Science.gov

Sample records for actin polymerization induced

  1. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  3. Thromboxane-induced actin polymerization in hypoxic neonatal pulmonary arterial myocytes involves Cdc42 signaling.

    PubMed

    Fediuk, Jena; Sikarwar, Anurag S; Nolette, Nora; Dakshinamurti, Shyamala

    2014-12-01

    In hypoxic pulmonary arterial (PA) myocytes, challenge with thromboxane mimetic U46619 induces marked actin polymerization and contraction, phenotypic features of persistent pulmonary hypertension of the newborn (PPHN). Rho GTPases regulate the actin cytoskeleton. We previously reported that U46619-induced actin polymerization in hypoxic PA myocytes occurs independently of the RhoA pathway and hypothesized involvement of the Cdc42 pathway. PA myocytes grown in normoxia or hypoxia for 72 h were stimulated with U46619, then analyzed for Rac/Cdc42 activation by affinity precipitation, phosphatidylinositide-3-kinase (PI3K) activity by phospho-Akt, phospho-p21-activated kinase (PAK) by immunoblot, and association of Cdc42 with neuronal Wiskott Aldrich Syndrome protein (N-WASp) by immunoprecipitation. The effect of Rac or PAK inhibition on filamentous actin was quantified by laser-scanning cytometry and by cytoskeletal fractionation; effects of actin-modifying agents were measured by isometric myography. Basal Cdc42 activity increased in hypoxia, whereas Rac activity decreased. U46619 challenge increased Cdc42 and Rac activity in hypoxic cells, independently of PI3K. Hypoxia increased phospho-PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia but increased after U46619 exposure. Hypoxia doubled filamentous-to-globular ratios of α- and γ-actin isoforms. Jasplakinolide stabilized γ-filaments, increasing force; cytochalasin D depolymerized all actin isoforms, decreasing force. Rac and PAK inhibition decreased filamentous actin in tissues although without decrease in force. Rho inhibition decreased myosin phosphorylation and force. Hypoxia induces actin polymerization in PA myocytes, particularly increasing filamentous α- and γ-actin, contributing to U46619-induced contraction. Hypoxic PA myocytes challenged with a thromboxane mimetic polymerize actin via the Cdc42 pathway, reflecting increased Cdc42 association with N-WASp. Mechanisms

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

    PubMed

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

    2016-03-21

    Actin molecules are among the three main cytoskeleton proteins of cells and undergo rapid cycling to regulate critical processes such as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. Although extensive studies have been carried out on the dynamics as well as biological functions of actin polymerization and depolymerization both in vivo and in vitro, the molecular mechanisms by which cells sense and respond to mechanical signals are not fully understood. In particular, little attention has been paid to the effect of a physical force that is exerted directly on the actin cytoskeleton. In this paper, we have explored how the mechanical force affects the actin polymerization and depolymerization behaviors at water/solid interfaces using an atomic force microscope (AFM) operated in liquid. By raster scanning an AFM probe on a substrate surface with a certain load, it was found that actin monomers could polymerize into filaments without the help of actin related proteins (ARPs). Further study indicated that actin monomers were inclined to form filaments only under a small scanning load. The polymerized actin filaments would be depolymerized when the mechanical force was stronger. A possible mechanism has been suggested to explain the mechanical force induced actin polymerization. PMID:26928199

  5. 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

  6. 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

  7. Inhibition of actin polymerization in the NAc shell inhibits morphine-induced CPP by disrupting its reconsolidation

    PubMed Central

    Li, Gongying; Wang, Yanmei; Yan, Min; Xu, Yunshuai; Song, Xiuli; Li, Qingqing; Zhang, Jinxiang; Ma, Hongxia; Wu, Yili

    2015-01-01

    Drug-associated contextual cues contribute to drug craving and relapse after abstinence, which is a major challenge to drug addiction treatment. Previous studies showed that disrupting memory reconsolidation impairs drug reward memory. However, the underlying mechanisms remain elusive. Although actin polymerization is involved in memory formation, its role in the reconsolidation of drug reward memory is unknown. In addition, the specific brain areas responsible for drug memory have not been fully identified. In the present study, we found that inhibiting actin polymerization in the nucleus accumbens (NAc) shell, but not the NAc core, abolishes morphine-induced conditioned place preference (CPP) by disrupting its reconsolidation in rats. Moreover, this effect persists for more than 2 weeks by a single injection of the actin polymerization inhibitor, which is not reversed by a morphine-priming injection. Furthermore, the application of actin polymerization inhibitor outside the reconsolidation window has no effect on morphine-associated contextual memory. Taken together, our findings first demonstrate that inhibiting actin polymerization erases morphine-induced CPP by disrupting its reconsolidation. Our study suggests that inhibition of actin polymerization during drug memory reconsolidation may be a potential approach to prevent drug relapse. PMID:26538334

  8. Colchicine activates actin polymerization by microtubule depolymerization.

    PubMed

    Jung, H I; Shin, I; Park, Y M; Kang, K W; Ha, K S

    1997-06-30

    Swiss 3T3 fibroblasts were treated with the microtubule-disrupting agent colchicine to study any interaction between microtubule dynamics and actin polymerization. Colchicine increased the amount of filamentous actin (F-actin), in a dose- and time-dependent manner with a significant increase at 1 h by about 130% over control level. Confocal microscopic observation showed that colchicine increased F-actin contents by stress fiber formation without inducing membrane ruffling. Colchicine did not activate phospholipase C and phospholipase D, whereas lysophosphatidic acid did, indicating that colchicine may have a different mechanism of actin polymerization regulation from LPA. A variety of microtubule-disrupting agents stimulated actin polymerization in Swiss 3T3 and Rat-2 fibroblasts as did colchicine, but the microtubule-stabilizing agent taxol inhibited actin polymerization induced by the above microtubule-disrupting agents. In addition, colchicine-induced actin polymerization was blocked by two protein phosphatase inhibitors, okadaic acid and calyculin A. These results suggest that microtubule depolymerization activates stress fiber formation by serine/threonine dephosphorylation in fibroblasts. PMID:9264034

  9. Detection of adenosine triphosphate through polymerization-induced aggregation of actin-conjugated gold/silver nanorods

    NASA Astrophysics Data System (ADS)

    Liao, Yu-Ju; Shiang, Yen-Chun; Chen, Li-Yi; Hsu, Chia-Lun; Huang, Chih-Ching; Chang, Huan-Tsung

    2013-11-01

    We have developed a simple and selective nanosensor for the optical detection of adenosine triphosphate (ATP) using globular actin-conjugated gold/silver nanorods (G-actin-Au/Ag NRs). By simply mixing G-actin and Au/Ag NRs (length ˜56 nm and diameter ˜12 nm), G-actin-Au/Ag NRs were prepared which were stable in physiological solutions (25 mM Tris-HCl, 150 mM NaCl, 5.0 mM KCl, 3.0 mM MgCl2 and 1.0 mM CaCl2; pH 7.4). Introduction of ATP into the G-actin-Au/Ag NR solutions in the presence of excess G-actin induced the formation of filamentous actin-conjugated Au/Ag NR aggregates through ATP-induced polymerization of G-actin. When compared to G-actin-modified spherical Au nanoparticles having a size of 13 nm or 56 nm, G-actin-Au/Ag NRs provided better sensitivity for ATP, mainly because the longitudinal surface plasmon absorbance of the Au/Ag NR has a more sensitive response to aggregation. This G-actin-Au/Ag NR probe provided high sensitivity (limit of detection 25 nM) for ATP with remarkable selectivity (>10-fold) over other adenine nucleotides (adenosine, adenosine monophosphate and adenosine diphosphate) and nucleoside triphosphates (guanosine triphosphate, cytidine triphosphate and uridine triphosphate). It also allowed the determination of ATP concentrations in plasma samples without conducting tedious sample pretreatments; the only necessary step was simple dilution. Our experimental results are in good agreement with those obtained from a commercial luciferin-luciferase bioluminescence assay. Our simple, sensitive and selective approach appears to have a practical potential for the clinical diagnosis of diseases (e.g. cystic fibrosis) associated with changes in ATP concentrations.

  10. PLCβ3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration.

    PubMed

    Janjanam, Jagadeesh; Chandaka, Giri Kumar; Kotla, Sivareddy; Rao, Gadiparthi N

    2015-12-15

    Monocyte chemotactic protein 1 (MCP1) stimulates vascular smooth muscle cell (VSMC) migration in vascular wall remodeling. However, the mechanisms underlying MCP1-induced VSMC migration have not been understood. Here we identify the signaling pathway associated with MCP1-induced human aortic smooth muscle cell (HASMC) migration. MCP1, a G protein-coupled receptor agonist, activates phosphorylation of cortactin on S405 and S418 residues in a time-dependent manner, and inhibition of its phosphorylation attenuates MCP1-induced HASMC G-actin polymerization, F-actin stress fiber formation, and migration. Cortactin phosphorylation on S405/S418 is found to be critical for its interaction with WAVE2, a member of the WASP family of cytoskeletal regulatory proteins required for cell migration. In addition, the MCP1-induced cortactin phosphorylation is dependent on PLCβ3-mediated PKCδ activation, and siRNA-mediated down-regulation of either of these molecules prevents cortactin interaction with WAVE2, affecting G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Upstream, MCP1 activates CCR2 and Gαq/11 in a time-dependent manner, and down-regulation of their levels attenuates MCP1-induced PLCβ3 and PKCδ activation, cortactin phosphorylation, cortactin-WAVE2 interaction, G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Together these findings demonstrate that phosphorylation of cortactin on S405 and S418 residues is required for its interaction with WAVE2 in MCP1-induced cytoskeleton remodeling, facilitating HASMC migration. PMID:26490115

  11. PLCβ3 mediates cortactin interaction with WAVE2 in MCP1-induced actin polymerization and cell migration

    PubMed Central

    Janjanam, Jagadeesh; Chandaka, Giri Kumar; Kotla, Sivareddy; Rao, Gadiparthi N.

    2015-01-01

    Monocyte chemotactic protein 1 (MCP1) stimulates vascular smooth muscle cell (VSMC) migration in vascular wall remodeling. However, the mechanisms underlying MCP1-induced VSMC migration have not been understood. Here we identify the signaling pathway associated with MCP1-induced human aortic smooth muscle cell (HASMC) migration. MCP1, a G protein–coupled receptor agonist, activates phosphorylation of cortactin on S405 and S418 residues in a time-dependent manner, and inhibition of its phosphorylation attenuates MCP1-induced HASMC G-actin polymerization, F-actin stress fiber formation, and migration. Cortactin phosphorylation on S405/S418 is found to be critical for its interaction with WAVE2, a member of the WASP family of cytoskeletal regulatory proteins required for cell migration. In addition, the MCP1-induced cortactin phosphorylation is dependent on PLCβ3-mediated PKCδ activation, and siRNA-mediated down-regulation of either of these molecules prevents cortactin interaction with WAVE2, affecting G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Upstream, MCP1 activates CCR2 and Gαq/11 in a time-dependent manner, and down-regulation of their levels attenuates MCP1-induced PLCβ3 and PKCδ activation, cortactin phosphorylation, cortactin–WAVE2 interaction, G-actin polymerization, F-actin stress fiber formation, and HASMC migration. Together these findings demonstrate that phosphorylation of cortactin on S405 and S418 residues is required for its interaction with WAVE2 in MCP1-induced cytoskeleton remodeling, facilitating HASMC migration. PMID:26490115

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. Chondramides, novel cyclodepsipeptides from myxobacteria, influence cell development and induce actin filament polymerization in the green alga Micrasterias.

    PubMed

    Holzinger, A; Lütz-Meindl, U

    2001-02-01

    The effects of chondramides A-D, new actin targeting cyclodepsipeptides from the myxobacterium Chondromyces crocatus, are probed on the unicellular green alga Micrasterias denticulata, a model organism for studies on cytomorphogenesis. All four chondramides readily enter the cells and cause severe shape malformations when applied during growth. However, the four derivatives have different lowest effective concentrations. Chondramide A: 20 microM, chondramide B: 15 microM, chondramide C: 5 microM chondramide D: 10 microM. At the ultrastructural level, chondramide C, the most effective drug, causes the appearance of abnormal, dense F-actin bundles, and a substantial increase in ER, which covers large parts of the developing semicell. Also the secondary cell wall is malformed by the drug. When chondramide C effects are investigated by means of indirect immunofluorescence, alterations of the F-actin system are also visible. Instead of the cortical F-actin network of untreated controls, distinct parts of the cell are covered by abundant F-actin aggregations. Phalloidin staining of chondramide C treated cells results in a decreased fluorescence in a time-dependent manner due to binding competitions between these drugs. F-actin polymerizing and bundling capacities of chondramides A-D are presented in Micrasterias for the first time, and may in future make this substances a useful tool for cell biological research. PMID:11169761

  18. 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

  19. Impact of Carbon Nanomaterials on Actin Polymerization.

    PubMed

    Dong, Ying; Sun, Haiyan; Li, Xu; Li, Xin; Zhao, Lina

    2016-03-01

    Many nanomaterials have entered people's daily lives and impact the normal process of biological entities consequently. As one kind of the important nanomaterials, carbon based nanomaterials have invoked a lot of concerns from scientific researches because of their unique physicochemical properties. In eukaryotes, actin is the most abundantly distributed protein in both cytoplasm and cell nucleus, and closely controls the cell proliferation and mobility. Recently, many investigations have found some carbon based nanomaterials can affect actin cytoskeleton remarkably, including fullerenes derivatives, carbon nanotubes, graphene and its derivatives. However, these interaction processes are complicated and the underlying mechanism is far from being understood clearly. In this review, we discussed the different mechanisms of carbon nanomaterials impact on actin polymerization into three pathways, as triggering the signaling pathways from carbon nanomaterials outside of cells, increasing the production of reactive oxygen species from carbon nanomaterials inside of cells and direct interaction from carbon nanomaterials inside of cells. As a result, the dimension and size of carbon nanomaterials play a key role in regulation of actin cytoskeleton. Furthermore, we forecasted the possible investigation strategy for meeting the challenges of the future study on this topic. We hope the findings are helpful in understanding the molecular mechanism in carbon nanomaterials regulating actin polymerization, and provide new insight in novel nanomedicine development for inhibition tumor cell migration. PMID:27455649

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. Cytosolic pressure provides a propulsive force comparable to actin polymerization during lamellipod protrusion

    NASA Astrophysics Data System (ADS)

    Manoussaki, Daphne; Shin, William D.; Waterman, Clare M.; Chadwick, Richard S.

