significance is poorly understood. This study investigated the role of CLIC5 in postnatal development and maintenance of hair bundles. Confocal and scanning electron microscopy of CLIC5-deficient jitterbug (jbg) mice revealed progressive fusion of stereocilia as early as postnatal day 10. Radixin (RDX), protein tyrosine phosphatase receptor Q (PTPRQ), and taperin (TPRN), deafness-associated proteins that also concentrate at the base of stereocilia, were mislocalized in fused stereocilia of jbg mice. TPRQ and RDX were dispersed even prior to stereocilia fusion. Biochemical assays showed interaction of CLIC5 with ERM proteins, TPRN, and possibly myosin VI (MYO6). In addition, CLIC5 and RDX failed to localize normally in fused stereocilia of MYO6 mutant mice. Based on these findings, we propose a model in which these proteins work together as a complex to stabilize linkages between the plasma membrane and subjacent actin cytoskeleton at the base of stereocilia. PMID:24285636

  15. Las17p-Vrp1p but not Las17p-Arp2/3 interaction is important for actin patch polarization in yeast.

    PubMed

    Rajmohan, Rajamuthiah; Wong, Ming Hwa; Meng, Lei; Munn, Alan L; Thanabalu, Thirumaran

    2009-05-01

    The actin cytoskeleton plays a central role in many important cellular processes such as cell polarization, cell division and endocytosis. The dynamic changes to the actin cytoskeleton that accompany these processes are regulated by actin-associated proteins Wiskott-Aldrich Syndrome Protein (WASP) (known as Las17p in yeast) and WASP-Interacting Protein (WIP) (known as Vrp1p in yeast). Both yeast and human WASP bind to and stimulate the Arp2/3 complex which in turn nucleates assembly of actin monomers into filaments at polarized sites at the cortex. WASP-WIP interaction in yeast and humans are important for Arp2/3 complex stimulation in vitro. It has been proposed that these interactions are also important for polarized actin assembly in vivo. However, the redundancy of actin-associated proteins has made it difficult to test this hypothesis. We have identified two point mutations (L80T and H94L) in yeast WASP that in combination abolish WASP-WIP interaction in yeast. We also identify an N-terminal fragment of Las17p (N-Las17p1-368) able to interact with Vrp1p but not Arp2/3. Using these mutant and truncated forms of yeast WASP we provide novel evidence that WASP interaction with WIP is more important than interaction with Arp2/3 for polarized actin assembly and endocytosis in yeast. PMID:19272406

  16. Actinic Prurigo.

    PubMed

    Rodríguez-Carreón, Alma Angélica; Rodríguez-Lobato, Erika; Rodríguez-Gutiérrez, Georgina; Cuevas-González, Juan Carlos; Mancheno-Valencia, Alexandra; Solís-Arias, Martha Patricia; Vega-Memije, María Elisa; Hojyo-Tomoka, María Teresa; Domínguez-Soto, Luciano

    2015-01-01

    Actinic prurigo is an idiopathic photodermatosis that affects the skin, as well as the labial and conjunctival mucosa in indigenous and mestizo populations of Latin America. It starts predominantly in childhood, has a chronic course, and is exacerbated with solar exposure. Little is known of its pathophysiology, including the known mechanisms of the participation of HLA-DR4 and an abnormal immunologic response with increase of T CD4+ lymphocytes. The presence of IgE, eosinophils, and mast cells suggests that it is a hypersensitivity reaction (likely type IVa or b). The diagnosis is clinical, and the presence of lymphoid follicles in the mucosal histopathologic study of mucosa is pathognomonic. The best available treatment to date is thalidomide, despite its secondary effects. PMID:26861426

  17. [Actinic Keratosis].

    PubMed

    Dejaco, D; Hauser, U; Zelger, B; Riechelmann, H

    2015-07-01

    Actinic keratosis is a cutaneous lesion characterized by proliferation of atypical epidermal keratinocytes due to prolonged exposure to exogenous factors such as ultraviolet radiation. AKs are in-situ-squamous cell carcinomas (PEC) of the skin. AK typically presents as erythematous, scaly patch or papule (classic AK), occasionally as thick, adherent scale on an erythematous base. Mostly fair-skinned adults are affected. AKs typically occur in areas of frequent sun exposure (balding scalp, face, "H-region", lateral neck, décolleté, dorsum of the hand and lower extremities). Actinic Cheilitis is the term used for AKs appearing on the lips. The diagnosis of AK is based on clinical examination including inspection and palpation. The typical palpable rough surface of AK often precedes a visible lesion. Dermoscopy may provide additional information. If diagnosis is uncertain and invasion suspected, biopsy and histopathologic evaluation should be performed. The potential for progression to invasive PECs mandates therapeutic intervention. Treatment options include topical and systemic therapies. Topical therapies are classified into physical, medical and combined physical-chemical approaches and a sequential combination of treatment modalities is possible. Topical-physical cryotherapy is the treatment of choice for isolated, non-hypertrophic AK. Topical-medical treatment, e. g. 5-fluoruracil (5FU) cream or Imiquomod or Ingenolmebutat application is used for multiple, non-hypertrophic AKs. For hypertrophic AKs, a dehorning pretreatment with salicinated vaseline is recommended. Isolated hypertrophic AKs often need cryotherapy with prolonged freezing time or several consecutive applications. Sequentially combined approaches are recommended for multiple, hypertrophic AKs. Photodynamic therapy (PDT) as example for a combined physical-chemical approach is an established treatment for multiple, non-hypertrophic and hypertrophic AKs. Prevention includes avoidance of sun and

  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. Proteomics Analysis of the Non-Muscle Myosin Heavy Chain IIa-Enriched Actin-Myosin Complex Reveals Multiple Functions within the Podocyte

    PubMed Central

    Hays, Thomas; Ma’ayan, Avi; Clark, Neil R.; Tan, Christopher M.; Teixeira, Avelino; Teixeira, Angela; Choi, Jae W.; Burdis, Nora; Jung, Sung Yun; Bajaj, Amol O.; O’Malley, Bert W.; He, John C.; Hyink, Deborah P.; Klotman, Paul E.

    2014-01-01

    MYH9 encodes non-muscle myosin heavy chain IIA (NMMHCIIA), the predominant force-generating ATPase in non-muscle cells. Several lines of evidence implicate a role for MYH9 in podocytopathies. However, NMMHCIIA‘s function in podocytes remains unknown. To better understand this function, we performed immuno-precipitation followed by mass-spectrometry proteomics to identify proteins interacting with the NMMHCIIA-enriched actin-myosin complexes. Computational analyses revealed that these proteins belong to functional networks including regulators of cytoskeletal organization, metabolism and networks regulated by the HIV-1 gene nef. We further characterized the subcellular localization of NMMHCIIA within podocytes in vivo, and found it to be present within the podocyte major foot processes. Finally, we tested the effect of loss of MYH9 expression in podocytes in vitro, and found that it was necessary for cytoskeletal organization. Our results provide the first survey of NMMHCIIA-enriched actin-myosin-interacting proteins within the podocyte, demonstrating the important role of NMMHCIIA in organizing the elaborate cytoskeleton structure of podocytes. Our characterization of NMMHCIIA’s functions goes beyond the podocyte, providing important insights into its general molecular role. PMID:24949636

  20. Energetic modeling and single-molecule verification of dynamic regulation on receptor complexes by actin corrals and lipid raft domains

    NASA Astrophysics Data System (ADS)

    Lin, Chien Y.; Huang, Jung Y.; Lo, Leu-Wei

    2014-12-01

    We developed an energetic model by integrating the generalized Langevin equation with the Cahn-Hilliard equation to simulate the diffusive behaviors of receptor proteins in the plasma membrane of a living cell. Simulation results are presented to elaborate the confinement effects from actin corrals and protein-induced lipid domains. Single-molecule tracking data of epidermal growth factor receptors (EGFR) acquired on live HeLa cells agree with the simulation results and the mechanism that controls the diffusion of single-molecule receptors is clarified. We discovered that after ligand binding, EGFR molecules move into lipid nanodomains. The transition rates between different diffusion states of liganded EGFR molecules are regulated by the lipid domains. Our method successfully captures dynamic interactions of receptors at the single-molecule level and provides insight into the functional architecture of both the diffusing EGFR molecules and their local cellular environment.

  1. mDia1 and formins: screw cap of the actin filament

    PubMed Central

    Mizuno, Hiroaki; Watanabe, Naoki

    2012-01-01

    Formin homology proteins (formins) are actin nucleation factors which remain bound to the growing barbed end and processively elongate actin filament (F-actin). Recently, we have demonstrated that a mammalian formin mDia1 rotates along the long-pitch helix of F-actin during processive elongation (helical rotation) by single-molecule fluorescence polarization. We have also shown processive depolymerization of mDia1-bound F-actin during which helical rotation was visualized. In the cell where F-actins are highly cross-linked, formins should rotate during filament elongation. Therefore, when formins are tightly anchored to cellular structures, formins may not elongate F-actin. Adversely, helical rotation of formins might affect the twist of F-actin. Formins could thus control actin elongation and regulate stability of cellular actin filaments through helical rotation. On the other hand, ADP-actin elongation at the mDia1-bound barbed end turned out to become decelerated by profilin, in marked contrast to its remarkably positive effect on mDia1-mediated ATP-actin elongation. This deceleration is caused by enhancement of the off-rate of ADP-actin. While mDia1 and profilin enhance the ADP-actin off-rate, they do not apparently increase the ADP-actin on-rate at the barbed end. These results imply that G-actin-bound ATP and its hydrolysis may be part of the acceleration mechanism of formin-mediated actin elongation.

  2. Cytoplasmic Actin: Purification and Single Molecule Assembly Assays

    PubMed Central

    Hansen, Scott D.; Zuchero, J. Bradley; Mullins, R. Dyche

    2014-01-01

    The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility, cytokinesis, and endocytosis. Actin polymerization is tightly regulated by its numerous cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. Here we describe how to use the pyrene actin assembly assay to determine the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleation or capping factors. Secondly, we show how fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how proteins regulate actin filament structure. Finally, we describe a method for visualizing dynamic assembly and disassembly of single actin filaments and fluorescently labeled actin binding proteins using total internal reflection fluorescence (TIRF) microscopy. PMID:23868587

  3. Affinity chromatography of immobilized actin and myosin.

    PubMed Central

    Bottomley, R C; Trayer, I P

    1975-01-01

    Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder. PMID:241335

  4. The Cannabinoid Receptor CB1 Interacts with the WAVE1 Complex and Plays a Role in Actin Dynamics and Structural Plasticity in Neurons

    PubMed Central

    Njoo, Christian; Agarwal, Nitin; Lutz, Beat; Kuner, Rohini

    2015-01-01

    The molecular composition of the cannabinoid type 1 (CB1) receptor complex beyond the classical G-protein signaling components is not known. Using proteomics on mouse cortex in vivo, we pulled down proteins interacting with CB1 in neurons and show that the CB1 receptor assembles with multiple members of the WAVE1 complex and the RhoGTPase Rac1 and modulates their activity. Activation levels of CB1 receptor directly impacted on actin polymerization and stability via WAVE1 in growth cones of developing neurons, leading to their collapse, as well as in synaptic spines of mature neurons, leading to their retraction. In adult mice, CB1 receptor agonists attenuated activity-dependent remodeling of dendritic spines in spinal cord neurons in vivo and suppressed inflammatory pain by regulating the WAVE1 complex. This study reports novel signaling mechanisms for cannabinoidergic modulation of the nervous system and demonstrates a previously unreported role for the WAVE1 complex in therapeutic applications of cannabinoids. PMID:26496209

  5. Dynamic actin gene family evolution in primates.

    PubMed

    Zhu, Liucun; Zhang, Ying; Hu, Yijun; Wen, Tieqiao; Wang, Qiang

    2013-01-01

    Actin is one of the most highly conserved proteins and plays crucial roles in many vital cellular functions. In most eukaryotes, it is encoded by a multigene family. Although the actin gene family has been studied a lot, few investigators focus on the comparison of actin gene family in relative species. Here, the purpose of our study is to systematically investigate characteristics and evolutionary pattern of actin gene family in primates. We identified 233 actin genes in human, chimpanzee, gorilla, orangutan, gibbon, rhesus monkey, and marmoset genomes. Phylogenetic analysis showed that actin genes in the seven species could be divided into two major types of clades: orthologous group versus complex group. Codon usages and gene expression patterns of actin gene copies were highly consistent among the groups because of basic functions needed by the organisms, but much diverged within species due to functional diversification. Besides, many great potential pseudogenes were found with incomplete open reading frames due to frameshifts or early stop codons. These results implied that actin gene family in primates went through "birth and death" model of evolution process. Under this model, actin genes experienced strong negative selection and increased the functional complexity by reproducing themselves. PMID:23841080

  6. Arp2/3 Controls the Motile Behavior of N-WASP-Functionalized GUVs and Modulates N-WASP Surface Distribution by Mediating Transient Links with Actin Filaments

    PubMed Central

    Delatour, Vincent; Helfer, Emmanuèle; Didry, Dominique; Lê, Kim Hô Diêp; Gaucher, Jean-François; Carlier, Marie-France; Romet-Lemonne, Guillaume

    2008-01-01

    Spatially controlled assembly of actin in branched filaments generates cell protrusions or the propulsion of intracellular vesicles and pathogens. The propulsive movement of giant unilamellar vesicles (GUVs) functionalized by N-WASP (full-length or truncated) is reconstituted in a biochemically controlled medium, and analyzed using phase contrast and fluorescence microscopy to elucidate the links between membrane components and the actin cytoskeleton that determine motile behavior. Actin-based propulsion displays a continuous regime or a periodic saltatory regime. The transition between the two regimes is controlled by the concentration of Arp2/3 complex, which branches filaments by interacting with N-WASP at the liposome surface. Saltatory motion is linked to cycles in the distribution of N-WASP at the membrane between a homogeneous and a segregated state. Comparison of the changes in distribution of N-WASP, Arp2/3, and actin during propulsion demonstrates that actin filaments bind to N-WASP, and that these bonds are transitory. This interaction, mediated by Arp2/3, drives N-WASP segregation. VC-fragments of N-WASP, that interact more weakly than N-WASP with the Arp2/3 complex, segregate less than N-WASP at the rear of the GUVs. GUV propulsion is inhibited by the presence of VCA-actin covalent complex, showing that the release of actin from the nucleator is required for movement. The balance between segregation and free diffusion determines whether continuous movement can be sustained. Computed surface distributions of N-WASP, derived from a theoretical description of this segregation-diffusion mechanism, account satisfactorily for the measured density profiles of N-WASP, Arp2/3 complex, and actin. PMID:18326652

  7. The neuronal and actin commitment: Why do neurons need rings?

    PubMed

    Leite, Sérgio Carvalho; Sousa, Mónica Mendes

    2016-09-01

    The role of the actin cytoskeleton in neurons has been extensively studied in actin-enriched compartments such as the growth cone and dendritic spines. The recent discovery of actin rings in the axon shaft and in dendrites, together with the identification of axon actin trails, has advanced our understanding on actin organization and dynamics in neurons. However, specifically in the case of actin rings, the mechanisms regulating their nucleation and assembly, and the functions that they may exert in axons and dendrites remain largely unexplored. Here we discuss the possible structural, mechanistic and functional properties of the subcortical neuronal cytoskeleton putting the current knowledge in perspective with the information available on actin rings formed in other biological contexts, and with the organization of actin-spectrin lattices in other cell types. The detailed analysis of these novel neuronal actin ring structures, together with the elucidation of the function of actin-binding proteins in neuron biology, has a large potential to uncover new mechanisms of neuronal function under normal conditions that may have impact in our understanding of axon degeneration and regeneration. © 2016 Wiley Periodicals, Inc. PMID:26784007

  8. Pushing with actin: from cells to pathogens.

    PubMed

    Small, J Victor

    2015-02-01

    Actin polymerization is harnessed by cells to generate lamellipodia for movement and by a subclass of pathogens to facilitate invasion of their infected hosts. Using electron tomography (ET), we have shown that lamellipodia are formed via the generation of subsets of actin filaments joined by branch junctions. Image averaging produced a 2.9 nm resolution model of branch junctions in situ and revealed a close fit to the electron density map of the actin-related protein 2/3 (Arp2/3)-actin complex in vitro. Correlated live-cell imaging and ET was also used to determine how actin networks are created and remodelled during the initiation and inhibition of protrusion in lamellipodia. Listeria, Rickettsia and viruses, such as vaccinia virus and baculovirus, exploit the actin machinery of host cells to generate propulsive actin comet tails to disseminate their infection. By applying ET, we have shown that baculovirus generates at its rear a fishbone-like array of subsets of branched actin filaments, with an average of only four filaments engaged in pushing at any one time. In both of these studies, the application of ET of negatively stained cytoskeletons for higher filament resolution and cryo-ET for preserving overall 3D morphology was crucial for obtaining a complete structure-function analysis of actin-driven propulsion. PMID:25619250

  9. Characterisation of a multimeric protein complex associated with ERp57 within the nucleus in paclitaxel-sensitive and -resistant epithelial ovarian cancer cells: the involvement of specific conformational states of beta-actin.

    PubMed

    Cicchillitti, Lucia; Della Corte, Anna; Di Michele, Michela; Donati, Maria Benedetta; Rotilio, Domenico; Scambia, Giovanni

    2010-08-01

    Ovarian cancer is the second most frequently diagnosed malignancy of the reproductive system and is the leading cause of gynecological cancer mortality. Although the majority of advanced ovarian carcinomas initially respond successfully to taxane-based chemotherapy, resistance to chemotherapy remains the primary factor accounting for the low 5-year survival in this patient population. Recent data obtained by our group demonstrate that the disulphide isomerase ERp57 is strongly modulated in paclitaxel resistance suggesting that it may represent a chemoresistance biomarker in ovarian cancer. In the present study, we characterise a nuclear multimeric complex where ERp57 is associated with protein species involved in cell division and gene expression, as Nucleolin, Nucleophosmin, Vimentin, Aurora kinase C and beta-actin. In particular, we show that the occurrence of the interaction of nuclear ERp57 with beta-actin is associated with paclitaxel resistance and that specific actin conformations modulate this complex. We propose the involvement of the nuclear ERp57 complex in mechanisms associated with chromosome segregation in which specific conformational states of actin play a role in the pathway involved in paclitaxel resistance. PMID:20596672

  10. The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1).

    PubMed Central

    Waterman-Storer, C M; Karki, S; Holzbaur, E L

    1995-01-01

    p150Glued was first identified as a polypeptide that copurifies with cytoplasmic dynein, the minus-end-directed microtubule-based motor protein, and has more recently been shown to be present as a member of the oligomeric dynactin complex, which includes the actin-related protein centractin (Arp-1). Dynactin is thought to mediate dynein-driven vesicle motility, as well as nuclear transport, in lower eukaryotes. The mechanism by which dynactin may function in these cellular processes is unknown. To examine the role of the dynactin complex in vivo, we overexpressed the rat cDNA encoding p150Glued in Rat-2 fibroblasts. Overexpression of full-length, as well as C-terminal deletion, constructs resulted in the decoration of microtubules with the p150Glued polypeptides. This cellular evidence for microtubule association was corroborated by in vitro microtubule-binding assays. Amino acids 39-150 of p150Glued were determined to be sufficient for microtubule association. We also tested for a direct interaction between p150Glued and centractin. In vitro translated centractin was specifically retained by a p150Glued affinity column, and this interaction was blocked by a synthetic peptide which corresponds to a highly conserved motif from the C terminus of p150Glued. These results demonstrate that p150Glued, a protein implicated in cytoplasmic dynein-based microtubule motility, is capable of direct binding to both microtubules and centractin. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7878030

  11. The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells

    PubMed Central

    Yasunaga, Takayuki; Hoff, Sylvia; Schell, Christoph; Helmstädter, Martin; Kretz, Oliver; Kuechlin, Sebastian; Yakulov, Toma A.; Engel, Christina; Müller, Barbara; Bensch, Robert; Ronneberger, Olaf; Huber, Tobias B.; Lienkamp, Soeren S.

    2015-01-01

    Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton. PMID:26644512

  12. The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells.

    PubMed

    Yasunaga, Takayuki; Hoff, Sylvia; Schell, Christoph; Helmstädter, Martin; Kretz, Oliver; Kuechlin, Sebastian; Yakulov, Toma A; Engel, Christina; Müller, Barbara; Bensch, Robert; Ronneberger, Olaf; Huber, Tobias B; Lienkamp, Soeren S; Walz, Gerd

    2015-12-01

    Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton. PMID:26644512

  13. Phase nucleation in curved space

    NASA Astrophysics Data System (ADS)

    Gómez, Leopoldo; García, Nicolás; Vitelli, Vincenzo; Lorenzana, José; Daniel, Vega

    Nucleation and growth is the dominant relaxation mechanism driving first-order phase transitions. In two-dimensional flat systems, nucleation has been applied to a wide range of problems in physics, chemistry and biology. Here we study nucleation and growth of two-dimensional phases lying on curved surfaces and show that curvature modifies both critical sizes of nuclei and paths towards the equilibrium phase. In curved space, nucleation and growth becomes inherently inhomogeneous and critical nuclei form faster on regions of positive Gaussian curvature. Substrates of varying shape display complex energy landscapes with several geometry-induced local minima, where initially propagating nuclei become stabilized and trapped by the underlying curvature (Gómez, L. R. et al. Phase nucleation in curved space. Nat. Commun. 6:6856 doi: 10.1038/ncomms7856 (2015).).

  14. Colloids and Nucleation

    NASA Technical Reports Server (NTRS)

    Ackerson, Bruce

    1997-01-01

    The objectives of the work funded under this grant were to develop a microphotographic technique and use it to monitor the nucleation and growth of crystals of hard colloidal spheres. Special attention is given to the possible need for microgravity studies in future experiments. A number of persons have been involved in this work. A masters student, Keith Davis, began the project and developed a sheet illumination apparatus and an image processing system for detection and analysis. His work on a segmentation program for image processing was sufficient for his master's research and has been published. A post doctoral student Bernie Olivier and a graduate student Yueming He, who originally suggested the sheet illumination, were funded by another source but along with Keith made photographic series of several samples (that had been made by Keith Davis). Data extraction has been done by Keith, Bernie, Yueming and two undergraduates employed on the grant. Results are published in Langmuir. These results describe the sheet lighting technique as one which illuminates not only the Bragg scattering crystal, but all the crystals. Thus, accurate crystal counts can be made for nucleation rate measurements. The strange crystal length scale reduction, observed in small angle light scattering (SALS) studies, following the initial nucleation and growth period, has been observed directly. The Bragg scattering (and dark) crystal size decreases in the crossover region. This could be an effect due to gravitational forces or due to over- compression of the crystal during growth. Direct observations indicate a complex morphology for the resulting hard sphere crystals. The crystal edges are fairly sharp but the crystals have a large degree of internal structure. This structure is a result of (unstable) growth and not aggregation. As yet unpublished work compares growth exponents data with data obtained by SALS. The nucleation rate density is determined over a broad volume fraction range

  15. Actin-Regulator Feedback Interactions during Endocytosis.

    PubMed

    Wang, Xinxin; Galletta, Brian J; Cooper, John A; Carlsson, Anders E

    2016-03-29

    Endocytosis mediated by clathrin, a cellular process by which cells internalize membrane receptors and their extracellular ligands, is an important component of cell signaling regulation. Actin polymerization is involved in endocytosis in varying degrees depending on the cellular context. In yeast, clathrin-mediated endocytosis requires a pulse of polymerized actin and its regulators, which recruit and activate the Arp2/3 complex. In this article, we seek to identify the main protein-protein interactions that 1) cause actin and its regulators to appear in pulses, and 2) determine the effects of key mutations and drug treatments on actin and regulator assembly. We perform a joint modeling/experimental study of actin and regulator dynamics during endocytosis in the budding yeast Saccharomyces cerevisiae. We treat both a stochastic model that grows an explicit three-dimensional actin network, and a simpler two-variable Fitzhugh-Nagumo type model. The models include a negative-feedback interaction of F-actin onto the Arp2/3 regulators. Both models explain the pulse time courses and the effects of interventions on actin polymerization: the surprising increase in the peak F-actin count caused by reduced regulator branching activity, the increase in F-actin resulting from slowing of actin disassembly, and the increased Arp2/3 regulator lifetime resulting from latrunculin treatment. In addition, they predict that decreases in the regulator branching activity lead to increases in accumulation of regulators, and we confirmed this prediction with experiments on yeast harboring mutations in the Arp2/3 regulators, using quantitative fluorescence microscopy. Our experimental measurements suggest that the regulators act quasi-independently, in the sense that accumulation of a particular regulator is most strongly affected by mutations of that regulator, as opposed to the others. PMID:27028652

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

    PubMed

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

    2015-01-12

    During blood vessel formation, endothelial cells (ECs) establish cell-cell junctions and rearrange to form multicellular tubes. Here, we show that during lumen formation, the actin nucleator and elongation factor, formin-like 3 (fmnl3), localizes to EC junctions, where filamentous actin (F-actin) cables assemble. Fluorescent actin reporters and fluorescence recovery after photobleaching experiments in zebrafish embryos identified a pool of dynamic F-actin with high turnover at EC junctions in vessels. Knockdown of fmnl3 expression, chemical inhibition of formin function, and expression of dominant-negative fmnl3 revealed that formin activity maintains a stable F-actin content at EC junctions by continual polymerization of F-actin cables. Reduced actin polymerization leads to destabilized endothelial junctions and consequently to failure in blood vessel lumenization and lumen instability. Our findings highlight the importance of formin activity in blood vessel morphogenesis. PMID:25584798

  17. CNS myelin wrapping is driven by actin disassembly.

    PubMed

    Zuchero, J Bradley; Fu, Meng-Meng; Sloan, Steven A; Ibrahim, Adiljan; Olson, Andrew; Zaremba, Anita; Dugas, Jason C; Wienbar, Sophia; Caprariello, Andrew V; Kantor, Christopher; Leonoudakis, Dmitri; Leonoudakus, Dmitri; Lariosa-Willingham, Karen; Kronenberg, Golo; Gertz, Karen; Soderling, Scott H; Miller, Robert H; Barres, Ben A

    2015-07-27

    Myelin is essential in vertebrates for the rapid propagation of action potentials, but the molecular mechanisms driving its formation remain largely unknown. Here we show that the initial stage of process extension and axon ensheathment by oligodendrocytes requires dynamic actin filament assembly by the Arp2/3 complex. Unexpectedly, subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins and rapid disassembly of the oligodendrocyte actin cytoskeleton and does not require Arp2/3. Inducing loss of actin filaments drives oligodendrocyte membrane spreading and myelin wrapping in vivo, and the actin disassembly factor gelsolin is required for normal wrapping. We show that myelin basic protein, a protein essential for CNS myelin wrapping whose role has been unclear, is required for actin disassembly, and its loss phenocopies loss of actin disassembly proteins. Together, these findings provide insight into the molecular mechanism of myelin wrapping and identify it as an actin-independent form of mammalian cell motility. PMID:26166300

  18. Phosphatidylinositol 3-Kinase-, Actin-, and Microtubule-Dependent Transport of Semliki Forest Virus Replication Complexes from the Plasma Membrane to Modified Lysosomes▿ †

    PubMed Central

    Spuul, Pirjo; Balistreri, Giuseppe; Kääriäinen, Leevi; Ahola, Tero

    2010-01-01

    Like other positive-strand RNA viruses, alphaviruses replicate their genomes in association with modified intracellular membranes. Alphavirus replication sites consist of numerous bulb-shaped membrane invaginations (spherules), which contain the double-stranded replication intermediates. Time course studies with Semliki Forest virus (SFV)-infected cells were combined with live-cell imaging and electron microscopy to reveal that the replication complex spherules of SFV undergo an unprecedented large-scale movement between cellular compartments. The spherules first accumulated at the plasma membrane and were then internalized using an endocytic process that required a functional actin-myosin network, as shown by blebbistatin treatment. Wortmannin and other inhibitors indicated that the internalization of spherules also required the activity of phosphatidylinositol 3-kinase. The spherules therefore represent an unusual type of endocytic cargo. After endocytosis, spherule-containing vesicles were highly dynamic and had a neutral pH. These primary carriers fused with acidic endosomes and moved long distances on microtubules, in a manner prevented by nocodazole. The result of the large-scale migration was the formation of a very stable compartment, where the spherules were accumulated on the outer surfaces of unusually large and static acidic vacuoles localized in the pericentriolar region. Our work highlights both fundamental similarities and important differences in the processes that lead to the modified membrane compartments in cells infected by distinct groups of positive-sense RNA viruses. PMID:20484502

  19. In Silico Reconstitution of Actin-Based Symmetry Breaking and Motility

    PubMed Central

    Dayel, Mark J.; Akin, Orkun; Landeryou, Mark; Risca, Viviana; Mogilner, Alex; Mullins, R. Dyche

    2009-01-01

    Eukaryotic cells assemble viscoelastic networks of crosslinked actin filaments to control their shape, mechanical properties, and motility. One important class of actin network is nucleated by the Arp2/3 complex and drives both membrane protrusion at the leading edge of motile cells and intracellular motility of pathogens such as Listeria monocytogenes. These networks can be reconstituted in vitro from purified components to drive the motility of spherical micron-sized beads. An Elastic Gel model has been successful in explaining how these networks break symmetry, but how they produce directed motile force has been less clear. We have combined numerical simulations with in vitro experiments to reconstitute the behavior of these motile actin networks in silico using an Accumulative Particle-Spring (APS) model that builds on the Elastic Gel model, and demonstrates simple intuitive mechanisms for both symmetry breaking and sustained motility. The APS model explains observed transitions between smooth and pulsatile motion as well as subtle variations in network architecture caused by differences in geometry and conditions. Our findings also explain sideways symmetry breaking and motility of elongated beads, and show that elastic recoil, though important for symmetry breaking and pulsatile motion, is not necessary for smooth directional motility. The APS model demonstrates how a small number of viscoelastic network parameters and construction rules suffice to recapture the complex behavior of motile actin networks. The fact that the model not only mirrors our in vitro observations, but also makes novel predictions that we confirm by experiment, suggests that the model captures much of the essence of actin-based motility in this system. PMID:19771152

  20. Villin Severing Activity Enhances Actin-based Motility In Vivo

    PubMed Central

    Revenu, Céline; Courtois, Matthieu; Michelot, Alphée; Sykes, Cécile; Louvard, Daniel

    2007-01-01

    Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition. PMID:17182858

  1. Extracellular signaling cues for nuclear actin polymerization.

    PubMed

    Plessner, Matthias; Grosse, Robert

    2015-01-01

    Contrary to cytoplasmic actin structures, the biological functions of nuclear actin filaments remain largely enigmatic. Recent progress in the field, however, has determined nuclear actin structures in somatic cells either under steady state conditions or in response to extracellular signaling cues. These actin structures differ in size and shape as well as in their temporal appearance and dynamics. Thus, a picture emerges that suggests that mammalian cells may have different pathways and mechanisms to assemble nuclear actin filaments. Apart from serum- or LPA-triggered nuclear actin polymerization, integrin activation by extracellular matrix interaction was recently implicated in nuclear actin polymerization through the linker of nucleoskeleton and cytoskeleton (LINC) complex. Some of these extracellular cues known so far appear to converge at the level of nuclear formin activity and subsequent regulation of myocardin-related transcription factors. Nevertheless, as the precise signaling events are as yet unknown, the regulation of nuclear actin polymerization may be of significant importance for different cellular functions as well as disease conditions caused by altered nuclear dynamics and architecture. PMID:26059398

  2. Dynamics of an actin spring

    NASA Astrophysics Data System (ADS)

    Riera, Christophe; Mahadevan, L.; Shin, Jennifer; Matsudaira, Paul

    2003-03-01

    The acrosome of the sperm of the horseshoe crab (Limulus Polyphemus) is an unusual actin based system that shows a spectacular dynamical transition in the presence of Ca++ that is present in abundance in the neighborhood of the egg. During this process, the bundle, which is initially bent and twisted uncoils and becomes straight in a matter of a few seconds. Based on microstructural data, we propose a model for the dynamics of uncoiling that is best represented by a triple-well potential corresponding to the different structural arrangements of the supertwisted filaments. Each of the false, true and coiled states corresponds to a local minimum of the energy, with the true state being the one with the lowest energy. Using an evolution equation derived by balancing torques, we investigate the nucleation and propagation of the phase transition and compare the results with those of experiments. Our model quantifies the hypothesis that the acrosomal bundle behaves like a mechano-chemical spring.