    2015-07-01

    Does cytosolic pressure facilitate f-actin polymerization to push the leading edge of a cell forward during self-propelled motion? AFM force-distance (f-d) curves obtained from lamellipodia of live cells often exhibit a signal from which the tension, bending modulus, elastic modulus and thickness in the membrane-cortex complex can be estimated close to the contact point. These measurements permit an estimate of the cytosolic pressure via the canonical Laplace force balance. The deeper portion of the f-d curve allows estimation of the bulk modulus of the cytoskeleton after removal of the bottom effect artifact. These estimates of tension, pressure, cortex thickness and elastic moduli imply that cytosolic pressure both pushes the membrane forward and compresses the actin cortex rearward to facilitate f-actin polymerization. We also estimate that cytosolic pressure fluctuations, most likely induced by myosin, provide a propulsive force comparable to that provided by f-actin polymerization in a lamellipod.

  7. Probing polymerization forces by using actin-propelled lipid vesicles

    NASA Astrophysics Data System (ADS)

    Upadhyaya, Arpita; Chabot, Jeffrey R.; Andreeva, Albina; Samadani, Azadeh; van Oudenaarden, Alexander

    2003-04-01

    Actin polymerization provides a powerful propulsion force for numerous types of cell motility. Although tremendous progress has been made in identifying the biochemical components necessary for actin-based motility, the precise biophysical mechanisms of force generation remain unclear. To probe the polymerization forces quantitatively, we introduce an experimental system in which lipid vesicles coated with the Listeria monocytogenes virulence factor ActA are propelled by actin polymerization. The polymerization forces cause significant deformations of the vesicle. We have used these deformations to obtain a spatially resolved measure of the forces exerted on the membrane using a model based on the competition between osmotic pressure and membrane stretching. Our results indicate that actin exerts retractile or propulsive forces depending on the local membrane curvature and that the membrane is strongly bound to the actin gel. These results are consistent with the observed dynamics. After a slow elongation of the vesicle from a spherical shape, the strong bonds between the actin gel and the membrane rupture if the retractile forces exceed a critical value, leading to a rapid release of the vesicle's trailing edge.

  8. Regulation of actin polymerization by tropomodulin-3 controls megakaryocyte actin organization and platelet biogenesis.

    PubMed

    Sui, Zhenhua; Nowak, Roberta B; Sanada, Chad; Halene, Stephanie; Krause, Diane S; Fowler, Velia M

    2015-07-23

    The actin cytoskeleton is important for platelet biogenesis. Tropomodulin-3 (Tmod3), the only Tmod isoform detected in platelets and megakaryocytes (MKs), caps actin filament (F-actin) pointed ends and binds tropomyosins (TMs), regulating actin polymerization and stability. To determine the function of Tmod3 in platelet biogenesis, we studied Tmod3(-/-) embryos, which are embryonic lethal by E18.5. Tmod3(-/-) embryos often show hemorrhaging at E14.5 with fewer and larger platelets, indicating impaired platelet biogenesis. MK numbers are moderately increased in Tmod3(-/-) fetal livers, with only a slight increase in the 8N population, suggesting that MK differentiation is not significantly affected. However, Tmod3(-/-) MKs fail to develop a normal demarcation membrane system (DMS), and cytoplasmic organelle distribution is abnormal. Moreover, cultured Tmod3(-/-) MKs exhibit impaired proplatelet formation with a wide range of proplatelet bud sizes, including abnormally large proplatelet buds containing incorrect numbers of von Willebrand factor-positive granules. Tmod3(-/-) MKs exhibit F-actin disturbances, and Tmod3(-/-) MKs spreading on collagen fail to polymerize F-actin into actomyosin contractile bundles. Tmod3 associates with TM4 and the F-actin cytoskeleton in wild-type MKs, and confocal microscopy reveals that Tmod3, TM4, and F-actin partially colocalize near the membrane of proplatelet buds. In contrast, the abnormally large proplatelets from Tmod3(-/-) MKs show increased F-actin and redistribution of F-actin and TM4 from the cortex to the cytoplasm, but normal microtubule coil organization. We conclude that F-actin capping by Tmod3 regulates F-actin organization in mouse fetal liver-derived MKs, thereby controlling MK cytoplasmic morphogenesis, including DMS formation and organelle distribution, as well as proplatelet formation and sizing. PMID:25964668

  9. 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.

  10. 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

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

    PubMed Central

    Miyata, H; Kinosita, K

    1994-01-01

    Liposomes encapsulating actin filaments were prepared by swelling at 0 degrees C lipid film consisting of a mixture of dimyristoyl phosphatidylcholine and cardiolipin (equal amounts by weight) in 100 microM rabbit skeletal muscle actin and 0.5 mM CaCl2 followed by polymerization of actin at 30 degrees C. Liposomes initially assumed either disk or dumbbell shape, but when cytochalasin D was added to the medium surrounding the liposomes, they were found to become spindle shaped. Liposomes containing bovine serum albumin that were given cytochalasin D and actin-containing liposomes that were given dimethylformamide, the solvent for cytochalasin D, did not transform. These results indicated actin-cytochalasin interaction is involved in the transformation process. Falling-ball viscometry and sedimentation analysis of actin solution indicated that cytochalasin cleaved actin filaments and caused depolymerization. The observation of polarized fluorescence of encapsulated actin labeled with acrylodan indicated that the actin filaments in the transformed liposomes aligned along the long axis of the liposomes. Because the actin filaments in the disk- or dumbbell-shaped liposomes formed bundles running along the liposome contour, the transformation was likely to be accompanied by the change in the actin filament arrangement in the liposomes, which was induced by actin-cytochalasin interaction. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:7948706

  12. Intersectin-2L Regulates Caveola Endocytosis Secondary to Cdc42-mediated Actin Polymerization*

    PubMed Central

    Klein, Irene K.; Predescu, Dan N.; Sharma, Tiffany; Knezevic, Ivana; Malik, Asrar B.; Predescu, Sanda

    2009-01-01

    Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PHITSN-2L) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The “catalytically dead” DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PHITSN-2L-transfected cells compared with control and 1 μm jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane. PMID:19622753

  13. Intersectin-2L regulates caveola endocytosis secondary to Cdc42-mediated actin polymerization.

    PubMed

    Klein, Irene K; Predescu, Dan N; Sharma, Tiffany; Knezevic, Ivana; Malik, Asrar B; Predescu, Sanda

    2009-09-18

    Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PH(ITSN-2L)) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The "catalytically dead" DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PH(ITSN-2L)-transfected cells compared with control and 1 microM jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane. PMID:19622753

  14. 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

  15. 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

  16. 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

  17. Estrogen Regulates Protein Synthesis and Actin Polymerization in Hippocampal Neurons through Different Molecular Mechanisms

    PubMed Central

    Briz, Victor; Baudry, Michel

    2014-01-01

    Estrogen rapidly modulates hippocampal synaptic plasticity by activating selective membrane-associated receptors. Reorganization of the actin cytoskeleton and stimulation of mammalian target of rapamycin (mTOR)-mediated protein synthesis are two major events required for the consolidation of hippocampal long-term potentiation and memory. Estradiol regulates synaptic plasticity by interacting with both processes, but the underlying molecular mechanisms are not yet fully understood. Here, we used acute rat hippocampal slices to analyze the mechanisms underlying rapid changes in mTOR activity and actin polymerization elicited by estradiol. Estradiol-induced mTOR phosphorylation was preceded by rapid and transient activation of both extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) and by phosphatase and tensin homolog (PTEN) degradation. These effects were prevented by calpain and ERK inhibitors. Estradiol-induced mTOR stimulation did not require activation of classical estrogen receptors (ER), as specific ERα and ERβ agonists (PPT and DPN, respectively) failed to mimic this effect, and ER antagonists could not block it. Estradiol rapidly activated both RhoA and p21-activated kinase (PAK). Furthermore, a specific inhibitor of RhoA kinase (ROCK), H1152, and a potent and specific PAK inhibitor, PF-3758309, blocked estradiol-induced cofilin phosphorylation and actin polymerization. ER antagonists also blocked these effects of estrogen. Consistently, both PPT and DPN stimulated PAK and cofilin phosphorylation as well as actin polymerization. Finally, the effects of estradiol on actin polymerization were insensitive to protein synthesis inhibitors, but its stimulation of mTOR activity was impaired by latrunculin A, a drug that disrupts actin filaments. Taken together, our results indicate that estradiol regulates local protein synthesis and cytoskeletal reorganization via different molecular mechanisms and signaling pathways. PMID:24611062

  18. Fluvoxamine, an anti-depressant, inhibits human glioblastoma invasion by disrupting actin polymerization

    PubMed Central

    Hayashi, Keiichiro; Michiue, Hiroyuki; Yamada, Hiroshi; Takata, Katsuyoshi; Nakayama, Hiroki; Wei, Fan-Yan; Fujimura, Atsushi; Tazawa, Hiroshi; Asai, Akira; Ogo, Naohisa; Miyachi, Hiroyuki; Nishiki, Tei-ichi; Tomizawa, Kazuhito; Takei, Kohji; Matsui, Hideki

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common malignant brain tumor with a median survival time about one year. Invasion of GBM cells into normal brain is the major cause of poor prognosis and requires dynamic reorganization of the actin cytoskeleton, which includes lamellipodial protrusions, focal adhesions, and stress fibers at the leading edge of GBM. Therefore, we hypothesized that inhibitors of actin polymerization can suppress GBM migration and invasion. First, we adopted a drug repositioning system for screening with a pyrene-actin-based actin polymerization assay and identified fluvoxamine, a clinically used antidepressant. Fluvoxamine, selective serotonin reuptake inhibitor, was a potent inhibitor of actin polymerization and confirmed as drug penetration through the blood–brain barrier (BBB) and accumulation of whole brain including brain tumor with no drug toxicity. Fluvoxamine inhibited serum-induced ruffle formation, cell migration, and invasion of human GBM and glioma stem cells in vitro by suppressing both FAK and Akt/mammalian target of rapamycin signaling. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same dose of anti-depressant effect. In conclusion, fluvoxamine is a promising anti-invasive treatment against GBM with reliable approach. PMID:26988603

  19. Fluvoxamine, an anti-depressant, inhibits human glioblastoma invasion by disrupting actin polymerization.

    PubMed

    Hayashi, Keiichiro; Michiue, Hiroyuki; Yamada, Hiroshi; Takata, Katsuyoshi; Nakayama, Hiroki; Wei, Fan-Yan; Fujimura, Atsushi; Tazawa, Hiroshi; Asai, Akira; Ogo, Naohisa; Miyachi, Hiroyuki; Nishiki, Tei-ichi; Tomizawa, Kazuhito; Takei, Kohji; Matsui, Hideki

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common malignant brain tumor with a median survival time about one year. Invasion of GBM cells into normal brain is the major cause of poor prognosis and requires dynamic reorganization of the actin cytoskeleton, which includes lamellipodial protrusions, focal adhesions, and stress fibers at the leading edge of GBM. Therefore, we hypothesized that inhibitors of actin polymerization can suppress GBM migration and invasion. First, we adopted a drug repositioning system for screening with a pyrene-actin-based actin polymerization assay and identified fluvoxamine, a clinically used antidepressant. Fluvoxamine, selective serotonin reuptake inhibitor, was a potent inhibitor of actin polymerization and confirmed as drug penetration through the blood-brain barrier (BBB) and accumulation of whole brain including brain tumor with no drug toxicity. Fluvoxamine inhibited serum-induced ruffle formation, cell migration, and invasion of human GBM and glioma stem cells in vitro by suppressing both FAK and Akt/mammalian target of rapamycin signaling. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same dose of anti-depressant effect. In conclusion, fluvoxamine is a promising anti-invasive treatment against GBM with reliable approach. PMID:26988603

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

    PubMed

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

    2016-01-01

    Dendritic spines are mushroom-shaped protrusions of the postsynaptic membrane. Spines receive the majority of glutamatergic synaptic inputs. Their morphology, dynamics, and density have been related to synaptic plasticity and learning. The main determinant of spine shape is filamentous actin. Using FRAP, we have reexamined the actin dynamics of individual spines from pyramidal hippocampal neurons, both in cultures and in hippocampal organotypic slices. Our results indicate that, in cultures, the actin mobile fraction is independently regulated at the individual spine level, and mobile fraction values do not correlate with either age or distance from the soma. The most significant factor regulating actin mobile fraction was the presence of astrocytes in the culture substrate. Spines from neurons growing in the virtual absence of astrocytes have a more stable actin cytoskeleton, while spines from neurons growing in close contact with astrocytes show a more dynamic cytoskeleton. According to their recovery time, spines were distributed into two populations with slower and faster recovery times, while spines from slice cultures were grouped into one population. Finally, employing fast lineal acquisition protocols, we confirmed the existence of loci with high polymerization rates within the spine. PMID:26881098

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

    PubMed Central

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

    2016-01-01

    Dendritic spines are mushroom-shaped protrusions of the postsynaptic membrane. Spines receive the majority of glutamatergic synaptic inputs. Their morphology, dynamics, and density have been related to synaptic plasticity and learning. The main determinant of spine shape is filamentous actin. Using FRAP, we have reexamined the actin dynamics of individual spines from pyramidal hippocampal neurons, both in cultures and in hippocampal organotypic slices. Our results indicate that, in cultures, the actin mobile fraction is independently regulated at the individual spine level, and mobile fraction values do not correlate with either age or distance from the soma. The most significant factor regulating actin mobile fraction was the presence of astrocytes in the culture substrate. Spines from neurons growing in the virtual absence of astrocytes have a more stable actin cytoskeleton, while spines from neurons growing in close contact with astrocytes show a more dynamic cytoskeleton. According to their recovery time, spines were distributed into two populations with slower and faster recovery times, while spines from slice cultures were grouped into one population. Finally, employing fast lineal acquisition protocols, we confirmed the existence of loci with high polymerization rates within the spine. PMID:26881098

  2. Cytosolic pressure provides a propulsive force comparable to actin polymerization during lamellipod protrusion.

    PubMed

    Manoussaki, Daphne; Shin, William D; Waterman, Clare M; Chadwick, Richard S

    2015-01-01

    Does cytosolic pressure facilitate f-actin polymerization to push the leading edge of a cell forward during self-propelled motion? AFM force-distance (f-d) curves obtained from lamellipodia of live cells often exhibit a signal from which the tension, bending modulus, elastic modulus and thickness in the membrane-cortex complex can be estimated close to the contact point. These measurements permit an estimate of the cytosolic pressure via the canonical Laplace force balance. The deeper portion of the f-d curve allows estimation of the bulk modulus of the cytoskeleton after removal of the bottom effect artifact. These estimates of tension, pressure, cortex thickness and elastic moduli imply that cytosolic pressure both pushes the membrane forward and compresses the actin cortex rearward to facilitate f-actin polymerization. We also estimate that cytosolic pressure fluctuations, most likely induced by myosin, provide a propulsive force comparable to that provided by f-actin polymerization in a lamellipod. PMID:26197304

  3. Adhesion controls bacterial actin polymerization-based movement.

    PubMed

    Soo, Frederick S; Theriot, Julie A

    2005-11-01

    As part of its infectious life cycle, the bacterial pathogen Listeria monocytogenes propels itself through the host-cell cytoplasm by triggering the polymerization of host-cell actin near the bacterial surface, harnessing the activity of several cytoskeletal proteins used during actin-based cell crawling. To distinguish among several classes of biophysical models of actin-based bacterial movement, we used a high-throughput tracking technique to record the movement of many individual bacteria during temperature shifts. The speed of each bacterium varied strongly with temperature, closely following the Arrhenius rate law. Among bacteria, the prefactor A of the Arrhenius dependence unexpectedly varied exponentially with apparent activation energy, E(a), over a wide range (8-21 kcal/mol), reminiscent of the "rate compensation effect" of classical catalytic reactions. Average E(a) were increased for mutant bacteria deficient in binding Ena/VASP proteins and bacteria moving in diluted extract. These two effects were additive. The observed temperature and rate compensation effects are consistent with a class of simple kinetic models in which the bacterium advances through the thermally driven, cooperative breakage of groups of adhesive bonds on its surface. The estimated number of coupled adhesive bonds N on the bacterial surface varies between 10 and 40 bonds. In contrast to other models, this model correctly predicts an experimentally observed negative correlation between bacterial speed and actin gel density. The idea that speed depends on adhesion, rather than polymerization, suggests several alternative mechanisms by which known cytoskeletal regulatory proteins could control cellular movement. PMID:16251274

  4. Mechanical stimulation induces formin-dependent assembly of a perinuclear actin rim

    PubMed Central

    Shao, Xiaowei; Li, Qingsen; Mogilner, Alex; Bershadsky, Alexander D.; Shivashankar, G. V.

    2015-01-01

    Cells constantly sense and respond to mechanical signals by reorganizing their actin cytoskeleton. Although a number of studies have explored the effects of mechanical stimuli on actin dynamics, the immediate response of actin after force application has not been studied. We designed a method to monitor the spatiotemporal reorganization of actin after cell stimulation by local force application. We found that force could induce transient actin accumulation in the perinuclear region within ∼2 min. This actin reorganization was triggered by an intracellular Ca2+ burst induced by force application. Treatment with the calcium ionophore A23187 recapitulated the force-induced perinuclear actin remodeling. Blocking of actin polymerization abolished this process. Overexpression of Klarsicht, ANC-1, Syne Homology (KASH) domain to displace nesprins from the nuclear envelope did not abolish Ca2+-dependent perinuclear actin assembly. However, the endoplasmic reticulum- and nuclear membrane-associated inverted formin-2 (INF2), a potent actin polymerization activator (mutations of which are associated with several genetic diseases), was found to be important for perinuclear actin assembly. The perinuclear actin rim structure colocalized with INF2 on stimulation, and INF2 depletion resulted in attenuation of the rim formation. Our study suggests that cells can respond rapidly to external force by remodeling perinuclear actin in a unique Ca2+- and INF2-dependent manner. PMID:25941386

  5. Pearling instability of membrane tubes driven by curved proteins and actin polymerization

    NASA Astrophysics Data System (ADS)

    Jelerčič, U.; Gov, N. S.

    2015-12-01

    Membrane deformation inside living cells is crucial for the proper shaping of various intracellular organelles and is necessary during the fission/fusion processes that allow membrane recycling and transport (e.g. endocytosis). Proteins that induce membrane curvature play a key role in such processes, mostly by adsorbing to the membrane and forming a scaffold that deforms the membrane according to the curvature of the proteins. In this paper we explore the possibility of membrane tube destabilization through a pearling mechanism enabled by the combined effects of the adsorbed curved proteins and the actin polymerization that they recruit. The pearling instability can serve as the initiation for fission of the tube into vesicles. We find that adsorbed curved proteins are more likely to stabilize the tubes, while the actin polymerization can provide the additional constrictive force needed for the robust instability. We discuss the relevance of the theoretical results to in vivo and in vitro experiments.

  6. ERK reinforces actin polymerization to power persistent edge protrusion during motility

    PubMed Central

    Mendoza, Michelle C.; Vilela, Marco; Juarez, Jesus E.; Blenis, John; Danuser, Gaudenz

    2016-01-01

    Cells move through perpetual protrusion and retraction cycles at the leading edge. These cycles are coordinated with substrate adhesion and retraction of the cell rear. Here, we tracked spatial and temporal fluctuations in the molecular activities of individual moving cells to elucidate how extracellular regulated kinase (ERK) signaling controlled the dynamics of protrusion and retraction cycles. ERK is activated by many cell-surface receptors and we found that ERK signaling specifically reinforced cellular protrusions so that they translated into rapid, sustained forward motion of the leading edge. Using quantitative fluorescent speckle microscopy (qFSM) and cross-correlation analysis, we showed that ERK controlled the rate and timing of actin polymerization by promoting the recruitment of the actin nucleator Arp2/3 to the leading edge. Arp2/3 activity generates branched actin networks that can produce pushing force. These findings support a model in which surges in ERK activity induced by extracellular cues enhance Arp2/3-mediated actin polymerization to generate protrusion power phases with enough force to counteract increasing membrane tension and to promote sustained motility. PMID:25990957

  7. Plasma membrane restricted RhoGEF activity is sufficient for RhoA-mediated actin polymerization

    PubMed Central

    van Unen, Jakobus; Reinhard, Nathalie R.; Yin, Taofei; Wu, Yi I.; Postma, Marten; Gadella, Theodorus W.J.; Goedhart, Joachim

    2015-01-01

    The small GTPase RhoA is involved in cell morphology and migration. RhoA activity is tightly regulated in time and space and depends on guanine exchange factors (GEFs). However, the kinetics and subcellular localization of GEF activity towards RhoA are poorly defined. To study the mechanism underlying the spatiotemporal control of RhoA activity by GEFs, we performed single cell imaging with an improved FRET sensor reporting on the nucleotide loading state of RhoA. By employing the FRET sensor we show that a plasma membrane located RhoGEF, p63RhoGEF, can rapidly activate RhoA through endogenous GPCRs and that localized RhoA activity at the cell periphery correlates with actin polymerization. Moreover, synthetic recruitment of the catalytic domain derived from p63RhoGEF to the plasma membrane, but not to the Golgi apparatus, is sufficient to activate RhoA. The synthetic system enables local activation of endogenous RhoA and effectively induces actin polymerization and changes in cellular morphology. Together, our data demonstrate that GEF activity at the plasma membrane is sufficient for actin polymerization via local RhoA signaling. PMID:26435194

  8. 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

  9. Loss of CD73-mediated actin polymerization promotes endometrial tumor progression

    PubMed Central

    Bowser, Jessica L.; Blackburn, Michael R.; Shipley, Gregory L.; Molina, Jose G.; Dunner, Kenneth; Broaddus, Russell R.

    2015-01-01

    Ecto-5′-nucleotidase (CD73) is central to the generation of extracellular adenosine. Previous studies have highlighted a detrimental role for extracellular adenosine in cancer, as it dampens T cell–mediated immune responses. Here, we determined that, in contrast to other cancers, CD73 is markedly downregulated in poorly differentiated and advanced-stage endometrial carcinoma compared with levels in normal endometrium and low-grade tumors. In murine models, CD73 deficiency led to a loss of endometrial epithelial barrier function, and pharmacological CD73 inhibition increased in vitro migration and invasion of endometrial carcinoma cells. Given that CD73-generated adenosine is central to regulating tissue protection and physiology in normal tissues, we hypothesized that CD73-generated adenosine in endometrial carcinoma induces an innate reflex to protect epithelial integrity. CD73 associated with cell-cell contacts, filopodia, and membrane zippers, indicative of involvement in cell-cell adhesion and actin polymerization–dependent processes. We determined that CD73-generated adenosine induces cortical actin polymerization via adenosine A1 receptor (A1R) induction of a Rho GTPase CDC42–dependent conformational change of the actin-related proteins 2 and 3 (ARP2/3) actin polymerization complex member N-WASP. Cortical F-actin elevation increased membrane E-cadherin, β-catenin, and Na+K+ ATPase. Together, these findings reveal that CD73-generated adenosine promotes epithelial integrity and suggest why loss of CD73 in endometrial cancer allows for tumor progression. Moreover, our data indicate that the role of CD73 in cancer is more complex than previously described. PMID:26642367

  10. 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.

  11. 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

  12. Quantitative Analysis of Approaches to Measure Cooperative Phosphate Release in Polymerized Actin

    PubMed Central

    Burnett, Mark M.; Carlsson, Anders E.

    2012-01-01

    We use stochastic simulations that treat several experimental probes of actin dynamics to explore the extent to which phosphate dissociation in filamentous actin may be cooperative. Phosphate time-courses from polymerization and copolymerization experiments of ATP- and ADP-actin are studied, including the effects of variations in filament-number concentration as well as single-filament depolymerization time-courses. We find that highly cooperative models are consistent with the treated experimental data. We also find that some types of experiments that are believed to provide strong constraints on the cooperativity of actin hydrolysis models do not provide such constraints. PMID:23283236

  13. A Steric Antagonism of Actin Polymerization by a Salmonella Virulence Protein

    SciTech Connect

    Margarit,S.; Davidson, W.; Frego, L.; Stebbins, F.

    2006-01-01

    Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.

  14. Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization.

    PubMed

    Yi, Jinsoo; Schmidt, Jacob; Chien, Aichi; Montemagno, Carlo D

    2009-02-25

    We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present. PMID:19417437

  15. Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization

    NASA Astrophysics Data System (ADS)

    Yi, Jinsoo; Schmidt, Jacob; Chien, Aichi; Montemagno, Carlo D.

    2009-02-01

    We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present.

  16. Actin-curcumin interaction: insights into the mechanism of actin polymerization inhibition.

    PubMed

    Dhar, Gopa; Chakravarty, Devlina; Hazra, Joyita; Dhar, Jesmita; Poddar, Asim; Pal, Mahadeb; Chakrabarti, Pinak; Surolia, Avadhesha; Bhattacharyya, Bhabatarak

    2015-02-01

    Curcumin, derived from rhizomes of the Curcuma longa plant, is known to possess a wide range of medicinal properties. We have examined the interaction of curcumin with actin and determined their binding and thermodynamic parameters using isothermal titration calorimetry. Curcumin is weakly fluorescent in aqueous solution, and binding to actin enhances fluorescence several fold with a large blue shift in the emission maximum. Curcumin inhibits microfilament formation, which is similar to its role in inhibiting microtubule formation. We synthesized a series of stable curcumin analogues to examine their affinity for actin and their ability to inhibit actin self-assembly. Results show that curcumin is a ligand with two symmetrical halves, each of which possesses no activity individually. Oxazole, pyrazole, and acetyl derivatives are less effective than curcumin at inhibiting actin self-assembly, whereas a benzylidiene derivative is more effective. Cell biology studies suggest that disorganization of the actin network leads to destabilization of filaments in the presence of curcumin. Molecular docking reveals that curcumin binds close to the cytochalasin binding site of actin. Further molecular dynamics studies reveal a possible allosteric effect in which curcumin binding at the "barbed end" of actin is transmitted to the "pointed end", where conformational changes disrupt interactions with the adjacent actin monomer to interrupt filament formation. Finally, the recognition and binding of actin by curcumin is yet another example of its unique ability to target multiple receptors. PMID:25564154