  3. A dynamic phase-field model for structural transformations and twinning: Regularized interfaces with transparent prescription of complex kinetics and nucleation. Part II: Two-dimensional characterization and boundary kinetics

    NASA Astrophysics Data System (ADS)

    Agrawal, Vaibhav; Dayal, Kaushik

    2015-12-01

    A companion paper presented the formulation of a phase-field model - i.e., a model with regularized interfaces that do not require explicit numerical tracking - that allows for easy and transparent prescription of complex interface kinetics and nucleation. The key ingredients were a re-parametrization of the energy density to clearly separate nucleation from kinetics; and an evolution law that comes from a conservation statement for interfaces. This enables clear prescription of nucleation through the source term of the conservation law and of kinetics through an interfacial velocity field. This model overcomes an important shortcoming of existing phase-field models, namely that the specification of kinetics and nucleation is both restrictive and extremely opaque. In this paper, we present a number of numerical calculations - in one and two dimensions - that characterize our formulation. These calculations illustrate (i) highly-sensitive rate-dependent nucleation; (ii) independent prescription of the forward and backward nucleation stresses without changing the energy landscape; (iii) stick-slip interface kinetics; (iii) the competition between nucleation and kinetics in determining the final microstructural state; (iv) the effect of anisotropic kinetics; and (v) the effect of non-monotone kinetics. These calculations demonstrate the ability of this formulation to precisely prescribe complex nucleation and kinetics in a simple and transparent manner. We also extend our conservation statement to describe the kinetics of the junction lines between microstructural interfaces and boundaries. This enables us to prescribe an additional kinetic relation for the boundary, and we examine the interplay between the bulk kinetics and the junction kinetics.

  4. 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. PMID:26872971

  5. [Cytoskeletal actin and its associated proteins. Some examples in Protista].

    PubMed

    Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

    1998-06-01

    Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin

  6. Mto2p, a novel fission yeast protein required for cytoplasmic microtubule organization and anchoring of the cytokinetic actin ring.

    PubMed

    Venkatram, Srinivas; Jennings, Jennifer L; Link, Andrew; Gould, Kathleen L

    2005-06-01

    Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires gamma-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Delta cells fail to establish a stable EMTOC and localize gamma-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Delta cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Delta cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis. PMID:15800064

  7. Actin dynamics shape microglia effector functions.

    PubMed

    Uhlemann, Ria; Gertz, Karen; Boehmerle, Wolfgang; Schwarz, Tobias; Nolte, Christiane; Freyer, Dorette; Kettenmann, Helmut; Endres, Matthias; Kronenberg, Golo

    2016-06-01

    Impaired actin filament dynamics have been associated with cellular senescence. Microglia, the resident immune cells of the brain, are emerging as a central pathophysiological player in neurodegeneration. Microglia activation, which ranges on a continuum between classical and alternative, may be of critical importance to brain disease. Using genetic and pharmacological manipulations, we studied the effects of alterations in actin dynamics on microglia effector functions. Disruption of actin dynamics did not affect transcription of genes involved in the LPS-triggered classical inflammatory response. By contrast, in consequence of impaired nuclear translocation of phospho-STAT6, genes involved in IL-4 induced alternative activation were strongly downregulated. Functionally, impaired actin dynamics resulted in reduced NO secretion and reduced release of TNFalpha and IL-6 from LPS-stimulated microglia and of IGF-1 from IL-4 stimulated microglia. However, pathological stabilization of the actin cytoskeleton increased LPS-induced release of IL-1beta and IL-18, which belong to an unconventional secretory pathway. Reduced NO release was associated with decreased cytoplasmic iNOS protein expression and decreased intracellular arginine uptake. Furthermore, disruption of actin dynamics resulted in reduced microglia migration, proliferation and phagocytosis. Finally, baseline and ATP-induced [Ca(2+)]int levels were significantly increased in microglia lacking gelsolin, a key actin-severing protein. Together, the dynamic state of the actin cytoskeleton profoundly and distinctly affects microglia behaviours. Disruption of actin dynamics attenuates M2 polarization by inhibiting transcription of alternative activation genes. In classical activation, the role of actin remodelling is complex, does not relate to gene transcription and shows a major divergence between cytokines following conventional and unconventional secretion. PMID:25989853

  8. Actin in Herpesvirus Infection

    PubMed Central

    Roberts, Kari L.; Baines, Joel D.

    2011-01-01

    Actin is important for a variety of cellular processes, including uptake of extracellular material and intracellular transport. Several emerging lines of evidence indicate that herpesviruses exploit actin and actin-associated myosin motors for viral entry, intranuclear transport of capsids, and virion egress. The goal of this review is to explore these processes and to highlight potential future directions for this area of research. PMID:21994736

  9. Actin from Saccharomyces cerevisiae.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

    Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth. Images PMID:6217414

  10. Actin Rings of Power.

    PubMed

    Schwayer, Cornelia; Sikora, Mateusz; Slováková, Jana; Kardos, Roland; Heisenberg, Carl-Philipp

    2016-06-20

    Circular or ring-like actin structures play important roles in various developmental and physiological processes. Commonly, these rings are composed of actin filaments and myosin motors (actomyosin) that, upon activation, trigger ring constriction. Actomyosin ring constriction, in turn, has been implicated in key cellular processes ranging from cytokinesis to wound closure. Non-constricting actin ring-like structures also form at cell-cell contacts, where they exert a stabilizing function. Here, we review recent studies on the formation and function of actin ring-like structures in various morphogenetic processes, shedding light on how those different rings have been adapted to fulfill their specific roles. PMID:27326928

  11. Spiral actin-polymerization waves can generate amoeboidal cell crawling

    NASA Astrophysics Data System (ADS)

    Dreher, A.; Aranson, I. S.; Kruse, K.

    2014-05-01

    Amoeboidal cell crawling on solid substrates is characterized by protrusions that seemingly appear randomly along the cell periphery and drive the cell forward. For many cell types, it is known that the protrusions result from polymerization of the actin cytoskeleton. However, little is known about how the formation of protrusions is triggered and whether the appearance of subsequent protrusions is coordinated. Recently, the spontaneous formation of actin-polymerization waves was observed. These waves have been proposed to orchestrate the cytoskeletal dynamics during cell crawling. Here, we study the impact of cytoskeletal polymerization waves on cell migration using a phase-field approach. In addition to directionally moving cells, we find states reminiscent of amoeboidal cell crawling. In this framework, new protrusions are seen to emerge from a nucleation process, generating spiral actin waves in the cell interior. Nucleation of new spirals does not require noise, but occurs in a state that is apparently displaying spatio-temporal chaos.

  12. Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3 complex-driven cytoplasmic streaming in mouse oocytes

    PubMed Central

    Yi, Kexi; Unruh, Jay R.; Deng, Manqi; Slaughter, Brian D.; Rubinstein, Boris; Li, Rong

    2012-01-01

    Mature mammalian oocytes are poised for the completion of second polar body extrusion upon fertilization by positioning the metaphase spindle in close proximity to an actomyosin-rich cortical cap. Loss of this spindle position asymmetry is often associated with poor oocyte quality and infertility 1–3. Here, we report a novel role for the Arp2/3 actin nucleation complex in the maintenance of asymmetric spindle position in mature mouse oocytes. The Arp2/3 complex localizes to the cortical cap in a Ran GTPase-dependent manner and accounts for the nucleation of the majority of actin filaments in both the cortical cap and a cytoplasmic actin network. Inhibition of Arp2/3 complex activity or localization leads to rapid dissociation of the spindle from the cortex. High resolution live imaging and spatiotemporal image correlation spectroscopy (STICS) analysis reveal that in normal oocytes actin filaments flow continuously away from the Arp2/3-rich cortex, generating a cytoplamic streaming that results in a net pushing force on the spindle toward the actomyosin cap. Arp2/3 inhibition not only diminishes this actin flow and cytoplamic streaming but also enables a reverse streaming driven by myosin-II-based cortical contraction, leading to spindle movement away from the cortex. We conclude that the Arp2/3 complex maintains asymmetric meiotic spindle position by generating an actin polymerization-driven cytoplamic streaming and by suppressing a counteracting force from myosin-II-based contractility. PMID:21874009

  13. Nucleation and interfacial adsorption in ternary systems.

    PubMed

    Philippe, T

    2015-03-01

    Nucleation is studied in incompressible ternary fluids by examining the topology of the overall landscape of the energy surface. Minimum free energy paths for nucleation (MFEPs) of a single nucleus in an infinite matrix are computed with the string method in the framework of the continuum theory of nucleation for the regular solution. Properties of the critical nucleus are compared with the predictions of the classical nucleation theory. MFEPs are found to exhibit complex nucleation pathways with non-monotonic variations of compositions in the interfacial region, specifically adsorption of a component. In the symmetric regular solution, the minority component is found to segregate at the interface during nucleation with a concomitant depletion of the nucleus core, resulting in unpredicted partition of the non-selective component. Despite increasing the gradient energy, such inhomogeneity in composition is shown to lower the nucleation barrier. PMID:25747088

  14. Ice nucleation properties of agricultural soil dusts

    NASA Astrophysics Data System (ADS)

    Steinke, Isabelle; Funk, Roger; Busse, Jacqueline; Iturri, Antonela; Kirchen, Silke; Leue, Martin; Möhler, Ottmar; Schwartz, Thomas; Sierau, Berko; Toprak, Emre; Ulrich, Andreas; Hoose, Corinna; Leisner, Thomas

    2015-04-01

    Soil dust particles emitted from agricultural areas contain large amounts of organic material such as fungi, bacteria and plant debris. Being carrier for potentially highly ice-active biological particles, agricultural soil dusts are candidates for being very ice-active as well. In this work, we present ice nucleation experiments conducted in the AIDA cloud chamber. We investigated the ice nucleation efficiency of four types of soil dust from different regions of the world. Results are presented for the immersion freezing and the deposition nucleation mode: all soil dusts show higher ice nucleation efficiencies than desert dusts, especially at temperatures above 254 K. For one soil dust sample, the effect of heat treatments was investigated. Heat treatments did not affect the ice nucleation efficiency which presumably excludes primary biological particles as the only source of the increased ice nucleation efficiency. Therefore, organo-mineral complexes or organic compounds may contribute substantially to the high ice nucleation activity of agricultural soil dusts.

  15. Single-headed binding of a spin-labeled-HMM-ADP complex to F-actin. Saturation transfer electron paramagnetic resonance and sedimentation studies.

    PubMed Central

    Manuck, B A; Seidel, J C; Gergely, J

    1986-01-01

    The interaction of actin and spin-labeled heavy meromyosin (MSL-HMM) was studied in the presence and absence of adenosine diphosphate or 5'-adenyl-yl-imidodiphosphate (AMPPNP) to determine the contributions of single and double-headed binding. The extent of single-headed binding to actin was deduced from a comparison of the fraction of immobilized heads (fi) with the fraction of bound molecules (fs) determined by saturation-transfer EPR (ST-EPR) and sedimentation, respectively. The ST-EPR measurements depend on the reduced motion of the spin label rigidly bound to the HMM heads upon the interaction of the latter with actin. During titration of acto-MSL-HMM with nucleotide, we measured changes in fi and fs brought about by dissociation of MSL-HMM from actin. On titration with ADP, fs changed very little, remaining above 0.8, while fi decreased to approximately 0.5 at 10mM ADP, a result consistent with extensive single-headed binding of MSL-HMM to actin. On titration with AMPPNP, single-headed binding was not detected; viz., fi and fs decreased in parallel. It was not necessary to postulate a nucleotide induced state of the bound heads, differing in motional properties from that of rigor heads, to account for the results. PMID:3017466

  16. Mammalian target of rapamycin complex (mTOR) pathway modulates blood-testis barrier (BTB) function through F-actin organization and gap junction.

    PubMed

    Li, Nan; Cheng, C Yan

    2016-09-01

    mTOR (mammalian target of rapamycin) is one of the most important signaling molecules in mammalian cells which regulates an array of cellular events, ranging from cell metabolism to cell proliferation. Based on the association of mTOR with the core component proteins, such as Raptor or Rictor, mTOR can become the mTORC1 (mammalian target of rapamycin complex 1) or mTORC2, respectively. Studies have shown that during the epithelial cycle of spermatogenesis, mTORC1 promotes remodeling and restructuring of the blood-testis barrier (BTB) in vitro and in vivo, making the Sertoli cell tight junction (TJ)-permeability barrier "leaky"; whereas mTORC2 promotes BTB integrity, making the Sertoli cell TJ-barrier "tighter". These contrasting effects, coupled with the spatiotemporal expression of the core signaling proteins at the BTB that confer the respective functions of mTORC1 vs. mTORC2 thus provide a unique mechanism to modulate BTB dynamics, allowing or disallowing the transport of biomolecules and also preleptotene spermatocytes across the immunological barrier. More importantly, studies have shown that these changes to BTB dynamics conferred by mTORC1 and mTORC2 are mediated by changes in the organization of the actin microfilament networks at the BTB, and involve gap junction (GJ) intercellular communication. Since GJ has recently been shown to be crucial to reboot spermatogenesis and meiosis following toxicant-induced aspermatogenesis, these findings thus provide new insightful information regarding the integration of mTOR and GJ to regulate spermatogenesis. PMID:26957088

  17. Nuclear F-actin formation and reorganization upon cell spreading.

    PubMed

    Plessner, Matthias; Melak, Michael; Chinchilla, Pilar; Baarlink, Christian; Grosse, Robert

    2015-05-01

    We recently discovered signal-regulated nuclear actin network assembly. However, in contrast to cytoplasmic actin regulation, polymeric nuclear actin structures and functions remain only poorly understood. Here we describe a novel molecular tool to visualize real-time nuclear actin dynamics by targeting the Actin-Chromobody-TagGFP to the nucleus, thus establishing a nuclear Actin-Chromobody. Interestingly, we observe nuclear actin polymerization into dynamic filaments upon cell spreading and fibronectin stimulation, both of which appear to be triggered by integrin signaling. Furthermore, we show that nucleoskeletal proteins such as the LINC (linker of nucleoskeleton and cytoskeleton) complex and components of the nuclear lamina couple cell spreading or integrin activation by fibronectin to nuclear actin polymerization. Spreading-induced nuclear actin polymerization results in serum response factor (SRF)-mediated transcription through nuclear retention of myocardin-related transcription factor A (MRTF-A). Our results reveal a signaling pathway, which links integrin activation by extracellular matrix interaction to nuclear actin polymerization through the LINC complex, and therefore suggest a role for nuclear actin polymerization in the context of cellular adhesion and mechanosensing. PMID:25759381

  18. Arabidopsis contains ancient classes of differentially expressed actin-related protein genes.

    PubMed

    McKinney, Elizabeth Cohen; Kandasamy, Muthugapatti K; Meagher, Richard B

    2002-03-01

    Actin-related proteins (ARPs) share less than 60% amino acid sequence homology with conventional actins and have roles in diverse cytoskeletal processes in the cytoplasm and nucleus. The genome of Arabidopsis was explored for possible ARP gene family members. Eight potential ARP gene sequences were found dispersed on three of the five Arabidopsis chromosomes. AtARP2 and AtARP3 are protein orthologs of their similarly named counterparts in other kingdoms. AtARP4, AtARP5, and AtARP6 are orthologs of two classes of nuclear ARPs previously characterized in animals and fungi, BAF53s and ARP6s. AtARP7 and AtARP8 appear to be novel proteins that are not closely related to any known animal or fungal ARPs, and may be plant specific. The complex Arabidopsis ARP gene structures each contain from five to 20 exons. Expressed transcripts were identified and characterized for AtARP2 through AtARP8, but not for AtARP9, and transcripts representing two splice variants were found for AtARP8. The seven expressed genes are predicted to encode proteins ranging from 146 to 471 amino acids in length. Relative to conventional actin and the other ARPs, AtARP2 and AtARP3 transcripts are expressed at very low levels in all organs. AtARP5, AtARP6, and AtARP8 each have distinct transcript expression patterns in seedlings, roots, leaves, flowers, and siliques. Using isovariant-specific monoclonal antibodies, AtARP4 and AtARP7 proteins were shown to be most highly expressed in flowers. The likely involvement of plant ARPs in actin nucleation, branching of actin filaments, chromatin restructuring, and transcription are briefly discussed. PMID:11891255

  19. Regulators of Actin Dynamics in Gastrointestinal Tract Tumors

    PubMed Central

    Steinestel, Konrad; Wardelmann, Eva; Hartmann, Wolfgang; Grünewald, Inga

    2015-01-01

    Reorganization of the actin cytoskeleton underlies cell migration in a wide variety of physiological and pathological processes, such as embryonic development, wound healing, and tumor cell invasion. It has been shown that actin assembly and disassembly are precisely regulated by intracellular signaling cascades that respond to changes in the cell microenvironment, ligand binding to surface receptors, or oncogenic transformation of the cell. Actin-nucleating and actin-depolymerizing (ANFs/ADFs) and nucleation-promoting factors (NPFs) regulate cytoskeletal dynamics at the leading edge of migrating cells, thereby modulating cell shape; these proteins facilitate cellular movement and mediate degradation of the surrounding extracellular matrix by secretion of lytic proteases, thus eliminating barriers for tumor cell invasion. Accordingly, expression and activity of these actin-binding proteins have been linked to enhanced metastasis and poor prognosis in a variety of malignancies. In this review, we will summarize what is known about expression patterns and the functional role of actin regulators in gastrointestinal tumors and evaluate first pharmacological approaches to prevent invasion and metastatic dissemination of malignant cells. PMID:26345720

  20. Regulators of Actin Dynamics in Gastrointestinal Tract Tumors.

    PubMed

    Steinestel, Konrad; Wardelmann, Eva; Hartmann, Wolfgang; Grünewald, Inga

    2015-01-01

    Reorganization of the actin cytoskeleton underlies cell migration in a wide variety of physiological and pathological processes, such as embryonic development, wound healing, and tumor cell invasion. It has been shown that actin assembly and disassembly are precisely regulated by intracellular signaling cascades that respond to changes in the cell microenvironment, ligand binding to surface receptors, or oncogenic transformation of the cell. Actin-nucleating and actin-depolymerizing (ANFs/ADFs) and nucleation-promoting factors (NPFs) regulate cytoskeletal dynamics at the leading edge of migrating cells, thereby modulating cell shape; these proteins facilitate cellular movement and mediate degradation of the surrounding extracellular matrix by secretion of lytic proteases, thus eliminating barriers for tumor cell invasion. Accordingly, expression and activity of these actin-binding proteins have been linked to enhanced metastasis and poor prognosis in a variety of malignancies. In this review, we will summarize what is known about expression patterns and the functional role of actin regulators in gastrointestinal tumors and evaluate first pharmacological approaches to prevent invasion and metastatic dissemination of malignant cells. PMID:26345720

  1. The actin binding protein adseverin regulates osteoclastogenesis.

    PubMed

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  2. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    PubMed Central

    Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

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

    PubMed Central

    Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1–actin complex, we constructed a homology model of the AtADF1–actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson–Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

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

    PubMed

    Du, Juan; Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  5. To be or not to be assembled: progressing into nuclear actin filaments.

    PubMed

    Grosse, Robert; Vartiainen, Maria K

    2013-11-01

    The paradigm states that cytoplasmic actin operates as filaments and nuclear actin is mainly monomeric, acting as a scaffold in transcription complexes. However, why should a powerful function of actin, namely polymerization, not be used in the nucleus? Recent progress in the field forces us to rethink this issue, as many actin filament assembly proteins have been linked to nuclear functions and new experimental approaches have provided the first direct visualizations of polymerized nuclear actin. PMID:24088744

  6. Actin-based endosome and phagosome rocketing in macrophages: activation by the secretagogue antagonists lanthanum and zinc.

    PubMed

    Southwick, Frederick S; Li, Wei; Zhang, Fangliang; Zeile, William L; Purich, Daniel L

    2003-01-01

    Although motile endocytic vesicles form actin-rich rocket tails [Merrifield et al., 1999: Nature Cell Biol 1:72-74], the mechanism of intracellular organelle locomotion remains poorly understood. We now demonstrate that bone marrow macrophages treated with lanthanum and zinc ions, well-known secretagogue antagonists, reliably exhibit vesicle motility. This treatment results in accentuated membrane ruffling and the formation of phagosomes and early endosomes that move rapidly through the cytoplasm by assembling actin filament rocket tails. Protein-specific immunolocalization demonstrated the presence of Arp2/3 complex in the polymerization zone and throughout the actin-rich tail, whereas N-WASP was most abundant in the polymerization zone. Although Arp2/3 and N-WASP play essential roles in nucleating filament assembly, other processes (i.e., elongation and filament cross-linking) are required to produce forces needed for motility. Efficient elongation was found to require zyxin, VASP, and profilin, proteins that interact by means of their ABM-1 and ABM-2 proline-rich motifs. The functional significance of these motifs was demonstrated by inhibition of vesicle motility by the motif-specific ABM-1 and ABM-2 analogues. Furthermore, lanthanum/zinc treatment also facilitated the early onset of actin-based vaccinia motility, a process that also utilizes Arp2/3 and N-WASP for nucleation and the zyxin-VASP-profilin complex for efficient elongation. Although earlier studies using cell extracts clouded the role of oligoproline sequences in activating the polymerization zone, our studies emphasize the importance of evaluating motility in living cells. PMID:12451594

  7. Characterization of Two Classes of Small Molecule Inhibitors of Arp2/3 Complex

    SciTech Connect

    Nolen, B.; Tomasevic, N; Russell, A; Pierce, D; Jia, Z; McCormick, C; Hartman, J; Sakowicz, R; Pollard, T

    2009-01-01

    Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones; this is because of the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying the Arp2/3 complex in living cells.

  8. Excitable actin dynamics in lamellipodial protrusion and retraction.

    PubMed

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

    2012-04-01

    Many animal cells initiate crawling by protruding lamellipodia, consisting of a dense network of actin filaments, at their leading edge. We imaged XTC cells that exhibit flat lamellipodia on poly-L-lysine-coated coverslips. Using active contours, we tracked the leading edge and measured the total amount of F-actin by summing the pixel intensities within a 5-μm band. We observed protrusion and retraction with period 130-200 s and local wavelike features. Positive (negative) velocities correlated with minimum (maximum) integrated actin concentration. Approximately constant retrograde flow indicated that protrusions and retractions were driven by fluctuations of the actin polymerization rate. We present a model of these actin dynamics as an excitable system in which a diffusive, autocatalytic activator causes actin polymerization; F-actin accumulation in turn inhibits further activator accumulation. Simulations of the model reproduced the pattern of actin polymerization seen in experiments. To explore the model's assumption of an autocatalytic activation mechanism, we imaged cells expressing markers for both F-actin and the p21 subunit of the Arp2/3 complex. We found that integrated Arp2/3-complex concentrations spike several seconds before spikes of F-actin concentration. This suggests that the Arp2/3 complex participates in an activation mechanism that includes additional diffuse components. Response of cells to stimulation by fetal calf serum could be reproduced by the model, further supporting the proposed dynamical picture. PMID:22500749

  9. Differential Actin-regulatory Activities of Tropomodulin1 and Tropomodulin3 with Diverse Tropomyosin and Actin Isoforms*

    PubMed Central

    Yamashiro, Sawako; Gokhin, David S.; Sui, Zhenhua; Bergeron, Sarah E.; Rubenstein, Peter A.; Fowler, Velia M.

    2014-01-01

    Tropomodulins (Tmods) are F-actin pointed end capping proteins that interact with tropomyosins (TMs) and cap TM-coated filaments with higher affinity than TM-free filaments. Here, we tested whether differences in recognition of TM or actin isoforms by Tmod1 and Tmod3 contribute to the distinct cellular functions of these Tmods. We found that Tmod3 bound ∼5-fold more weakly than Tmod1 to α/βTM, TM5b, and TM5NM1. However, surprisingly, Tmod3 was as effective as Tmod1 at capping pointed ends of skeletal muscle α-actin (αsk-actin) filaments coated with α/βTM, TM5b, or TM5NM1. Tmod3 only capped TM-coated αsk-actin filaments more weakly than Tmod1 in the presence of recombinant αTM2, which is unacetylated at its NH2 terminus, binds F-actin weakly, and has a disabled Tmod-binding site. Moreover, both Tmod1 and Tmod3 were similarly effective at capping pointed ends of platelet β/cytoplasmic γ (γcyto)-actin filaments coated with TM5NM1. In the absence of TMs, both Tmod1 and Tmod3 had similarly weak abilities to nucleate β/γcyto-actin filament assembly, but only Tmod3 could sequester cytoplasmic β- and γcyto-actin (but not αsk-actin) monomers and prevent polymerization under physiological conditions. Thus, differences in TM binding by Tmod1 and Tmod3 do not appear to regulate the abilities of these Tmods to cap TM-αsk-actin or TM-β/γcyto-actin pointed ends and, thus, are unlikely to determine selective co-assembly of Tmod, TM, and actin isoforms in different cell types and cytoskeletal structures. The ability of Tmod3 to sequester β- and γcyto-actin (but not αsk-actin) monomers in the absence of TMs suggests a novel function for Tmod3 in regulating actin remodeling or turnover in cells. PMID:24644292

  10. Direct dynamin–actin interactions regulate the actin cytoskeleton

    PubMed Central

    Gu, Changkyu; Yaddanapudi, Suma; Weins, Astrid; Osborn, Teresia; Reiser, Jochen; Pollak, Martin; Hartwig, John; Sever, Sanja

    2010-01-01

    The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase-dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin-binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin-capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin-CPs. PMID:20935625

  11. Sensing actin dynamics: Structural basis for G-actin-sensitive nuclear import of MAL

    SciTech Connect

    Hirano, Hidemi; Matsuura, Yoshiyuki

    2011-10-22

    Highlights: {yields} MAL has a bipartite NLS that binds to Imp{alpha} in an extended conformation. {yields} Mutational analyses verified the functional significance of MAL-Imp{alpha} interactions. {yields} Induced folding and NLS-masking by G-actins inhibit nuclear import of MAL. -- Abstract: The coordination of cytoskeletal actin dynamics with gene expression reprogramming is emerging as a crucial mechanism to control diverse cellular processes, including cell migration, differentiation and neuronal circuit assembly. The actin-binding transcriptional coactivator MAL (also known as MRTF-A/MKL1/BSAC) senses G-actin concentration and transduces Rho GTPase signals to serum response factor (SRF). MAL rapidly shuttles between the cytoplasm and the nucleus in unstimulated cells but Rho-induced depletion of G-actin leads to MAL nuclear accumulation and activation of transcription of SRF:MAL-target genes. Although the molecular and structural basis of actin-regulated nucleocytoplasmic shuttling of MAL is not understood fully, it is proposed that nuclear import of MAL is mediated by importin {alpha}/{beta} heterodimer, and that G-actin competes with importin {alpha}/{beta} for the binding to MAL. Here we present structural, biochemical and cell biological evidence that MAL has a classical bipartite nuclear localization signal (NLS) in the N-terminal 'RPEL' domain containing Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs. The NLS residues of MAL adopt an extended conformation and bind along the surface groove of importin-{alpha}, interacting with the major- and minor-NLS binding sites. We also present a crystal structure of wild-type MAL RPEL domain in complex with five G-actins. Comparison of the importin-{alpha}- and actin-complexes revealed that the binding of G-actins to MAL is associated with folding of NLS residues into a helical conformation that is inappropriate for importin-{alpha} recognition.

  12. Mechanism of Actin Network Attachment to Moving Membranes

    PubMed Central

    Co, Carl; Wong, Derek T.; Gierke, Sarah; Chang, Vicky; Taunton, Jack

    2007-01-01

    Summary Actin filament networks exert protrusive and attachment forces on membranes and thereby drive membrane deformation and movement. Here, we show that N-WASP WH2 domains play a previously unanticipated role in vesicle movement by transiently attaching actin filament barbed ends to the membrane. To dissect the attachment mechanism, we reconstituted the propulsive motility of lipid-coated glass beads using purified soluble proteins. N-WASP WH2 mutants assembled actin comet tails and initiated movement, but the comet tails catastrophically detached from the membrane. When presented on the surface of a lipid-coated bead, WH2 domains were sufficient to maintain comet tail attachment. In v-Src-transformed fibroblasts, N-WASP WH2 mutants were severely defective in the formation of circular podosome arrays. In addition to creating an attachment force, interactions between WH2 domains and barbed ends may locally amplify signals for dendritic actin nucleation. PMID:17350575

  13. Neogenin recruitment of the WAVE regulatory complex maintains adherens junction stability and tension

    PubMed Central

    Lee, Natalie K.; Fok, Ka Wai; White, Amanda; Wilson, Nicole H.; O'Leary, Conor J.; Cox, Hayley L.; Michael, Magdalene; Yap, Alpha S.; Cooper, Helen M.

    2016-01-01

    To maintain tissue integrity during epithelial morphogenesis, adherens junctions (AJs) must resist the mechanical stresses exerted by dynamic tissue movements. Junctional stability is dependent on actomyosin contractility within the actin ring. Here we describe a novel function for the axon guidance receptor, Neogenin, as a key component of the actin nucleation machinery governing junctional stability. Loss of Neogenin perturbs AJs and attenuates junctional tension. Neogenin promotes actin nucleation at AJs by recruiting the Wave regulatory complex (WRC) and Arp2/3. A direct interaction between the Neogenin WIRS domain and the WRC is crucial for the spatially restricted recruitment of the WRC to the junction. Thus, we provide the first example of a functional WIRS–WRC interaction in epithelia. We further show that Neogenin regulates cadherin recycling at the AJ. In summary, we identify Neogenin as a pivotal component of the AJ, where it influences both cadherin dynamics and junctional tension. PMID:27029596

  14. 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

  15. 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. PMID:26272553

  16. Regulation of actin assembly by microtubules in fission yeast cell polarity.

    PubMed

    Chang, Fred; Feierbach, Becket; Martin, Sophie

    2005-01-01

    It has been speculated that microtubule plus ends function to regulate the actin cytoskeleton in processes such as cytokinesis, cell polarization and cell migration. In the fission yeast Schizosaccharomyces pombe, interphase microtubules regulate cell polarity through proteins such as tea1p, a kelch repeat protein, and for3p, a formin that nucleates actin cable assembly at cell tips. Here, we review recent progress on understanding tea1p regulation and function. Microtubules may govern the localization of tea1p by transporting it on the plus ends of microtubules and depositing it directly onto the cell tip when the microtubule catastrophes. The interaction of tea1p with the CLIP170 protein tip1p is responsible for its localization at growing microtubule plus ends. Tea1p may regulate cell polarity by associating with large 'polarisome' complexes that include for3p. For3p is present at both cell tips, but is not on the microtubules. Tea1p is needed to localize the formin to establish polarized cell growth at cell tips that have not grown previously. These studies begin to elucidate a molecular pathway for how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth. PMID:16355535

  17. Sequence and comparative genomic analysis of actin-related proteins.

    PubMed

    Muller, Jean; Oma, Yukako; Vallar, Laurent; Friederich, Evelyne; Poch, Olivier; Winsor, Barbara

    2005-12-01

    Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of approximately 700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4. PMID:16195354

  18. 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

  19. Nuclear actin and protein 4.1: essential interactions during nuclear assembly in vitro.

    PubMed

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

    2003-09-16

    Structural protein 4.1, which has crucial interactions within the spectrin-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 detergent-extracted 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 nonperturbing conditions, the total nuclear actin population is retained and visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As nuclear lamina assembled, but preceding DNA synthesis, actin distributed in a reticulated pattern 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. PMID:12960380

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

    PubMed Central

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

    2003-01-01

    Structural protein 4.1, which has crucial interactions within the spectrin–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 detergent-extracted 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 nonperturbing conditions, the total nuclear actin population is retained and visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As nuclear lamina assembled, but preceding DNA synthesis, actin distributed in a reticulated pattern 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. PMID:12960380

  1. 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.