  17. Increased beta-actin and tubulin polymerization in regrowing axons: relationship to the conditioning lesion effect.

    PubMed

    Lund, Linda M; Machado, Victor M; McQuarrie, Irvine G

    2002-12-01

    Spinal motor neurons of Sprague-Dawley rats were examined to determine which of the neuronal isoforms of actin (beta or gamma) upregulate following axon injury. In situ hybridization studies showed greater beta-actin mRNA levels but no change in gamma-actin mRNA levels-suggesting that axon regrowth utilizes beta-actin. We radiolabeled the newly synthesized actin and tubulin that are subsequently transported in the axon to the site of an axotomizing injury. This allowed us to evaluate changes in polymerization as new cytoskeletal elements approach the injury site. Previous studies had shown that the rate of the most rapid subcomponent of actin and tubulin transport (called SCb) accelerates following axotomy (J. Jacob and I. McQuarrie, J. Neurobiol. 22: 570-583, 1991). This rate increase is associated with an increased proportion of SCb tubulin and actin in polymer (vs monomer) form (J. Jacob and I. McQuarrie, J. Neurosci, Res. 43: 412-419, 1996). However, in that study newly synthesized proteins were radiolabeled at 7 days after axotomy-which is at the peak of increased protein synthesis. This time-course did not examine actin and tubulin that were already in transit in axons when the injury occurred. This actin and tubulin would enter the regrowing axons first. Here, we have radiolabeled newly synthesized proteins 3 days prior to axotomy. For beta-tubulin, the ratio of monomer to polymer was unaffected. For actin, the equilibrium shifted strongly toward polymerization. We conclude that the acceleration of axonal outgrowth seen after the second of two serial axotomies (the "conditioning lesion effect") is related to the ability of actin that is already in transit to polymerize in response to the first axotomy. PMID:12504890

  18. Macromolecular crowding gives rise to microviscosity, anomalous diffusion and accelerated actin polymerization

    NASA Astrophysics Data System (ADS)

    Rashid, Rafi; Chee, Stella Min Ling; Raghunath, Michael; Wohland, Thorsten

    2015-05-01

    Macromolecular crowding (MMC) has been used in various in vitro experimental systems to mimic in vivo physiology. This is because the crowded cytoplasm of cells contains many different types of solutes dissolved in an aqueous medium. MMC in the extracellular microenvironment is involved in maintaining stem cells in their undifferentiated state (niche) as well as in aiding their differentiation after they have travelled to new locations outside the niche. MMC at physiologically relevant fractional volume occupancies (FVOs) significantly enhances the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells during chemically induced adipogenesis. The mechanism by which MMC produces this enhancement is not entirely known. In the context of extracellular collagen deposition, we have recently reported the importance of optimizing the FVO while minimizing the bulk viscosity. Two opposing properties will determine the net rate of a biochemical reaction: the negative effect of bulk viscosity and the positive effect of the excluded volume, the latter being expressed by the FVO. In this study we have looked more closely at the effect of viscosity on reaction rates. We have used fluorimetry to measure the rate of actin polymerization and fluorescence correlation spectroscopy (FCS) to measure diffusion of various probes in solutions containing the crowder Ficoll at physiological concentrations. Similar to its effect on collagen, Ficoll enhanced the actin polymerization rate despite increasing the bulk viscosity. Our FCS measurements reveal a relatively minor component of anomalous diffusion. In addition, our measurements do suggest that microviscosity becomes relevant in a crowded environment. We ruled out bulk viscosity as a cause of the rate enhancement by performing the actin polymerization assay in glycerol. These opposite effects of Ficoll and glycerol led us to conclude that microviscosity becomes relevant at the length scale of the reacting

  19. 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

  20. Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

    PubMed Central

    Carnell, Michael; Zech, Tobias; Calaminus, Simon D.; Ura, Seiji; Hagedorn, Monica; Johnston, Simon A.; May, Robin C.; Soldati, Thierry; Machesky, Laura M.

    2011-01-01

    WASP and SCAR homologue (WASH) is a recently identified and evolutionarily conserved regulator of actin polymerization. In this paper, we show that WASH coats mature Dictyostelium discoideum lysosomes and is essential for exocytosis of indigestible material. A related process, the expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles, and cells expressing dominant negative WASH mutants inefficiently expel C. neoformans. D. discoideum WASH causes filamentous actin (F-actin) patches to form on lysosomes, leading to the removal of vacuolar adenosine triphosphatase (V-ATPase) and the neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coats are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase. PMID:21606208

  1. Mena–GRASP65 interaction couples actin polymerization to Golgi ribbon linking

    PubMed Central

    Tang, Danming; Zhang, Xiaoyan; Huang, Shijiao; Yuan, Hebao; Li, Jie; Wang, Yanzhuang

    2016-01-01

    In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking. PMID:26538023

  2. Actin polymerization stabilizes α4β1 integrin anchors that mediate monocyte adhesion

    PubMed Central

    Becker, Henry; Hyduk, Sharon J.; Wong, Janice C.; Digby, Genevieve; Arora, Pamma D.; Cano, Adrianet Puig; Hartwig, John; McCulloch, Christopher A.

    2012-01-01

    Leukocytes arrested on inflamed endothelium via integrins are subjected to force imparted by flowing blood. How leukocytes respond to this force and resist detachment is poorly understood. Live-cell imaging with Lifeact-transfected U937 cells revealed that force triggers actin polymerization at upstream α4β1 integrin adhesion sites and the adjacent cortical cytoskeleton. Scanning electron microscopy revealed that this culminates in the formation of structures that anchor monocyte adhesion. Inhibition of actin polymerization resulted in cell deformation, displacement, and detachment. Transfection of dominant-negative constructs and inhibition of function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. These included activation of Rap1, phosphoinositide 3-kinase γ isoform, and Rac but not Cdc42. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces. PMID:22472442

  3. F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation

    PubMed Central

    Babich, Alexander; Li, Shuixing; O'Connor, Roddy S.; Milone, Michael C.; Freedman, Bruce D.

    2012-01-01

    Activation of T cells by antigen-presenting cells involves assembly of signaling molecules into dynamic microclusters (MCs) within a specialized membrane domain termed the immunological synapse (IS). Actin and myosin IIA localize to the IS, and depletion of F-actin abrogates MC movement and T cell activation. However, the mechanisms that coordinate actomyosin dynamics and T cell receptor signaling are poorly understood. Using pharmacological inhibitors that perturb individual aspects of actomyosin dynamics without disassembling the network, we demonstrate that F-actin polymerization is the primary driver of actin retrograde flow, whereas myosin IIA promotes long-term integrity of the IS. Disruption of F-actin retrograde flow, but not myosin IIA contraction, arrested MC centralization and inhibited sustained Ca2+ signaling at the level of endoplasmic reticulum store release. Furthermore, perturbation of retrograde flow inhibited PLCγ1 phosphorylation within MCs but left Zap70 activity intact. These studies highlight the importance of ongoing actin polymerization as a central driver of actomyosin retrograde flow, MC centralization, and sustained Ca2+ signaling. PMID:22665519

  4. Microcephaly-dystonia due to mutated PLEKHG2 with impaired actin polymerization.

    PubMed

    Edvardson, Simon; Wang, Haibo; Dor, Talya; Atawneh, Osamah; Yaacov, Barak; Gartner, Jutta; Cinnamon, Yuval; Chen, Songhai; Elpeleg, Orly

    2016-01-01

    Rearrangement of the actin cytoskeleton is controlled by RhoGTPases which are activated by RhoGEFs. We identified homozygosity for Arg204Trp mutation in the Rho guanidine exchange factor (RhoGEF) PLEKHG2 gene in five patients with profound mental retardation, dystonia, postnatal microcephaly, and distinct neuroimaging pattern. The activity of the mutant PLEKHG2 was significantly decreased, both in basal state and when Gβγ- or lysophosphatidic acid (LPA)-stimulated. SDF1a-stimulated actin polymerization was significantly impaired in patient cells, and this abnormality was duplicated in control cells when PLEKHG2 expression was downregulated. These results underscore the role of PLEKHG2 in actin polymerization and delineate the clinical and radiological findings in PLEKHG2 deficiency. PMID:26573021

  5. 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

  6. LeftyA decreases Actin Polymerization and Stiffness in Human Endometrial Cancer Cells

    PubMed Central

    Salker, Madhuri S.; Schierbaum, Nicolas; Alowayed, Nour; Singh, Yogesh; Mack, Andreas F.; Stournaras, Christos; Schäffer, Tilman E.; Lang, Florian

    2016-01-01

    LeftyA, a cytokine regulating stemness and embryonic differentiation, down-regulates cell proliferation and migration. Cell proliferation and motility require actin reorganization, which is under control of ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1). The present study explored whether LeftyA modifies actin cytoskeleton, shape and stiffness of Ishikawa cells, a well differentiated endometrial carcinoma cell line. The effect of LeftyA on globular over filamentous actin ratio was determined utilizing Western blotting and flow cytometry. Rac1 and PAK1 transcript levels were measured by qRT-PCR as well as active Rac1 and PAK1 by immunoblotting. Cell stiffness (quantified by the elastic modulus), cell surface area and cell volume were studied by atomic force microscopy (AFM). As a result, 2 hours treatment with LeftyA (25 ng/ml) significantly decreased Rac1 and PAK1 transcript levels and activity, depolymerized actin, and decreased cell stiffness, surface area and volume. The effect of LeftyA on actin polymerization was mimicked by pharmacological inhibition of Rac1 and PAK1. In the presence of the Rac1 or PAK1 inhibitor LeftyA did not lead to significant further actin depolymerization. In conclusion, LeftyA leads to disruption of Rac1 and Pak1 activity with subsequent actin depolymerization, cell softening and cell shrinkage. PMID:27404958

  7. Dissociation of F-actin induced by hydrostatic pressure.

    PubMed

    Garcia, C R; Amaral Júnior, J A; Abrahamsohn, P; Verjovski-Almeida, S

    1992-11-01

    F-actin purified from rabbit skeletal muscle undergoes reversible dissociation when subjected to hydrostatic pressures up to 240 MPa. Dissociation and reversibility were detected by the following procedures: fluorescence spectral changes observed under pressure, when either intrinsic tryptophan or pyrenyl emission of N-(1-pyrenyl)iodoacetamide-labeled actin were monitored; electron microscopy of samples fixed under pressure; size-exclusion HPLC of pressurized actin. The effect of pressure upon F-actin that had been polymerized in the presence of either Mg2+, Ca2+ or K+ was studied. The standard volume changes for the association of actin subunits, calculated from pressure/dissociation curves were 74 +/- 14 ml/mol for Mg-F-actin, 79 +/- 12 ml/mol for Ca-F-actin and 328 +/- 63 ml/mol for K-F-actin, indicating that actin subunits are packed differently in the polymer depending on which cation is present. All pressure/dissociation data could be fitted by a model for dissociation of a dimer, which suggests that in the F-actin filament there is a predominant intersubunit interaction interface, most likely the head-to-tail intrastrand interaction between two subunits which repeats itself along the polymer. A tenfold change in total protein concentration from 20 micrograms to 200 micrograms/ml Mg-F-actin did not cause a change in the pressure required for half-maximal dissociation. This indicates a heterogeneity of free energy of association among actin monomers in the Mg-F-actin polymer, suggesting that, in addition to the predominant intersubunit interaction, the disordered interactions in the filament significantly contribute to the heterogeneity of microenvironments in the interface between the subunits. PMID:1425683

  8. The Deficiency of PIP2 5-Phosphatase in Lowe Syndrome Affects Actin Polymerization

    PubMed Central

    Suchy, Sharon F.; Nussbaum, Robert L.

    2002-01-01

    Lowe syndrome is a rare X-linked disorder characterized by bilateral congenital cataracts, renal Fanconi syndrome, and mental retardation. Lowe syndrome results from mutations in the OCRL1 gene, which encodes a phosphatidylinositol 4,5 bisphosphate 5-phosphatase located in the trans-Golgi network. As a first step in identifying the link between ocrl1 deficiency and the clinical disorder, we have identified a reproducible cellular abnormality of the actin cytoskeleton in fibroblasts from patients with Lowe syndrome. The cellular abnormality is characterized by a decrease in long actin stress fibers, enhanced sensitivity to actin depolymerizing agents, and an increase in punctate F-actin staining in a distinctly anomalous distribution in the center of the cell. We also demonstrate an abnormal distribution of two actin-binding proteins, gelsolin and α-actinin, proteins regulated by both PIP2 and Ca+2 that would be expected to be altered in Lowe cells. Actin polymerization plays a key role in the formation, maintenance, and proper function of tight junctions and adherens junctions, which have been demonstrated to be critical in renal proximal tubule function, and in the differentiation of the lens. These findings point to a general mechanism to explain how this PIP2 5-phosphatase deficiency might produce the Lowe syndrome phenotype. PMID:12428211

  9. Palmitoylation of LIM Kinase-1 ensures spine-specific actin polymerization and morphological plasticity

    PubMed Central

    George, Joju; Soares, Cary; Montersino, Audrey; Beique, Jean-Claude; Thomas, Gareth M

    2015-01-01

    Precise regulation of the dendritic spine actin cytoskeleton is critical for neurodevelopment and neuronal plasticity, but how neurons spatially control actin dynamics is not well defined. Here, we identify direct palmitoylation of the actin regulator LIM kinase-1 (LIMK1) as a novel mechanism to control spine-specific actin dynamics. A conserved palmitoyl-motif is necessary and sufficient to target LIMK1 to spines and to anchor LIMK1 in spines. ShRNA knockdown/rescue experiments reveal that LIMK1 palmitoylation is essential for normal spine actin polymerization, for spine-specific structural plasticity and for long-term spine stability. Palmitoylation is critical for LIMK1 function because this modification not only controls LIMK1 targeting, but is also essential for LIMK1 activation by its membrane-localized upstream activator PAK. These novel roles for palmitoylation in the spatial control of actin dynamics and kinase signaling provide new insights into structural plasticity mechanisms and strengthen links between dendritic spine impairments and neuropathological conditions. DOI: http://dx.doi.org/10.7554/eLife.06327.001 PMID:25884247

  10. Cofilin-induced cooperative conformational changes of actin subunits revealed using cofilin-actin fusion protein

    PubMed Central

    Umeki, Nobuhisa; Hirose, Keiko; Uyeda, Taro Q. P.