  2. H2O2-treated actin: assembly and polymer interactions with cross-linking proteins.

    PubMed Central

    DalleDonne, I; Milzani, A; Colombo, R

    1995-01-01

    During inflammation, hydrogen peroxide, produced by polymorphonuclear leukocytes, provokes cell death mainly by disarranging filamentous (polymerized) actin (F-actin). To show the molecular mechanism(s) by which hydrogen peroxide could alter actin dynamics, we analyzed the ability of H2O2-treated actin samples to polymerize as well as the suitability of actin polymers (from oxidized monomers) to interact with cross-linking proteins. H2O2-treated monomeric (globular) actin (G-actin) shows an altered time course of polymerization. The increase in the lag phase and the lowering in both the polymerization rate and the polymerization extent have been evidenced. Furthermore, steady-state actin polymers, from oxidized monomers, are more fragmented than control polymers. This seems to be ascribable to the enhanced fragility of oxidized filaments rather than to the increase in the nucleation activity, which markedly falls. These facts; along with the unsuitability of actin polymers from oxidized monomers to interact with both filamin and alpha-actinin, suggest that hydrogen peroxide influences actin dynamics mainly by changing the F-actin structure. H2O2, via the oxidation of actin thiols (in particular, the sulfhydryl group of Cys-374), likely alters the actin C-terminus, influencing both subunit/subunit interactions and the spatial structure of the binding sites for cross-linking proteins in F-actin. We suggest that most of the effects of hydrogen peroxide on actin could be explained in the light of the "structural connectivity," demonstrated previously in actin. Images FIGURE 3 FIGURE 9 PMID:8599677

  3. Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin

    PubMed Central

    Kudryashova, Elena; Kalda, Caitlin; Kudryashov, Dmitri S.

    2012-01-01

    Actin Crosslinking Domain (ACD) is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K0.5 = 30 µM) reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg2+-GTP to support crosslinking, but the kinetic parameters (KM = 8 µM and 50 µM for ATP and GTP, respectively) suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0–9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions. PMID:23029200

  4. Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

    PubMed Central

    Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T.; Rao, Madan; Mayor, Satyajit

    2015-01-01

    Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface. PMID:26378258

  5. The Bacterial Actin-Like Cytoskeleton

    PubMed Central

    Carballido-López, Rut

    2006-01-01

    Recent advances have shown conclusively that bacterial cells possess distant but true homologues of actin (MreB, ParM, and the recently uncovered MamK protein). Despite weak amino acid sequence similarity, MreB and ParM exhibit high structural homology to actin. Just like F-actin in eukaryotes, MreB and ParM assemble into highly dynamic filamentous structures in vivo and in vitro. MreB-like proteins are essential for cell viability and have been implicated in major cellular processes, including cell morphogenesis, chromosome segregation, and cell polarity. ParM (a plasmid-encoded actin homologue) is responsible for driving plasmid-DNA partitioning. The dynamic prokaryotic actin-like cytoskeleton is thought to serve as a central organizer for the targeting and accurate positioning of proteins and nucleoprotein complexes, thereby (and by analogy to the eukaryotic cytoskeleton) spatially and temporally controlling macromolecular trafficking in bacterial cells. In this paper, the general properties and known functions of the actin orthologues in bacteria are reviewed. PMID:17158703

  6. Insights into COPll Coat Nucleation from the Structure of Sec23 Sar1 Complexed with the Active Fragment of Sec31

    SciTech Connect

    Bi,X.; Mancias, J.; Goldberg, J.

    2007-01-01

    The COPII vesicular coat forms on the endoplasmic reticulum from Sar1-GTP, Sec23/24 and Sec13/31 protein subunits. Here, we define the interaction between Sec23/24{center_dot}Sar1 and Sec13/31, involving a 40 residue Sec31 fragment. In the crystal structure of the ternary complex, Sec31 binds as an extended polypeptide across a composite surface of the Sec23 and Sar1-GTP molecules, explaining the stepwise character of Sec23/24{center_dot}Sar1 and Sec13/31 recruitment to the membrane. The Sec31 fragment stimulates GAP activity of Sec23/24, and a convergence of Sec31 and Sec23 residues at the Sar1 GTPase active site explains how GTP hydrolysis is triggered leading to COPII coat disassembly. The Sec31 active fragment is accommodated in a binding groove supported in part by Sec23 residue Phe380. Substitution of the corresponding residue F382L in human Sec23A causes cranio-lenticulo-sutural dysplasia, and we suggest that this mutation disrupts the nucleation of COPII coat proteins at endoplasmic reticulum exit sites.

  7. Actin Automata with Memory

    NASA Astrophysics Data System (ADS)

    Alonso-Sanz, Ramón; Adamatzky, Andy

    Actin is a globular protein which forms long polar filaments in eukaryotic. The actin filaments play the roles of cytoskeleton, motility units, information processing and learning. We model actin filament as a double chain of finite state machines, nodes, which take states “0” and “1”. The states are abstractions of absence and presence of a subthreshold charge on actin units corresponding to the nodes. All nodes update their state in parallel to discrete time. A node updates its current state depending on states of two closest neighbors in the node chain and two closest neighbors in the complementary chain. Previous models of actin automata consider momentary state transitions of nodes. We enrich the actin automata model by assuming that states of nodes depend not only on the current states of neighboring node but also on their past states. Thus, we assess the effect of memory of past states on the dynamics of acting automata. We demonstrate in computational experiments that memory slows down propagation of perturbations, decrease entropy of space-time patterns generated, transforms traveling localizations to stationary oscillators, and stationary oscillations to still patterns.

  8. The Molecular Evolution of Actin

    PubMed Central

    Hightower, Robin C.; Meagher, Richard B.

    1986-01-01

    We have investigated the molecular evolution of plant and nonplant actin genes comparing nucleotide and amino acid sequences of 20 actin genes. Nucleotide changes resulting in amino acid substitutions (replacement substitutions) ranged from 3–7% for all pairwise comparisons of animal actin genes with the following exceptions. Comparisons between higher animal muscle actin gene sequences and comparisons between higher animal cytoplasmic actin gene sequences indicated <3% divergence. Comparisons between plant and nonplant actin genes revealed, with two exceptions, 11–15% replacement substitution. In the analysis of plant actins, replacement substitution between soybean actin genes SAc1, SAc3, SAc4 and maize actin gene MAc1 ranged from 8–10%, whereas these members within the soybean actin gene family ranged from 6–9% replacement substitution. The rate of sequence divergence of plant actin sequences appears to be similar to that observed for animal actins. Furthermore, these and other data suggest that the plant actin gene family is ancient and that the families of soybean and maize actin genes have diverged from a single common ancestral plant actin gene that originated long before the divergence of monocots and dicots. The soybean actin multigene family encodes at least three classes of actin. These classes each contain a pair of actin genes that have been designated kappa (SAc1, SAc6), lambda (SAc2, SAc4) and mu (SAc3, SAc7). The three classes of soybean actin are more divergent in nucleotide sequence from one another than higher animal cytoplasmic actin is divergent from muscle actin. The location and distribution of amino acid changes were compared between actin proteins from all sources. A comparison of the hydropathy of all actin sequences, except from Oxytricha, indicated a strong similarity in hydropathic character between all plant and nonplant actins despite the greater number of replacement substitutions in plant actins. These protein sequence

  9. Actin is required for IFT regulation in Chlamydomonas reinhardtii

    PubMed Central

    Avasthi, Prachee; Onishi, Masayuki; Karpiak, Joel; Yamamoto, Ryosuke; Mackinder, Luke; Jonikas, Martin C.; Sale, Winfield S.; Shoichet, Brian; Pringle, John R.; Marshall, Wallace F.

    2014-01-01

    Summary Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Since actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here, we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation, and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor suggesting actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length. PMID:25155506

  10. Actin is required for IFT regulation in Chlamydomonas reinhardtii.

    PubMed

    Avasthi, Prachee; Onishi, Masayuki; Karpiak, Joel; Yamamoto, Ryosuke; Mackinder, Luke; Jonikas, Martin C; Sale, Winfield S; Shoichet, Brian; Pringle, John R; Marshall, Wallace F

    2014-09-01

    Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length. PMID:25155506

  11. Structure and Mechanics of Actin Cortex Contained in Vesicles

    NASA Astrophysics Data System (ADS)

    Limozin, Laurent; Roth, Alexander; Sackmann, Erich

    2003-03-01

    We designed giant phospholipid vesicles containing actin filaments as an elementary mechanical cell model. G-actin is polymerized inside the vesicles through ionophore-mediated Mg++ entry and the filaments are bound electrostatically to the membrane through lipids with amino-polyethyleneglycol (PEG) headgroups forming a shell beneath the membrane. The density of this cortex is varied by changing the initial actin concentration. A magnetic micrometric bead attached on the top of a sedimented vesicle is pulled vertically while horizontal and vertical displacements of the bead are simulatenously tracked by microscopy. Linear response allows to determine the bending and shear moduli of the actin-membrane complexe.

  12. Arp2/3-mediated F-actin formation controls regulated exocytosis in vivo

    PubMed Central

    Tran, Duy T.; Masedunskas, Andrius; Weigert, Roberto; Ten Hagen, Kelly G.

    2015-01-01

    The actin cytoskeleton plays crucial roles in many cellular processes, including regulated secretion. However, the mechanisms controlling F-actin dynamics in this process are largely unknown. Through 3D time-lapse imaging in a secreting organ, we show that F-actin is actively disassembled along the apical plasma membrane at the site of secretory vesicle fusion and re-assembled directionally on vesicle membranes. Moreover, we show that fusion pore formation and PIP2 redistribution precedes actin and myosin recruitment to secretory vesicle membranes. Finally, we show essential roles for the branched actin nucleators Arp2/3- and WASp in the process of secretory cargo expulsion and integration of vesicular membranes with the apical plasma membrane. Our results highlight previously unknown roles for branched actin in exocytosis and provide a genetically tractable system to image the temporal and spatial dynamics of polarized secretion in vivo. PMID:26639106

  13. Arp2/3-mediated F-actin formation controls regulated exocytosis in vivo.

    PubMed

    Tran, Duy T; Masedunskas, Andrius; Weigert, Roberto; Ten Hagen, Kelly G

    2015-01-01

    The actin cytoskeleton plays crucial roles in many cellular processes, including regulated secretion. However, the mechanisms controlling F-actin dynamics in this process are largely unknown. Through 3D time-lapse imaging in a secreting organ, we show that F-actin is actively disassembled along the apical plasma membrane at the site of secretory vesicle fusion and re-assembled directionally on vesicle membranes. Moreover, we show that fusion pore formation and PIP2 redistribution precedes actin and myosin recruitment to secretory vesicle membranes. Finally, we show essential roles for the branched actin nucleators Arp2/3- and WASp in the process of secretory cargo expulsion and integration of vesicular membranes with the apical plasma membrane. Our results highlight previously unknown roles for branched actin in exocytosis and provide a genetically tractable system to image the temporal and spatial dynamics of polarized secretion in vivo. PMID:26639106

  14. Characterizing protein crystal nucleation

    NASA Astrophysics Data System (ADS)

    Akella, Sathish V.

    We developed an experimental microfluidic based technique to measure the nucleation rates and successfully applied the technique to measure nucleation rates of lysozyme crystals. The technique involves counting the number of samples which do not have crystals as a function of time. Under the assumption that nucleation is a Poisson process, the fraction of samples with no crystals decays exponentially with the decay constant proportional to nucleation rate and volume of the sample. Since nucleation is a random and rare event, one needs to perform measurements on large number of samples to obtain good statistics. Microfluidics offers the solution of producing large number of samples at minimal material consumption. Hence, we developed a microfluidic method and measured nucleation rates of lysozyme crystals in supersaturated protein drops, each with volume of ˜ 1 nL. Classical Nucleation Theory (CNT) describes the kinetics of nucleation and predicts the functional form of nucleation rate in terms of the thermodynamic quantities involved, such as supersaturation, temperature, etc. We analyzed the measured nucleation rates in the context of CNT and obtained the activation energy and the kinetic pre-factor characterizing the nucleation process. One conclusion is that heterogeneous nucleation dominates crystallization. We report preliminary studies on selective enhancement of nucleation in one of the crystal polymorprhs of lysozyme (spherulite) using amorphous mesoporous bioactive gel-glass te{naomi06, naomi08}, CaO.P 2O5.SiO2 (known as bio-glass) with 2-10 nm pore-size diameter distribution. The pores act as heterogeneous nucleation centers and claimed to enhance the nucleation rates by molecular confinement. The measured kinetic profiles of crystal fraction of spherulites indicate that the crystallization of spherulites may be proceeding via secondary nucleation pathways.

  15. Rickettsia parkeri invasion of diverse host cells involves an Arp2/3 complex, WAVE complex and Rho-family GTPase-dependent pathway.

    PubMed

    Reed, Shawna C O; Serio, Alisa W; Welch, Matthew D

    2012-04-01

    Rickettsiae are obligate intracellular pathogens that are transmitted to humans by arthropod vectors and cause diseases such as spotted fever and typhus. Although rickettsiae require the host cell actin cytoskeleton for invasion, the cytoskeletal proteins that mediate this process have not been completely described. To identify the host factors important during cell invasion by Rickettsia parkeri, a member of the spotted fever group (SFG), we performed an RNAi screen targeting 105 proteins in Drosophila melanogaster S2R+ cells. The screen identified 21 core proteins important for invasion, including the GTPases Rac1 and Rac2, the WAVE nucleation-promoting factor complex and the Arp2/3 complex. In mammalian cells, including endothelial cells, the natural targets of R. parkeri, the Arp2/3 complex was also crucial for invasion, while requirements for WAVE2 as well as Rho GTPases depended on the particular cell type. We propose that R. parkeri invades S2R+ arthropod cells through a primary pathway leading to actin nucleation, whereas invasion of mammalian endothelial cells occurs via redundant pathways that converge on the host Arp2/3 complex. Our results reveal a key role for the WAVE and Arp2/3 complexes, as well as a higher degree of variation than previously appreciated in actin nucleation pathways activated during Rickettsia invasion. PMID:22188208

  16. DISTORTED3/SCAR2 is a putative arabidopsis WAVE complex subunit that activates the Arp2/3 complex and is required for epidermal morphogenesis.

    PubMed

    Basu, Dipanwita; Le, Jie; El-Essal, Salah El-Din; Huang, Shanjin; Zhang, Chunhua; Mallery, Eileen L; Koliantz, Gregore; Staiger, Christopher J; Szymanski, Daniel B

    2005-02-01

    In a plant cell, a subset of actin filaments function as a scaffold that positions the endomembrane system and acts as a substrate on which organelle motility occurs. Other actin filament arrays appear to be more dynamic and reorganize in response to growth signals and external cues. The distorted group of trichome morphology mutants provides powerful genetic tools to study the control of actin filament nucleation in the context of morphogenesis. In this article, we report that DISTORTED3 (DIS3) encodes a plant-specific SCAR/WAVE homolog. Null alleles of DIS3, like those of other Arabidopsis thaliana WAVE and Actin-Related Protein (ARP) 2/3 subunit genes, cause trichome distortion, defects in cell-cell adhesion, and reduced hypocotyl growth in etiolated seedlings. DIS3 efficiently activates the actin filament nucleation and branching activity of vertebrate Arp2/3 and functions within a WAVE-ARP2/3 pathway in vivo. DIS3 may assemble into a WAVE complex via a physical interaction with a highly diverged Arabidopsis Abi-1-like bridging protein. These results demonstrate the utility of the Arabidopsis trichome system to understand how the WAVE and ARP2/3 complexes translate signaling inputs into a coordinated morphogenetic response. PMID:15659634

  17. 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. PMID:26498789

  18. 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

  19. Intranuclear Actin Regulates Osteogenesis

    PubMed Central

    Sen, Buer; Xie, Zhihui; Uzer, Gunes; Thompson, William R.; Styner, Maya; Wu, Xin; Rubin, Janet

    2016-01-01

    Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and global effects on gene transcription. We here show that in mesenchymal stem cells (MSCs), cytochalasin D treatment causes rapid cofilin-/importin-9-dependent transfer of G-actin into the nucleus. The continued presence of intranuclear actin, which forms rod-like structures that stain with phalloidin, is associated with induction of robust expression of the osteogenic genes osterix and osteocalcin in a Runx2-dependent manner, and leads to acquisition of osteogenic phenotype. Adipogenic differentiation also occurs, but to a lesser degree. Intranuclear actin leads to nuclear export of Yes-associated protein (YAP); maintenance of nuclear YAP inhibits Runx2 initiation of osteogenesis. Injection of cytochalasin into the tibial marrow space of live mice results in abundant bone formation within the space of 1 week. In sum, increased intranuclear actin forces MSC into osteogenic lineage through controlling Runx2 activity; this process may be useful for clinical objectives of forming bone. PMID:26140478

  20. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53

    PubMed Central

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1–393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using “hot-spot” p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  1. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53.

    PubMed

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1-393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using "hot-spot" p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  2. Nucleation and growth of cadherin adhesions

    SciTech Connect

    Lambert, Mireille; Thoumine, Olivier; Brevier, Julien; Choquet, Daniel; Riveline, Daniel; Mege, Rene-Marc

    2007-11-15

    Cell-cell contact formation relies on the recruitment of cadherin molecules and their anchoring to actin. However, the precise chronology of events from initial cadherin trans-interactions to adhesion strengthening is unclear, in part due to the lack of access to the distribution of cadherins within adhesion zones. Using N-cadherin expressing cells interacting with N-cadherin coated surfaces, we characterized the formation of cadherin adhesions at the ventral cell surface. TIRF and RIC microscopies revealed streak-like accumulations of cadherin along actin fibers. FRAP analysis indicated that engaged cadherins display a slow turnover at equilibrium, compatible with a continuous addition and removal of cadherin molecules within the adhesive contact. Association of cadherin cytoplasmic tail to actin as well as actin cables and myosin II activity are required for the formation and maintenance of cadherin adhesions. Using time lapse microscopy we deciphered how cadherin adhesions form and grow. As lamellipodia protrude, cadherin foci stochastically formed a few microns away from the cell margin. Neo-formed foci coalesced aligned and coalesced with preformed foci either by rearward sliding or gap filling to form cadherin adhesions. Foci experienced collapse at the rear of cadherin adhesions. Based on these results, we present a model for the nucleation, directional growth and shrinkage of cadherin adhesions.

  3. Transient nucleation in glasses

    NASA Technical Reports Server (NTRS)

    Kelton, K. F.

    1991-01-01

    Nucleation rates in condensed systems are frequently not at their steady state values. Such time dependent (or transient) nucleation is most clearly observed in devitrification studies of metallic and silicate glasses. The origin of transient nucleation and its role in the formation and stability of desired phases and microstructures are discussed. Numerical models of nucleation in isothermal and nonisothermal situations, based on the coupled differential equations describing cluster evolution within the classical theory, are presented. The importance of transient nucleation in glass formation and crystallization is discussed.

  4. Membrane related dynamics and the formation of actin in cells growing on micro-topographies: a spatial computational model

    PubMed Central

    2014-01-01

    Background Intra-cellular processes of cells at the interface to an implant surface are influenced significantly by their extra-cellular surrounding. Specifically, when growing osteoblasts on titanium surfaces with regular micro-ranged geometry, filaments are shorter, less aligned and they concentrate at the top of the geometric structures. Changes to the cytoskeleton network, i. e., its localization, alignment, orientation, and lengths of the filaments, as well as the overall concentration and distribution of key-actors are induced. For example, integrin is distributed homogeneously, whereas integrin in activated state and vinculin, both components of focal adhesions, have been found clustered on the micro-ranged geometries. Also, the concentration of Rho, an intracellular signaling protein related to focal adhesion regulation, was significantly lower. Results To explore whether regulations associated with the focal adhesion complex can be responsible for the changed actin filament patterns, a spatial computational model has been developed using ML-Space, a rule-based model description language, and its associated Brownian-motion-based simulator. The focus has been on the deactivation of cofilin in the vicinity of the focal adhesion complex. The results underline the importance of sensing mechanisms to support a clustering of actin filament nucleations on the micro-ranged geometries, and of intracellular diffusion processes, which lead to spatially heterogeneous distributions of active (dephosphorylated) cofilin, which in turn influences the organization of the actin network. We find, for example, that the spatial heterogeneity of key molecular actors can explain the difference in filament lengths in cells on different micro-geometries partly, but to explain the full extent, further model assumptions need to be added and experimentally validated. In particular, our findings and hypothesis referring to the role, distribution, and amount of active cofilin have still

  5. Actin Recruitment to the Chlamydia Inclusion Is Spatiotemporally Regulated by a Mechanism That Requires Host and Bacterial Factors

    PubMed Central

    Chin, Elizabeth; Kirker, Kelly; Zuck, Meghan; James, Garth; Hybiske, Kevin

    2012-01-01

    The ability to exit host cells at the end of their developmental growth is a critical step for the intracellular bacterium Chlamydia. One exit strategy, extrusion, is mediated by host signaling pathways involved with actin polymerization. Here, we show that actin is recruited to the chlamydial inclusion as a late event, occurring after 20 hours post-infection (hpi) and only within a subpopulation of cells. This event increases significantly in prevalence and extent from 20 to 68 hpi, and actin coats strongly correlated with extrusions. In contrast to what has been reported for other intracellular pathogens, actin nucleation on Chlamydia inclusions did not ‘flash’, but rather exhibited moderate depolymerization dynamics. By using small molecule agents to selectively disrupt host signaling pathways involved with actin nucleation, modulate actin polymerization dynamics and also to disable the synthesis and secretion of chlamydial proteins, we further show that host and bacterial proteins are required for actin coat formation. Transient disruption of either host or bacterial signaling pathways resulted in rapid loss of coats in all infected cells and a reduction in extrusion formation. Inhibition of Chlamydia type III secretion also resulted in rapid loss of actin association on inclusions, thus implicating chlamydial effector proteins(s) as being central factors for engaging with host actin nucleating factors, such as formins. In conclusion, our data illuminate the host and bacterial driven process by which a dense actin matrix is dynamically nucleated and maintained on the Chlamydia inclusion. This late stage event is not ubiquitous for all infected cells in a population, and escalates in prevalence and extent throughout the developmental cycle of Chlamydia, culminating with their exit from the host cell by extrusion. The initiation of actin recruitment by Chlamydia appears to be novel, and may serve as an upstream determinant of the extrusion mechanism. PMID

  6. Filamentous actin is a substrate for protealysin, a metalloprotease of invasive Serratia proteamaculans.

    PubMed

    Tsaplina, Olga; Efremova, Tatiana; Demidyuk, Ilya; Khaitlina, Sofia

    2012-01-01

    Homologous bacterial metalloproteases ECP32/grimelysin from Serratia grimesii and protealysin from Serratia proteamaculans are involved in the invasion of the nonpathogenic bacteria in eukaryotic cells and are suggested to translocate into the cytoplasm [Bozhokina ES et al. (2011) Cell Biol Int35, 111-118]. The proteases have been characterized as actin-hydrolyzing enzymes with a narrow specificity toward intact cell proteins. However, cleavage of filamentous actin (F-actin) (i.e. the main actin species in the cell) and the properties of the cleaved F-actin have not been investigated previously. In the present study, we revealed the presence of protealysin in the cytoplasm of 3T3-SV40 cells infected with S. proteamaculans or recombinant Escherichia coli expressing the protealysin gene. We also show for the first time that purified protealysin and the lysates of the recombinant E. coli producing protealysin cleave 20-40% of F-actin. Cleavage limited predominantly to the bond Gly42-Val43 efficiently increases the steady-state ATPase activity (dynamics) of F-actin. abolishes this effect and promotes the nucleation of protealysin-cleaved Mg-globular-actin even in the absence of 0.1 m KCl, most likely as a result of the stabilization of lateral intermonomer contacts of actin subunits. The results obtained in the present study suggest that F-actin can be a target for protealysin upon its translocation into the host cell. PMID:22077798

  7. Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA

    PubMed Central

    Bell, Jason C.; Plank, Jody L.; Dombrowski, Christopher C.; Kowalczykowski, Stephen C.

    2012-01-01

    Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA1. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly2,3. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA-family relative to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts2–5. Previous single-molecule assays have measured nucleation and growth of RecA—and its eukaryotic homolog RAD51—on naked dsDNA and ssDNA6–12; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA3. Using single-molecule microscopy, we directly visualized RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5′→3′ direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism where RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (i.e., DNA unwrapping) and then grows. We further demonstrate that the recombination mediator protein pair, Rec

  8. VASP Governs Actin Dynamics by Modulating Filament Anchoring

    PubMed Central

    Trichet, Léa; Campàs, Otger; Sykes, Cécile; Plastino, Julie

    2007-01-01

    Actin filament dynamics at the cell membrane are important for cell-matrix and cell-cell adhesions and the protrusion of the leading edge. Since actin filaments must be connected to the cell membrane to exert forces but must also detach from the membrane to allow it to move and evolve, the balance between actin filament tethering and detachment at adhesion sites and the leading edge is key for cell shape changes and motility. How this fine tuning is performed in cells remains an open question, but possible candidates are the Drosophila enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins, which localize to dynamic actin structures in the cell. Here we study VASP-mediated actin-related proteins 2/3 (Arp2/3) complex-dependent actin dynamics using a substrate that mimics the fluid properties of the cell membrane: an oil-water interface. We show evidence that polymerization activators undergo diffusion and convection on the fluid surface, due to continual attachment and detachment to the actin network. These dynamics are enhanced in the presence of VASP, and we observe cycles of catastrophic detachment of the actin network from the surface, resulting in stop-and-go motion. These results point to a role for VASP in the modulation of filament anchoring, with implications for actin dynamics at cell adhesions and at the leading edge of the cell. PMID:17098798

  9. Electrophoresis and orientation of F-actin in agarose gels.

    PubMed Central

    Borejdo, J; Ortega, H

    1989-01-01

    F-Actin was electrophoresed on agarose gels. In the presence of 2 mM MgCl2 and above pH 8.5 F-actin entered 1% agarose; when the electric field was 2.1 V/cm and the pH was 8.8, F-actin migrated through a gel as a single band at a rate of 2.5 mm/h. Labeling of actin with fluorophores did not affect its rate of migration, but an increase in ionic strength slowed it down. After the electrophoresis actin was able to bind phalloidin and heavy meromyosin (HMM) and it activated Mg2+-dependent ATPase activity of HMM. The mobility of F-actin increased with the rise in pH. Acto-S-1 complex was also able to migrate in agarose at basic pH, but at a lower rate than F-actin alone. The orientation of fluorescein labeled F-actin and of fluorescein labeled S-1 which formed rigor bonds with F-actin was measured during the electrophoresis by the fluorescence detected linear dichroism method. The former showed little orientation, probably because the dye was mobile on the surface of actin, but we were able to measure the orientation of the absorption dipole of the dye bound to S-1 which was attached to F-actin, and found that it assumed an orientation largely parallel to the direction of the electric field. These results show that actin can migrate in agarose gels in the F form and that it is oriented during the electrophoresis. Images FIGURE 1 FIGURE 3 FIGURE 4 PMID:2528384

  10. A dynamic phase-field model for structural transformations and twinning: Regularized interfaces with transparent prescription of complex kinetics and nucleation. Part I: Formulation and one-dimensional characterization

    NASA Astrophysics Data System (ADS)

    Agrawal, Vaibhav; Dayal, Kaushik

    2015-12-01

    The motion of microstructural interfaces is important in modeling twinning and structural phase transformations. Continuum models fall into two classes: sharp-interface models, where interfaces are singular surfaces; and regularized-interface models, such as phase-field models, where interfaces are smeared out. The former are challenging for numerical solutions because the interfaces need to be explicitly tracked, but have the advantage that the kinetics of existing interfaces and the nucleation of new interfaces can be transparently and precisely prescribed. In contrast, phase-field models do not require explicit tracking of interfaces, thereby enabling relatively simple numerical calculations, but the specification of kinetics and nucleation is both restrictive and extremely opaque. This prevents straightforward calibration of phase-field models to experiment and/or molecular simulations, and breaks the multiscale hierarchy of passing information from atomic to continuum. Consequently, phase-field models cannot be confidently used in dynamic settings. This shortcoming of existing phase-field models motivates our work. We present the formulation of a phase-field model - i.e., a model with regularized interfaces that do not require explicit numerical tracking - that allows for easy and transparent prescription of complex interface kinetics and nucleation. The key ingredients are a re-parametrization of the energy density to clearly separate nucleation from kinetics; and an evolution law that comes from a conservation statement for interfaces. This enables clear prescription of nucleation - through the source term of the conservation law - and kinetics - through a distinct interfacial velocity field. A formal limit of the kinetic driving force recovers the classical continuum sharp-interface driving force, providing confidence in both the re-parametrized energy and the evolution statement. We present some 1D calculations characterizing the formulation; in a

  11. Calcium-actin waves and oscillations of cellular membranes.

    PubMed

    Veksler, Alex; Gov, Nir S

    2009-09-16

    We propose a mechanism for the formation of membrane oscillations and traveling waves, which arise due to the coupling between the actin cytoskeleton and the calcium flux through the membrane. In our model, the fluid cell membrane has a mobile but constant population of proteins with a convex spontaneous curvature, which act as nucleators of actin polymerization and adhesion. Such a continuum model couples the forces of cell-substrate adhesion, actin polymerization, membrane curvature, and the flux of calcium through the membrane. Linear stability analysis shows that sufficiently strong coupling among the calcium, membrane, and protein dynamics may induce robust traveling waves on the membrane. This result was checked for a reduced feedback scheme and is compared to the results without the effects of calcium, where permanent phase separation without waves or oscillations is obtained. The model results are compared to the published observations of calcium waves in cell membranes, and a number of testable predictions are proposed. PMID:19751660

  12. Tailor-Made Ezrin Actin Binding Domain to Probe Its Interaction with Actin In-Vitro

    PubMed Central

    Shrivastava, Rohini; Köster, Darius; Kalme, Sheetal; Mayor, Satyajit; Neerathilingam, Muniasamy

    2015-01-01

    Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well. PMID:25860910

  13. Ice-Nucleating Bacteria

    NASA Astrophysics Data System (ADS)

    Obata, Hitoshi

    Since the discovery of ice-nucleating bacteria in 1974 by Maki et al., a large number of studies on the biological characteristics, ice-nucleating substance, ice nucleation gene and frost damage etc. of the bacteria have been carried out. Ice-nucleating bacteria can cause the freezing of water at relatively warm temperature (-2.3°C). Tween 20 was good substrates for ice-nucleating activity of Pseudomonas fluorescens KUIN-1. Major fatty acids of Isolate (Pseudomonas fluorescens) W-11 grown at 30°C were palmitic, cis-9-hexadecenoic and cis-11-octadecenoic which amounted to 90% of the total fatty acids. Sequence analysis shows that an ice nucleation gene from Pseudomonas fluorescens is related to the gene of Pseudomonas syringae.

  14. Actin and Arp2/3 localize at the centrosome of interphase cells

    SciTech Connect

    Hubert, Thomas; Vandekerckhove, Joel; Gettemans, Jan

    2011-01-07

    Research highlights: {yields} Actin was detected at the centrosome with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. {yields} Centrosomal actin was found in interphase but not mitotic MDA-MB-231 cells. {yields} Neither the anti-actin antibody C4 that binds to globular, monomer actin, nor the anti-actin antibody 2G2 that recognizes the nuclear conformation of actin detect actin at the centrosome. {yields} The Arp2/3 complex transiently localizes at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. -- Abstract: Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel ('lower dimer') actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

  15. Force of an actin spring

    NASA Astrophysics Data System (ADS)

    Shin, Jennifer; Mahadevan, L.; Matsudaira, Paul

    2003-03-01

    The acrosomal process of the horseshoe crab sperm is a novel mechanochemical molecular spring that converts its elastic stain energy to mechanical work upon the chemical activation by Ca2+. Twisted and bent, the initial state of the acrosomal bundle features a high degree of complexity in its structure and the energy is believed to be stored in the highly strained actin filaments as an elastic potential energy. When activated, the bundle relaxes from the coil of the highly twisted and bent filaments to its straight conformation at a mean velocity of 15um/s. The mean extension velocity increases dramatically from 3um/s to 27um/s when temperature of the medium is changed from 9.6C to 32C (respective viscosities of 1.25-0.75cp), yet it exhibits a very weak dependence on changes in the medium viscosity (1cp-33cp). These experiments suggest that the uncoiling of the actin spring should be limited not by the viscosity of the medium but by the unlatching events of involved proteins at a molecular level. Unlike the viscosity-limited processes, where force is directly related to the rate of the reaction, a direct measurement is required to obtain the spring force of the acrosomal process. The extending acrosomal bundle is forced to push against a barrier and its elastic buckling response is analyzed to measure the force generated during the uncoiling.

  16. Characterization of the cadmium complex of peptide 49-61: a putative nucleation center for cadmium-induced folding in rabbit liver metallothionein IIA.

    PubMed

    Muñoz, A; Laib, F; Petering, D H; Shaw, C F

    1999-08-01

    The synthetic peptide fragment containing residues 49-61 of rabbit liver metallothionein II (MT-II) (Ac-Ile-Cys-Lys-Gly-Ala-Ser-Asp-Lys-Cys-Ser-Cys-Cys-Ala-COOH), which includes the only sequential four cysteines bound to the same metal ion in Cd7MT, forms a stable, monomeric Cd-peptide complex with 1:1 stoichiometry (Cd:peptide) via Cd-thiolate interactions. This represents the first synthesis of a single metal-binding site of MT independent of the domains. The 111Cd NMR chemical shift at 716 ppm indicates that the 111Cd2+ in the metal site is terminally coordinated to four side-chain thiolates of the cysteine residues. The pH of half dissociation for this Cd-peptide derivative, approximately 3.3, demonstrates an affinity similar to that for Cd7MT. Molecular mechanics calculations show that the thermodynamically most stable folding for this isolated Cd2+ center has the same counterclockwise chirality (lambda or S) observed in the native holo-protein. These properties are consistent with its proposed role as a nucleation center for cadmium-induced protein folding. However, the kinetic reactivity of the CdS4 structure toward 5,5'-dithiobis(5-nitrobenzoate) (DTNB) and EDTA is greatly increased compared to the complete cluster (a-domain or holo-protein). The rate law for the reaction with DTNB is rate = (k(uf) + k(1,f) + k(2,f) [DTNB])[peptide], where k(uf) = 0.15 s(-1), k(1,f)= 2.59x10(-3) s(-1), and k(2,f) = 0.88 M(-1) s(-1). The ultrafast step (uf), observable only by stopped-flow measurement, is unprecedented for mammalian (M7MT) and crustacean (M6MT) holo-proteins or the isolated domains. The accommodation of other metal ions by the peptide indicates a rich coordination chemistry, including stoichiometries of M-peptide for Hg2+, Cd2+, and Zn2+, M2-peptide for Hg2+ and Au+, and (Et3PAu)2-peptide. PMID:10555583

  17. Actin-associated Proteins in the Pathogenesis of Podocyte Injury

    PubMed Central

    He, Fang-Fang; Chen, Shan; Su, Hua; Meng, Xian-Fang; Zhang, Chun

    2013-01-01

    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. PMID:24396279

  18. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins

    NASA Astrophysics Data System (ADS)

    Loisel, Thomas P.; Boujemaa, Rajaa; Pantaloni, Dominique; Carlier, Marie-France

    1999-10-01

    Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, α-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

  19. Role of actin in auxin transport and transduction of gravity

    NASA Astrophysics Data System (ADS)

    Hu, S.; Basu, S.; Brady, S.; Muday, G.

    Transport of the plant hormone auxin is polar and the direction of the hormone movement appears to be controlled by asymmetric distribution of auxin transport protein complexes. Changes in the direction of auxin transport are believed to drive asymmetric growth in response to changes in the gravity vector. To test the possibility that asymmetric distribution of the auxin transport protein complex is mediated by attachment to the actin cytoskeleton, a variety of experimental approaches have been used. The most direct demonstration of the role of the actin cytoskeleton in localization of the protein complex is the ability of one protein in this complex to bind to affinity columns containing actin filaments. Additionally, treatments of plant tissues with drugs that fragment the actin c toskeleton reducey polar transport. In order to explore this actin interaction and the affect of gravity on auxin transport and developmental polarity, embryos of the brown alga, Fucus have been examined. Fucus zygotes are initially symmetrical, but develop asymmetry in response to environmental gradients, with light gradients being the best- characterized signal. Gravity will polarize these embryos and gravity-induced polarity is randomized by clinorotation. Auxin transport also appears necessary for environmental controls of polarity, since auxin efflux inhibitors perturb both photo- and gravity-polarization at a very discrete temporal window within six hours after fertilization. The actin cytoskeleton has previously been shown to reorganize after fertilization of Fucus embryos leading to formation of an actin patch at the site of polar outgrowth. These actin patches still form in Fucus embryos treated with auxin efflux inhibitors, yet the position of these patches is randomized. Together, these results suggest that there are connections between the actin cytoskeleton, auxin transport, and gravity oriented growth and development. (Supported by NASA Grant: NAG2-1203)

  20. Thoracic Aortic Aneurysm (TAAD)-causing Mutation in Actin Affects Formin Regulation of Polymerization*

    PubMed Central

    Malloy, Lindsey E.; Wen, Kuo-Kuang; Pierick, Alyson R.; Wedemeyer, Elesa W.; Bergeron, Sarah E.; Vanderpool, Nicole D.; McKane, Melissa; Rubenstein, Peter A.; Bartlett, Heather L.

    2012-01-01

    More than 30 mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause autosomal dominant thoracic aortic aneurysm and dissection. The mutation R256H is of particular interest because it also causes patent ductus arteriosus and moyamoya disease. R256H is one of the more prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament, may affect F-actin stability. To understand the molecular ramifications of the R256H mutation, we generated Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells displayed abnormal cytoskeletal morphology and increased sensitivity to latrunculin A. After cable disassembly induced by transient exposure to latrunculin A, mutant cells were delayed in reestablishing the actin cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed nucleation. Consequently, we investigated regulation of mutant actin by formin, a potent facilitator of nucleation and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was inhibited by the FH1-FH2 fragment of the yeast formin, Bni1. This fragment strongly capped the filament rather than facilitating polymerization. Interestingly, phalloidin or the presence of wild type actin reversed the strong capping behavior of Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation resulting in filament instability and misregulation by formin. These biochemical effects may contribute to abnormal histology identified in diseased arterial samples from affected patients. PMID:22753406

  1. Thoracic aortic aneurysm (TAAD)-causing mutation in actin affects formin regulation of polymerization.

    PubMed

    Malloy, Lindsey E; Wen, Kuo-Kuang; Pierick, Alyson R; Wedemeyer, Elesa W; Bergeron, Sarah E; Vanderpool, Nicole D; McKane, Melissa; Rubenstein, Peter A; Bartlett, Heather L

    2012-08-17

    More than 30 mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause autosomal dominant thoracic aortic aneurysm and dissection. The mutation R256H is of particular interest because it also causes patent ductus arteriosus and moyamoya disease. R256H is one of the more prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament, may affect F-actin stability. To understand the molecular ramifications of the R256H mutation, we generated Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells displayed abnormal cytoskeletal morphology and increased sensitivity to latrunculin A. After cable disassembly induced by transient exposure to latrunculin A, mutant cells were delayed in reestablishing the actin cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed nucleation. Consequently, we investigated regulation of mutant actin by formin, a potent facilitator of nucleation and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was inhibited by the FH1-FH2 fragment of the yeast formin, Bni1. This fragment strongly capped the filament rather than facilitating polymerization. Interestingly, phalloidin or the presence of wild type actin reversed the strong capping behavior of Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation resulting in filament instability and misregulation by formin. These biochemical effects may contribute to abnormal histology identified in diseased arterial samples from affected patients. PMID:22753406

  2. Diamond nucleation using polyethene

    DOEpatents

    Morell, Gerardo; Makarov, Vladimir; Varshney, Deepak; Weiner, Brad

    2013-07-23

    The invention presents a simple, non-destructive and non-abrasive method of diamond nucleation using polyethene. It particularly describes the nucleation of diamond on an electrically viable substrate surface using polyethene via chemical vapor deposition (CVD) technique in a gaseous environment.

  3. Diamond Nucleation Using Polyethene

    NASA Technical Reports Server (NTRS)

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

    2013-01-01

    The invention presents a simple, non-destructive and non-abrasive method of diamond nucleation using polyethene. It particularly describes the nucleation of diamond on an electrically viable substrate surface using polyethene via chemical vapor deposition (CVD) technique in a gaseous environment.

  4. Chloride intracellular channel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms.

    PubMed Central

    Suginta, W; Karoulias, N; Aitken, A; Ashley, R H

    2001-01-01

    Mammalian chloride intracellular channel (CLIC) (p64-related) proteins are widely expressed, with an unusual dual localization as both soluble and integral membrane proteins. The molecular basis for their cellular localization and ion channel activity remains unclear. To help in addressing these problems, we identified novel rat brain CLIC4 (p64H1) binding partners by affinity chromatography, mass spectrometric analysis and microsequencing. Brain CLIC4 binds dynamin I, alpha-tubulin, beta-actin, creatine kinase and two 14-3-3 isoforms; the interactions are confirmed in vivo by immunoprecipitation. Gel overlay and reverse pull-down assays indicate that the binding of CLIC4 to dynamin I and 14-3-3zeta is direct. In HEK-293 cells, biochemical and immunofluorescence analyses show partial co-localization of recombinant CLIC4 with caveolin and with functional caveolae, which is consistent with a dynamin-associated role for CLIC4 in caveolar endocytosis. We speculate that brain CLIC4 might be involved in the dynamics of neuronal plasma membrane microdomains (micropatches) containing caveolin-like proteins and might also have other cellular roles related to membrane trafficking. Our results provide the basis for new hypotheses concerning novel ways in which CLIC proteins might be associated with cell membrane remodelling, the control of cell shape, and anion channel activity. PMID:11563969

  5. Nucleation of amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Kashchiev, Dimo; Auer, Stefan

    2010-06-01

    We consider nucleation of amyloid fibrils in the case when the process occurs by the mechanism of direct polymerization of practically fully extended protein segments, i.e., β-strands, into β-sheets. Applying the classical nucleation theory, we derive a general expression for the work to form a nanosized amyloid fibril (protofilament) constituted of successively layered β-sheets. Analysis of this expression reveals that with increasing its size, the fibril transforms from one-dimensional to two-dimensional aggregate in order to preserve the equilibrium shape corresponding to minimal formation work. We determine the size of the fibril nucleus, the fibril nucleation work, and the fibril nucleation rate as explicit functions of the concentration and temperature of the protein solution. The results obtained are applicable to homogeneous nucleation, which occurs when the solution is sufficiently pure and/or strongly supersaturated.

  6. Mechanics of Biomimetic Liposomes Encapsulating an Actin Shell.

    PubMed

    Guevorkian, Karine; Manzi, John; Pontani, Léa-Lætitia; Brochard-Wyart, Françoise; Sykes, Cécile

    2015-12-15

    Cell-shape changes are insured by a thin, dynamic, cortical layer of cytoskeleton underneath the plasma membrane. How this thin cortical structure impacts the mechanical properties of the whole cell is not fully understood. Here, we study the mechanics of liposomes or giant unilamellar vesicles, when a biomimetic actin cortex is grown at the inner layer of the lipid membrane via actin-nucleation-promoting factors. Using a hydrodynamic tube-pulling technique, we show that tube dynamics is clearly affected by the presence of an actin shell anchored to the lipid bilayer. The same force pulls much shorter tubes in the presence of the actin shell compared to bare membranes. However, in both cases, we observe that the dynamics of tube extrusion has two distinct features characteristic of viscoelastic materials: rapid elastic elongation, followed by a slower elongation phase at a constant rate. We interpret the initial elastic regime by an increase of membrane tension due to the loss of lipids into the tube. Tube length is considerably shorter for cortex liposomes at comparable pulling forces, resulting in a higher spring constant. The presence of the actin shell seems to restrict lipid mobility, as is observed in the corral effect in cells. The viscous regime for bare liposomes corresponds to a leakout of the internal liquid at constant membrane tension. The presence of the actin shell leads to a larger friction coefficient. As the tube is pulled from a patchy surface, membrane tension increases locally, leading to a Marangoni flow of lipids. As a conclusion, the presence of an actin shell is revealed by its action that alters membrane mechanics. PMID:26682806

  7. Nonequilibrium thermodynamics of nucleation.

    PubMed

    Schweizer, M; Sagis, L M C

    2014-12-14

    We present a novel approach to nucleation processes based on the GENERIC framework (general equation for the nonequilibrium reversible-irreversible coupling). Solely based on the GENERIC structure of time-evolution equations and thermodynamic consistency arguments of exchange processes between a metastable phase and a nucleating phase, we derive the fundamental dynamics for this phenomenon, based on continuous Fokker-Planck equations. We are readily able to treat non-isothermal nucleation even when the nucleating cores cannot be attributed intensive thermodynamic properties. In addition, we capture the dynamics of the time-dependent metastable phase being continuously expelled from the nucleating phase, and keep rigorous track of the volume corrections to the dynamics. Within our framework the definition of a thermodynamic nuclei temperature is manifest. For the special case of nucleation of a gas phase towards its vapor-liquid coexistence, we illustrate that our approach is capable of reproducing recent literature results obtained by more microscopic considerations for the suppression of the nucleation rate due to nonisothermal effects. PMID:25494727

  8. Nonequilibrium thermodynamics of nucleation

    SciTech Connect

    Schweizer, M.; Sagis, L. M. C.

    2014-12-14

    We present a novel approach to nucleation processes based on the GENERIC framework (general equation for the nonequilibrium reversible-irreversible coupling). Solely based on the GENERIC structure of time-evolution equations and thermodynamic consistency arguments of exchange processes between a metastable phase and a nucleating phase, we derive the fundamental dynamics for this phenomenon, based on continuous Fokker-Planck equations. We are readily able to treat non-isothermal nucleation even when the nucleating cores cannot be attributed intensive thermodynamic properties. In addition, we capture the dynamics of the time-dependent metastable phase being continuously expelled from the nucleating phase, and keep rigorous track of the volume corrections to the dynamics. Within our framework the definition of a thermodynamic nuclei temperature is manifest. For the special case of nucleation of a gas phase towards its vapor-liquid coexistence, we illustrate that our approach is capable of reproducing recent literature results obtained by more microscopic considerations for the suppression of the nucleation rate due to nonisothermal effects.

  9. Interactions Between DNA and Actin in Model Cystic Fibrosis Sputum

    NASA Astrophysics Data System (ADS)

    Kyung, Hee; Sanders, Lori; Angelini, Thomas; Butler, John; Wong, Gerard

    2003-03-01

    Cystic fibrosis sputum is a complex fluid which has a high concentration of DNA and F-actin, two anionic biological polyelectrolytes. In this work, we study the interactions between DNA and actin in an aqueous environment over a wide range of polyelectrolyte lengths and salt levels, using synchrotron Small Angle X-ray Scattering(SAXS) and confocal microscopy. Perliminary results indicate the existence of a compressed phase of nematic F-actin in the presence of DNA. This work was supported by NSF DMR-0071761, the Beckman Young Investigator Program, and the Cystic Fibrosis Foundation.

  10. Ice nucleation terminology

    NASA Astrophysics Data System (ADS)

    Vali, G.; DeMott, P.; Möhler, O.; Whale, T. F.

    2014-08-01

    Progress in the understanding of ice nucleation is being hampered by the lack of uniformity in how some terms are used in the literature. This even extends to some ambiguity of meanings attached to some terms. Suggestions are put forward here for common use of terms. Some are already well established and clear of ambiguities. Others are less engrained and will need a conscious effort in adoption. Evolution in the range of systems where ice nucleation is being studied enhances the need for a clear nomenclature. The ultimate limit in the clarity of definitions is, of course, the limited degree to which ice nucleation processes are understood.

  11. Characterization of actin filament deformation in response to actively driven microspheres propagated through entangled actin networks

    NASA Astrophysics Data System (ADS)

    Falzone, Tobias; Blair, Savanna; Robertson-Anderson, Rae

    2014-03-01

    The semi-flexible biopolymer actin is a ubiquitous component of nearly all biological organisms, playing an important role in many biological processes such as cell structure and motility, cancer invasion and metastasis, muscle contraction, and cell signaling. Concentrated actin networks possess unique viscoelastic properties that have been the subject of much theoretical and experimental work. However, much is still unknown regarding the correlation of the applied stress on the network to the induced filament strain at the molecular level. Here, we use dual optical traps alongside fluorescence microscopy to carry out active microrheology measurements that link mechanical stress to structural response at the micron scale. Specifically, we actively drive microspheres through entangled actin networks while simultaneously measuring the force the surrounding filaments exert on the sphere and visualizing the deformation and subsequent relaxation of fluorescent labeled filaments within the network. These measurements, which provide much needed insight into the link between stress and strain in actin networks, are critical for clarifying our theoretical understanding of the complex viscoelastic behavior exhibited in actin networks.

  12. CryoEM reveals different coronin binding modes for ADP- and ADP-BeFx- actin filaments

    PubMed Central

    Ge, Peng; Oztug Durer, Zeynep A.; Kudryashov, Dmitri; Zhou, Z. Hong; Reisler, Emil

    2015-01-01

    Essential cellular processes involving the actin cytoskeleton are regulated by auxiliary proteins which can sense the nucleotide state of actin. Here we report cryo electron microscopy (cryoEM) structures at 8.6 Å resolution for ADP- and ADP-BeFx- (mimicking ADP-Pi) bound actin filaments in complex with the β-propeller domain (residues 1–600) of yeast coronin 1 (crn1). Our structures identify the main differences in the interaction of coronin with the two nucleotide states of F-actin. We derived pseudo-atomic models by fitting the atomic structures of actin and coronin into these structures. The identified binding interfaces on actin were confirmed by chemical crosslinking, fluorescence spectroscopy and actin mutagenesis. Importantly, the structures of actin and coronin mapped in this study offer a structural explanation for the nucleotide-dependent effects of coronin on cofilin-assisted remodeling of F-actin. PMID:25362487

  13. Nuclear F-actin Formation and Reorganization upon Cell Spreading*♦

    PubMed Central

    Plessner, Matthias; Melak, Michael; Chinchilla, Pilar; Baarlink, Christian; Grosse, Robert

    2015-01-01

    We recently discovered signal-regulated nuclear actin network assembly. However, in contrast to cytoplasmic actin regulation, polymeric nuclear actin structures and functions remain only poorly understood. Here we describe a novel molecular tool to visualize real-time nuclear actin dynamics by targeting the Actin-Chromobody-TagGFP to the nucleus, thus establishing a nuclear Actin-Chromobody. Interestingly, we observe nuclear actin polymerization into dynamic filaments upon cell spreading and fibronectin stimulation, both of which appear to be triggered by integrin signaling. Furthermore, we show that nucleoskeletal proteins such as the LINC (linker of nucleoskeleton and cytoskeleton) complex and components of the nuclear lamina couple cell spreading or integrin activation by fibronectin to nuclear actin polymerization. Spreading-induced nuclear actin polymerization results in serum response factor (SRF)-mediated transcription through nuclear retention of myocardin-related transcription factor A (MRTF-A). Our results reveal a signaling pathway, which links integrin activation by extracellular matrix interaction to nuclear actin polymerization through the LINC complex, and therefore suggest a role for nuclear actin polymerization in the context of cellular adhesion and mechanosensing. PMID:25759381

  14. Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast

    PubMed Central

    Huang, Junqi; Huang, Yinyi; Yu, Haochen; Subramanian, Dhivya; Padmanabhan, Anup; Thadani, Rahul; Tao, Yaqiong; Tang, Xie; Wedlich-Soldner, Roland

    2012-01-01

    In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly. PMID:23185032

  15. Nonstationary homogeneous nucleation

    NASA Technical Reports Server (NTRS)

    Harstad, K. G.

    1974-01-01

    The theory of homogeneous condensation is reviewed and equations describing this process are presented. Numerical computer solutions to transient problems in nucleation (relaxation to steady state) are presented and compared to a prior computation.

  16. Bidirectional actin transport is influenced by microtubule and actin stability.

    PubMed

    Chetta, Joshua; Love, James M; Bober, Brian G; Shah, Sameer B

    2015-11-01

    Local and long-distance transport of cytoskeletal proteins is vital to neuronal maintenance and growth. Though recent progress has provided insight into the movement of microtubules and neurofilaments, mechanisms underlying the movement of actin remain elusive, in large part due to rapid transitions between its filament states and its diverse cellular localization and function. In this work, we integrated live imaging of rat sensory neurons, image processing, multiple regression analysis, and mathematical modeling to perform the first quantitative, high-resolution investigation of GFP-actin identity and movement in individual axons. Our data revealed that filamentous actin densities arise along the length of the axon and move short but significant distances bidirectionally, with a net anterograde bias. We directly tested the role of actin and microtubules in this movement. We also confirmed a role for actin densities in extension of axonal filopodia, and demonstrated intermittent correlation of actin and mitochondrial movement. Our results support a novel mechanism underlying slow component axonal transport, in which the stability of both microtubule and actin cytoskeletal components influence the mobility of filamentous actin. PMID:26043972

  17. Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins.

    PubMed

    Courtemanche, Naomi; Pollard, Thomas D; Chen, Qian

    2016-06-01

    When tagged with a fluorescent protein, actin is not fully functional, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly in vitro and in fission yeast cells. mEGFP-LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP-LifeAct and LifeAct-mCherry bind actin filaments with Kd values of ∼10 μM. LifeAct-mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct-mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ∼190,000 actin molecules (∼500 μm) in the fission yeast contractile ring. These filaments shorten from ∼500 to ∼100 subunits as the ring constricts. PMID:27159499

  18. Self-organizing actin waves as planar phagocytic cup structures

    PubMed Central

    Ecke, Mary; Schroth-Diez, Britta; Gerwig, Silke; Engel, Ulrike; Maddera, Lucinda; Clarke, Margaret

    2009-01-01

    Actin waves that travel on the planar membrane of a substrate-attached cell underscore the capability of the actin system to assemble into dynamic structures by the recruitment of proteins from the cytoplasm. The waves have no fixed shape, can reverse their direction of propagation and can fuse or divide. Actin waves separate two phases of the plasma membrane that are distinguished by their lipid composition. The area circumscribed by a wave resembles in its phosphoinositide content the interior of a phagocytic cup, leading us to explore the possibility that actin waves are in-plane phagocytic structures generated without the localized stimulus of an attached particle. Consistent with this view, wave-forming cells were found to exhibit a high propensity for taking up particles. Cells fed rod-shaped particles produced elongated phagocytic cups that displayed a zonal pattern that reflected in detail the actin and lipid pattern of free-running actin waves. Neutrophils and macrophages are known to spread on surfaces decorated with immune complexes, a process that has been interpreted as “frustrated” phagocytosis. We suggest that actin waves enable a phagocyte to scan a surface for particles that might be engulfed. PMID:19855162

  19. Nuclear Actin Extends, with No Contraction in Sight

    PubMed Central

    Pederson, Thoru; Aebi, Ueli

    2005-01-01

    Within the past two years, actin has been implicated in eukaryotic gene transcription by all three classes of RNA polymerase. Moreover, within just the past year, actin has been identified as a constituent of filaments attached to the nuclear pore complexes and extending into the nucleus. This review summarizes these and other very recent advances in the nuclear actin field and emphasizes the key present issues. On the one hand, we are confronted with a body of evidence for a role of actin in gene transcription but with no known structural basis; on the other hand, there is now evidence for polymeric actin—not likely in the classical F-actin conformation—in the nuclear periphery with no known function. In addition, numerous proteins that interact with either G- or F-actin are increasingly being detected in the nucleus, suggesting that both monomeric and oligomeric or polymeric forms of actin are at play and raising the possibility that the equilibrium between them, perhaps differentially regulated at various intranuclear sites, may be a major determinant of nuclear function. PMID:16148048

  20. Identification of Actin-Binding Proteins from Maize Pollen

    SciTech Connect

    Staiger, C.J.

    2004-01-13

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

  1. Cdc42 and PI(4,5)P2-induced actin assembly in Xenopus egg extracts.

    PubMed

    Lebensohn, Andres M; Ma, Le; Ho, Hsin-Yi Henry; Kirschner, Marc W

    2006-01-01

    Xenopus egg cytoplasmic extracts have been used to study a variety of complex cellular processes. Given their amenability to biochemical manipulation and physiological balance of regulatory proteins, these extracts are an ideal system to dissect signal transduction pathways leading to actin assembly. We have developed methods to study Cdc42 and PI(4,5)P2-induced actin assembly in Xenopus egg extracts. In this chapter, we describe detailed procedures to prepare Xenopus egg extracts, Cdc42, and PI(4,5)P2 for use in actin assembly experiments. We also describe a fluorometric pyrene actin assay for quantitative kinetic analysis of actin polymerization and a microscopic rhodamine actin assay for quick measurement of actin rearrangements in extracts. Finally we provide a protocol for immunodepletion of proteins and discuss the use of immunodepletion and rescue experiments for functional analysis of components in the extracts. PMID:16472657

  2. A new link between the retrograde actin flow and focal adhesions.

    PubMed

    Yamashiro, Sawako; Watanabe, Naoki

    2014-11-01

    The retrograde actin flow, continuous centripetal movement of the cell peripheral actin networks, is widely observed in adherent cells. The retrograde flow is believed to facilitate cell migration when linked to cell adhesion molecules. In this review, we summarize our current knowledge regarding the functional relationship between the retrograde actin flow and focal adhesions (FAs). We also introduce our recent study in which single-molecule speckle (SiMS) microscopy dissected the complex interactions between FAs and the local actin flow. FAs do not simply impede the actin flow, but actively attract and remodel the local actin network. Our findings provide a new insight into the mechanisms for protrusion and traction force generation at the cell leading edge. Furthermore, we discuss possible roles of the actin flow-FA interaction based on the accumulated knowledge and our SiMS study. PMID:25190817

  3. WHAMY is a novel actin polymerase promoting myoblast fusion, macrophage cell motility and sensory organ development in Drosophila.

    PubMed

    Brinkmann, Klaus; Winterhoff, Moritz; Önel, Susanne-Filiz; Schultz, Jörg; Faix, Jan; Bogdan, Sven

    2016-02-01

    Wiskott-Aldrich syndrome proteins (WASPs) are nucleation-promoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR (also known as WAVE), WASP and WASH (also known as CG13176), have been analyzed so far. Here, we characterized WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, we found that WHAMY specifically interacted with activated Rac1 through its two CRIB domains, which were sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promoted exceptionally fast actin filament elongation, although it did not activate the Arp2/3 complex. Loss- and gain-of-function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further implied synergistic functions between WHAMY and WASP during morphogenesis. Double mutants were late-embryonic lethal and showed severe defects in myoblast fusion. Trans-heterozygous mutant animals showed strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution. PMID:26675239

  4. Characterization of the Cadherin-Catenin Complex of the Sea Anemone Nematostella vectensis and Implications for the Evolution of Metazoan Cell-Cell Adhesion.

    PubMed

    Clarke, Donald Nathaniel; Miller, Phillip W; Lowe, Christopher J; Weis, William I; Nelson, William James

    2016-08-01

    The cadherin-catenin complex (CCC) mediates cell-cell adhesion in bilaterian animals by linking extracellular cadherin-based adhesions to the actin cytoskeleton. However, it is unknown whether the basic organization of the complex is conserved across all metazoans. We tested whether protein interactions and actin-binding properties of the CCC are conserved in a nonbilaterian animal, the sea anemone Nematostella vectensis We demonstrated that N. vectensis has a complete repertoire of cadherin-catenin proteins, including two classical cadherins, one α-catenin, and one β-catenin. Using size-exclusion chromatography and multi-angle light scattering, we showed that α-catenin and β-catenin formed a heterodimer that bound N. vectensis Cadherin-1 and -2. Nematostella vectensis α-catenin bound F-actin with equivalent affinity as either a monomer or an α/β-catenin heterodimer, and its affinity for F-actin was, in part, regulated by a novel insert between the N- and C-terminal domains. Nematostella vectensis α-catenin inhibited Arp2/3 complex-mediated nucleation of actin filaments, a regulatory property previously thought to be unique to mammalian αE-catenin. Thus, despite significant differences in sequence, the key interactions of the CCC are conserved between bilaterians and cnidarians, indicating that the core function of the CCC as a link between cell adhesions and the actin cytoskeleton is ancestral in the eumetazoans. PMID:27189570

  5. Characterization of the Cadherin–Catenin Complex of the Sea Anemone Nematostella vectensis and Implications for the Evolution of Metazoan Cell–Cell Adhesion

    PubMed Central

    Clarke, Donald Nathaniel; Miller, Phillip W.; Lowe, Christopher J.; Weis, William I.; Nelson, William James

    2016-01-01

    The cadherin–catenin complex (CCC) mediates cell–cell adhesion in bilaterian animals by linking extracellular cadherin-based adhesions to the actin cytoskeleton. However, it is unknown whether the basic organization of the complex is conserved across all metazoans. We tested whether protein interactions and actin-binding properties of the CCC are conserved in a nonbilaterian animal, the sea anemone Nematostella vectensis. We demonstrated that N. vectensis has a complete repertoire of cadherin–catenin proteins, including two classical cadherins, one α-catenin, and one β-catenin. Using size-exclusion chromatography and multi-angle light scattering, we showed that α-catenin and β-catenin formed a heterodimer that bound N. vectensis Cadherin-1 and -2. Nematostella vectensis α-catenin bound F-actin with equivalent affinity as either a monomer or an α/β-catenin heterodimer, and its affinity for F-actin was, in part, regulated by a novel insert between the N- and C-terminal domains. Nematostella vectensis α-catenin inhibited Arp2/3 complex-mediated nucleation of actin filaments, a regulatory property previously thought to be unique to mammalian αE-catenin. Thus, despite significant differences in sequence, the key interactions of the CCC are conserved between bilaterians and cnidarians, indicating that the core function of the CCC as a link between cell adhesions and the actin cytoskeleton is ancestral in the eumetazoans. PMID:27189570

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

    PubMed

    Muday, G K; Hu, S; Brady, S R

    2000-06-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport. PMID:11543284

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  8. Arabidopsis BRICK1/HSPC300 is an essential WAVE-complex subunit that selectively stabilizes the Arp2/3 activator SCAR2.

    PubMed

    Le, Jie; Mallery, Eileen L; Zhang, Chunhua; Brankle, Steven; Szymanski, Daniel B

    2006-05-01

    The actin cytoskeleton dynamically reorganizes the cytoplasm during cell morphogenesis. The actin-related protein (Arp)2/3 complex is a potent nucleator of actin filaments that controls a variety of endomembrane functions including the endocytic internalization of plasma membrane , vacuole biogenesis , plasma-membrane protrusion in crawling cells , and membrane trafficking from the Golgi . Therefore, Arp2/3 is an important signaling target during morphogenesis. The evolutionarily conserved Rac-WAVE-Arp2/3 pathway links actin filament nucleation to cell morphogenesis . WAVE translates Rac-GTP signals into Arp2/3 activation by regulating the stability and/or localization of the activator subunit Scar/WAVE . The WAVE complex includes Sra1/PIR121/CYFIP1, Nap1/NAP125, Abi-1/Abi-2, Brick1(Brk1)/HSPC300, and Scar/WAVE : Defining the in vivo function of each subunit is an important step toward understanding this complicated signaling pathway. Brk1/HSPC300 has been the most recalcitrant WAVE-complex protein and has no known function. In this paper, we report that Arabidopsis brick1 (brk1) is a member of the "distorted group" of trichome morphology mutants, a group that defines a WAVE-ARP2/3 morphogenesis pathway . In this paper we provide the first strong genetic and biochemical evidence that BRK1 is a critical WAVE-complex subunit that selectively stabilizes the Arp2/3 activator SCAR2. PMID:16584883

  9. Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum

    PubMed Central

    Lin, Wei-Chi; Wang, Liang-Chen; Pang, Te-Ling; Chen, Mei-Yu

    2015-01-01

    Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971–amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG− cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401–600 and aa 501–550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501–550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG− cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG− cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion. PMID:25609090

  10. Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum.