    2016-01-01

    To investigate cooperative conformational changes of actin filaments induced by cofilin binding, we engineered a fusion protein made of Dictyostelium cofilin and actin. The filaments of the fusion protein were functionally similar to actin filaments bound with cofilin in that they did not bind rhodamine-phalloidin, had quenched fluorescence of pyrene attached to Cys374 and showed enhanced susceptibility of the DNase loop to cleavage by subtilisin. Quantitative analyses of copolymers made of different ratios of the fusion protein and control actin further demonstrated that the fusion protein affects the structure of multiple neighboring actin subunits in copolymers. Based on these and other recent related studies, we propose a mechanism by which conformational changes induced by cofilin binding is propagated unidirectionally to the pointed ends of the filaments, and cofilin clusters grow unidirectionally to the pointed ends following this path. Interestingly, the fusion protein was unable to copolymerize with control actin at pH 6.5 and low ionic strength, suggesting that the structural difference between the actin moiety in the fusion protein and control actin is pH-sensitive. PMID:26842224

  11. Steric Effects Induce Geometric Remodeling of Actin Bundles in Filopodia.

    PubMed

    Dobramysl, Ulrich; Papoian, Garegin A; Erban, Radek

    2016-05-10

    Filopodia are ubiquitous fingerlike protrusions, spawned by many eukaryotic cells, to probe and interact with their environments. Polymerization dynamics of actin filaments, comprising the structural core of filopodia, largely determine their instantaneous lengths and overall lifetimes. The polymerization reactions at the filopodial tip require transport of G-actin, which enter the filopodial tube from the filopodial base and diffuse toward the filament barbed ends near the tip. Actin filaments are mechanically coupled into a tight bundle by cross-linker proteins. Interestingly, many of these proteins are relatively short, restricting the free diffusion of cytosolic G-actin throughout the bundle and, in particular, its penetration into the bundle core. To investigate the effect of steric restrictions on G-actin diffusion by the porous structure of filopodial actin filament bundle, we used a particle-based stochastic simulation approach. We discovered that excluded volume interactions result in partial and then full collapse of central filaments in the bundle, leading to a hollowed-out structure. The latter may further collapse radially due to the activity of cross-linking proteins, hence producing conical-shaped filament bundles. Interestingly, electron microscopy experiments on mature filopodia indeed frequently reveal actin bundles that are narrow at the tip and wider at the base. Overall, our work demonstrates that excluded volume effects in the context of reaction-diffusion processes in porous networks may lead to unexpected geometric growth patterns and complicated, history-dependent dynamics of intermediate metastable configurations. PMID:27166814

  12. Post-polymerization crosstalk between the actin cytoskeleton and microtubule network.

    PubMed

    Joo, E Emily; Yamada, Kenneth M

    2016-05-01

    Cellular cytoskeletal systems play many pivotal roles in living organisms by controlling cell shape, division, and migration, which ultimately govern morphology, physiology, and functions of animals. Although the cytoskeletal systems are distinct and play different roles, there is growing evidence that these diverse cytoskeletal systems coordinate their functions with each other. This coordination between cytoskeletal systems, often termed cytoskeletal crosstalk, has been identified when the dynamic state of one individual system affects the other system. In this review, we briefly describe some well-established examples of crosstalk between cytoskeletal systems and then introduce a newly discovered form of crosstalk between the actin cytoskeleton and microtubule network that does not appear to directly alter polymerization or depolymerization of either system. The biological impact and possible significance of this post-polymerization crosstalk between actin and microtubules will be discussed in detail. PMID:27058810

  13. Observation and Kinematic Description of Long Actin Tracks Induced by Spherical Beads

    PubMed Central

    Kang, Hyeran; Perlmutter, David S.; Shenoy, Vivek B.; Tang, Jay X.

    2010-01-01

    We report an in vitro study comparing the growth of long actin tails induced by spherical beads coated with the verprolin central acidic domain of the polymerization enzyme N-WASP to that induced by Listeria monocytogenes in similar cellular extracts. The tracks behind the beads show characteristic differences in shape and curvature from those left by the bacteria, which have an elongated shape and a similar polymerization-inducing enzyme distributed only on the rear surface of the cell. The experimental tracks are simulated using a generalized kinematic model, which incorporates three modes of bead rotation with respect to the tail. The results show that the trajectories of spherical beads are mechanically deterministic rather than random, as suggested by stochastic models. Assessment of the bead rotation and its mechanistic basis offers insights into the biological function of actin-based motility. PMID:21044576

  14. 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

  15. A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization

    PubMed Central

    Guo, Zhenhua; Stephenson, Robert; Qiu, Jiazhang; Zheng, Shijun; Luo, Zhao-Qing

    2014-01-01

    Successful infection by the opportunistic pathogen Legionella pneumophila requires the collective activity of hundreds of virulence proteins delivered into the host cell by the Dot/Icm type IV secretion system. These virulence proteins, also called effectors modulate distinct host cellular processes to create a membrane-bound niche called the Legionella containing vacuole (LCV) supportive of bacterial growth. We found that Ceg14(Lpg0437), a Dot/Icm substrate is toxic to yeast and such toxicity can be alleviated by overexpression of profilin, a protein involved in cytoskeletal structure in eukaryotes. We further showed that mutations in profilin affect actin binding but not other functions such as interactions with poly-L-proline or phosphatidylinositol, abolish its suppressor activity. Consistent with the fact the profilin suppresses its toxicity, expression of Ceg14 but not its non-toxic mutants in yeast affects actin distribution and budding of daughter cells. Although Ceg14 does not detectably interact with profilin, it co-sediments with filamentous actin and inhibits actin polymerization, causing the accumulation of short actin filaments. These results reveal that multiple L. pneumophila effectors target components of the host cytoskeleton. PMID:24286927

  16. 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

  17. CaMKII prevents spontaneous acrosomal exocytosis in sperm through induction of actin polymerization.

    PubMed

    Shabtay, Ortal; Breitbart, Haim

    2016-07-01

    In order to interact with the egg and undergo acrosomal exocytosis or the acrosome reaction (AR), mammalian spermatozoa must undergo a series of biochemical changes in the female reproductive tract, collectively called capacitation. We showed that F-actin is formed during sperm capacitation and fast depolymerization occurs prior to the AR. We hypothesized that F-actin protects the sperm from undergoing spontaneous-AR (sAR) which decreases fertilization rate. We show that activation of the actin-severing protein gelsolin induces a significant increase in sAR. Moreover, inhibition of CaMKII or PLD during sperm capacitation, caused an increase in sAR and inhibition of F-actin formation. Spermine, which leads to PLD activation, was able to reverse the effects of CaMKII inhibition on sAR-increase and F-actin-decrease. Furthermore, the increase in sAR and the decrease in F-actin caused by the inactivation of the PLD-pathway, were reversed by activation of CaMKII using H2O2 or by inhibiting protein phosphatase 1 which enhance the phosphorylation and oxidation states of CaMKII. These results indicate that two distinct pathways lead to F-actin formation in the sperm capacitation process which prevents the occurrence of sAR. PMID:27178669

  18. Actin disruption agents induce phosphorylation of histone H2AX in human breast adenocarcinoma MCF-7 cells.

    PubMed

    Shin, Ik Jae; Ahn, Yong-Tae; Kim, Yongkuk; Kim, Jong-Myoung; An, Won G

    2011-05-01

    Modified actin dynamics are a unique feature of transformed cancer cells and thereby promising targets for cancer chemotherapy. While latrunculin B (LB) and pectenotoxin-2 (PTX-2), both derived from natural sources, inhibit actin polymerization, jasplakinolide (JSP) prevents actin depolymerization. The purpose of this study was to examine the detailed molecular action of actin disruption inducing apoptosis via double strand breaks (DSBs). Actin disruption induced phosphorylation of H2AX, a well known DSB marker leading to G2 arrest and consequently resulted in apoptosis on MCF-7 cancer cells. Cells impaired by actin disruption activated Erk (extracellular signal-related kinase) and p53 protein was involved in DNA damage responses, but did not change the levels of p21Cip1/WAF1 protein in MCF-7 cells. To overcome the DSBs by actin disruption, MCF-7 cells set the repair system through the homologous recombination (HR) pathway. These results indicate that actin is involved in the signaling inducing DSBs and HR repair as well as G2 cell cycle arrest in human cancer. Therefore, the results suggest that actin disruption might be a potential candidate for developing anti-cancer therapies in human breast cancer. PMID:21399880

  19. Plasmodium falciparum aldolase and the C-terminal cytoplasmic domain of certain apical organellar proteins promote actin polymerization.

    PubMed

    Diaz, Suraya A; Martin, Stephen R; Grainger, Munira; Howell, Steven A; Green, Judith L; Holder, Anthony A

    2014-10-01

    The current model of Apicomplexan motility and host cell invasion is that both processes are driven by an actomyosin motor located beneath the plasma membrane, with the force transduced to the outside of the cell via coupling through aldolase and the cytoplasmic tail domains (CTDs) of certain type 1 membrane proteins. In Plasmodium falciparum (Pf), aldolase is thought to bind to the CTD of members of the thrombospondin-related anonymous protein (TRAP) family, which are micronemal proteins and represented by MTRAP in merozoites. Other type 1 membrane proteins including members of the erythrocyte binding antigen (EBA) and reticulocyte binding protein homologue (RH) protein families, which are also apical organellar proteins, have also been implicated in host cell binding in erythrocyte invasion. However, recent studies with Toxoplasma gondii have questioned the importance of aldolase in these processes. Using biolayer interferometry we show that Pf aldolase binds with high affinity to both rabbit and Pf actin, with a similar affinity for filamentous (F-) actin and globular (G-) actin. The interaction between Pf aldolase and merozoite actin was confirmed by co-sedimentation assays. Aldolase binding was shown to promote rabbit actin polymerization indicating that the interaction is more complicated than binding alone. The CTDs of some but not all type 1 membrane proteins also promoted actin polymerization in the absence of aldolase; MTRAP and RH1 CTDs promoted actin polymerization but EBA175 CTD did not. Direct actin polymerization mediated by membrane protein CTDs may contribute to actin recruitment, filament formation and stability during motor assembly, and actin-mediated movement, independent of aldolase. PMID:25261592

  20. Stretch-dependent smooth muscle differentiation in the portal vein-role of actin polymerization, calcium signaling, and microRNAs.

    PubMed

    Albinsson, Sebastian; Bhattachariya, Anirban; Hellstrand, Per

    2014-04-01

    The mechanical forces acting on SMC in the vascular wall are known to regulate processes such as vascular remodeling and contractile differentiation. However, investigations to elucidate the underlying mechanisms of mechanotransduction in smooth muscle have been hampered by technical limitations associated with mechanical studies on pressurized small arteries, due primarily to the small amount of available tissue. The murine portal vein is a relatively large vessel showing myogenic tone that in many respects recapitulates the properties of small resistance vessels. Studies on stretched portal veins to elucidate mechanisms of mechanotransduction in the vascular wall have shown that stretch-sensitive regulation of contractile differentiation is mediated via Rho-activation and actin polymerization, while stretch-induced growth is regulated by the MAPK pathway. In this review, we have summarized findings on mechanotransduction in the portal vein with focus on stretch-induced contractile differentiation and the role of calcium, actin polymerization and miRNAs in this response. PMID:24238368

  1. A POROELASTIC MODEL FOR CELL CRAWLING INCLUDING MECHANICAL COUPLING BETWEEN CYTOSKELETAL CONTRACTION AND ACTIN POLYMERIZATION.

    PubMed

    Taber, L A; Shi, Y; Yang, L; Bayly, P V

    2011-01-01

    Much is known about the biophysical mechanisms involved in cell crawling, but how these processes are coordinated to produce directed motion is not well understood. Here, we propose a new hypothesis whereby local cytoskeletal contraction generates fluid flow through the lamellipodium, with the pressure at the front of the cell facilitating actin polymerization which pushes the leading edge forward. The contraction, in turn, is regulated by stress in the cytoskeleton. To test this hypothesis, finite element models for a crawling cell are presented. These models are based on nonlinear poroelasticity theory, modified to include the effects of active contraction and growth, which are regulated by mechanical feedback laws. Results from the models agree reasonably well with published experimental data for cell speed, actin flow, and cytoskeletal deformation in migrating fish epidermal keratocytes. The models also suggest that oscillations can occur for certain ranges of parameter values. PMID:21765817

  2. Drebrin inhibits cofilin-induced severing of F-actin.

    PubMed

    Grintsevich, Elena E; Reisler, Emil

    2014-08-01

    Molecular cross-talk between neuronal drebrin A and cofilin is believed to be a part of the activity-dependent cytoskeleton-modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimer's disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1-300) inhibits, but do not abolish cofilin-induced severing of actin filaments. Cosedimentation experiments showed that F-actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F-actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F-actin induced by the competitor's binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre-steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments. PMID:25047716

  3. Nuclear actin polymerization from faster growing ends in the initial activation of Hox gene transcription are nuclear speckles involved?

    PubMed

    Naum-Onganía, Gabriela; Díaz, Víctor M; Blasi, Francesco; Rivera-Pomar, Rolando

    2013-01-01

    The HoxB cluster expression is activated by retinoic acid and transcribed in a collinear manner. The DNA-binding Pknox1-Pbx1 complex modulates Hox protein activity. Here, NT2-D1 teratocarcinoma cells -a model of Hox gene expression- were used to show that upon retinoic acid induction, Pknox1 co-localizes with polymeric nuclear actin. We have found that globular actin aggregates, polymeric actin, the elongating RNA polymerase II and THOC match euchromatic regions corresponding to nuclear speckles. Moreover, RNA polymerase II, N-WASP, and transcription/splicing factors p54(nrb) and PSF were validated as Pknox1 interactors by tandem affinity purification. PSF pulled down with THOC and nuclear actin, both of which co-localize in nuclear speckles. Although latrunculin A slightly decreases the general level of HoxB gene expression, inhibition of nuclear actin polymerization by cytochalasin D blocks the expression of HoxB transcripts in a collinear manner. Thus, our results support the hypothesis that nuclear actin polymerization is involved in the activation of HoxB gene expression by means of nuclear speckles. PMID:24406343

  4. Bacterial lipopolysaccharide induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in pulmonary vascular endothelial cells: concurrent F-actin depolymerization and new actin synthesis.

    PubMed

    Goldblum, S E; Ding, X; Brann, T W; Campbell-Washington, J

    1993-10-01

    Bacterial lipopolysaccharide (LPS) influences pulmonary vascular endothelial barrier function in vitro. We studied whether LPS regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to Escherichia coli 0111:B4 LPS 10 ng/ml or media for up to 6 h and evaluated for: 1) transendothelial 14C-albumin flux, 2) F-actin organization with fluorescence microscopy, 3) F-actin quantitation by spectrofluorometry, and 4) monomeric G-actin levels by the DNAse 1 inhibition assay. LPS induced increments in 14C-albumin flux (P < 0.001) and intercellular gap formation at > or = 2-6 h. During this same time period the endothelial F-actin pool was not significantly changed compared to simultaneous media controls. Mean (+/- SE) G-actin (micrograms/mg total protein) was significantly (P < 0.002) increased compared to simultaneous media controls at 2, 4, and 6 h but not at 0.5 or 1 h. Prior F-actin stabilization with phallicidin protected against the LPS-induced increments in G-actin (P = 0.040) as well as changes in barrier function (P < 0.0001). Prior protein synthesis inhibition unmasked an LPS-induced decrement in F-actin (P = 0.0044), blunted the G-actin increment (P = 0.010), and increased LPS-induced changes in endothelial barrier function (P < 0.0001). Therefore, LPS induces pulmonary vascular endothelial F-actin depolymerization, intercellular gap formation, and barrier dysfunction. Over the same time period, LPS increased total actin (P < 0.0001) and new actin synthesis (P = 0.0063) which may be a compensatory endothelial cell response to LPS-induced F-actin depolymerization. PMID:8408232

  5. Polymerization of actin in RBL-2H3 cells can be triggered through either the IgE receptor or the adenosine receptor but different signaling pathways are used.

    PubMed Central

    Apgar, J R

    1994-01-01

    Crosslinking of the IgE receptor on rat basophilic leukemia (RBL) cells using the multivalent antigen DNP-BSA leads to a rapid and sustained increase in the filamentous actin content of the cells. Stimulation of RBL cells through the adenosine receptor also induces a very rapid polymerization of actin, which peaks in 45-60 s and is equivalent in magnitude to the F-actin response elicited through stimulation of the IgE receptor. However, in contrast to the IgE mediated response, which remains elevated for over 30 min, the F-actin increase induced by the adenosine analogue 5'-(N-ethylcarboxamido)-adenosine (NECA) is relatively transient and returns to baseline values within 5-10 min. While previous work has shown that the polymerization of actin in RBL cells stimulated through the IgE receptor is mediated by protein kinase C (PKC), protein kinase inhibitors have no effect on the F-actin response activated through the adenosine receptor. In contrast, pretreatment of the cells with pertussis toxin completely inhibits the F-actin response to NECA but has relatively little effect on the response induced through the IgE receptor. Stimulation of RBL cells through either receptor causes increased production of phosphatidylinositol mono-phosphate (PIP) and phosphatidylinositol bis-phosphate (PIP2), which correlates with the F-actin response. Production of PIP and PIP2 may be important downstream signals since these polyphosphoinositides are able to regulate the interaction of gelsolin and profilin with actin. Thus the polymerization of actin can be triggered through either the adenosine receptor or the IgE receptor, but different upstream signaling pathways are being used. The IgE mediated response requires the activation of PKC while stimulation through the adenosine receptor is PKC independent but involves a G protein. PMID:8049523

  6. Antiobesity Action of ACAM by Modulating the Dynamics of Cell Adhesion and Actin Polymerization in Adipocytes.

    PubMed

    Murakami, Kazutoshi; Eguchi, Jun; Hida, Kazuyuki; Nakatsuka, Atsuko; Katayama, Akihiro; Sakurai, Miwa; Choshi, Haruki; Furutani, Masumi; Ogawa, Daisuke; Takei, Kohji; Otsuka, Fumio; Wada, Jun

    2016-05-01

    Coxsackie virus and adenovirus receptor-like membrane protein (CLMP) was identified as the tight junction-associated transmembrane protein of epithelial cells with homophilic binding activities. CLMP is also recognized as adipocyte adhesion molecule (ACAM), and it is upregulated in mature adipocytes in rodents and humans with obesity. Here, we present that aP2 promoter-driven ACAM transgenic mice are protected from obesity and diabetes with the prominent reduction of adipose tissue mass and smaller size of adipocytes. ACAM is abundantly expressed on plasma membrane of mature adipocytes and associated with formation of phalloidin-positive polymerized form of cortical actin (F-actin). By electron microscopy, the structure of zonula adherens with an intercellular space of ∼10-20 nm was observed with strict parallelism of the adjoining cell membranes over distances of 1-20 μm, where ACAM and γ-actin are abundantly expressed. The formation of zonula adherens may increase the mechanical strength, inhibit the adipocyte hypertrophy, and improve the insulin sensitivity. PMID:26956488

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

    NASA Astrophysics Data System (ADS)

    van Oudenaarden, Alexander

    2000-03-01

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

  8. GLUTAMATE-INDUCED Ca2+ INFLUX IN THIRD-ORDER NEURONS OF SALAMANDER RETINA IS REGULATED BY THE ACTIN CYTOSKELETON

    PubMed Central

    AKOPIAN, A.; SZIKRA, T.; CRISTOFANILLI, M.; KRIZAJ, D.

    2010-01-01

    Ligand-gated ion channels (ionotropic receptors) link to the cortical cytoskeleton via specialized scaffold proteins and thereby to appropriate signal transduction pathways in the cell. We studied the role of filamentous actin in the regulation of Ca influx through glutamate receptor-activated channels in third-order neurons of salamander retina. Staining by Alexa-Fluor 488-phalloidin, to visualize polymerized actin, we show localization of filamentous actin in neurites, and the membrane surrounding the cell soma. With Ca2+ imaging we found that in dissociated neurons, depolymerization of filamentous actin by latrunculin A, or cytochalasin D significantly reduced glutamate-induced intracellular Ca2+ accumulation to 53±7% of control value. Jasplakinolide, a stabilizer of filamentous actin, by itself slightly increased the glutamate-induced Ca2+ signal and completely attenuated the inhibitory effect when applied in combination with actin depolymerizing agents. These results indicate that in salamander retinal neurons the actin cytoskeleton regulates Ca2+ influx through ionotropic glutamate receptor-activated channels, suggesting regulatory roles for filamentous actin in a number of Ca2+-dependent physiological and pathological processes. PMID:16359816

  9. Cadmium-induced glutathionylation of actin occurs through a ROS-independent mechanism: Implications for cytoskeletal integrity

    SciTech Connect

    Choong, Grace; Liu, Ying; Xiao, Weiqun; Templeton, Douglas M.

    2013-10-15

    Cadmium disrupts the actin cytoskeleton in rat mesangial cells, and we have previously shown that this involves a complex interplay involving activation of kinase signaling, protein translocation, and disruption of focal adhesions. Here we investigate the role that glutathionylation of actin plays in Cd{sup 2+}-associated cytoskeletal reorganization. Low concentrations of Cd{sup 2+} (0.5–2 μM) caused an increase in actin glutathionylation by 6 h, whereas at higher concentrations glutathionylation remained at basal levels. Although oxidation with diamide increased glutathionylation, reactive oxygen species (ROS) were not involved in the Cd{sup 2+}-dependent effect, as only Cd{sup 2+} concentrations above 2 μM were sufficient to increase ROS. However, low [Cd{sup 2+}] increased total glutathione levels without affecting the ratio of reduced/oxidized glutathione, and inhibition of glutathione synthesis suppressed actin glutathionylation. Cadmium increased the activity of the enzyme glutaredoxin, which influences the equilibrium between glutathionylated and deglutathionylated proteins and thus may influence levels of glutathionylated actin. Together these observations show that cadmium-dependent effects on actin glutathionylation are affected by glutathione metabolism and not by direct effects of ROS on thiol chemistry. In vitro polymerization assays with glutathionylated actin show a decreased rate of polymerization. In contrast, immunofluorescence of cytoskeletal structure in intact cells suggests that increases in actin glutathionylation accompanying increased glutathione levels occurring under low Cd{sup 2+} exposure are protective in vivo, with cytoskeletal disruption ensuing only when higher Cd{sup 2+} concentrations increase ROS levels and prevent an increase in actin–glutathione conjugates. - Highlights: • Cadmium disrupts the actin cytoskeleton in mesangial cells. • Cadmium induces glutathionylation of actin at low concentrations.

  10. 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

  11. Actin-cytoskeleton rearrangement modulates proton-induced uptake

    SciTech Connect

    Ben-Dov, Nadav; Korenstein, Rafi

    2013-04-15

    Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction of inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process. - Highlights: ► Acidification of cells' exterior enhances uptake of macromolecules by the cells. ► Disruption of actin stress fibers leads to enhancement of proton induced uptake. ► Extensive depolymerization of cellular actin attenuates proton-induced uptake.

  12. Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking.

    PubMed

    Bugalhão, Joana N; Mota, Luís Jaime; Franco, Irina S

    2016-02-01

    The Legionella pneumophila effector protein VipA is an actin nucleator that co-localizes with actin filaments and early endosomes in infected macrophages and which interferes with organelle trafficking when expressed in yeast. To identify the regions of VipA involved in its subcellular localization and functions, we ectopically expressed specific VipA mutant proteins in eukaryotic cells. This indicated that the characteristic punctate distribution of VipA depends on its NH2 -terminal (amino acid residues 1-133) and central coiled-coil (amino acid residues 133-206) regions, and suggested a role for the COOH-terminal (amino acid residues 206-339) region in association with actin filaments and for the NH2 -terminal in co-localization with early endosomes. Co-immunoprecipitation and in vitro assays showed that the COOH-terminal region of VipA is necessary and sufficient to mediate actin binding, and is essential but insufficient to induce microfilament formation. Assays in yeast revealed that the NH2 and the COOH-terminal regions, and possibly an NPY motif within the NH2 region of VipA, are necessary for interference with organelle trafficking. Overall, this suggests that subversion of eukaryotic vesicular trafficking by VipA involves both its ability to associate with early endosomes via its NH2 -terminal region and its capacity to bind and polymerize actin through its COOH-terminal region. PMID:26626407

  13. Analysis of the aplyronine A-induced protein-protein interaction between actin and tubulin by surface plasmon resonance.

    PubMed

    Hirayama, Yuichiro; Yamagishi, Kota; Suzuki, Tomohiro; Kawagishi, Hirokazu; Kita, Masaki; Kigoshi, Hideo

    2016-06-15

    The antitumor macrolide aplyronine A induces protein-protein interaction (PPI) between actin and tubulin to exert highly potent biological activities. The interactions and binding kinetics of these molecules were analyzed by the surface plasmon resonance with biotinylated aplyronines or tubulin as ligands. Strong binding was observed for tubulin and actin with immobilized aplyronine A. These PPIs were almost completely inhibited by one equivalent of either aplyronine A or C, or mycalolide B. In contrast, a non-competitive actin-depolymerizing agent, latrunculin A, highly accelerated their association. Significant binding was also observed for immobilized tubulin with an actin-aplyronine A complex, and the dissociation constant KD was 1.84μM. Our method could be used for the quantitative analysis of the PPIs between two polymerizing proteins stabilized with small agents. PMID:27161875

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

    PubMed Central

    Hocking, Kyle M.; Putumbaka, Gowthami; Wise, Eric S.; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2016-01-01

    Objective Papaverine is used to prevent vasospasm in human saphenous veins (HSV) during vein graft preparation prior to implantation as a bypass conduit. Papaverine is a nonspecific inhibitor of phosphodiesterases, leading to increases in both intracellular cGMP and cAMP. We hypothesized that papaverine reduces force by decreasing intracellular calcium concentrations ([Ca2+]i) and myosin light chain phosphorylation, and increasing actin depolymerization via regulation of actin regulatory protein phosphorylation. Approach and Results HSV was equilibrated in a muscle bath, pre-treated with 1 mM papaverine followed by 5 μM norepinephrine, and force along with [Ca2+]i levels were concurrently measured. Filamentous actin (F-actin) level was measured by an in vitro actin assay. Tissue was snap frozen to measure myosin light chain and actin regulatory protein phosphorylation. Pre-treatment with papaverine completely inhibited norepinephrine-induced force generation, blocked increases in [Ca2+]i and led to a decrease in the phosphorylation of myosin light chain. Papaverine pre-treatment also led to increased phosphorylation of the heat shock-related protein 20 (HSPB6) and the vasodilator stimulated phosphoprotein (VASP), as well as decreased filamentous actin (F-actin) levels suggesting depolymerization of actin. Conclusions These results suggest that papaverine-induced force inhibition of HSV involves [Ca2+]i-mediated inhibition of myosin light chain phosphorylation and actin regulatory protein phosphorylation-mediated actin depolymerization. Thus, papaverine induces sustained inhibition of contraction of HSV by the modulation of both myosin cross-bridge formation and actin cytoskeletal dynamics and is a pharmacological alternative to high pressure distention to prevent vasospasm. PMID:27136356

  15. 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

  16. THE POLYMERIZATION OF ACTIN: ITS ROLE IN THE GENERATION OF THE ACROSOMAL PROCESS OF CERTAIN ECHINODERM SPERM

    PubMed Central

    Tilney, Lewis G.; Hatano, Sadashi; Ishikawa, Harunori; Mooseker, Mark S.