    PubMed

    Lin, Wei-Chi; Wang, Liang-Chen; Pang, Te-Ling; Chen, Mei-Yu

    2015-03-15

    Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971-amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG(-) cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401-600 and aa 501-550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501-550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG(-) cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG(-) cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion. PMID:25609090

  11. Gcn1 and Actin Binding to Yih1

    PubMed Central

    Sattlegger, Evelyn; Barbosa, João A. R. G.; Moraes, Maria Carolina S.; Martins, Rafael M.; Hinnebusch, Alan G.; Castilho, Beatriz A.

    2011-01-01

    Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1–132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68–258, encompassing part of the RWD and the C-terminal “ancient domain”; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68–258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation. PMID:21239490

  12. 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

  13. 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

  14. Probing the Flexibility of Tropomyosin and Its Binding to Filamentous Actin Using Molecular Dynamics Simulations

    PubMed Central

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

    2013-01-01

    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. PMID:24138864

  15. Direct interaction of beta-dystroglycan with F-actin.

    PubMed Central

    Chen, Yun-Ju; Spence, Heather J; Cameron, Jacqueline M; Jess, Thomas; Ilsley, Jane L; Winder, Steven J

    2003-01-01

    Dystroglycans are essential transmembrane adhesion receptors for laminin. Alpha-dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of beta-dystroglycan. Beta-dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton. As a part of the dystrophin-glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity. Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype. Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of beta-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells. PMID:12892561

  16. The budding yeast amphiphysin complex is required for contractile actin ring (CAR) assembly and post-contraction GEF-independent accumulation of Rho1-GTP.

    PubMed

    Cundell, Michael John; Price, Clive

    2014-01-01

    The late events of the budding yeast cell division cycle, cytokinesis and cell separation, require the assembly of a contractile actomyosin ring (CAR), primary and secondary septum formation followed by enzymatic degradation of the primary septum. Here we present evidence that demonstrates a role for the budding yeast amphiphysin complex, a heterodimer comprising Rvs167 and Rvs161, in CAR assembly and cell separation. The iqg1-1 allele is synthetically lethal with both rvs167 and rvs161 null mutations. We show that both Iqg1 and the amphiphysin complex are required for CAR assembly in early anaphase but cells are able to complete assembly in late anaphase when these activities are, respectively, either compromised or absent. Amphiphysin dependent CAR assembly is dependent upon the Rvs167 SH3 domain, but this function is insufficient to explain the observed synthetic lethality. Dosage suppression of the iqg1-1 allele demonstrates that endocytosis is required for the default cell separation pathway in the absence of CAR contraction but is unlikely to be required to maintain viability. The amphiphysin complex is required for normal, post-mitotic, localization of Chs3 and the Rho1 GEF, Rom2, which are responsible for secondary septum deposition and the accumulation of GTP bound Rho1 at the bud neck. It is concluded that a failure of polarity establishment in the absence of CAR contraction and amphiphysin function leads to loss of viability as a result of the consequent cell separation defect. PMID:24874185

  17. A Two-step Mechanism for the Folding of Actin by the Yeast Cytosolic Chaperonin

    PubMed Central

    Stuart, Sarah F.; Leatherbarrow, Robin J.; Willison, Keith R.

    2011-01-01

    Actin requires the chaperonin containing TCP1 (CCT), a hexadecameric ATPase essential for cell viability in eukaryotes, to fold to its native state. Following binding of unfolded actin to CCT, the cavity of the chaperone closes and actin is folded and released in an ATP-dependent folding cycle. In yeast, CCT forms a ternary complex with the phosducin-like protein PLP2p to fold actin, and together they can return nascent or chemically denatured actin to its native state in a pure in vitro folding assay. The complexity of the CCT-actin system makes the study of the actin folding mechanism technically challenging. We have established a novel spectroscopic assay through selectively labeling the C terminus of yeast actin with acrylodan and observe significant changes in the acrylodan fluorescence emission spectrum as actin is chemically unfolded and then refolded by the chaperonin. The variation in the polarity of the environment surrounding the fluorescent probe during the unfolding/folding processes has allowed us to monitor actin as it folds on CCT. The rate of actin folding at a range of temperatures and ATP concentrations has been determined for both wild type CCT and a mutant CCT, CCT4anc2, defective in folding actin in vivo. Binding of the non-hydrolysable ATP analog adenosine 5′-(β,γ-imino)triphosphate to the ternary complex leads to 3-fold faster release of actin from CCT following addition of ATP, suggesting a two-step folding process with a conformational change occurring upon closure of the cavity and a subsequent final folding step involving packing of the C terminus to the native-like state. PMID:21056978

  18. Actin-based phagosome motility.

    PubMed

    Zhang, Fangliang; Southwick, Frederick S; Purich, Daniel L

    2002-10-01

    Despite abundant evidence of actin's involvement at the particle internalization stage of phagocytosis, little is known about whether phagosomes undergo the same type of actin-based motility as observed with endocytic vesicles or such intracellular pathogens as Listeria and Shigella. By employing video microscopy to follow the fate of latex bead-containing phagosomes within the cytoplasm of bone marrow macrophages, we have made the novel observation of actin-based phagosome motility. Immunofluorescence microscopy confirmed that phagosomes containing IgG-opsonized, bovine serum albumin (or BSA) -coated or uncoated latex beads all formed actin-rich rocket tails that persisted only during a brief, 1-2 min period of actin-based motility. Average speeds of actin-based phagosome motility were 0.13 +/- 0.06 microm/s for IgG-coated beads, 0.14 +/- 0.04 microm/s for BSA-coated beads, and 0.11+/- 0.03 microm/s for uncoated beads. Moreover, the speeds and motile-phase duration of each type of phagosome were comparable to the behavior of pinosomes [Merrifield et al., 1999: Nat. Cell Biol. 1:72-74.]. Determination of optimal conditions for observing and analyzing actin-based phagosome motility should facilitate future investigations of phagocytosis and phagosome maturation. PMID:12211106

  19. Three-Dimensional Structure of Cofilin Bound to Monomeric Actin Derived by Structural Mass Spectrometry Data

    SciTech Connect

    Amisha Kamal,J.; Benchaar, S.; Takamoto, K.; Reisler, E.; Chance, M.

    2007-01-01

    The cytoskeletal protein, actin, has its structure and function regulated by cofilin. In the absence of an atomic resolution structure for the actin/cofilin complex, the mechanism of cofilin regulation is poorly understood. Theoretical studies based on the similarities of cofilin and gelsolin segment 1 proposed the cleft between subdomains 1 and 3 in actin as the cofilin binding site. We used radiolytic protein footprinting with mass spectrometry and molecular modeling to provide an atomic model of how cofilin binds to monomeric actin. Footprinting data suggest that cofilin binds to the cleft between subdomains 1 and 2 in actin and that cofilin induces further closure of the actin nucleotide cleft. Site-specific fluorescence data confirm these results. The model identifies key ionic and hydrophobic interactions at the binding interface, including hydrogen-bonding between His-87 of actin to Ser-89 of cofilin that may control the charge dependence of cofilin binding. This model and its implications fill an especially important niche in the actin field, owing to the fact that ongoing crystallization efforts of the actin/cofilin complex have so far failed. This 3D binary complex structure is derived from a combination of solution footprinting data and computational approaches and outlines a general method for determining the structure of such complexes.

  20. The effect of mouse twinfilin-1 on the structure and dynamics of monomeric actin.

    PubMed

    Takács-Kollár, Veronika; Nyitrai, Miklós; Hild, Gábor

    2016-07-01

    The effect of twinfilin-1 on the structure and dynamics of monomeric actin was investigated with fluorescence spectroscopy and differential scanning calorimetry experiments. Fluorescence anisotropy measurements proved that G-actin and twinfilin-1 could form a complex. Due to the formation of the complexes the dissociation of the nucleotide slowed down from the nucleotide-binding pocket of actin. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of twinfilin-1. Temperature dependent fluorescence resonance energy transfer and differential scanning calorimetry experiments revealed that the protein matrix of actin becomes more rigid and more heat resistant in the presence of twinfilin-1. The results suggest that the nucleotide binding cleft shifted into a more closed and stable conformational state of actin in the presence of twinfilin-1. PMID:27079635

  1. Crystallization and preliminary structural characterization of the two actin isoforms of the malaria parasite

    PubMed Central

    Bhargav, Saligram Prabhakar; Vahokoski, Juha; Kumpula, Esa-Pekka; Kursula, Inari

    2013-01-01

    Malaria is a devastating disease caused by apicomplexan parasites of the genus Plasmodium that use a divergent actin-powered molecular motor for motility and invasion. Plasmodium actin differs from canonical actins in sequence, structure and function. Here, the purification, crystallization and secondary-structure analysis of the two Plasmodium actin isoforms are presented. The recombinant parasite actins were folded and could be purified to homogeneity. Plasmodium actins I and II were crystallized in complex with the gelsolin G1 domain; the crystals diffracted to resolutions of 1.19 and 2.2 Å and belonged to space groups P212121 and P21, respectively, each with one complex in the asymmetric unit. PMID:24100575

  2. Bacterial actins and their diversity

    PubMed Central

    Ozyamak, Ertan; Kollman, Justin M.; Komeili, Arash

    2015-01-01

    For many years bacteria were considered rather simple organisms, but the dogmatic notion that subcellular organization is a eukaryotic trait has been overthrown for more than a decade. The discovery of homologs of the eukaryotic cytoskeletal proteins actin, tubulin, and intermediate filaments in bacteria has been instrumental in changing this view. Over the recent years we gained an incredible level of insight into the diverse family of bacterial actins and their molecular workings. Here we review the functional, biochemical and structural features of the most well-studied bacterial actins. PMID:24015924

  3. The role of actin networks in cellular mechanosensing

    NASA Astrophysics Data System (ADS)

    Azatov, Mikheil

    behavior as in cancer metastasis. In addition to stiffness, the local geometry or topography of the surface has been shown to modulate the movement, morphology, and cytoskeletal organization of cells. However, the effect of topography on fluctuations of intracellular structures, which arise from motor driven activity on a viscoelastic actin network are not known. I have used nanofabricated substrates with parallel ridges to show that the cell shape, the actin cytoskeleton and focal adhesions all align along the direction of the ridges, exhibiting a biphasic dependence on the spacing between ridges. I further demonstrated that palladin bands along actin stress fibers undergo a complex diffusive motion with velocities aligned along the direction of ridges. These results provide insight into the mechanisms of cellular mechanosensing of the environment, suggesting a complex interplay between the actin cytoskeleton and cellular adhesions in coordinating cellular response to surface topography. Overall, this work has advanced our understanding of mechanisms that govern cellular responses to their physical environment.

  4. Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

    PubMed Central

    Belyy, Alexander; Tabakova, Irina; Lang, Alexander E.; Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-01-01

    Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. PMID:26713879

  5. Combining Classical MD and QM calculations to elucidate complex system nucleation: a twisted, three-stranded, parallel β-sheet seeds amyloid fibril conception.

    PubMed

    Mompeán, Miguel; González, Carlos; Lomba, Enrique; Laurents, Douglas V

    2014-07-01

    The crystal structure of the Sup35 prion segment, GNNQQNY, revealed precise side chain packing and an extensive H-bond network. However, the conformers and stabilizing interactions involved at nascent amyloid formation are still unclear. Here, long molecular dynamics simulations and quantum mechanical calculations have been utilized to study the conformation and energetics of the initial structure that acts to nucleate further growth. Considering all the plausible intermediates that may act as stepping stones, we find that the initial nucleus is a twisted single-layer, three-stranded parallel β-sheet. H-bonds between β-strands in this twisted sheet, some of which differ from those of the crystal structure's nontwisted β-strands, are key for the nucleus' formation and stability. High level theoretical calculations of these H-bonds' energetics can account for this amyloid-like trimer's remarkable stability. The intermeshing of facing sheets to form the dry interface provides less stability and would occur between two three-stranded β-sheets without metastable water nanowires. PMID:24918329

  6. ACD toxin-produced actin oligomers poison formin-controlled actin polymerization

    PubMed Central

    Heisler, David B.; Kudryashova, Elena; Grinevich, Dmitry O.; Suarez, Cristian; Winkelman, Jonathan D.; Birukov, Konstantin G.; Kotha, Sainath R.; Parinandi, Narasimham L.; Vavylonis, Dimitrios; Kovar, David R.; Kudryashov, Dmitri S.

    2015-01-01

    The actin crosslinking domain (ACD) is an actin-specific toxin produced by several pathogens, including life-threatening spp. of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Actin crosslinking by ACD is thought to lead to slow cytoskeleton failure owing to a gradual sequestration of actin in the form of nonfunctional oligomers. Here we found that ACD converted cytoplasmic actin into highly toxic oligomers that potently “poisoned” the ability of major actin assembly proteins, formins, to sustain actin polymerization. Thus, ACD can target the most abundant cellular protein by employing actin oligomers as secondary toxins to efficiently subvert cellular functions of actin while functioning at very low doses. PMID:26228148

  7. Epidemiology of actinic keratoses.

    PubMed

    Green, Adèle C

    2015-01-01

    The epidemiology of actinic keratoses (AKs) reflects their causation by cumulative sun exposure, with the highest prevalence seen in pale-skinned people living at low latitudes and on the most sun-exposed body sites, namely the hands, forearms and face. AKs are markers of increased risk of basal cell carcinoma, squamous cell carcinoma and melanoma, especially when they are numerous and have coalesced into an area of 'field cancerisation'. The major risk factors are male sex, advanced age, sun-sensitive complexion, high lifetime sun exposure and prolonged immunosuppression. Clinical counts of AKs enable the assessment and monitoring of AK burden, but accurate counting is notoriously difficult, especially when skin is severely sun damaged. AK counting has been repeatedly shown to be unreliable, even among expert dermatologists. Notwithstanding these challenges, qualitative assessment of the natural history of AKs shows a high turnover, with new lesions developing and with other lesions regressing. A very small proportion of AKs undergo malignant transformation, but the precise rate of transformation is unknown due to the inaccuracies in monitoring AK lesions over time. Primary prevention of AKs is achieved by limiting intense sun exposure through sun-protective behaviour, including seeking deep shade, wearing sun-protective clothing and applying sunscreen regularly to exposed skin, from an early age. PMID:25561199

  8. Chemotaxis and Actin Oscillations

    NASA Astrophysics Data System (ADS)

    Bodenschatz, Eberhard; Hsu, Hsin-Fang; Negrete, Jose; Beta, Carsten; Pumir, Alain; Gholami, Azam; Tarantola, Marco; Westendorf, Christian; Zykov, Vladimir

    Recently, self-oscillations of the cytoskeletal actin have been observed in Dictyostelium, a model system for studying chemotaxis. Here we report experimental results on the self-oscillation mechanism and the role of regulatory proteins and myosin II. We stimulate cells rapidly and periodically by using photo un-caging of the chemoattractant in a micro-fluidic device and measured the cellular responses. We found that the response amplitude grows with stimulation strength only in a very narrow region of stimulation, after which the response amplitude reaches a plateau. Moreover, the frequency-response is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and de-polymerization time in the single cell level. Despite of the large cell-to-cell variability, we found that the polymerization time is independent of external stimuli and the de-polymerization time is prolonged as the stimulation strength increases. Our conclusions will be summarized and the role of noise in the signaling network will be discussed. German Science Foundation CRC 937.

  9. Distinct impact of targeted actin cytoskeleton reorganization on mechanical properties of normal and malignant cells.

    PubMed

    Efremov, Yu M; Dokrunova, A A; Efremenko, A V; Kirpichnikov, M P; Shaitan, K V; Sokolova, O S

    2015-11-01

    The actin cytoskeleton is substantially modified in cancer cells because of changes in actin-binding protein abundance and functional activity. As a consequence, cancer cells have distinctive motility and mechanical properties, which are important for many processes, including invasion and metastasis. Here, we studied the effects of actin cytoskeleton alterations induced by specific nucleation inhibitors (SMIFH2, CK-666), cytochalasin D, Y-27632 and detachment from the surface by trypsinization on the mechanical properties of normal Vero and prostate cancer cell line DU145. The Young's modulus of Vero cells was 1300±900 Pa, while the prostate cancer cell line DU145 exhibited significantly lower Young's moduli (600±400 Pa). The Young's moduli exhibited a log-normal distribution for both cell lines. Unlike normal cells, cancer cells demonstrated diverse viscoelastic behavior and different responses to actin cytoskeleton reorganization. They were more resistant to specific formin-dependent nucleation inhibition, and reinforced their cortical actin after detachment from the substrate. This article is part of a Special Issue entitled: Mechanobiology. PMID:25970206

  10. Kinetic studies of the cooperative binding of subfragment 1 to regulated actin.

    PubMed Central

    Trybus, K M; Taylor, E W

    1980-01-01

    The transient-state kinetics of binding of myosin subfragment 1 (SF-1) to regulated actin in the presence and absence of Ca2+ were investigated. The binding of SF-1 to pure actin, to actin-tropomyosin (actin-TM), or to actin-tropomyosin-troponin (actin-TM-TN) in the presence of Ca2+ was kinetically the same. In each case, the light-scattering transients were biphasic, suggesting a two-step binding of SF-1 to actin. Binding of SF-1 to regulated actin in the absence of Ca2+ was different from binding in its presence and also varied depending on whether SF-1 or regulated actin was in excess. The kinetic results in the absence of CA2+ are explained by a cooperative binding model, in which the initial binding of SF-1 molecules to open (active) actin sites increases the number of open sites. TN-I labeled with the fluorophore 4-(N-iodoacetoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3 diazole (TN*) was used to probe the state of the actin-TM-TN complex. Binding of SF-1 or CA2+ to regulated actin (in the absence of Ca2+) decreased the fluorescence of actin-TM-TN* by 30%, suggesting that binding of SF-1 or CA2+ induces a similar change in state. The change in fluorescence of TN* was also used to measure the rate of the transition from the active to the relaxed state in the absence of CA2+, which was 430 sec-1 at 4 degrees C in 0.1 M KCl. The lag prior to association of SF-1 with regulated actin (in the absence of Ca2+) was abolished when three SF-1 molecules were prebound per seven G-actin monomers. Similarly, a titration of actin-TM-TN* (in the absence of Ca2+) with SF-1 or SF-1-ADP showed that most actin sites are open, as measured by the fluorescence change, when the occupancy of actin-TM-TN* by SF-1-ADP or SF-1 is approximately 50%. The evidence shows that partial occupancy of a block of G-actin sites (possibly seven) by SF-1 or SF-1-ADP stabilizes the open (active) conformation. PMID:6938966

  11. Nucleation of Ice

    NASA Astrophysics Data System (ADS)

    Molinero, Valeria

    2009-03-01

    The freezing of water into ice is a ubiquitous transformation in nature, yet the microscopic mechanism of homogeneous nucleation of ice has not yet been elucidated. One of the reasons is that nucleation happens in time scales that are too fast for an experimental characterization and two slow for a systematic study with atomistic simulations. In this work we use coarse-grained molecular dynamics simulations with the monatomic model of water mW[1] to shed light into the mechanism of homogeneous nucleation of ice and its relationship to the thermodynamics of supercooled water. Cooling of bulk water produces either crystalline ice or low- density amorphous ice (LDA) depending on the quenching rate. We find that ice crystallization occurs faster at temperatures close to the liquid-liquid transition, defined as the point of maximum inflection of the density with respect to the temperature. At the liquid-liquid transition, the time scale of nucleation becomes comparable to the time scale of relaxation within the liquid phase, determining --effectively- the end of the metastable liquid state. Our results imply that no ultraviscous liquid water can exist at temperatures just above the much disputed glass transition of water. We discuss how the scenario is changed when water is in confinement, and the relationship of the mechanism of ice nucleation to that of other liquids that present the same phase behavior, silicon [2] and germanium [3]. [4pt] [1] Molinero, V. & Moore, E. B. Water modeled as an intermediate element between carbon and silicon. Journal of Physical Chemistry B (2008). Online at http://pubs.acs.org/cgi- bin/abstract.cgi/jpcbfk/asap/abs/jp805227c.html [0pt] [2] Molinero, V., Sastry, S. & Angell, C. A. Tuning of tetrahedrality in a silicon potential yields a series of monatomic (metal-like) glass formers of very high fragility. Physical Review Letters 97, 075701 (2006).

  12. Microtubule nucleation and organization in dendrites.

    PubMed

    Delandre, Caroline; Amikura, Reiko; Moore, Adrian W

    2016-07-01

    Dendrite branching is an essential process for building complex nervous systems. It determines the number, distribution and integration of inputs into a neuron, and is regulated to create the diverse dendrite arbor branching patterns characteristic of different neuron types. The microtubule cytoskeleton is critical to provide structure and exert force during dendrite branching. It also supports the functional requirements of dendrites, reflected by differential microtubule architectural organization between neuron types, illustrated here for sensory neurons. Both anterograde and retrograde microtubule polymerization occur within growing dendrites, and recent studies indicate that branching is enhanced by anterograde microtubule polymerization events in nascent branches. The polarities of microtubule polymerization events are regulated by the position and orientation of microtubule nucleation events in the dendrite arbor. Golgi outposts are a primary microtubule nucleation center in dendrites and share common nucleation machinery with the centrosome. In addition, pre-existing dendrite microtubules may act as nucleation sites. We discuss how balancing the activities of distinct nucleation machineries within the growing dendrite can alter microtubule polymerization polarity and dendrite branching, and how regulating this balance can generate neuron type-specific morphologies. PMID:27097122

  13. The Effect of Crosslinking on the Microscale Stress Response and Molecular Deformations in Actin Networks

    NASA Astrophysics Data System (ADS)

    Gurmessa, Bekele; Fitzpatrick, Robert; Valdivia, Jonathon; Anderson, Rae M. R.

    Actin, the most abundant protein in eukaryotic cells, is a semi-flexible biopolymer in the cytoskeleton that plays a crucial structural and mechanical role in cell stability, motion and replication, as well as muscle contraction. Most of these mechanically driven structural changes in cells stem from the complex viscoelastic nature of entangled actin networks and the presence of a myriad of proteins that cross-link actin filaments. Despite their importance, the mechanical response of actin networks is not yet well understood, particularly at the molecular level. Here, we use optical trapping - coupled with fluorescence microscopy - to characterize the microscale stress response and induced filament deformations in entangled and cross-linked actin networks subject to localized mechanical perturbations. In particular, we actively drive a microsphere 10 microns through an entangled or cross- linked actin network at a constant speed and measure the resistive force that the deformed actin filaments exert on the bead during and following strain. We simultaneously visualize and track individual sparsely-labeled actin filaments to directly link force response to molecular deformations, and map the propagation of the initially localized perturbation field throughout the rest of the network (~100 um). By varying the concentration of actin and cross-linkers we directly determine the role of crosslinking and entanglements on the length and time scales of stress propagation, molecular deformation and relaxation mechanisms in actin networks.

  14. Isolation of a 5-Kilodalton Actin-Sequestering Peptide from Human Blood Platelets

    NASA Astrophysics Data System (ADS)

    Safer, Daniel; Golla, Rajasree; Nachmias, Vivianne T.

    1990-04-01

    Resting human platelets contain ≈0.3 mM unpolymerized actin. When freshly drawn and washed platelets are treated with saponin, 85-90% of the unpolymerized actin diffuses out. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions shows that the bulk of this unpolymerized actin migrates with a higher mobility than does pure G-actin, profilactin, or actin-gelsolin complex. When muscle G-actin is added to fresh or boiled saponin extract, the added muscle actin is shifted to the high-mobility form. The saponin extract contains an acidic peptide having a molecular mass in the range of 5 kDa, which has been purified to homogeneity by reverse-phase HPLC. This peptide also shifts muscle actin to the high-mobility form. Addition of either boiled saponin extract or the purified peptide to muscle G-actin also strongly and stoichiometrically inhibits salt-induced polymerization, as assayed by falling-ball viscometry and by sedimentation. We conclude that this peptide binds to the bulk of the unpolymerized actin in platelets and prevents it from polymerizing.

  15. Ha-VP39 binding to actin and the influence of F-actin on assembly of progeny virions.

    PubMed

    Lu, S; Ge, G; Qi, Y

    2004-11-01

    We present evidence that actin is necessary for the successful assembly of HaNPV virions. Purified nucleocapsid protein Ha-VP39 of Heliothis armigera nuclear polyhedrosis virus (HaNPV) was found to be able to bind to actin in vitro without assistance, as demonstrated by Western blot and isothermal titration calorimeter. DeltaH and binding constants (K) detected by isothermal titration calorimeter strongly suggested that Ha-VP39 first binds actin to seed the formation of hexamer complex of actin, and the hexamers then link to each other to form filaments, and the filaments finally twist into cable structures. The proliferation of HaNPV was completely inhibited in Hz-AM1 cells cultivated in the medium containing 0.5 microg/ml cytochalasin D (CD) to prevent polymerization of actin, while its yield was reduced to 10(-4) in the presence of 0.1 microg/ml CD. Actin concentration and the viral DNA synthesis were not significantly affected by CD even though the progeny virions assembled in the CD treated cells were morphologically different from normal ones and resulted in fewer plaques in plaque assay. PMID:15503206

  16. The multi-faceted role of the actin cap in cellular mechanosensation and mechanotransduction.

    PubMed

    Kim, Dong-Hwee; Chambliss, Allison B; Wirtz, Denis

    2013-06-21

    The perinuclear actin cap (or actin cap) is a recently characterized cytoskeletal organelle composed of thick, parallel, and highly contractile acto-myosin filaments that are specifically anchored to the apical surface of the interphase nucleus. The actin cap is present in a wide range of adherent eukaryotic cells, but is disrupted in several human diseases, including laminopathies and cancer. Through its large terminating focal adhesions and anchorage to the nuclear lamina and nuclear envelope through LINC complexes, the perinuclear actin cap plays a critical role both in mechanosensation and mechanotransduction, the ability of cells to sense changes in matrix compliance and to respond to mechanical forces, respectively. PMID:23930135

  17. Proteomic Analysis Identifies an NADPH Oxidase 1 (Nox1)-Mediated Role for Actin-Related Protein 2/3 Complex Subunit 2 (ARPC2) in Promoting Smooth Muscle Cell Migration

    PubMed Central

    Al Ghouleh, Imad; Rodríguez, Andrés; Pagano, Patrick J.; Csányi, Gábor

    2013-01-01

    A variety of vascular pathologies, including hypertension, restenosis and atherosclerosis, are characterized by vascular smooth muscle cell (VSMC) hypertrophy and migration. NADPH oxidase 1 (Nox1) plays a pivotal role in these phenotypes via distinct downstream signaling. However, the mediators differentiating these distinct phenotypes and their precise role in vascular disease are still not clear. The present study was designed to identify novel targets of VSMC Nox1 signaling using 2D Differential In-Gel Electrophoresis and Mass Spectrometry (2D-DIGE/MS). VSMC treatment with scrambled (Scrmb) or Nox1 siRNA and incubation with the oxidant hydrogen peroxide (H2O2; 50 μM, 3 h) followed by 2D-DIGE/MS on cell lysates identified 10 target proteins. Among these proteins, actin-related protein 2/3 complex subunit 2 (ARPC2) with no previous link to Nox isozymes, H2O2, or other reactive oxygen species (ROS), was identified and postulated to play an intermediary role in VSMC migration. Western blot confirmed that Nox1 mediates H2O2-induced ARPC2 expression in VSMC. Treatment with a p38 MAPK inhibitor (SB203580) resulted in reduced ARPC2 expression in H2O2-treated VSMC. Additionally, wound-healing “scratch” assay confirmed that H2O2 stimulates VSMC migration via Nox1. Importantly, gene silencing of ARPC2 suppressed H2O2-stimulated VSMC migration. These results demonstrate for the first time that Nox1-mediated VSMC migration involves ARPC2 as a downstream signaling target. PMID:24152438

  18. Caveolin 1 (Cav-1) and actin-related protein 2/3 complex, subunit 1B (ARPC1B) expressions as prognostic indicators for oral squamous cell carcinoma (OSCC).

    PubMed

    Auzair, Lukman Bin Md; Vincent-Chong, Vui King; Ghani, Wan Maria Nabillah; Kallarakkal, Thomas George; Ramanathan, Anand; Lee, Chia Ee; Rahman, Zainal Ariff Abdul; Ismail, Siti Mazlipah; Abraham, Mannil Thomas; Zain, Rosnah Binti

    2016-07-01

    Caveolin-1 (Cav-1) and Actin-Related Protein 2/3 Complex, Subunit 1B (ARPC1B) have been implicated in various human cancers, yet its role in tumorigenesis remains controversial. Therefore, this study aims to determine the protein expression of these two genes in oral squamous cell carcinomas (OSCCs) and to evaluate the clinical and prognostic impact of these genes in OSCC. Protein expressions of these two genes were determined by immunohistochemistry technique. The association between Cav-1 and ARPC1B with clinico-pathological parameters was evaluated by Chi-square test (or Fisher exact test where appropriate). Correlation between the protein expressions of these 2 genes with survival was analyzed using Kaplan-Meier and Cox regression models. Cav-1 and ARPC1B were found to be significantly over-expressed in OSCC compared to normal oral mucosa (p = 0.002 and p = 0.033, respectively). Low level of ARPC1B protein expression showed a significant correlation with lymph node metastasis (LNM) (p = 0.010) and advanced tumor staging (p = 0.003). Kaplan-Meier survival analyses demonstrated that patients with over-expression of Cav-1 protein were associated with poor prognosis (p = 0.030). Adjusted multivariate Cox regression model revealed that over-expression of Cav-1 remained as an independent significant prognostic factor for OSCC (HRR = 2.700, 95 % CI 1.013-7.198, p = 0.047). This study demonstrated that low-expression of ARPC1B is significantly associated with LNM and advanced tumor staging whereas high expression of Cav-1 can be a prognostic indicator for poor prognosis in OSCC patients. PMID:26138391

  19. Regulation of water flow by actin-binding protein-induced actin gelatin.

    PubMed Central

    Ito, T; Suzuki, A; Stossel, T P

    1992-01-01

    Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by actin filaments corresponds to the gel point measured viscometrically and because gelsolin, which noncovalently severs actin filaments, solates actin gels and restores water flow in a solution of actin cross-linked by ABP. Since ABP gels actin filaments in the periphery of many eukaryotic cells, such actin networks may contribute to physiological cell volume regulation. PMID:1318095

  20. Drosophila quail, a villin-related protein, bundles actin filaments in apoptotic nurse cells.

    PubMed

    Matova, N; Mahajan-Miklos, S; Mooseker, M S; Cooley, L

    1999-12-01

    Drosophila Quail protein is required for the completion of fast cytoplasm transport from nurse cells to the oocyte, an event critical for the production of viable oocytes. The abundant network of cytoplasmic filamentous actin, established at the onset of fast transport, is absent in quail mutant egg chambers. Previously, we showed that Quail is a germline-specific protein with sequence homology to villin, a vertebrate actin-regulating protein. In this study, we combined biochemical experiments with observations in egg chambers to define more precisely the function of this protein in the regulation of actin-bundle assembly in nurse cells. We report that recombinant Quail can bind and bundle filamentous actin in vitro in a manner similar to villin at a physiological calcium concentration. In contrast to villin, Quail is unable to sever or cap filamentous actin, or to promote nucleation of new actin filaments at a high calcium concentration. Instead, Quail bundles the filaments regardless of the calcium concentration. In vivo, the assembly of nurse-cell actin bundles is accompanied by extensive perforation of the nurse-cell nuclear envelopes, and both of these phenomena are manifestations of nurse-cell apoptosis. To investigate whether free calcium levels are affected during apoptosis, we loaded egg chambers with the calcium indicator Indo-1. Our observations indicate a rise in free calcium in the nurse-cell cytoplasm coincident with the permeabilization of the nuclear envelopes. We also show that human villin expressed in the Drosophila germline could sense elevated cytoplasmic calcium; in nurse cells with reduced levels of Quail protein, villin interfered with actin-bundle stability. We conclude that Quail efficiently assembles actin filaments into bundles in nurse cells and maintains their stability under fluctuating free calcium levels. We also propose a developmental model for the fast phase of cytoplasm transport incorporating findings presented in this study

  1. Yeast Rsp5 ubiquitin ligase affects the actin cytoskeleton in vivo and in vitro.

    PubMed

    Kaminska, Joanna; Spiess, Matthias; Stawiecka-Mirota, Marta; Monkaityte, Rasa; Haguenauer-Tsapis, Rosine; Urban-Grimal, Daniele; Winsor, Barbara; Zoladek, Teresa

    2011-12-01

    Yeast Rsp5 ubiquitin ligase is involved in several cellular processes, including endocytosis. Actin patches are sites of endocytosis, a process involving actin assembly and disassembly. Here we show Rsp5 localization in cortical patches and demonstrate its involvement in actin cytoskeleton organization and dynamics. We found that the Rsp5-F1-GFP2 N-terminal fragment and full length GFP-Rsp5 were recruited to peripheral patches that temporarily co-localized with Abp1-mCherry, a marker of actin patches. Actin cytoskeleton organization was defective in a strain lacking RSP5 or overexpressing RSP5, and this phenotype was accompanied by morphological abnormalities. Overexpression of RSP5 caused hypersensitivity of cells to Latrunculin A, an actin-depolymerizing drug and was toxic to cells lacking Las17, an activator of actin nucleation. Moreover, Rsp5 was required for efficient actin polymerization in a whole cell extract based in vitro system. Rsp5 interacted with Las17 and Las17-binding proteins, Lsb1 and Lsb2, in a GST-Rsp5-WW2/3 pull down assay. Rsp5 ubiquitinated Lsb1-HA and Lsb2-HA without directing them for degradation. Overexpression of RSP5 increased the cellular level of HA-Las17 in wild type and in lsb1Δ lsb2Δ strains in which the basal level of Las17 was already elevated. This increase was prevented in a strain devoid of Las17-binding protein Sla1 which is also a target of Rsp5 ubiquitination. Thus, Rsp5 together with Lsb1, Lsb2 and Sla1 regulate the level of Las17, an important activator of actin polymerization. PMID:22000681

  2. Coronin 1B antagonizes Cortactin and remodels Arp2/3-containing actin branches in lamellipodia

    PubMed Central

    Cai, Liang; Makhov, Alexander M.; Schafer, Dorothy A.; Bear, James E.