    1973-01-01

    When Asterias or Thyone sperm come in contact with egg jelly, a long process which in Thyone measures up to 90 µm in length is formed from the acrosomal region. This process can be generated in less than 30 s. Within this process is a bundle of microfilaments. Water extracts prepared from acetone powders of Asterias sperm contain a protein which binds rabbit skeletal muscle myosin forming a complex whose viscosity is reduced by ATP. Within this extract is a protein with the same molecular weight as muscle actin. It can be purified either by collecting the pellet produced after the addition of Mg++ or by reextracting an acetone powder of actomyosin prepared by the addition of highly purified muscle myosin to the extract. The sperm actin can be polymerized and by electron microscopy the polymer is indistinguishable from muscle F-actin. The sperm actin was shown to be localized in the microfilaments in the acrosomal processes by: (a) heavy meromyosin binding in situ, (b) sodium dodecyl sulfate (SDS) gel electrophoresis of the isolated acrosomal processes and a comparison to gels of flagella which contain no band corresponding to the molecular weight of actin, and (c) SDS gel electrophoresis of the extract from isolated acrosomal caps. Since the precursor for the microfilaments in the unreacted sperm appears amorphous, we suspected that the force for the generation of the acrosomal process is brought about by the polymerization of the sperm actin. This supposition was confirmed, for when unreacted sperm were lysed with the detergent Triton X-100 and the state of the actin in the sperm extract was analyzed by centrifugation, we determined that at least 80% of the actin in the unreacted sperm was in the monomeric state. PMID:4356568

  17. 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.

  18. 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

  19. Ratiometric Imaging of the T-Cell Actin Cytoskeleton Reveals the Nature of Receptor-Induced Cytoskeletal Enrichment

    PubMed Central

    Smoligovets, Alexander A.; Smith, Adam W.; Groves, Jay T.

    2013-01-01

    The T-cell actin cytoskeleton mediates adaptive immune system responses to peptide antigens by physically directing the motion and clustering of T-cell receptors (TCRs) on the cell surface. When TCR movement is impeded by externally applied physical barriers, the actin network exhibits transient enrichment near the trapped receptors. The coordinated nature of the actin density fluctuations suggests that they are composed of filamentous actin, but it has not been possible to eliminate de novo polymerization at TCR-associated actin polymerizing factors as an alternative cause. Here, we use a dual-probe cytoskeleton labeling strategy to distinguish between stable and polymerizing pools of actin. Our results suggest that TCR-associated actin consists of a relatively high proportion of the stable cytoskeletal fraction and extends away from the cell membrane into the cell. This implies that actin enrichment at mechanically trapped TCRs results from three-dimensional bunching of the existing filamentous actin network. PMID:23931330

  20. Actin Grips: Circular Actin-Rich Cytoskeletal Structures that Mediate the Wrapping of Polymeric Microfibers by Endothelial Cells

    PubMed Central

    Jones, Desiree; Park, DoYoung; Anghelina, Mirela; Pecot, Thierry; Machiraju, Raghu; Xue, Ruipeng; Lannutti, John; Thomas, Jessica; Cole, Sara; Moldovan, Leni; Moldovan, Nicanor I.

    2015-01-01

    Interaction of endothelial-lineage cells with three-dimensional substrates was much less studied than that with flat culture surfaces. We investigated the in vitro attachment of both mature endothelial cells (ECs) and of less differentiated EC colony-forming cells to poly-e-capro-lactone (PCL) fibers with diameters in 5–20 μm range (‘scaffold microfibers’, SMFs). We found that notwithstanding the poor intrinsic adhesiveness to PCL, both cell types completely wrapped the SMFs after long-term cultivation, thus attaining a cylindrical morphology. In this system, both EC types grew vigorously for more than a week and became increasingly more differentiated, as shown by multiplexed gene expression. Three-dimensional reconstructions from multiphoton confocal microscopy images using custom software showed that the filamentous (F) actin bundles took a conspicuous ring-like organization around the SMFs. Unlike the classical F-actin-containing stress fibers, these rings were not associated with either focal adhesions or intermediate filaments. We also demonstrated that plasma membrane boundaries adjacent to these circular cytoskeletal structures were tightly yet dynamically apposed to the SMFs, for which reason we suggest to call them ‘actin grips’. In conclusion, we describe a particular form of F-actin assembly with relevance for cytoskeletal organization in response to biomaterials, for endothelial-specific cell behavior in vitro and in vivo, and for tissue engineering. PMID:25818446

  1. 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

  2. Wound closure in the lamellipodia of single cells: mediation by actin polymerization in the absence of an actomyosin purse string.

    PubMed

    Henson, John H; Nazarian, Ronniel; Schulberg, Katrina L; Trabosh, Valerie A; Kolnik, Sarah E; Burns, Andrew R; McPartland, Kenneth J

    2002-03-01

    The actomyosin purse string is an evolutionarily conserved contractile structure that is involved in cytokinesis, morphogenesis, and wound healing. Recent studies suggested that an actomyosin purse string is crucial for the closure of wounds in single cells. In the present study, morphological and pharmacological methods were used to investigate the role of this structure in the closure of wounds in the peripheral cytoplasm of sea urchin coelomocytes. These discoidal shaped cells underwent a dramatic form of actin-based centripetal/retrograde flow and occasionally opened and closed spontaneous wounds in their lamellipodia. Fluorescent phalloidin staining indicated that a well defined fringe of actin filaments assembles from the margin of these holes, and drug studies with cytochalasin D and latrunculin A indicated that actin polymerization is required for wound closure. Additional evidence that actin polymerization is involved in wound closure was provided by the localization of components of the Arp2/3 complex to the wound margin. Significantly, myosin II immunolocalization demonstrated that it is not associated with wound margins despite being present in the perinuclear region. Pharmacological evidence for the lack of myosin II involvement in wound closure comes from experiments in which a microneedle was used to produce wounds in cells in which actomyosin contraction was inhibited by treatment with kinase inhibitors. Wounds produced in kinase inhibitor-treated cells closed in a manner similar to that seen with control cells. Taken together, our results suggest that an actomyosin purse string mechanism is not responsible for the closure of lamellar wounds in coelomocytes. We hypothesize that the wounds heal by means of a combination of the force produced by actin polymerization alone and centripetal flow. Interestingly, these cells did assemble an actomyosin structure around the margin of phagosome-like membrane invaginations, indicating that myosin is not simply

  3. Waves of actin and microtubule polymerization drive microtubule-based transport and neurite growth before single axon formation

    PubMed Central

    Winans, Amy M; Collins, Sean R; Meyer, Tobias

    2016-01-01

    Many developing neurons transition through a multi-polar state with many competing neurites before assuming a unipolar state with one axon and multiple dendrites. Hallmarks of the multi-polar state are large fluctuations in microtubule-based transport into and outgrowth of different neurites, although what drives these fluctuations remains elusive. We show that actin waves, which stochastically migrate from the cell body towards neurite tips, direct microtubule-based transport during the multi-polar state. Our data argue for a mechanical control system whereby actin waves transiently widen the neurite shaft to allow increased microtubule polymerization to direct Kinesin-based transport and create bursts of neurite extension. Actin waves also require microtubule polymerization, arguing that positive feedback links these two components. We propose that actin waves create large stochastic fluctuations in microtubule-based transport and neurite outgrowth, promoting competition between neurites as they explore the environment until sufficient external cues can direct one to become the axon. DOI: http://dx.doi.org/10.7554/eLife.12387.001 PMID:26836307

  4. ACTG2 variants impair actin polymerization in sporadic Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

    PubMed

    Halim, Danny; Hofstra, Robert M W; Signorile, Luca; Verdijk, Rob M; van der Werf, Christine S; Sribudiani, Yunia; Brouwer, Rutger W W; van IJcken, Wilfred F J; Dahl, Niklas; Verheij, Joke B G M; Baumann, Clarisse; Kerner, John; van Bever, Yolande; Galjart, Niels; Wijnen, Rene M H; Tibboel, Dick; Burns, Alan J; Muller, Françoise; Brooks, Alice S; Alves, Maria M

    2016-02-01

    Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS) is a rare congenital disorder, in which heterozygous missense variants in the Enteric Smooth Muscle actin γ-2 (ACTG2) gene have been recently identified. To investigate the mechanism by which ACTG2 variants lead to MMIHS, we screened a cohort of eleven MMIHS patients, eight sporadic and three familial cases, and performed immunohistochemistry, molecular modeling and molecular dynamics (MD) simulations, and in vitro assays. In all sporadic cases, a heterozygous missense variant in ACTG2 was identified. ACTG2 expression was detected in all intestinal layers where smooth muscle cells are present in different stages of human development. No histopathological abnormalities were found in the patients. Using molecular modeling and MD simulations, we predicted that ACTG2 variants lead to significant changes to the protein function. This was confirmed by in vitro studies, which showed that the identified variants not only impair ACTG2 polymerization, but also contribute to reduced cell contractility. Taken together, our results confirm the involvement of ACTG2 in sporadic MMIHS, and bring new insights to MMIHS pathogenesis. PMID:26647307

  5. Fz2 and Cdc42 Mediate Melanization and Actin Polymerization but Are Dispensable for Plasmodium Killing in the Mosquito Midgut

    PubMed Central

    Zachary, Daniel; Hoffmann, Jules A; Levashina, Elena A

    2006-01-01

    The midgut epithelium of the mosquito malaria vector Anopheles is a hostile environment for Plasmodium, with most parasites succumbing to host defenses. This study addresses morphological and ultrastructural features associated with Plasmodium berghei ookinete invasion in Anopheles gambiae midguts to define the sites and possible mechanisms of parasite killing. We show by transmission electron microscopy and immunofluorescence that the majority of ookinetes are killed in the extracellular space. Dead or dying ookinetes are surrounded by a polymerized actin zone formed within the basal cytoplasm of adjacent host epithelial cells. In refractory strain mosquitoes, we found that formation of this zone is strongly linked to prophenoloxidase activation leading to melanization. Furthermore, we identify two factors controlling both phenomena: the transmembrane receptor frizzled-2 and the guanosine triphosphate–binding protein cell division cycle 42. However, the disruption of actin polymerization and melanization by double-stranded RNA inhibition did not affect ookinete survival. Our results separate the mechanisms of parasite killing from subsequent reactions manifested by actin polymerization and prophenoloxidase activation in the A. gambiae–P. berghei model. These latter processes are reminiscent of wound healing in other organisms, and we propose that they represent a form of wound-healing response directed towards a moribund ookinete, which is perceived as damaged tissue. PMID:17196037

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

    PubMed Central

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

    2014-01-01

    Thymosin-β4 (Tβ4) and profilin are the two major sequestering proteins that maintain the pool of monomeric actin (G-actin) within cells of higher eukaryotes. Tβ4 prevents G-actin from joining a filament, whereas profilin:actin only supports barbed-end elongation. Here, we report two Tβ4:actin structures. The first structure shows that Tβ4 has two helices that bind at the barbed and pointed faces of G-actin, preventing the incorporation of the bound G-actin into a filament. The second structure displays a more open nucleotide binding cleft on G-actin, which is typical of profilin:actin structures, with a concomitant disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, show that the exchange of bound actin between Tβ4 and profilin involves both steric and allosteric components. The sensitivity of profilin to the conformational state of actin indicates a similar allosteric mechanism for the dissociation of profilin during filament elongation. PMID:25313062

  7. Septins guide microtubule protrusions induced by actin-depolymerizing toxins like Clostridium difficile transferase (CDT).

    PubMed

    Nölke, Thilo; Schwan, Carsten; Lehmann, Friederike; Østevold, Kristine; Pertz, Olivier; Aktories, Klaus

    2016-07-12

    Hypervirulent Clostridium difficile strains, which are associated with increased morbidity and mortality, produce the actin-ADP ribosylating toxin Clostridium difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here, we show that septins (SEPT) are essential for CDT-induced protrusion formation. SEPT2, -6, -7, and -9 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. The septin inhibitor forchlorfenuron or knockdown of septins inhibits protrusion formation. At protrusion sites, septins colocalize with the GTPase Cdc42 (cell division control protein 42) and its effector Borg (binder of Rho GTPases), which act as up-stream regulators of septin polymerization. Precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to the microtubule plus-end tracking protein EB1, thereby guiding incoming microtubules. The data suggest that CDT usurps conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, Cdc42, Borgs, and restructuring of the actin cytoskeleton. PMID:27339141

  8. Active microrheology of entangled blends of DNA and Actin link polymer flexibility to induced molecular deformations and stress propagation

    NASA Astrophysics Data System (ADS)

    Fitzpatrick, Robert; Robertson-Anderson, Rae; Anderson Research Team

    Actin is a ubiquitous structural protein in the cytoskeleton that gives cells shape and rigidity, and plays important roles in mechanical processes such as cell motility and division. Actin's diverse roles stem from its ability to polymerize into semiflexible filaments that are less than one persistence length (17 µm) in length, and form entangled networks that display unique viscoelastic properties. We previously found that entangled actin networks propagate microscale forces over several persistence lengths (>60 m) and takes minutes to relax. DNA, oppositely, has thousands of persistence lengths (50 nm) per chain, exhibits minimal force propagation, and takes only seconds to re-equilibrate. To directly determine the role of flexibility in mechanical response and force propagation of entangled networks, we use optical tweezers and fluorescence microscopy to investigate blends of actin and DNA. We use optically driven microspheres to perturb the network far from equilibrium and measure the force the network creates in response to the induced force. We simultaneously track partially labeled actin filaments during the perturbation and subsequent relaxation period. We characterize filament deformation and show explicitly how induced microscale forces propagate through the network.

  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. The leukotriene B4 receptor BLT2 protects barrier function via actin polymerization with phosphorylation of myosin phosphatase target subunit 1 in human keratinocytes.