    2008-01-01

    Summary The dendritic actin network generated by Arp2/3 complex in lamellipodia underlies formation of protrusions, directional sensing and migration. While the generation of this network is well studied, the mechanisms regulating network disassembly are poorly understood. We report that Coronin 1B disassembles Arp2/3-containing actin filament branches by inducing Arp2/3 dissociation. This activity is antagonized by Cortactin, a filament branch stabilizer. Consistent with this biochemical competition, depletion of both proteins partially rescues defects in lamellipodial dynamics observed upon depletion of either protein alone. Coronin 1B targets actin branches in a manner that is mutually exclusive with Arp2/3 complex and alters the branch angle. We conclude that Coronin 1B replaces Arp2/3 complex at actin filament branches as the dendritic network matures and drives the turnover of branched actin networks. PMID:18775315

  3. Propagating waves separate two states of actin organization in living cells

    PubMed Central

    Schroth-Diez, Britta; Gerwig, Silke; Ecke, Mary; Hegerl, Reiner; Diez, Stefan; Gerisch, Günther

    2009-01-01

    Propagating actin waves are dynamic supramolecular structures formed by the self-assembly of proteins within living cells. They are built from actin filaments together with single-headed myosin, the Arp2∕3 complex, and coronin in a defined three-dimensional order. The function of these waves in structuring the cell cortex is studied on the substrate-attached surface of Dictyostelium cells by the use of total internal reflection fluorescence (TIRF) microscopy. Actin waves separate two areas of the cell cortex from each other, which are distinguished by the arrangement of actin filaments. The Arp2∕3 complex dominates in the area enclosed by a wave, where it has the capacity of building dendritic structures, while the proteins prevailing in the external area, cortexillin I and myosin-II, bundle actin filaments and arrange them in antiparallel direction. Wave propagation is accompanied by transitions in the state of actin with a preferential period of 5 min. Wave generation is preceded by local fluctuations in actin assembly, some of the nuclei of polymerized actin emanating from clathrin-coated structures, others emerging independently. The dynamics of phase transitions has been analyzed to provide a basis for modeling the nonlinear interactions that produce spatio-temporal patterns in the actin system of living cells. PMID:20514132

  4. Surface Bubble Nucleation Stability

    NASA Astrophysics Data System (ADS)

    Seddon, James R. T.; Kooij, E. Stefan; Poelsema, Bene; Zandvliet, Harold J. W.; Lohse, Detlef

    2011-02-01

    Recent research has revealed several different techniques for nanoscopic gas nucleation on submerged surfaces, with findings seemingly in contradiction with each other. In response to this, we have systematically investigated the occurrence of surface nanobubbles on a hydrophobized silicon substrate for various different liquid temperatures and gas concentrations, which we controlled independently. We found that nanobubbles occupy a distinct region of this parameter space, occurring for gas concentrations of approximately 100%-110%. Below the nanobubble region we did not detect any gaseous formations on the substrate, whereas micropancakes (micron wide, nanometer high gaseous domains) were found at higher temperatures and gas concentrations. We moreover find that supersaturation of dissolved gases is not a requirement for nucleation of bubbles.

  5. WIP Remodeling Actin behind the Scenes: How WIP Reshapes Immune and Other Functions

    PubMed Central

    Noy, Elad; Fried, Sophia; Matalon, Omri; Barda-Saad, Mira

    2012-01-01

    Actin polymerization is a fundamental cellular process regulating immune cell functions and the immune response. The Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor, which is exclusively expressed in hematopoietic cells, where it plays a key regulatory role in cytoskeletal dynamics. WASp interacting protein (WIP) was first discovered as the binding partner of WASp, through the use of the yeast two hybrid system. WIP was later identified as a chaperone of WASp, necessary for its stability. Mutations occurring at the WASp homology 1 domain (WH1), which serves as the WIP binding site, were found to cause the Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). WAS manifests as an immune deficiency characterized by eczema, thrombocytopenia, recurrent infections, and hematopoietic malignancies, demonstrating the importance of WIP for WASp complex formation and for a proper immune response. WIP deficiency was found to lead to different abnormalities in the activity of various lymphocytes, suggesting differential cell-dependent roles for WIP. Additionally, WIP deficiency causes cellular abnormalities not found in WASp-deficient cells, indicating that WIP fulfills roles beyond stabilizing WASp. Indeed, WIP was shown to interact with various binding partners, including the signaling proteins Nck, CrkL and cortactin. Recent studies have demonstrated that WIP also takes part in non immune cellular processes such as cancer invasion and metastasis, in addition to cell subversion by intracellular pathogens. Understanding of numerous functions of WIP can enhance our current understanding of activation and function of immune and other cell types. PMID:22837718

  6. Bleb Nucleation through Membrane Peeling

    NASA Astrophysics Data System (ADS)

    Alert, Ricard; Casademunt, Jaume

    2016-02-01

    We study the nucleation of blebs, i.e., protrusions arising from a local detachment of the membrane from the cortex of a cell. Based on a simple model of elastic linkers with force-dependent kinetics, we show that bleb nucleation is governed by membrane peeling. By this mechanism, the growth or shrinkage of a detached membrane patch is completely determined by the linker kinetics, regardless of the energetic cost of the detachment. We predict the critical nucleation radius for membrane peeling and the corresponding effective energy barrier. These may be typically smaller than those predicted by classical nucleation theory, implying a much faster nucleation. We also perform simulations of a continuum stochastic model of membrane-cortex adhesion to obtain the statistics of bleb nucleation times as a function of the stress on the membrane. The determinant role of membrane peeling changes our understanding of bleb nucleation and opens new directions in the study of blebs.

  7. 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.

  8. Nucleation in Synoptically Forced Cirrostratus

    NASA Technical Reports Server (NTRS)

    Lin, R.-F.; Starr, D. OC.; Reichardt, J.; DeMott, P. J.

    2004-01-01

    Formation and evolution of cirrostratus in response to weak, uniform and constant synoptic forcing is simulated using a one-dimensional numerical model with explicit microphysics, in which the particle size distribution in each grid box is fully resolved. A series of tests of the model response to nucleation modes (homogeneous-freezing-only/heterogeneous nucleation) and heterogeneous nucleation parameters are performed. In the case studied here, nucleation is first activated in the prescribed moist layer. A continuous cloud-top nucleation zone with a depth depending on the vertical humidity gradient and one of the nucleation parameters is developed afterward. For the heterogeneous nucleation cases, intermittent nucleation zones in the mid-upper portion of the cloud form where the relative humidity is on the rise, because existent ice crystals do not uptake excess water vapor efficiently, and ice nuclei (IN) are available. Vertical resolution as fine as 1 m is required for realistic simulation of the homogeneous-freezing-only scenario, while the model resolution requirement is more relaxed in the cases where heterogeneous nucleation dominates. Bulk microphysical and optical properties are evaluated and compared. Ice particle number flux divergence, which is due to the vertical gradient of the gravity-induced particle sedimentation, is constantly and rapidly changing the local ice number concentration, even in the nucleation zone. When the depth of the nucleation zone is shallow, particle number concentration decreases rapidly as ice particles grow and sediment away from the nucleation zone. When the depth of the nucleation zone is large, a region of high ice number concentration can be sustained. The depth of nucleation zone is an important parameter to be considered in parametric treatments of ice cloud generation.

  9. Hierarchical nanostructure and synergy of multimolecular signalling complexes.

    PubMed

    Sherman, Eilon; Barr, Valarie A; Merrill, Robert K; Regan, Carole K; Sommers, Connie L; Samelson, Lawrence E

    2016-01-01

    Signalling complexes are dynamic, multimolecular structures and sites for intracellular signal transduction. Although they play a crucial role in cellular activation, current research techniques fail to resolve their structure in intact cells. Here we present a multicolour, photoactivated localization microscopy approach for imaging multiple types of single molecules in fixed and live cells and statistical tools to determine the nanoscale organization, topology and synergy of molecular interactions in signalling complexes downstream of the T-cell antigen receptor. We observe that signalling complexes nucleated at the key adapter LAT show a hierarchical topology. The critical enzymes PLCγ1 and VAV1 localize to the centre of LAT-based complexes, and the adapter SLP-76 and actin molecules localize to the periphery. Conditional second-order statistics reveal a hierarchical network of synergic interactions between these molecules. Our results extend our understanding of the nanostructure of signalling complexes and are relevant to studying a wide range of multimolecular complexes. PMID:27396911

  10. Hierarchical nanostructure and synergy of multimolecular signalling complexes

    NASA Astrophysics Data System (ADS)

    Sherman, Eilon; Barr, Valarie A.; Merrill, Robert K.; Regan, Carole K.; Sommers, Connie L.; Samelson, Lawrence E.

    2016-07-01

    Signalling complexes are dynamic, multimolecular structures and sites for intracellular signal transduction. Although they play a crucial role in cellular activation, current research techniques fail to resolve their structure in intact cells. Here we present a multicolour, photoactivated localization microscopy approach for imaging multiple types of single molecules in fixed and live cells and statistical tools to determine the nanoscale organization, topology and synergy of molecular interactions in signalling complexes downstream of the T-cell antigen receptor. We observe that signalling complexes nucleated at the key adapter LAT show a hierarchical topology. The critical enzymes PLCγ1 and VAV1 localize to the centre of LAT-based complexes, and the adapter SLP-76 and actin molecules localize to the periphery. Conditional second-order statistics reveal a hierarchical network of synergic interactions between these molecules. Our results extend our understanding of the nanostructure of signalling complexes and are relevant to studying a wide range of multimolecular complexes.

  11. 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.

  12. A Combination of Actin Treadmilling and Cross-Linking Drives Contraction of Random Actomyosin Arrays.

    PubMed

    Oelz, Dietmar B; Rubinstein, Boris Y; Mogilner, Alex

    2015-11-01

    We investigate computationally the self-organization and contraction of an initially random actomyosin ring. In the framework of a detailed physical model for a ring of cross-linked actin filaments and myosin-II clusters, we derive the force balance equations and solve them numerically. We find that to contract, actin filaments have to treadmill and to be sufficiently cross linked, and myosin has to be processive. The simulations reveal how contraction scales with mechanochemical parameters. For example, they show that the ring made of longer filaments generates greater force but contracts slower. The model predicts that the ring contracts with a constant rate proportional to the initial ring radius if either myosin is released from the ring during contraction and actin filaments shorten, or if myosin is retained in the ring, while the actin filament number decreases. We demonstrate that a balance of actin nucleation and compression-dependent disassembly can also sustain contraction. Finally, the model demonstrates that with time pattern formation takes place in the ring, worsening the contractile process. The more random the actin dynamics are, the higher the contractility will be. PMID:26536259

  13. Bacterial Actins? An Evolutionary Perspective

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.; York, Amanda L.

    2003-01-01

    According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life. An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles. Two recent papers present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin. Sequence comparisons reveml that eukaryotic actin and the bacterial homolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories.

  14. Actin engine in immunological synapse.

    PubMed

    Piragyte, Indre; Jun, Chang-Duk

    2012-06-01

    T cell activation and function require physical contact with antigen presenting cells at a specialized junctional structure known as the immunological synapse. Once formed, the immunological synapse leads to sustained T cell receptor-mediated signalling and stabilized adhesion. High resolution microscopy indeed had a great impact in understanding the function and dynamic structure of immunological synapse. Trends of recent research are now moving towards understanding the mechanical part of immune system, expanding our knowledge in mechanosensitivity, force generation, and biophysics of cell-cell interaction. Actin cytoskeleton plays inevitable role in adaptive immune system, allowing it to bear dynamic and precise characteristics at the same time. The regulation of mechanical engine seems very complicated and overlapping, but it enables cells to be very sensitive to external signals such as surface rigidity. In this review, we focus on actin regulators and how immune cells regulate dynamic actin rearrangement process to drive the formation of immunological synapse. PMID:22916042

  15. Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation

    PubMed Central

    Chambers, Joseph E; Dalton, Lucy E; Clarke, Hanna J; Malzer, Elke; Dominicus, Caia S; Patel, Vruti; Moorhead, Greg; Ron, David; Marciniak, Stefan J

    2015-01-01

    Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR. DOI: http://dx.doi.org/10.7554/eLife.04872.001 PMID:25774599

  16. Live imaging provides new insights on dynamic F-actin filopodia and differential endocytosis during myoblast fusion in Drosophila.

    PubMed

    Haralalka, Shruti; Shelton, Claude; Cartwright, Heather N; Guo, Fengli; Trimble, Rhonda; Kumar, Ram P; Abmayr, Susan M

    2014-01-01

    The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the

  17. Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis.

    PubMed

    Tang, Elizabeth I; Lee, Will M; Cheng, C Yan

    2016-04-01

    Germ cell transport across the seminiferous epithelium during spermatogenesis requires the intricate coordination of cell junctions, signaling proteins, and both actin- and microtubule (MT)-based cytoskeletons. Although the involvement of cytoskeletons in germ cell transport has been suggested, the precise mechanism(s) remains elusive. Based on growing evidence that actin and MT interactions underlie fundamental cellular processes, such as cell motility, it is unlikely that actin- and MT-based cytoskeletons work independently to regulate germ cell transport in the testis. Using rats treated with adjudin, a potential male contraceptive that disrupts spermatid adhesion and transport in the testis, as a study model, we show herein that actin- and MT-based cytoskeletons are both necessary for transport of spermatids and residual bodies/phagosomes across the seminiferous epithelium in adult rat testes. Analysis of intratubular expression of F-actin and tubulin revealed disruption of both actin and MT networks, concomitant with misdirected spermatids and phagosomes in rats treated with adjudin. Actin regulatory proteins, epidermal growth factor receptor pathway substrate 8 and actin-related protein 3, were mislocalized and down-regulated at the actin-rich anchoring junction between germ and Sertoli cells (apical ectoplasmic specialization) after adjudin treatment. Nonreceptor tyrosine kinase p-FAK-Tyr(407), known to regulate F-actin nucleation via actin-related protein 3, was also mislocalized and down-regulated at the apical ectoplasmic specialization, corroborating the observation of actin cytoskeleton disruption. Additionally, spatiotemporal expression of MT regulatory protein end-binding protein 1, shown to be involved in MT-actin cross talk herein, was also disrupted after adjudin treatment. In summary, spermatid/phagosome transport across the epithelium during spermatogenesis requires the coordination between actin- and MT-based cytoskeletons. PMID:26894662

  18. On the Ice Nucleation Spectrum

    NASA Technical Reports Server (NTRS)

    Barahona, D.

    2012-01-01

    This work presents a novel formulation of the ice nucleation spectrum, i.e. the function relating the ice crystal concentration to cloud formation conditions and aerosol properties. The new formulation is physically-based and explicitly accounts for the dependency of the ice crystal concentration on temperature, supersaturation, cooling rate, and particle size, surface area and composition. This is achieved by introducing the concepts of ice nucleation coefficient (the number of ice germs present in a particle) and nucleation probability dispersion function (the distribution of ice nucleation coefficients within the aerosol population). The new formulation is used to generate ice nucleation parameterizations for the homogeneous freezing of cloud droplets and the heterogeneous deposition ice nucleation on dust and soot ice nuclei. For homogeneous freezing, it was found that by increasing the dispersion in the droplet volume distribution the fraction of supercooled droplets in the population increases. For heterogeneous ice nucleation the new formulation consistently describes singular and stochastic behavior within a single framework. Using a fundamentally stochastic approach, both cooling rate independence and constancy of the ice nucleation fraction over time, features typically associated with singular behavior, were reproduced. Analysis of the temporal dependency of the ice nucleation spectrum suggested that experimental methods that measure the ice nucleation fraction over few seconds would tend to underestimate the ice nuclei concentration. It is shown that inferring the aerosol heterogeneous ice nucleation properties from measurements of the onset supersaturation and temperature may carry significant error as the variability in ice nucleation properties within the aerosol population is not accounted for. This work provides a simple and rigorous ice nucleation framework where theoretical predictions, laboratory measurements and field campaign data can be

  19. Crucial roles of the Arp2/3 complex during mammalian corticogenesis

    PubMed Central

    Wang, Pei-Shan; Chou, Fu-Sheng; Ramachandran, Sreekumar; Xia, Sheng; Chen, Huei-Ying; Guo, Fengli; Suraneni, Praveen; Maher, Brady J.

    2016-01-01

    The polarity and organization of radial glial cells (RGCs), which serve as both stem cells and scaffolds for neuronal migration, are crucial for cortical development. However, the cytoskeletal mechanisms that drive radial glial outgrowth and maintain RGC polarity remain poorly understood. Here, we show that the Arp2/3 complex – the unique actin nucleator that produces branched actin networks – plays essential roles in RGC polarity and morphogenesis. Disruption of the Arp2/3 complex in murine RGCs retards process outgrowth toward the basal surface and impairs apical polarity and adherens junctions. Whereas the former is correlated with an abnormal actin-based leading edge, the latter is consistent with blockage in membrane trafficking. These defects result in altered cell fate, disrupted cortical lamination and abnormal angiogenesis. In addition, we present evidence that the Arp2/3 complex is a cell-autonomous regulator of neuronal migration. Our data suggest that Arp2/3-mediated actin assembly might be particularly important for neuronal cell motility in a soft or poorly adhesive matrix environment. PMID:27385014

  20. Crucial roles of the Arp2/3 complex during mammalian corticogenesis.

    PubMed

    Wang, Pei-Shan; Chou, Fu-Sheng; Ramachandran, Sreekumar; Xia, Sheng; Chen, Huei-Ying; Guo, Fengli; Suraneni, Praveen; Maher, Brady J; Li, Rong

    2016-08-01

    The polarity and organization of radial glial cells (RGCs), which serve as both stem cells and scaffolds for neuronal migration, are crucial for cortical development. However, the cytoskeletal mechanisms that drive radial glial outgrowth and maintain RGC polarity remain poorly understood. Here, we show that the Arp2/3 complex - the unique actin nucleator that produces branched actin networks - plays essential roles in RGC polarity and morphogenesis. Disruption of the Arp2/3 complex in murine RGCs retards process outgrowth toward the basal surface and impairs apical polarity and adherens junctions. Whereas the former is correlated with an abnormal actin-based leading edge, the latter is consistent with blockage in membrane trafficking. These defects result in altered cell fate, disrupted cortical lamination and abnormal angiogenesis. In addition, we present evidence that the Arp2/3 complex is a cell-autonomous regulator of neuronal migration. Our data suggest that Arp2/3-mediated actin assembly might be particularly important for neuronal cell motility in a soft or poorly adhesive matrix environment. PMID:27385014

  1. 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.

  2. HopW1 from Pseudomonas syringae Disrupts the Actin Cytoskeleton to Promote Virulence in Arabidopsis

    PubMed Central

    Cecchini, Nicolas M.; Li, Yujie; Lee, Min Woo; Kovar, David R.; Greenberg, Jean T.

    2014-01-01

    A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis. PMID:24968323

  3. A detailed study of ice nucleation by feldspar minerals

    NASA Astrophysics Data System (ADS)

    Whale, T. F.; Murray, B. J.; Wilson, T. W.; Carpenter, M. A.; Harrison, A.; Holden, M. A.; Vergara Temprado, J.; Morris, J.; O'Sullivan, D.

    2015-12-01

    Immersion mode heterogeneous ice nucleation plays a crucial role in controlling the composition of mixed phase clouds, which contain both supercooled liquid water and ice particles. The amount of ice in mixed phase clouds can affect cloud particle size, lifetime and extent and so affects radiative properties and precipitation. Feldspar minerals are probably the most important minerals for ice nucleation in mixed phase clouds because they nucleate ice more efficiently than other components of atmospheric mineral dust (Atkinson et al. 2013). The feldspar class of minerals is complex, containing numerous chemical compositions, several crystal polymorphs and wide variations in microscopic structure. Here we present the results of a study into ice nucleation by a wide range of different feldspars. We found that, in general, alkali feldspars nucleate ice more efficiently than plagioclase feldspars. However, we also found that particular alkali feldspars nucleate ice relatively inefficiently, suggesting that chemical composition is not the only important factor that dictates the ice nucleation efficiency of feldspar minerals. Ice nucleation by feldspar is described well by the singular model and is probably site specific in nature. The alkali feldspars that do not nucleate ice efficiently possess relatively homogenous structure on the micrometre scale suggesting that the important sites for nucleation are related to surface topography. Ice nucleation active site densities for the majority of tested alkali feldspars are similar to those found by Atkinson et al (2013), meaning that the validity of global aerosol modelling conducted in that study is not affected. Additionally, we have found that ice nucleation by feldspars is strongly influenced, both positively and negatively, by the solute content of droplets. Most other nucleants we have tested are unaffected by solutes. This provides insight into the mechanism of ice nucleation by feldspars and could be of importance

  4. Surface Nanobubbles Nucleate Microdroplets

    NASA Astrophysics Data System (ADS)

    Zhang, Xuehua; Lhuissier, Henri; Sun, Chao; Lohse, Detlef

    2014-04-01

    When a hydrophobic solid is in contact with water, surface nanobubbles often form at the interface. They have a lifetime many orders of magnitude longer than expected. Here, we show that they even withstand a temperature increase to temperatures close to the boiling point of bulk water; i.e., they do not nucleate larger bubbles ("superstability"). On the contrary, when the vapor-liquid contact line passes a nanobubble, a liquid film remains around it, which, after pinch-off, results in a microdroplet in which the nanobubbles continue to exist. Finally, the microdroplet evaporates and the nanobubble consequently bursts. Our results support that pinning plays a crucial role for nanobubble stability.

  5. F-actin mechanics control spindle centring in the mouse zygote

    NASA Astrophysics Data System (ADS)

    Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S.; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition.

  6. F-actin mechanics control spindle centring in the mouse zygote.

    PubMed

    Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition. PMID:26727405

  7. F-actin mechanics control spindle centring in the mouse zygote

    PubMed Central

    Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S.; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition. PMID:26727405

  8. Actuators Acting without Actin.

    PubMed

    Geitmann, Anja

    2016-06-30

    Plant actuators move organs, allowing the plant to respond to environmental cues or perform other mechanical tasks. In Cardamine hursuta the dispersal of seeds is accomplished by explosive opening of the fruit. The biomechanical mechanism relies on a complex interplay between turgor regulation and cell wall mechanical properties. PMID:27368097

  9. Direct interaction of microtubule- and actin-based transport motors

    NASA Technical Reports Server (NTRS)

    Huang, J. D.; Brady, S. T.; Richards, B. W.; Stenolen, D.; Resau, J. H.; Copeland, N. G.; Jenkins, N. A.

    1999-01-01

    The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.

  10. 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

  11. Transient nucleation in condensed systems

    NASA Technical Reports Server (NTRS)

    Kelton, K. F.; Greer, A. L.; Thompson, C. V.

    1983-01-01

    Using classical nucleation theory we consider transient nucleation occurring in a one-component, condensed system under isothermal conditions. We obtain an exact closed-form expression for the time dependent cluster populations. In addition, a more versatile approach is developed: a numerical simulation technique which models directly the reactions by which clusters are produced. This simulation demonstrates the evolution of cluster populations and nucleation rate in the transient regime. Results from the simulation are verified by comparison with exact analytical solutions for the steady state. Experimental methods for measuring transient nucleation are assessed, and it is demonstrated that the observed behavior depends on the method used. The effect of preexisting cluster distributions is studied. Previous analytical and numerical treatments of transient nucleation are compared to the solutions obtained from the simulation. The simple expressions of Kashchiev are shown to give good descriptions of the nucleation behavior.

  12. Association of actin with alpha crystallins

    NASA Technical Reports Server (NTRS)

    Gopalakrishnan, S.; Boyle, D.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The alpha crystallins are cytosolic proteins that co-localize and co-purify with actin-containing microfilaments. Affinity column chromatography employing both covalently-coupled actin or alpha crystallin was used to demonstrate specific and saturable binding of actin with alpha crystallin. This conclusion was confirmed by direct visualization of alpha aggregates bound to actin polymerized in vitro. The significance of this interaction in relation to the functional properties of these two polypeptides will be discussed.

  13. 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

  14. Actin dynamics: from nanoscale to microscale.

    PubMed

    Carlsson, Anders E

    2010-01-01

    The dynamic nature of actin in cells manifests itself constantly. Polymerization near the cell edge is balanced by depolymerization in the interior, externally induced actin polymerization is followed by depolymerization, and spontaneous oscillations of actin at the cell periphery are frequently seen. I discuss how mathematical modeling relates quantitative measures of actin dynamics to the rates of underlying molecular level processes. The dynamic properties addressed include the rate of actin assembly at the leading edge of a moving cell, the disassembly rates of intracellular actin networks, the polymerization time course in externally stimulated cells, and spontaneous spatiotemporal patterns formed by actin. Although several aspects of actin assembly have been clarified by increasingly sophisticated models, our understanding of rapid actin disassembly is limited, and the origins of nonmonotonic features in externally stimulated actin polymerization remain unclear. Theory has generated several concrete, testable hypotheses for the origins of spontaneous actin waves and cell-edge oscillations. The development and use of more biomimetic systems applicable to the geometry of a cell will be key to obtaining a quantitative understanding of actin dynamics in cells. PMID:20462375

  15. Actin Interacting Protein1 and Actin Depolymerizing Factor Drive Rapid Actin Dynamics in Physcomitrella patens[W

    PubMed Central

    Augustine, Robert C.; Pattavina, Kelli A.; Tüzel, Erkan; Vidali, Luis; Bezanilla, Magdalena

    2011-01-01

    The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics. PMID:22003077

  16. Nucleation Rate Analysis of Methane Hydrate from Molecular Dynamics Simulations

    DOE PAGESBeta

    Yuhara, Daisuke; Barnes, Brian C.; Suh, Donguk; Knott, Brandon C.; Beckham, Gregg T.; Yasuoka, Kenji; Wu, David T.; Amadeu K. Sum

    2015-01-06

    Clathrate hydrates are solid crystalline structures most commonly formed from solutions that have nucleated to form a mixed solid composed of water and gas. Understanding the mechanism of clathrate hydrate nucleation is essential to grasp the fundamental chemistry of these complex structures and their applications. Molecular dynamics (MD) simulation is an ideal method to study nucleation at the molecular level because the size of the critical nucleus and formation rate occur on the nano scale. Moreover, various analysis methods for nucleation have been developed through MD to analyze nucleation. In particular, the mean first-passage time (MFPT) and survival probability (SP)more » methods have proven to be effective in procuring the nucleation rate and critical nucleus size for monatomic systems. This study assesses the MFPT and SP methods, previously used for monatomic systems, when applied to analyzing clathrate hydrate nucleation. Because clathrate hydrate nucleation is relatively difficult to observe in MD simulations (due to its high free energy barrier), these methods have yet to be applied to clathrate hydrate systems. In this study, we have analyzed the nucleation rate and critical nucleus size of methane hydrate using MFPT and SP methods from data generated by MD simulations at 255 K and 50 MPa. MFPT was modified for clathrate hydrate from the original version by adding the maximum likelihood estimate and growth effect term. The nucleation rates were calculated by MFPT and SP methods and are within 5%; the critical nucleus size estimated by the MFPT method was 50% higher, than values obtained through other more rigorous but computationally expensive estimates. These methods can also be extended to the analysis of other clathrate hydrates.« less

  17. Nucleation Rate Analysis of Methane Hydrate from Molecular Dynamics Simulations

    SciTech Connect

    Yuhara, Daisuke; Barnes, Brian C.; Suh, Donguk; Knott, Brandon C.; Beckham, Gregg T.; Yasuoka, Kenji; Wu, David T.; Amadeu K. Sum

    2015-01-06

    Clathrate hydrates are solid crystalline structures most commonly formed from solutions that have nucleated to form a mixed solid composed of water and gas. Understanding the mechanism of clathrate hydrate nucleation is essential to grasp the fundamental chemistry of these complex structures and their applications. Molecular dynamics (MD) simulation is an ideal method to study nucleation at the molecular level because the size of the critical nucleus and formation rate occur on the nano scale. Moreover, various analysis methods for nucleation have been developed through MD to analyze nucleation. In particular, the mean first-passage time (MFPT) and survival probability (SP) methods have proven to be effective in procuring the nucleation rate and critical nucleus size for monatomic systems. This study assesses the MFPT and SP methods, previously used for monatomic systems, when applied to analyzing clathrate hydrate nucleation. Because clathrate hydrate nucleation is relatively difficult to observe in MD simulations (due to its high free energy barrier), these methods have yet to be applied to clathrate hydrate systems. In this study, we have analyzed the nucleation rate and critical nucleus size of methane hydrate using MFPT and SP methods from data generated by MD simulations at 255 K and 50 MPa. MFPT was modified for clathrate hydrate from the original version by adding the maximum likelihood estimate and growth effect term. The nucleation rates were calculated by MFPT and SP methods and are within 5%; the critical nucleus size estimated by the MFPT method was 50% higher, than values obtained through other more rigorous but computationally expensive estimates. These methods can also be extended to the analysis of other clathrate hydrates.

  18. Probing Individual Ice Nucleation Events with Environmental Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Bingbing; China, Swarup; Knopf, Daniel; Gilles, Mary; Laskin, Alexander

    2016-04-01

    Heterogeneous ice nucleation is one of the processes of critical relevance to a range of topics in the fundamental and the applied science and technologies. Heterogeneous ice nucleation initiated by particles proceeds where microscopic properties of particle surfaces essentially control nucleation mechanisms. Ice nucleation in the atmosphere on particles governs the formation of ice and mixed phase clouds, which in turn influence the Earth's radiative budget and climate. Heterogeneous ice nucleation is still insufficiently understood and poses significant challenges in predictive understanding of climate change. We present a novel microscopy platform allowing observation of individual ice nucleation events at temperature range of 193-273 K and relative humidity relevant for ice formation in the atmospheric clouds. The approach utilizes a home built novel ice nucleation cell interfaced with Environmental Scanning Electron Microscope (IN-ESEM system). The IN-ESEM system is applied for direct observation of individual ice formation events, determining ice nucleation mechanisms, freezing temperatures, and relative humidity onsets. Reported microanalysis of the ice nucleating particles (INP) include elemental composition detected by the energy dispersed analysis of X-rays (EDX), and advanced speciation of the organic content in particles using scanning transmission x-ray microscopy with near edge X-ray absorption fine structure spectroscopy (STXM/NEXAFS). The performance of the IN-ESEM system is validated through a set of experiments with kaolinite particles with known ice nucleation propensity. We demonstrate an application of the IN-ESEM system to identify and characterize individual INP within a complex mixture of ambient particles.

  19. Actin Foci Adhesion of D. discoideum

    NASA Astrophysics Data System (ADS)

    Flanders, Bret; Paneru, Govind

    2014-03-01

    Amoeboid migration is a fast (10 μm min-1) integrin-independent mode of migration that is important with D. discoideum, leukocytes, and breast cancer cells. It is poorly understood, but depends on the establishment of adhesive contacts to the substrate where the cell transmits traction forces. In pre-aggregative D. discoideum, a model system for learning about amoeboid migration, these adhesive contacts are discrete complexes that are known as actin-foci. They have an area of ~ 0.5 μm2 and a lifetime of ~ 20 s. This talk will present measurements of the adhesive character of actin foci that have been obtained using a submicron force transducer that was designed for this purpose. Results on the rupture stresses and lifetimes of individual acting foci under nano-newton level forces will be described in the context of a general theory for cellular adhesion. This theory depends on, essentially, three cellular properties: the membrane-medium surface tension, the number density of adhesion receptors in the membrane, and the receptor-substrate potential energy surface. Therefore, the use of the transducer to determine the surface tension will be presented, as well.

  20. How cofilin severs an actin filament.

    PubMed

    De La Cruz, Enrique M

    2009-05-15

    The actin regulatory protein, cofilin, promotes actin assembly dynamics by severing filaments and increasing the number of ends from which subunits add and dissociate. Recent studies provide biophysical descriptions of cooperative filament interactions in energetic, mechanical and structural terms. A one-dimensional Ising model with nearest-neighbor interactions permits thermodynamic analysis of cooperative binding and indicates that one or a few cofilin molecules can sever a filament. Binding and cooperative interactions are entropically driven. A significant fraction of the binding free energy results from the linked dissociation of filament-associated ions (polyelectrolyte effect), which modulate filament structure, stability and mechanics. The remaining binding free energy and essentially all of the cooperative free energy arise from the enhanced conformational dynamics of the cofilactin complex. Filament mechanics are modulated by cofilin such that cofilin-saturated filaments are approximately 10- to 20-fold more compliant in bending and twisting than bare filaments. Cofilin activity is well described by models in which discontinuities in topology, mechanics and conformational dynamics generate stress concentration and promote fracture at junctions of bare and decorated segments, analogous to the grain boundary fracture of crystalline materials and the thermally driven formation of shear transformation zones in colloidal glass. PMID:20700473

  1. Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter Functions

    SciTech Connect

    Quintero-Monzon,O.; Rodal, A.; Strokopytov, B.; Almo, S.; Goode, B.

    2005-01-01

    Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.

  2. Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells

    PubMed Central

    Chen, Xiaorui; Ni, Fengyun; Kondrashkina, Elena; Ma, Jianpeng; Wang, Qinghua

    2015-01-01

    Leiomodin (Lmod) is a class of potent tandem-G-actin–binding nucleators in muscle cells. Lmod mutations, deletion, or instability are linked to lethal nemaline myopathy. However, the lack of high-resolution structures of Lmod nucleators in action severely hampered our understanding of their essential cellular functions. Here we report the crystal structure of the actin–Lmod2162–495 nucleus. The structure contains two actin subunits connected by one Lmod2162–495 molecule in a non–filament-like conformation. Complementary functional studies suggest that the binding of Lmod2 stimulates ATP hydrolysis and accelerates actin nucleation and polymerization. The high level of conservation among Lmod proteins in sequence and functions suggests that the mechanistic insights of human Lmod2 uncovered here may aid in a molecular understanding of other Lmod proteins. Furthermore, our structural and mechanistic studies unraveled a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin–binding nucleators. PMID:26417072

  3. Mechanism of Actin-Based Motility

    NASA Astrophysics Data System (ADS)

    Pantaloni, Dominique; Le Clainche, Christophe; Carlier, Marie-France

    2001-05-01

    Spatially controlled polymerization of actin is at the origin of cell motility and is responsible for the formation of cellular protrusions like lamellipodia. The pathogens Listeria monocytogenes and Shigella flexneri, which undergo actin-based propulsion, are acknowledged models of the leading edge of lamellipodia. Actin-based motility of the bacteria or of functionalized microspheres can be reconstituted in vitro from only five pure proteins. Movement results from the regulated site-directed treadmilling of actin filaments, consistent with observations of actin dynamics in living motile cells and with the biochemical properties of the components of the synthetic motility medium.

  4. Nucleation and transients at the onset of vortex turbulence

    NASA Technical Reports Server (NTRS)

    Huber, Greg; Alstrom, Preben; Bohr, Tomas

    1992-01-01

    We present analytical and numerical results that explain the transient turbulent dynamics observed in the complex Ginzburg-Landau equation. Just below the transition to turbulence, we observe that metastable turbulent states break down by the nucleation and growth of single-vortex droplets, leading to a 'frozen' state with a low (but finite) density of stationary vortices. We derive the relation between nucleation time and radius, and determine their dependence on the distance to the turbulence transition line.

  5. Surface nanobubbles nucleate microdroplets.

    PubMed

    Zhang, Xuehua; Lhuissier, Henri; Sun, Chao; Lohse, Detlef

    2014-04-11

    When a hydrophobic solid is in contact with water, surface nanobubbles often form at the interface. They have a lifetime many orders of magnitude longer than expected. Here, we show that they even withstand a temperature increase to temperatures close to the boiling point of bulk water; i.e., they do not nucleate larger bubbles ("superstability"). On the contrary, when the vapor-liquid contact line passes a nanobubble, a liquid film remains around it, which, after pinch-off, results in a microdroplet in which the nanobubbles continue to exist. Finally, the microdroplet evaporates and the nanobubble consequently bursts. Our results support that pinning plays a crucial role for nanobubble stability. PMID:24765973

  6. On Capillary Rise and Nucleation

    ERIC Educational Resources Information Center

    Prasad, R.

    2008-01-01

    A comparison of capillary rise and nucleation is presented. It is shown that both phenomena result from a balance between two competing energy factors: a volume energy and a surface energy. Such a comparison may help to introduce nucleation with a topic familiar to the students, capillary rise. (Contains 1 table and 3 figures.)

  7. The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

    PubMed

    Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

    2016-08-15

    Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends. PMID:27307584

  8. Kv3.3 Channels Bind Hax-1 and Arp2/3 to Assemble a Stable Local Actin Network that Regulates Channel Gating.

    PubMed

    Zhang, Yalan; Zhang, Xiao-Feng; Fleming, Matthew R; Amiri, Anahita; El-Hassar, Lynda; Surguchev, Alexei A; Hyland, Callen; Jenkins, David P; Desai, Rooma; Brown, Maile R; Gazula, Valeswara-Rao; Waters, Michael F; Large, Charles H; Horvath, Tamas L; Navaratnam, Dhasakumar; Vaccarino, Flora M; Forscher, Paul; Kaczmarek, Leonard K

    2016-04-01

    Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons. PMID:26997484

  9. Actin cap associated focal adhesions and their distinct role in cellular mechanosensing

    PubMed Central

    Kim, Dong-Hwee; Khatau, Shyam B.; Feng, Yunfeng; Walcott, Sam; Sun, Sean X.; Longmore, Gregory D.; Wirtz, Denis

    2012-01-01

    The ability for cells to sense and adapt to different physical microenvironments plays a critical role in development, immune responses, and cancer metastasis. Here we identify a small subset of focal adhesions that terminate fibers in the actin cap, a highly ordered filamentous actin structure that is anchored to the top of the nucleus by the LINC complexes; these differ from conventional focal adhesions in morphology, subcellular organization, movements, turnover dynamics, and response to biochemical stimuli. Actin cap associated focal adhesions (ACAFAs) dominate cell mechanosensing over a wide range of matrix stiffness, an ACAFA-specific function regulated by actomyosin contractility in the actin cap, while conventional focal adhesions are restrictively involved in mechanosensing for extremely soft substrates. These results establish the perinuclear actin cap and associated ACAFAs as major mediators of cellular mechanosensing and a critical element of the physical pathway that transduce mechanical cues all the way to the nucleus. PMID:22870384

  10. Nucleation rate in monotectic alloys

    NASA Astrophysics Data System (ADS)

    Falk, F.

    Cooling a melt of a monotectic system into the miscibility gap results in nucleation of fluid droplets in a fluid matrix prior to solidification. For homogeneous nucleation the temperature dependence of the nucleation rate is calculated. As material parameters the chemical potential of the species involved, the diffusion constant of the fluid, and the surface tension between adjacent phases are important. Since their temperature dependence is not well known from experiments, different theoretical models are used and their influence is discussed. The surface tension turns out to be the most crucial parameter in determining the nucleation rate. For AlIn numerical results are presented. In this system the undercooling with respect to homogeneous nucleation increases from zero at the critical point to 100 K at a composition near the monotectic point.

  11. Preferential Nucleation during Polymorphic Transformations

    PubMed Central

    Sharma, H.; Sietsma, J.; Offerman, S. E.

    2016-01-01

    Polymorphism is the ability of a solid material to exist in more than one phase or crystal structure. Polymorphism may occur in metals, alloys, ceramics, minerals, polymers, and pharmaceutical substances. Unresolved are the conditions for preferential nucleation during polymorphic transformations in which structural relationships or special crystallographic orientation relationships (OR’s) form between the nucleus and surrounding matrix grains. We measured in-situ and simultaneously the nucleation rates of grains that have zero, one, two, three and four special OR’s with the surrounding parent grains. These experiments show a trend in which the activation energy for nucleation becomes smaller – and therefore nucleation more probable - with increasing number of special OR’s. These insights contribute to steering the processing of polymorphic materials with tailored properties, since preferential nucleation affects which crystal structure forms, the average grain size and texture of the material, and thereby - to a large extent - the final properties of the material. PMID:27484579

  12. Preferential Nucleation during Polymorphic Transformations

    NASA Astrophysics Data System (ADS)

    Sharma, H.; Sietsma, J.; Offerman, S. E.

    2016-08-01

    Polymorphism is the ability of a solid material to exist in more than one phase or crystal structure. Polymorphism may occur in metals, alloys, ceramics, minerals, polymers, and pharmaceutical substances. Unresolved are the conditions for preferential nucleation during polymorphic transformations in which structural relationships or special crystallographic orientation relationships (OR’s) form between the nucleus and surrounding matrix grains. We measured in-situ and simultaneously the nucleation rates of grains that have zero, one, two, three and four special OR’s with the surrounding parent grains. These experiments show a trend in which the activation energy for nucleation becomes smaller – and therefore nucleation more probable - with increasing number of special OR’s. These insights contribute to steering the processing of polymorphic materials with tailored properties, since preferential nucleation affects which crystal structure forms, the average grain size and texture of the material, and thereby - to a large extent - the final properties of the material.

  13. Preferential Nucleation during Polymorphic Transformations.

    PubMed

    Sharma, H; Sietsma, J; Offerman, S E

    2016-01-01

    Polymorphism is the ability of a solid material to exist in more than one phase or crystal structure. Polymorphism may occur in metals, alloys, ceramics, minerals, polymers, and pharmaceutical substances. Unresolved are the conditions for preferential nucleation during polymorphic transformations in which structural relationships or special crystallographic orientation relationships (OR's) form between the nucleus and surrounding matrix grains. We measured in-situ and simultaneously the nucleation rates of grains that have zero, one, two, three and four special OR's with the surrounding parent grains. These experiments show a trend in which the activation energy for nucleation becomes smaller - and therefore nucleation more probable - with increasing number of special OR's. These insights contribute to steering the processing of polymorphic materials with tailored properties, since preferential nucleation affects which crystal structure forms, the average grain size and texture of the material, and thereby - to a large extent - the final properties of the material. PMID:27484579

  14. Reactive oxygen species (ROS)-induced actin glutathionylation controls actin dynamics in neutrophils

    PubMed Central

    Sakai, Jiro; Li, Jingyu; Subramanian, Kulandayan K.; Mondal, Subhanjan; Bajrami, Besnik; Hattori, Hidenori; Jia, Yonghui; Dickinson, Bryan C.; Zhong, Jia; Ye, Keqiang; Chang, Christopher J; Ho, Ye-Shih; Zhou, Jun; Luo, Hongbo R.

    2012-01-01

    Summary The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis, and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, however the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils. PMID:23159440

  15. The Design of MACs (Minimal Actin Cortices)

    PubMed Central

    Vogel, Sven K; Heinemann, Fabian; Chwastek, Grzegorz; Schwille, Petra

    2013-01-01

    The actin cell cortex in eukaryotic cells is a key player in controlling and maintaining the shape of cells, and in driving major shape changes such as in cytokinesis. It is thereby constantly being remodeled. Cell shape changes require forces acting on membranes that are generated by the interplay of membrane coupled actin filaments and assemblies of myosin motors. Little is known about how their interaction regulates actin cell cortex remodeling and cell shape changes. Because of the vital importance of actin, myosin motors and the cell membrane, selective in vivo experiments and manipulations are often difficult to perform or not feasible. Thus, the intelligent design of minimal in vitro systems for actin-myosin-membrane interactions could pave a way for investigating actin cell cortex mechanics in a detailed and quantitative manner. Here, we present and discuss the design of several bottom-up in vitro systems accomplishing the coupling of actin filaments to artificial membranes, where key parameters such as actin densities and membrane properties can be varied in a controlled manner. Insights gained from these in vitro systems may help to uncover fundamental principles of how exactly actin-myosin-membrane interactions govern actin cortex remodeling and membrane properties for cell shape changes. © 2013 Wiley Periodicals, Inc. PMID:24039068

  16. Surface area controlled heterogeneous nucleation

    NASA Astrophysics Data System (ADS)

    Steer, Brian; Gorbunov, Boris; Rowles, Jonathan; Green, David

    2012-02-01

    Heterogeneous nucleation of liquid from a gas phase on nanoparticles has been studied under various saturation ratios and nuclei size. The probability of liquid droplet nucleation, especially at a low degree of deviation from equilibrium, was measured for both atmospheric aerosol particles and engineered nanoparticles Cr2O3. The concept of a critical saturation ratio and the validity of the one-to-one relationship between the nuclei number and the number of droplets were examined. A transient zone between no nucleation and established nucleation termed the surface area controlled nucleation was observed. In this zone, the probability of stable phase formation is determined by the surface area of nuclei. There are two distinctive features of the surface area controlled nucleation: the nucleation probability is much less than 1 and is proportional to the surface area of nuclei. For condensation particle counters (CPCs) counting nanoparticles, these features mean that counts measured are proportional to the surface area of nanoparticles and, therefore, the CPCs counts can be calibrated to measure the surface area.

  17. The interaction of vinculin with actin.

    PubMed

    Golji, Javad; Mofrad, Mohammad R K

    2013-04-01

    Vinculin can interact with F-actin both in recruitment of actin filaments to the growing focal adhesions and also in capping of actin filaments to regulate actin dynamics. Using molecular dynamics, both interactions are simulated using different vinculin conformations. Vinculin is simulated either with only its vinculin tail domain (Vt), with all residues in its closed conformation, with all residues in an open I conformation, and with all residues in an open II conformation. The open I conformation results from movement of domain 1 away from Vt; the open II conformation results from complete dissociation of Vt from the vinculin head domains. Simulation of vinculin binding along the actin filament showed that Vt alone can bind along the actin filaments, that vinculin in its closed conformation cannot bind along the actin filaments, and that vinculin in its open I conformation can bind along the actin filaments. The simulations confirm that movement of domain 1 away from Vt in formation of vinculin 1 is sufficient for allowing Vt to bind along the actin filament. Simulation of Vt capping actin filaments probe six possible bound structures and suggest that vinculin would cap actin filaments by interacting with both S1 and S3 of the barbed-end, using the surface of Vt normally occluded by D4 and nearby vinculin head domain residues. Simulation of D4 separation from Vt after D1 separation formed the open II conformation. Binding of open II vinculin to the barbed-end suggests this conformation allows for vinculin capping. Three binding sites on F-actin are suggested as regions that could link to vinculin. Vinculin is suggested to function as a variable switch at the focal adhesions. The conformation of vinculin and the precise F-actin binding conformation is dependent on the level of mechanical load on the focal adhesion. PMID:23633939

  18. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls.

    PubMed

    Butler, J H; Hu, S; Brady, S R; Dixon, M W; Muday, G K

    1998-02-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo. PMID:11536873

  19. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

    NASA Technical Reports Server (NTRS)

    Butler, J. H.; Hu, S.; Brady, S. R.; Dixon, M. W.; Muday, G. K.

    1998-01-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

  20. Climate Impacts of Ice Nucleation

    NASA Technical Reports Server (NTRS)

    Gettelman, Andrew; Liu, Xiaohong; Barahona, Donifan; Lohmann, Ulrike; Chen, Celia

    2012-01-01

    Several different ice nucleation parameterizations in two different General Circulation Models (GCMs) are used to understand the effects of ice nucleation on the mean climate state, and the Aerosol Indirect Effects (AIE) of cirrus clouds on climate. Simulations have a range of ice microphysical states that are consistent with the spread of observations, but many simulations have higher present-day ice crystal number concentrations than in-situ observations. These different states result from different parameterizations of ice cloud nucleation processes, and feature different balances of homogeneous and heterogeneous nucleation. Black carbon aerosols have a small (0.06 Wm(exp-2) and not statistically significant AIE when included as ice nuclei, for nucleation efficiencies within the range of laboratory measurements. Indirect effects of anthropogenic aerosols on cirrus clouds occur as a consequence of increasing anthropogenic sulfur emissions with different mechanisms important in different models. In one model this is due to increases in homogeneous nucleation fraction, and in the other due to increases in heterogeneous nucleation with coated dust. The magnitude of the effect is the same however. The resulting ice AIE does not seem strongly dependent on the balance between homogeneous and heterogeneous ice nucleation. Regional effects can reach several Wm2. Indirect effects are slightly larger for those states with less homogeneous nucleation and lower ice number concentration in the base state. The total ice AIE is estimated at 0.27 +/- 0.10 Wm(exp-2) (1 sigma uncertainty). This represents a 20% offset of the simulated total shortwave AIE for ice and liquid clouds of 1.6 Wm(sup-2).

  1. Climate Impacts of Ice Nucleation

    SciTech Connect

    Gettelman, A.; Liu, Xiaohong; Barahona, Donifan; Lohmann, U.; Chen, Chih-Chieh

    2012-10-19

    [1] Several different ice nucleation parameterizations in two different General Circulation Models (GCMs) are used to understand the effects of ice nucleation on the mean climate state, and the Aerosol Indirect Effects (AIE) of cirrus clouds on climate. Simulations have a range of ice microphysical states that are consistent with the spread of observations, but many simulations have higher present-day ice crystal number concentrations than in-situ observations. These different states result from different parameterizations of ice cloud nucleation processes, and feature different balances of homogeneous and heterogeneous nucleation. Black carbon aerosols have a small (-0.06 Wm-2) and not statistically significant AIE when included as ice nuclei, for nucleation efficiencies within the range of laboratory measurements. Indirect effects of anthropogenic aerosols on cirrus clouds occur as a consequence of increasing anthropogenic sulfur emissions with different mechanisms important in different models. In one model this is due to increases in homogeneous nucleation fraction, and in the other due to increases in heterogeneous nucleation with coated dust. The magnitude of the effect is the same however. The resulting ice AIE does not seem strongly dependent on the balance between homogeneous and heterogeneous ice nucleation. Regional effects can reach several Wm-2. Indirect effects are slightly larger for those states with less homogeneous nucleation and lower ice number concentration in the base state. The total ice AIE is estimated at 0.27 ± 0.10 Wm-2 (1σ uncertainty). Finally, this represents a 20% offset of the simulated total shortwave AIE for ice and liquid clouds of -1.6 Wm-2.

  2. The impacts of cation stoichiometry and substrate surface quality on nucleation, structure, defect formation, and intermixing in complex oxide heteroepitaxy LaCrO3 on SrTiO3(001)

    SciTech Connect

    Qiao, Liang; Zhang, K. H. L; Bowden, Mark E; Varga, Tamas; Shutthanandan, Vaithiyalingam; Colby, Robert; Du, Yingge; Kabius, Bernd; Sushko, Peter V; Biegalski, Michael D; Chambers, S. A.

    2013-01-01

    Our ability to design and fabricate electronic devices with reproducible properties using complex oxides is critically dependent on our ability to controllably synthesize these materials in thin-film form. Structure-property relationships are intimately tied to film and interface composition Here we report on the effect of cation stoichiometry on structural quality and defect formation in LaCrO3 heteroepitaxial films prepared using molecular beam epitaxy. We calculate from first principles the regions of stability of various candidate defects as a function of Cr and O chemical potential, along with the predicted effects of these defects on structural parameters. We show that epitaxial LaCrO3 films readily nucleate and remain coherently strained on SrTiO3(001) over a wide range of La-to-Cr atom ratios, but that La-rich films are of considerably lower structural quality than stoichiometric and Cr-rich films. Cation imbalances are accompanied by anti-site defect formation, as deduced by comparing experimental trends in the c lattice parameter with those from first-principles calculations. Cation mixing occurs at the interface for all La-to-Cr ratios investigated, and is not quenched by deposition on SrTiO3(001) at ambient temperature. Indiffused La atoms occupy Sr sites, most likely facilitated by Sr vacancy formation in STO resulting from high-temperature oxygen annealing required to prepare the substrate. Intermixing is effectively quenched by using molecular beam epitaxy to deposit LaCrO3 at ambient temperature on defect free Si(001). However, analogous pulsed laser deposition on Si is accompanied by cation mixing.

  3. Nucleation dynamics of water nanodroplets.

    PubMed

    Bhattacharya, Dipanjan; Bosman, Michel; Mokkapati, Venkata R S S; Leong, Fong Yew; Mirsaidov, Utkur

    2014-04-01

    The origin of the condensation of water begins at the nanoscale, a length-scale that is challenging to probe for liquids. In this work we directly image heterogeneous nucleation of water nanodroplets by in situ transmission electron microscopy. Using gold nanoparticles bound to a flat surface as heterogeneous nucleation sites, we observe nucleation and growth of water nanodroplets. The growth of nanodroplet radii follows the power law: R(t)~(t-t 0) β , where β~0.2-0.3. PMID:24667092

  4. Metadynamics studies of crystal nucleation

    PubMed Central

    Giberti, Federico; Salvalaglio, Matteo; Parrinello, Michele

    2015-01-01

    Crystallization processes are characterized by activated events and long timescales. These characteristics prevent standard molecular dynamics techniques from being efficiently used for the direct investigation of processes such as nucleation. This short review provides an overview on the use of metadynamics, a state-of-the-art enhanced sampling technique, for the simulation of phase transitions involving the production of a crystalline solid. In particular the principles of metadynamics are outlined, several order parameters are described that have been or could be used in conjunction with metadynamics to sample nucleation events and then an overview is given of recent metadynamics results in the field of crystal nucleation. PMID:25866662

  5. Dew nucleation and growth

    NASA Astrophysics Data System (ADS)

    Beysens, Daniel

    2006-11-01

    Dew is the condensation of water vapor into liquid droplets on a substrate. It is characterized by an initial heterogeneous nucleation on a substrate and a further growth of droplets. The presence of a substrate that geometrically constrains the growth is the origin of the peculiarities and richness of the phenomenon. A key point is the drop interaction through drop fusion or coalescence, which leads to scaling in the growth and gives universality to the process. As a matter of fact, growth dynamics are only dependent on substrate and drop dimensionality. Coalescence events lead to temporal and spatio-temporal fluctuations in the substrate coverage, drop configuration, etc., which give rise to a very peculiar dynamics. When the substrate is a liquid or a liquid crystal, the drop pattern can exhibit special spatial order, such as crystalline, hexatic phases and fractal contours. Condensation on a solid substrate near its melting point can make the drop jump. The applications of monitoring dew formation are manifold. Examples can be found in medicine (sterilization process), agriculture (green houses) and hydrology (production of drinkable water). To cite this article: D. Beysens, C. R. Physique 7 (2006).

  6. Mechanosensitive kinetic preference of actin-binding protein to actin filament

    NASA Astrophysics Data System (ADS)

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

    The kinetic preference of actin-binding proteins to actin filaments is altered by external forces on the filament. Such an altered kinetic preference is largely responsible for remodeling the actin cytoskeletal structure in response to intracellular forces. During remodeling, actin-binding proteins and actin filaments interact under isothermal conditions, because the cells are homeostatic. In such a temperature homeostatic state, we can rigorously and thermodynamically link the chemical potential of actin-binding proteins to stresses on the actin filaments. From this relationship, we can construct a physical model that explains the force-dependent kinetic preference of actin-binding proteins to actin filaments. To confirm the model, we have analyzed the mechanosensitive alternation of the kinetic preference of Arp2/3 and cofilin to actin filaments. We show that this model captures the qualitative responses of these actin-binding proteins to the forces, as observed experimentally. Moreover, our theoretical results demonstrate that, depending on the structural parameters of the binding region, actin-binding proteins can show different kinetic responses even to the same mechanical signal tension, in which the double-helix nature of the actin filament also plays a critical role in a stretch-twist coupling of the filament.

  7. A Role for Nuclear Actin in HDAC 1 and 2 Regulation

    PubMed Central

    Serebryannyy, Leonid A.; Cruz, Christina M.; de Lanerolle, Primal

    2016-01-01

    Class I histone deacetylases (HDACs) are known to remove acetyl groups from histone tails. This liberates positive charges on the histone tail and allows for tighter winding of DNA, preventing transcription factor binding and gene activation. Although the functions of HDAC proteins are becoming apparent both biochemically and clinically, how this class of proteins is regulated remains poorly understood. We identified a novel interaction between nuclear actin and HDAC 1 and HDAC 2. Nuclear actin has been previously shown to interact with a growing list of nuclear proteins including chromatin remodeling complexes, transcription factors and RNA polymerases. We find that monomeric actin is able to bind the class I HDAC complex. Furthermore, increasing the concentration of actin in HeLa nuclear extracts was able to suppress overall HDAC function. Conversely, polymerizing nuclear actin increased HDAC activity and decreased histone acetylation. Moreover, the interaction between class I HDACs and nuclear actin was found to be activity dependent. Together, our data suggest nuclear actin is able to regulate HDAC 1 and 2 activity. PMID:27345839

  8. A yeast TCP-1-like protein is required for actin function in vivo.

    PubMed Central

    Vinh, D B; Drubin, D G

    1994-01-01

    We previously identified the ANC2 gene in a screen for mutations that enhance the defects caused by yeast actin mutations. Here we report that ANC2 is an essential gene that encodes a member of the TCP-1 family. TCP-1-related proteins are subunits of cytosolic heteromeric protein complexes referred to as chaperonins. These complexes can bind to newly synthesized actin and tubulin in vitro and can convert these proteins into an assembly-competent state. We show that anc2-1 mutants contain abnormal and disorganized actin structures, are defective in cellular morphogenesis, and are hypersensitive to the microtubule inhibitor benomyl. Furthermore, overexpression of wild-type Anc2p ameliorates defects in actin organization and cell growth caused by actin overproduction. Mutations in BIN2 and BIN3, two other genes that encode TCP-1-like proteins, also enhance the phenotypes of actin mutants. Taken together, these findings demonstrate that TCP-1-like proteins are required for actin and tubulin function in vivo. Images PMID:7916461

  9. Induction of HoxB Transcription by Retinoic Acid Requires Actin Polymerization

    PubMed Central

    Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M.; Crippa, Massimo P.; Scita, Giorgio

    2009-01-01

    We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of “elongating” RNAPII, Prep1, β-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes. PMID:19477923

  10. Quantifying morphological features of actin cytoskeletal filaments in plant cells based on mathematical morphology.

    PubMed

    Kimori, Yoshitaka; Hikino, Kazumi; Nishimura, Mikio; Mano, Shoji

    2016-01-21

    By quantifying the morphological properties of biological structures, we can better evaluate complex shapes and detect subtle morphological changes in organisms. In this paper, we propose a shape analysis method based on morphological image processing, and apply it to image analysis of actin cytoskeletal filaments in root hair cells of Arabidopsis thaliana. In plant cells, the actin cytoskeletal filaments have critical roles in various cellular processes such as vesicle trafficking and organelle motility. The dynamics of vesicles and organelles in plant cells depend on actin cytoskeletal filaments, regulating cell division and cell enlargement. To better understand the actin-dependent organelle motility, we attempted to quantify the organization of actin filaments in the root hair cells of the root hair defective 3 (rhd3) mutant. RHD3 is involved in actin organization, and its defect has been reported to affect the dynamics of various vesicles and organelles. We measured three shape features of the actin filaments in wild-type and mutant plants. One feature (thickness) was depicted on a grayscale; the others (describing the complexity of the filament network patterns in two-dimensional space) were depicted as binary features. The morphological phenotypes of the cytoskeletal filaments clearly differed between wild-type and mutant. Subtle variations of filament morphology among the mutants were detected and statistically quantified. PMID:26551157

  11. Induction of HoxB transcription by retinoic acid requires actin polymerization.

    PubMed

    Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M; Crippa, Massimo P; Scita, Giorgio; Blasi, Francesco

    2009-08-01

    We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of "elongating" RNAPII, Prep1, beta-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes. PMID:19477923

  12. Elasticity, adhesion and actin based propulsion

    NASA Astrophysics Data System (ADS)

    Gopinathan, Ajay

    2006-03-01

    When a cells crawls, its shape re-organizes via polymerization and depolymerization of actin filaments. The growing ends of the filaments are oriented towards the outside of the cell, and their polymerization pushes the cell membrane forwards. The same mechanism comes into play when the bacterial pathogen Listeria monocytogenes infects a cell. The bacterium hijacks the host cell's actin machinery to create an actin network (the actin comet tail) that propels the bacterium through cells and into neighboring cells. We propose a mechanism for how polymerization gives rise to motility that incorporates the effects of inhomogeneous polymerization. We treat the actin comet tail as an elastic continuum tethered to the rear of the bacterium. The interplay of polymerization and tethering gives rise to inhomogeneous stresses calculated with a finite element analysis. We quantitatively reproduce many distinctive features of actin propulsion that have been observed experimentally, including stepped motion, hopping, tail shape and the propulsion of flat surfaces.

  13. Aluminum Induces Rigor within the Actin Network of Soybean Cells.

    PubMed Central

    Grabski, S.; Schindler, M.

    1995-01-01

    Aluminum is toxic to both plants and animals. Root growth and pollen-tube extension are inhibited after aluminum stress in acidic environments. Incubation of cultured neurons with aluminum results in the formation of neurofibrillar tangles reminiscent of the neural pathology observed in Alzheimer's disease. The present communication demonstrates that aluminum induces a rapid and dramatic increase in the rigidity of the actin network in soybean (Glycine max) root cells. This rigidity can be prevented by either co-incubation with sodium fluoride or magnesium, or pretreatment with cytochalasin D. It is proposed that the growth-inhibitory activity and cytotoxicity of aluminum in plants may be a consequence of a global rigor that is induced within the actin network. This rigor may result from the formation of nonhydrolyzable [Al3+-ADP] or [Al3+-ATP] complexes whose binding to actin/myosin can modify contraction. Additionally, Al3+-mediated interference with the normal kinetics of F-actin filament assembly/disassembly could precipitate subsequent disorganization of associated cytoskeletal structures and promote altered expression of cytoskeletal proteins. PMID:12228515

  14. Myosin-Va and dynamic actin oppose microtubules to drive long-range organelle transport.

    PubMed

    Evans, Richard D; Robinson, Christopher; Briggs, Deborah A; Tooth, David J; Ramalho, Jose S; Cantero, Marta; Montoliu, Lluis; Patel, Shyamal; Sviderskaya, Elena V; Hume, Alistair N

    2014-08-01

    In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the "highways and local roads" model for transport along microtubule and actin tracks. The "cooperative capture" model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning. PMID:25065759

  15. Rapid Glucose Depletion Immobilizes Active Myosin-V on Stabilized Actin Cables

    PubMed Central

    Xu, Li; Bretscher, Anthony

    2014-01-01

    Summary Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton [1]. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles [2, 3]. This is mediated by myosin-Vs transporting cargos along F-actin bundles known as actin cables [4]. Actin cables are dynamic structures regulated by assembly, stabilization and disassembly [5]. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage [6]. Thus upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase [7, 8], translation [9] and phosphoinositide metabolism [10]. Here we show that glucose withdrawal rapidly (<1 min) depletes ATP levels and the yeast myosin V, Myo2, responds by relocalizing to actin cables, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigor-like state of a myosin-V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Glucose depletion has differential effects on the actin cytoskeleton resulting in disassembly of actin patches with concomitant inhibition of endocytosis, and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin-V in a newly described state on selectively stabilized actin cables. PMID:25308080

  16. Nonclassical nucleation and growth of inorganic nanoparticles

    NASA Astrophysics Data System (ADS)

    Lee, Jisoo; Yang, Jiwoong; Kwon, Soon Gu; Hyeon, Taeghwan

    2016-08-01

    The synthesis of nanoparticles with particular compositions and structures can lead to nanoparticles with notable physicochemical properties, thus promoting their use in various applications. In this area of nanoscience, the focus is shifting from size- and shape-uniform single-component nanoparticles to multicomponent nanoparticles with enhanced performance and/or multifunctionality. With the increasing complexity of synthetic reactions, an understanding of the formation mechanisms of the nanoparticles is needed to enable a systematic synthetic approach. This Review highlights mechanistic studies underlying the synthesis of nanoparticles, with an emphasis on nucleation and growth behaviours that are not expected from classical theories. We discuss the structural properties of nanoclusters that are of a size that bridges molecules and solids. We then describe the role of nanoclusters in the prenucleation process as well as in nonclassical nucleation models. The growth of nanoparticles via the assembly and merging of primary particles is also overviewed. Finally, we present the heterogeneous nucleation mechanisms behind the synthesis of multicomponent nanoparticles.

  17. Biomineralization: Ion binding and nucleation

    NASA Astrophysics Data System (ADS)

    Kröger, Roland

    2015-04-01

    The visualization of organic-acid-induced crystal growth by means of liquid-cell transmission electron microscopy can provide key insights into the nucleation of calcium carbonate in an organic matrix.

  18. Bubble nucleation in stout beers

    NASA Astrophysics Data System (ADS)

    Lee, W. T.; McKechnie, J. S.; Devereux, M. G.

    2011-05-01

    Bubble nucleation in weakly supersaturated solutions of carbon dioxide—such as champagne, sparkling wines, and carbonated beers—is well understood. Bubbles grow and detach from nucleation sites: gas pockets trapped within hollow cellulose fibers. This mechanism appears not to be active in stout beers that are supersaturated solutions of nitrogen and carbon dioxide. In their canned forms these beers require additional technology (widgets) to release the bubbles which will form the head of the beer. We extend the mathematical model of bubble nucleation in carbonated liquids to the case of two gases and show that this nucleation mechanism is active in stout beers, though substantially slower than in carbonated beers and confirm this by observation. A rough calculation suggests that despite the slowness of the process, applying a coating of hollow porous fibers to the inside of a can or bottle could be a potential replacement for widgets.

  19. Functional interdependence between septin and actin cytoskeleton

    PubMed Central

    Schmidt, Katja; Nichols, Benjamin J

    2004-01-01

    Background Septin2 is a member of a highly conserved GTPase family found in fungi and animals. Septins have been implicated in a diversity of cellular processes including cytokinesis, formation of diffusion barriers and vesicle trafficking. Septin2 partially co-localises with actin bundles in mammalian interphase cells and Septin2-filamentmorphology depends upon an intact actin cytoskeleton. How this interaction is regulated is not known. Moreover, evidence that Septin2 is remodelled or redistributed in response to other changes in actin organisation is lacking. Results Septin2 filaments are associated with actin fibres, but Septin2 is not associated with actin at the leading edge of moving cells or in ruffles where actin is highly dynamic. Rather, Septin2 is spatially segregated from these active areas and forms O- and C-shaped structures, similar to those previously observed after latrunculin treatment. FRAP experiments showed that all assemblies formed by Septin2 are highly dynamic with a constant exchange of Septin2 in and out of these structures, and that this property is independent of actin. A combination of RNAi experiments and expression of truncated forms of Septin2 showed that Septin2 plays a significant role in stabilising or maintaining actin bundles. Conclusion We show that Septin2 can form dynamic structures with differing morphologies in living cells, and that these morphologies are dependent on the functional state of the actin cytoskeleton. Our data provide a link between the different morphological states of Septin2 and functions of Septin2 in actin-dynamics, and are consistent with the model proposed by Kinoshita and colleagues, that Septin2 filaments play a role in stabilisation of actin stress fibres thus preventing actin turnover. PMID:15541171

  20. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression

    PubMed Central

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the “status” of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  1. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression.

    PubMed

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the "status" of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  2. The catalytic domain of inositol-1,4,5-trisphosphate 3-kinase-a contributes to ITPKA-induced modulation of F-actin.

    PubMed

    Ashour, Dina Julia; Pelka, Benjamin; Jaaks, Patricia; Wundenberg, Torsten; Blechner, Christine; Zobiak, Bernd; Failla, Antonio Virgilio; Windhorst, Sabine

    2015-02-01

    Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has been considered as an actin bundling protein because its N-terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross-linked actin filaments, here we analyzed if full length-ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F-actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C-terminal ABD, we exclude that ITPKA cross-links actin filaments by simultaneous F-actin binding with two different ABDs. Instead, stimulated-emission-depletion-microscopy and measurement of InsP3 Kinase activity give evidence that that N-terminal ABD-homodimers of ITPKA bind to F-actin while the monomeric C-termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3 Kinase activity is doubled and the product of InsP3 Kinase activity, Ins(1,3,4,5)P4 , inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3 Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3 Kinase domain substantially contributes to this process. PMID:25620569

  3. Actin depolymerization mediated loss of SNTA1 phosphorylation and Rac1 activity has implications on ROS production, cell migration and apoptosis.

    PubMed

    Bhat, Sehar Saleem; Parray, Arif Ali; Mushtaq, Umar; Fazili, Khalid Majid; Khanday, Firdous Ahmad

    2016-06-01

    Alpha-1-syntrophin (SNTA1) and Rac1 are part of a signaling pathway via the dystrophin glycoprotein complex (DGC). Both SNTA1 and Rac1 proteins are over-expressed in various carcinomas. It is through the DGC signaling pathway that SNTA1 has been shown to act as a link between the extra cellular matrix, the internal cell signaling apparatus and the actin cytoskeleton. SNTA1 is involved in the modulation of the actin cytoskeleton and actin reorganization. Rac1 also controls actin cytoskeletal organization in the cell. In this study, we present the interplay between f-actin, SNTA1 and Rac1. We analyzed the effect of actin depolymerization on SNTA1 tyrosine phosphorylation and Rac1 activity using actin depolymerizing drugs, cytochalasin D and latrunculin A. Our results indicate a marked decrease in the tyrosine phosphorylation of SNTA1 upon actin depolymerization. Results suggest that actin depolymerization mediated loss of SNTA1 phosphorylation leads to loss of interaction between SNTA1 and Rac1, with a concomitant loss of Rac1 activation. The loss of SNTA1tyrosine phosphorylation and Rac1 activity by actin depolymerization results in increased apoptosis, decreased cell migration and decreased reactive oxygen species (ROS) levels in breast carcinoma cells. Collectively, our results present a possible role of f-actin in the SNTA1-Rac1 signaling pathway and implications of actin depolymerization on cell migration, ROS production and apoptosis. PMID:27048259

  4. Calcium control of Saccharomyces cerevisiae actin assembly.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

    Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo. Images PMID:6757718

  5. Architecture and Connectivity Govern Actin Network Contractility.

    PubMed

    Ennomani, Hajer; Letort, Gaëlle; Guérin, Christophe; Martiel, Jean-Louis; Cao, Wenxiang; Nédélec, François; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2016-03-01

    Actomyosin contractility plays a central role in a wide range of cellular processes, including the establishment of cell polarity, cell migration, tissue integrity, and morphogenesis during development. The contractile response is variable and depends on actomyosin network architecture and biochemical composition. To determine how this coupling regulates actomyosin-driven contraction, we used a micropatterning method that enables the spatial control of actin assembly. We generated a variety of actin templates and measured how defined actin structures respond to myosin-induced forces. We found that the same actin filament crosslinkers either enhance or inhibit the contractility of a network, depending on the organization of actin within the network. Numerical simulations unified the roles of actin filament branching and crosslinking during actomyosin contraction. Specifically, we introduce the concept of "network connectivity" and show that the contractions of distinct actin architectures are described by the same master curve when considering their degree of connectivity. This makes it possible to predict the dynamic response of defined actin structures to transient changes in connectivity. We propose that, depending on the connectivity and the architecture, network contraction is dominated by either sarcomeric-like or buckling mechanisms. More generally, this study reveals how actin network contractility depends on its architecture under a defined set of biochemical conditions. PMID:26898468

  6. Stochastic model of profilin-actin polymerization

    NASA Astrophysics Data System (ADS)

    Horan, Brandon; Vavylonis, Dimitrios

    A driving factor in cell motility and other processes that involve changes of cell shape is the rapid polymerization of actin subunits into long filaments. This process is regulated by profilin, a protein which binds to actin subunits and regulates elongation of actin filaments. Whether profilin stimulates polymerization by coupling to hydrolysis of ATP-bound actin is debated. Previous studies have proposed indirect coupling to ATP hydrolysis using rate equations, but did not include the effects of fluctuations that are important near the critical concentration. We developed stochastic simulations using the Gillespie algorithm to study single filament elongation at the barbed end in the presence of profilin. We used recently measured rate constants and estimated the rate of profilin binding to the barbed end such that detailed balance is satisfied. Fast phosphate release at the tip of the filament was accounted for. The elongation rate and length diffusivity as functions of profilin and actin concentration were calculated and used to extract the critical concentrations of free actin and of total actin. We show under what conditions profilin leads to an increase in the critical concentration of total actin but a decrease in the critical concentration of free actin.

  7. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks. PMID:26915738

  8. In Situ Investigations into CaCO3 Nucleation

    NASA Astrophysics Data System (ADS)

    Nielsen, Michael Harold

    Classical theories of nucleation were developed over a hundred years ago starting with Gibbs. However, much remains unknown about the process of phase transition in aqueous electrolyte solutions due to the lack of experimental tools able to probe dynamic processes at the time and length scales of the phase transformation. In the calcium carbonate system, recent discovery of an amorphous phase, as well as the suggested existence of potential precursor states such as so-called 'pre-nucleation clusters' or dense liquid droplets, has called into question the utility of the classical framework in making accurate predictions of nucleation. Added to these questions are those regarding the effects that chemical templates have on nucleating calcium carbonate. Many organisms use complex organic matrices to form architecturally complex functional structures out of sea water at ambient temperatures. By contrast, laboratory methods to materials synthesis often require extreme conditions yet maintain at best a low level of control over the development of the resulting material. With the goal of tightly controlling formation of functional materials, scientists have looked to such biomineral systems for inspiration. Self-assembled monolayers (SAMs) of functionalized alkanethiols have been found to act as idealized chemical templates for calcium carbonate nucleation, controlling the nucleating plane of the calcite phase for many surface functionalities. Yet there remain many open questions as to the fundamental mechanisms by which these templates achieve this control. In this dissertation many investigations of calcium carbonate nucleation are discussed, which examine the nucleation pathways of calcium carbonate and mechanisms of control by which alkanethiol surfaces direct the oriented formation of calcite. Traditional in situ microscopy techniques are used to make nucleation rate measurements of templated calcite nucleation on alkanethiol SAMs to test the applicability of the

  9. Docking, molecular dynamics and QM/MM studies to delineate the mode of binding of CucurbitacinE to F-actin.

    PubMed

    Kumar, R Pravin; Roopa, L; Nongthomba, Upendra; Sudheer Mohammed, M M; Kulkarni, Naveen

    2016-01-01

    CucurbitacinE (CurE) has been known to bind covalently to F-actin and inhibit depolymerization. However, the mode of binding of CurE to F-actin and the consequent changes in the F-actin dynamics have not been studied. Through quantum mechanical/molecular mechanical (QM/MM) and density function theory (DFT) simulations after the molecular dynamics (MD) simulations of the docked complex of F-actin and CurE, a detailed transition state (TS) model for the Michael reaction is proposed. The TS model shows nucleophilic attack of the sulphur of Cys257 at the β-carbon of Michael Acceptor of CurE producing an enol intermediate that forms a covalent bond with CurE. The MD results show a clear difference between the structure of the F-actin in free form and F-actin complexed with CurE. CurE affects the conformation of the nucleotide binding pocket increasing the binding affinity between F-actin and ADP, which in turn could affect the nucleotide exchange. CurE binding also limits the correlated displacement of the relatively flexible domain 1 of F-actin causing the protein to retain a flat structure and to transform into a stable "tense" state. This structural transition could inhibit depolymerization of F-actin. In conclusion, CurE allosterically modulates ADP and stabilizes F-actin structure, thereby affecting nucleotide exchange and depolymerization of F-actin. PMID:26615469

  10. A mechanism of leading-edge protrusion in the absence of Arp2/3 complex

    PubMed Central

    Suraneni, Praveen; Fogelson, Ben; Rubinstein, Boris; Noguera, Philippe; Volkmann, Niels; Hanein, Dorit; Mogilner, Alex; Li, Rong

    2015-01-01

    Cells employ protrusive leading edges to navigate and promote their migration in diverse physiological environments. Classical models of leading-edge protrusion rely on a treadmilling dendritic actin network that undergoes continuous assembly nucleated by the Arp2/3 complex, forming ruffling lamellipodia. Recent work demonstrated, however, that, in the absence of the Arp2/3 complex, fibroblast cells adopt a leading edge with filopodia-like protrusions (FLPs) and maintain an ability to move, albeit with altered responses to different environmental signals. We show that formin-family actin nucleators are required for the extension of FLPs but are insufficient to produce a continuous leading edge in fibroblasts lacking Arp2/3 complex. Myosin II is concentrated in arc-like regions of the leading edge in between FLPs, and its activity is required for coordinated advancement of these regions with formin-generated FLPs. We propose that actomyosin contraction acting against membrane tension advances the web of arcs between FLPs. Predictions of this model are verified experimentally. The dependence of myosin II in leading-edge advancement helps explain the previously reported defect in directional movement in the Arpc3-null fibroblasts. We provide further evidence that this defect is cell autonomous during chemotaxis. PMID:25568333

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

    PubMed Central

    2010-01-01

    Background Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton. Results Here, we in addition to toxins use conditional expression of the major actin regulatory protein LIM kinase-1 (LIMK1), and shRNA knock-down of cofilin to modulate the cellular F/G-actin ratio in the Ra2 microglia cell line, and we use Fluorescence Recovery after Photobleaching (FRAP) in β-actin-YFP-transduced cells to obtain a dynamic measure of actin recovery rates (actin turn-over rates) in different F/G-actin states of the actin cytoskeleton. Our data demonstrate that stimulated NADPH oxidase function was severely impaired only at extreme actin recovery rates and F/G-actin ratios, and surprisingly, that any moderate changes of these parameters of the actin cytoskeleton invariably resulted in an increased NADPH oxidase activity. Conclusion moderate actin polymerization and depolymerization both increase the FMLP and PMA-stimulated NADPH oxidase activity of microglia, which is directly correlated with neither actin recovery rate nor F/G- actin ratio. Our results indicate that NADPH oxidase functions in an enhanced state of activity in stimulated phagocytes despite widely different states of the actin cytoskeleton. PMID:20825680

  12. Dynamics of active actin networks

    NASA Astrophysics Data System (ADS)

    Koehler, Simone

    2014-03-01

    Local mechanical and structural properties of a eukaryotic cell are determined by its cytoskeleton. To adapt to their environment, cells rely on constant self-organized rearrangement processes of their actin cytoskeleton. To shed light on the principles underlying these dynamic self-organization processes we investigate a minimal reconstituted active system consisting of actin filaments, crosslinking molecules and molecular motor filaments. Using quantitative fluorescence microscopy and image analysis, we show, that these minimal model systems exhibit a generic structure formation mechanism. The competition between force generation by molecular motors and the stabilization of the network by crosslinking proteins results in a highly dynamic reorganization process which is characterized by anomalous transport dynamics with a superdiffusive behavior also found in intracellular dynamics. In vitro, these dynamics are governed by chemical and physical parameters that alter the balance of motor and crosslinking proteins, such as pH. These findings can be expected to have broad implications in our understanding of cytoskeletal regulation in vivo.

  13. Reverse actin sliding triggers strong myosin binding that moves tropomyosin

    SciTech Connect

    Bekyarova, T.I.; Reedy, M.C.; Baumann, B.A.J.; Tregear, R.T.; Ward, A.; Krzic, U.; Prince, K.M.; Perz-Edwards, R.J.; Reconditi, M.; Gore, D.; Irving, T.C.; Reedy, M.K.

    2008-09-03

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the 'steric blocking' mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca{sup 2+} with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca{sup 2+}], and stretch activation, at lower [Ca{sup 2+}], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored 'actin target zones.' Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca{sup 2+}] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca{sup 2+}], Vi-'paralyzed' fibers produce force substantially above passive response at pCa {approx} 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding 'brakes' by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

  14. Reverse actin sliding triggers strong myosin binding that moves tropomyosin.

    PubMed

    Bekyarova, T I; Reedy, M C; Baumann, B A J; Tregear, R T; Ward, A; Krzic, U; Prince, K M; Perz-Edwards, R J; Reconditi, M; Gore, D; Irving, T C; Reedy, M K

    2008-07-29

    Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the "steric blocking" mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca(2+) with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca(2+)], and stretch activation, at lower [Ca(2+)], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored "actin target zones." Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca(2+)] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca(2+)], Vi-"paralyzed" fibers produce force substantially above passive response at pCa approximately 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding "brakes" by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions. PMID:18658238

  15. Nucleation and growth of zinc from chloride concentrated solutions

    SciTech Connect

    Trejo, G.; Ortega B, R.; Meas V, Y.; Ozil, P.; Chainet, E.; Nguyen, B.

    1998-12-01

    The electrodeposition of metals is a complex phenomenon influenced by a number of factors that modify the rates of nucleation and growth and determine the properties of the deposits. In this work the authors study the influence of the zinc chloride (ZnCl{sub 2}) concentration on the zinc nucleation process on glassy carbon, in a KCl electrolyte under conditions close to those employed in commercial acid deposition baths for zinc. The electrochemical study was performed using cyclic voltammetry and potentiostatic current-time transients. The charge-transfer coefficient and the formal potential for ZnCl{sub 2} reduction were evaluated from cyclic voltammetry experiments. The nucleation process was analyzed by comparing the transients obtained with the known dimensionless (i/i{sub m}){sup 2} vs. t/t{sub m} response for instantaneous or progressive nucleation. The results show that the nucleation process and the number density of sites are dependent on ZnCl{sub 2} concentration. Scanning electron microscopy analysis of the deposits shows that the deposits are homogeneous and compact although a change in the morphology is observed as a function of ZnCl{sub 2} concentration. Evaluation of the corrosion resistance reveals the influence of the nucleation process on the subsequent corrosion resistance of the zinc deposits.

  16. Determination of critical nucleation number for a single nucleation amyloid-β aggregation model.

    PubMed

    Ghosh, Preetam; Vaidya, Ashwin; Kumar, Amit; Rangachari, Vijayaraghavan

    2016-03-01

    Aggregates of amyloid-β (Aβ) peptide are known to be the key pathological agents in Alzheimer disease (AD). Aβ aggregates to form large, insoluble fibrils that deposit as senile plaques in AD brains. The process of aggregation is nucleation-dependent in which the formation of a nucleus is the rate-limiting step, and controls the physiochemical fate of the aggregates formed. Therefore, understanding the properties of nucleus and pre-nucleation events will be significant in reducing the existing knowledge-gap in AD pathogenesis. In this report, we have determined the plausible range of critical nucleation number (n(*)), the number of monomers associated within the nucleus for a homogenous aggregation model with single unique nucleation event, by two independent methods: A reduced-order stability analysis and ordinary differential equation based numerical analysis, supported by experimental biophysics. The results establish that the most likely range of n(*) is between 7 and 14 and within, this range, n(*) = 12 closely supports the experimental data. These numbers are in agreement with those previously reported, and importantly, the report establishes a new modeling framework using two independent approaches towards a convergent solution in modeling complex aggregation reactions. Our model also suggests that the formation of large protofibrils is dependent on the nature of n(*), further supporting the idea that pre-nucleation events are significant in controlling the fate of larger aggregates formed. This report has re-opened an old problem with a new perspective and holds promise towards revealing the molecular events in amyloid pathologies in the future. PMID:26774039

  17. Effect of alpha-actinin on actin structure. Actin ATPase activity.

    PubMed

    Singh, I; Goll, D E; Robson, R M

    1981-08-28

    Alpha-Actinin increases the ATPase activity of actin by up to 84%, depending un pH, divalent cations present and the added Mg2+: ATP ratio. Dithiothreitol decreases actin ATPase activity approx. 20% but does not reduce the ability of alpha-actinin to increase actin ATP activity. Increasing amounts of added alpha-actinin up to 1 mos alpha-actinin to 49 mol actin cause in increasing increment in actin ATPase activity, but adding alpha-actinin beyond 1 mol alpha-actinin to 49 mol actin elicits only small additional increments in activity. Actin ATPase activity ranges from approx 100 nmol Pi/mg actin per h (4.3 mol Pi/mol actin per h) at high levels (10 mM) of ATP in the presence of lower amounts (1 mM) of added mg2+ to approx. 12.5 nmol Pi/mg actin per h (0.52 mol Pi/mol actin per h) at high pH (8.5) or at low levels (0.5-1.0 mM) of ATP in the presence of higher amounts (10 mM) of added Mg2+ ATp uncomplexed with Mg2+ inhibits the ability of alpha-actinin to increase F-actin ATPase activity. Activities with different divalent cations showed that the actin ATPase in these studies, which was 1/100 as great as Mg2+-modified actomyosin ATPase activity, was not due to trace amounts of myosin contaminating the actin preparations. The results are consistent with the concept that alpha-actinin can alter the structure of actin monomers. PMID:6456018

  18. Hydrocarbon nucleation and aerosol formation in Neptune's atmosphere.

    PubMed

    Moses, J I; Allen, M; Yung, Y L

    1992-10-01

    Photodissociation of methane at high altitude levels in Neptune's atmosphere leads to the production of complex hydrocarbon species such as acetylene (C2H2), ethane (C2H6), methylacetylene (CH3C2H), propane (C3H8), diacetylene (C4H2), and butane (C4H8). These gases diffuse to the lower stratosphere where temperatures are low enough to initiate condensation. Particle formation may not occur readily, however, as the vapor species become supersaturated. We present a theoretical analysis of particle formation mechanisms at conditions relevant to Neptune's troposphere and stratosphere and show that hydrocarbon nucleation is very inefficient under Neptunian conditions: saturation ratios much greater than unity are required for aerosol formation by either homogeneous, heterogeneous, or ion-induced nucleation. Homogeneous nucleation will not be important for any of the hydrocarbon species considered; however, both heterogeneous and ion-induced nucleation should be possible on Neptune for most of the above species. The relative effectiveness of heterogeneous and ion-induced nucleation depends on the physical and thermodynamic properties of the particular species, the abundance of the condensable species, the temperature at which the vapor becomes supersaturated, and the number and type of condensation nuclei or ions available. Typical saturation ratios required for observable particle formation rates on Neptune range from approximately 3 for heterogeneous nucleation of methane in the upper troposphere to greater than 1000 for heterogeneous nucleation of methylacetylene, diacetylene, and butane in the lower stratosphere. Thus, methane clouds may form slightly above, and stratospheric hazes far below, their saturation levels. When used in conjunction with the results of detailed models of atmospheric photochemistry, our nucleation models place realistic constraints on the altitude levels at which we expect hydrocarbon hazes or clouds to form on Neptune. PMID:11538166

  19. Rest frame of bubble nucleation

    SciTech Connect

    Garriga, Jaume; Kanno, Sugumi; Tanaka, Takahiro E-mail: sugumi@cosmos.phy.tufts.edu

    2013-06-01

    Vacuum bubbles nucleate at rest with a certain critical size and subsequently expand. But what selects the rest frame of nucleation? This question has been recently addressed in [1] in the context of Schwinger pair production in 1+1 dimensions, by using a model detector in order to probe the nucleated pairs. The analysis in [1] showed that, for a constant external electric field, the adiabatic ''in'' vacuum of charged particles is Lorentz invariant, (and in this) case pairs tend to nucleate preferentially at rest with respect to the detector. Here, we sharpen this picture by showing that the typical relative velocity between the frame of nucleation and that of the detector is at most of order Δv ∼ S{sub E}{sup −1/3} << 1. Here, S{sub E} >> 1 is the action of the instanton describing pair creation. The bound Δv coincides with the minimum uncertainty in the velocity of a non-relativistic charged particle embedded in a constant electric field. A velocity of order Δv is reached after a time interval of order Δt ∼ S{sub E}{sup −1/3}r{sub 0} << r{sub 0} past the turning point in the semiclassical trajectory, where r{sub 0} is the size of the instanton. If the interaction takes place in the vicinity of the turning point, the semiclassical description of collision does not apply. Nonetheless, we find that even in this case there is still a strong asymmetry in the momentum transferred from the nucleated particles to the detector, in the direction of expansion after the turning point. We conclude that the correlation between the rest frame of nucleation and that of the detector is exceedingly sharp.

  20. Mathematical modelling and numerical simulations of actin dynamics in the eukaryotic cell.

    PubMed

    George, Uduak Z; Stéphanou, Angélique; Madzvamuse, Anotida

    2013-02-01

    The aim of this article is to study cell deformation and cell movement by considering both the mechanical and biochemical properties of the cortical network of actin filaments and its concentration. Actin is a polymer that can exist either in filamentous form (F-actin) or in monometric form (G-actin) (Chen et al. in Trends Biochem Sci 25:19-23, 2000) and the filamentous form is arranged in a paired helix of two protofilaments (Ananthakrishnan et al. in Recent Res Devel Biophys 5:39-69, 2006). By assuming that cell deformations are a result of the cortical actin dynamics in the cell cytoskeleton, we consider a continuum mathematical model that couples the mechanics of the network of actin filaments with its bio-chemical dynamics. Numerical treatment of the model is carried out using the moving grid finite element method (Madzvamuse et al. in J Comput Phys 190:478-500, 2003). Furthermore, by assuming slow deformations of the cell, we use linear stability theory to validate the numerical simulation results close to bifurcation points. Far from bifurcation points, we show that the mathematical model is able to describe the complex cell deformations typically observed in experimental results. Our numerical results illustrate cell expansion, cell contraction, cell translation and cell relocation as well as cell protrusions. In all these results, the contractile tonicity formed by the association of actin filaments to the myosin II motor proteins is identified as a key bifurcation parameter. PMID:22434394

  1. Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy.

    PubMed

    Ceccaldi, P E; Grohovaz, F; Benfenati, F; Chieregatti, E; Greengard, P; Valtorta, F

    1995-03-01

    Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles to the actin cytoskeleton of the nerve terminal. Using video-enhanced microscopy, we have now obtained experimental evidence in support of this model: the presence of dephosphorylated synapsin I is necessary for synaptic vesicles to bind actin; synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles; the ability to cross-link synaptic vesicles and actin is specific for synapsin I and is not shared by other basic proteins; the cross-linking between synaptic vesicles and actin is specific for the membrane of synaptic vesicles and does not reflect either a non-specific binding of membranes to the highly surface active synapsin I molecule or trapping of vesicles within the thick bundles of actin filaments; the formation of the ternary complex is virtually abolished when synapsin I is phosphorylated by CaM kinase II. The data indicate that synapsin I markedly affects synaptic vesicle traffic and cytoskeleton assembly in the nerve terminal and provide a molecular basis for the ability of synapsin I to regulate the availability of synaptic vesicles for exocytosis and thereby the efficiency of neurotransmitter release. PMID:7876313

  2. Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis.

    PubMed

    Cheng, Jackie; Grassart, Alexandre; Drubin, David G

    2012-08-01

    Myosin 1E (Myo1E) is recruited to sites of clathrin-mediated endocytosis coincident with a burst of actin assembly. The recruitment dynamics and lifetime of Myo1E are similar to those of tagged actin polymerization regulatory proteins. Like inhibition of actin assembly, depletion of Myo1E causes reduced transferrin endocytosis and a significant delay in transferrin trafficking to perinuclear compartments, demonstrating an integral role for Myo1E in these actin-mediated steps. Mistargeting of GFP-Myo1E or its src-homology 3 domain to mitochondria results in appearance of WIP, WIRE, N-WASP, and actin filaments at the mitochondria, providing evidence for Myo1E's role in actin assembly regulation. These results suggest for mammalian cells, similar to budding yeast, interdependence in the recruitment of type I myosins, WIP/WIRE, and N-WASP to endocytic sites for Arp2/3 complex activation to assemble F-actin as endocytic vesicles are being formed. PMID:22675027

  3. Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis

    PubMed Central

    Cheng, Jackie; Grassart, Alexandre; Drubin, David G.

    2012-01-01

    Myosin 1E (Myo1E) is recruited to sites of clathrin-mediated endocytosis coincident with a burst of actin assembly. The recruitment dynamics and lifetime of Myo1E are similar to those of tagged actin polymerization regulatory proteins. Like inhibition of actin assembly, depletion of Myo1E causes reduced transferrin endocytosis and a significant delay in transferrin trafficking to perinuclear compartments, demonstrating an integral role for Myo1E in these actin-mediated steps. Mistargeting of GFP-Myo1E or its src-homology 3 domain to mitochondria results in appearance of WIP, WIRE, N-WASP, and actin filaments at the mitochondria, providing evidence for Myo1E's role in actin assembly regulation. These results suggest for mammalian cells, similar to budding yeast, interdependence in the recruitment of type I myosins, WIP/WIRE, and N-WASP to endocytic sites for Arp2/3 complex activation to assemble F-actin as endocytic vesicles are being formed. PMID:22675027

  4. The actin binding site of thymosin beta 4 mapped by mutational analysis.

    PubMed Central

    Van Troys, M; Dewitte, D; Goethals, M; Carlier, M F; Vandekerckhove, J; Ampe, C

    1996-01-01

    We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin. Images PMID:8617195

  5. Modulation of nuclear localization of the influenza virus nucleoprotein through interaction with actin filaments.

    PubMed

    Digard, P; Elton, D; Bishop, K; Medcalf, E; Weeds, A; Pope, B

    1999-03-01

    The influenza virus genome is transcribed in the nuclei of infected cells but assembled into progeny virions in the cytoplasm. This is reflected in the cellular distribution of the virus nucleoprotein (NP), a protein which encapsidates genomic RNA to form ribonucleoprotein structures. At early times postinfection NP is found in the nucleus, but at later times it is found predominantly in the cytoplasm. NP contains several sequences proposed to act as nuclear localization signals (NLSs), and it is not clear how these are overridden to allow cytoplasmic accumulation of the protein. We find that NP binds tightly to filamentous actin in vitro and have identified a cluster of residues in NP essential for the interaction. Complexes containing RNA, NP, and actin could be formed, suggesting that viral ribonucleoproteins also bind