    PubMed

    Chiba, Takahito; Nakahara, Takeshi; Hashimoto-Hachiya, Akiko; Yokomizo, Takehiko; Uchi, Hiroshi; Furue, Masutaka

    2016-07-01

    Leukotriene B4 (LTB4 ) receptor type 2 (BLT2) is a novel G-protein-coupled receptor, which selectively binds to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) with stronger affinity than to LTB4 . Recently, 12-HHT has been shown to have a protective effect on the epidermal barrier in human keratinocytes or transfectant cells overexpressing BLT2. Because the protective activity of BLT2 in high-calcium conditions, which occurs in well-differentiated cells, is exerted through increasing the integrity of tight junctions, we investigated the effects of 12-HHT on the barrier function of human keratinocytes in low-calcium conditions that mimic the basal layer; to our knowledge, this has not been reported previously. After stimulation with or without 12-HHT, barrier function was measured using transepithelial electrical resistance (TER) and dextran permeability assay. Expression levels of adhesion molecules and actin polymerization were also evaluated. Treatment with 12-HHT increased TER, along with decreased epidermal permeability of dextran in human keratinocytes. Furthermore, 12-HHT induced actin polymerization with phosphorylation of myosin phosphatase target subunit 1. These results suggest that the ligation of BLT2 protects permeability barrier function by enhancing cell-cell contact, even under low-calcium conditions, and indicate that a BLT2 agonist could be a novel therapeutic target for barrier-disrupted skin diseases. PMID:26896822

  11. CFTR surface expression and chloride currents are decreased by inhibitors of N-WASP and actin polymerization

    PubMed Central

    Ganeshan, Radhika; Nowotarski, Krzysztof; Di, Anke; Nelson, Deborah J.; Kirk, Kevin L.

    2007-01-01

    Summary The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid turnover at the plasma membrane in various cell types. The ubiquitously expressed N-WASP promotes actin polymerization and regulates endocytic trafficking of other proteins in response to signaling molecules such as Rho-GTPases. In the present study we investigated the effects of wiskostatin, an N-WASP inhibitor, on the surface expression and activity of CFTR. We demonstrate, using surface biotinylation methods, that the steady-state surface CFTR pool in stably transfected BHK cells was dramatically decreased following wiskostatin treatment with a corresponding increase in the amount of intracellular CFTR. Similar effects were observed for latrunculin B, a specific actin-disrupting reagent. Both reagents strongly inhibited macroscopic CFTR-mediated Cl− currents in two cell types including HT29-Cl19A colonic epithelial cells. As previously reported, CFTR internalization from the cell surface was strongly inhibited by a cyclic-AMP cocktail. This effect of cyclic-AMP was only partially blunted in the presence of wiskostatin, which raises the possibility that these two factors modulate different steps in CFTR traffic. In kinetic studies wiskostatin appeared to accelerate the initial rate of CFTR endocytosis as well as inhibit its recycling back to the cell surface over longer time periods. Our studies implicate a role for N-WASP-mediated actin polymerization in regulating CFTR surface expression and channel activity. PMID:17084917

  12. 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

  13. Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

    PubMed Central

    Caution, Kyle; Gavrilin, Mikhail A.; Tazi, Mia; Kanneganti, Apurva; Layman, Daniel; Hoque, Sheshadri; Krause, Kathrin; Amer, Amal O.

    2015-01-01

    Inflammasomes are multiprotein complexes that include members of the NOD-like receptor family and caspase-1. Caspase-1 is required for the fusion of the Legionella vacuole with lysosomes. Caspase-11, independently of the inflammasome, also promotes phagolysosomal fusion. However, it is unclear how these proteases alter intracellular trafficking. Here, we show that caspase-11 and caspase-1 function in opposing manners to phosphorylate and dephosphorylate cofilin, respectively upon infection with Legionella. Caspase-11 targets cofilin via the RhoA GTPase, whereas caspase-1 engages the Slingshot phosphatase. The absence of either caspase-11 or caspase-1 maintains actin in the polymerized or depolymerized form, respectively and averts the fusion of pathogen-containing vacuoles with lysosomes. Therefore, caspase-11 and caspase-1 converge on the actin machinery with opposing effects to promote vesicular trafficking. PMID:26686473

  14. Ampakines promote spine actin polymerization, long-term potentiation, and learning in a mouse model of Angelman Syndrome

    PubMed Central

    Baudry, Michel; Kramar, Eniko; Xu, Xiaobo; Zadran, Homera; Moreno, Stephanie; Lynch, Gary; Gall, Christine; Bi, Xiaoning

    2012-01-01

    Angelman syndrome (AS) is a neurodevelopmental disorder largely due to abnormal maternal expression of the UBE3A gene leading to the deletion of E6-associated protein. AS subjects have severe cognitive impairments for which there are no therapeutic interventions. Mouse models (knockouts of the maternal Ube3a gene: ‘AS mice’) of the disorder have substantial deficits in long-term potentiation (LTP) and learning. Here we report a clinically plausible pharmacological treatment that ameliorates both deficits. AS mice were injected ip twice daily for 5 days with vehicle or the ampakine CX929; drugs of this type enhance fast EPSCs by positively modulating AMPA receptors. Theta burst stimulation (TBS) produced a normal enhancement of field EPSPs in hippocampal slices prepared from vehicle-treated AS mice but LTP decreased steadily to baseline; however, LTP in slices from ampakine-treated AS mice stabilized at levels found in wild-type controls. TBS-induced actin polymerization within dendritic spines, an essential event for stabilizing LTP, was severely impaired in slices from vehicle-treated AS mice but not in those from ampakine-treated AS mice. Long-term memory scores in a fear conditioning paradigm were reduced by 50% in vehicle-treated AS mice but were comparable to values for littermate controls in the ampakine-treated AS mice. We propose that AS is associated with a profound defect in activity-driven spine cytoskeletal reorganization, resulting in a loss of the synaptic plasticity required for the encoding of long-term memory. Notably, the spine abnormality along with the LTP and learning impairments can be reduced by a minimally invasive drug treatment. PMID:22525571

  15. Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

    PubMed

    De Maio, Anna; Natale, Emiliana; Rotondo, Sergio; Di Cosmo, Anna; Faraone-Mennella, Maria Rosaria

    2013-09-01

    Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity. PMID:23831359

  16. 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.

  17. 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 ...

  18. Formation of cofilin-actin rods following cucurbitacin-B-induced actin aggregation depends on Slingshot homolog 1-mediated cofilin hyperactivation.

    PubMed

    Zhang, Yan-Ting; Ouyang, Dong-Yun; Xu, Li-Hui; Zha, Qing-Bing; He, Xian-Hui

    2013-10-01

    Accumulating evidence indicates that cucurbitacin B (CuB), as well as other cucurbitacins, damages the actin cytoskeleton in a variety of cell types. However, the underlying mechanism of such an effect is not well understood. In this study, we showed that CuB rapidly induced actin aggregation followed by actin rod formation in melanoma cells. Cofilin, a critical regulator of actin dynamics, was dramatically dephosphorylated (i.e., activated) upon CuB treatment. Notably, the activated cofilin subsequently formed rod-like aggregates, which were highly colocalized with actin rods, indicating the formation of cofilin-actin rods. Cofilin knockdown significantly suppressed rod formation but did not prevent actin aggregation. Furthermore, knockdown of the cofilin phosphatase Slingshot homolog 1 (SSH1), but not chronophin (CIN), alleviated CuB-induced cofilin hyperactivation and cofilin-actin rod formation. The activity of Rho kinase and LIM kinase, two upstream regulators of cofilin activation, was downregulated after cofilin hyperactivation. Pretreatment with a thiol-containing reactive oxygen species (ROS) scavenger N-acetyl cysteine, but not other ROS inhibitors without thiol groups, suppressed CuB-induced actin aggregation, cofilin hyperactivation and cofilin-actin rod formation, suggesting that thiol oxidation might be involved in these processes. Taken together, our results demonstrated that CuB-induced formation of cofilin-actin rods was mediated by SSH1-dependent but CIN-independent cofilin hyperactivation. PMID:23695982

  19. Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro.

    PubMed

    Lv, Zhimei; Hu, Mengsi; Ren, Xiaoxu; Fan, Minghua; Zhen, Junhui; Chen, Liqun; Lin, Jiangong; Ding, Nannan; Wang, Qun; Wang, Rong

    2016-01-01

    Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin) rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation. PMID:26881253

  20. Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro

    PubMed Central

    Lv, Zhimei; Hu, Mengsi; Ren, Xiaoxu; Fan, Minghua; Zhen, Junhui; Chen, Liqun; Lin, Jiangong; Ding, Nannan; Wang, Qun; Wang, Rong

    2016-01-01

    Fyn, a member of the Src family of tyrosine kinases, is a key regulator in cytoskeletal remodeling in a variety of cell types. Recent studies have demonstrated that Fyn is responsible for nephrin tyrosine phosphorylation, which will result in polymerization of actin filaments and podocyte damage. Thus detailed involvement of Fyn in podocytes is to be elucidated. In this study, we investigated the potential role of Fyn/ROCK signaling and its interactions with paxillin. Our results presented that high glucose led to filamentous actin (F-actin) rearrangement in podocytes, accompanied by paxillin phosphorylation and increased cell motility, during which Fyn and ROCK were markedly activated. Gene knockdown of Fyn by siRNA showed a reversal effect on high glucose-induced podocyte damage and ROCK activation; however, inhibition of ROCK had no significant effects on Fyn phosphorylation. These observations demonstrate that in vitro Fyn mediates high glucose-induced actin cytoskeleton remodeling of podocytes via promoting ROCK activation and paxillin phosphorylation. PMID:26881253

  1. Chronologic and actinically induced aging in human facial skin

    SciTech Connect

    Gilchrest, B.A.; Szabo, G.; Flynn, E.; Goldwyn, R.M.

    1983-06-01

    Clinical and histologic stigmata of aging are much more prominent in habitually sun-exposed skin than in sun-protected skin, but other possible manifestations of actinically induced aging are almost unexplored. We have examined the interrelation of chronologic and actinic aging using paired preauricular (sun-exposed) and postauricular (sun-protected) skin specimens. Keratinocyte cultures derived from sun-exposed skin consistently had a shorter in vitro lifespan but increased plating efficiency compared with cultures derived from adjacent sun-protected skin of the same individual, confirming a previous study of different paired body sites. Electron microscopic histologic sections revealed focal abnormalities of keratinocyte proliferation and alignment in vitro especially in those cultures derived from sun-exposed skin and decreased intercellular contact in stratified colonies at late passage, regardless of donor site. One-micron histologic sections of the original biopsy specimens revealed no striking site-related keratinocyte alterations, but sun-exposed specimens had fewer epidermal Langerhans cells (p less than 0.001), averaging approximately 50 percent the number in sun-protected skin, a possible exaggeration of the previously reported age-associated decrease in this cell population. These data suggest that sun exposure indeed accelerates aging by several criteria and that, regardless of mechanism, environmental factors may adversely affect the appearance and function of aging skin in ways amenable to experimental quantitation.

  2. Intracellular calcium rise is not a necessary step for the stimulated actin polymerization

    SciTech Connect

    Yassin, R.

    1986-03-01

    Stimulation of rabbit peritoneal neutrophils by many chemotactic (formyl Methionyl-Leucyl-Phenylalanine (fMLP), Leukotriene B/sub 4/ (LTB/sub 4/)) and non-chemotactic (phorbol 12-myristate, 13-acetate (PMA), platelet activating factor (PAF), and the calcium ionophore A23187) factors produces rapid and dose dependent increases in the amount of actin associated with the cytoskeleton. The stimulated increase in cytoskeletal actin does not appear to require a rise in the intracellular concentration of free calcium. The increase in cytoskeletal actin produced by A23187 is transient and does not depend on the presence of calcium in the suspending medium. In the presence of extracellular calcium, the effect of the ionophore is biphasic with respect to concentration. The increases in actin association with cytoskeletal produced by fMLP, LTB/sub 4/, and A23187 but not by PMA, are inhibited by hyperosmolarity and pertussis toxin pretreatment. On the other hand, the addition of hyperosmolarity or pertussis toxin has small effect on the rise in the intracellular calcium produced by A23187. The results presented here suggest that an increase in the intracellular concentration of free calcium is not necessary for the stimulated increases in cytoskeletal actin.

  3. Fullerenol Nanoparticles with Structural Activity Induce Variable Intracellular Actin Filament Morphologies.

    PubMed

    Jin, Junjiang; Dong, Ying; Wang, Ying; Xia, Lin; Gu, Weihong; Bai, Xue; Chang, Yanan; Zhang, Mingyi; Chen, Kui; Li, Juan; Zhao, Lina; Xing, Gengmei

    2016-06-01

    Fullerenol nanoparticles are promising for various biological applications; many studies have shown that they induce variable and diverse biological effects including side effects. Separation and purification of two fractions of fullerenols has demonstrated that they have varied chemical structures on the surfaces of their carbon cages. Actin is an important structural protein that is able to transform functional structures under varied physiological conditions. We assessed the abilities of the two fractions of fullerenols to attach to actin and induce variable morphological features in actin filament structures. Specifically the fullerenol fraction with a surface electric charge of -1.913 ± 0.008q (x10(-6) C) has percentages of C-OH and C=O on the carbon cage of 16.14 ± 0.60 and 17.55 ± 0.69. These features allow it to form intermolecular hydrogen bonds with actin at a stoichiometric ratio of four fullerenols per actin subunit. Molecular simulations revealed these specific binding sites and binding modes in atomic details in the interaction between the active fullerenol and actin filament. Conversely, these interactions were not possible for the other fraction of fullerenol with that percentages of C-OH and C=O on the carbon cage were 15.59 ± 0.01 and 1.94 ± 0.11. Neither sample induced appreciable cytotoxicity or acute cell death. After entering cells, active fullerenol binding to actin induces variable morphological features and may transform ATP-actin to ADP-actin. These changes facilitate the binding of ADF/cofilin, allowing cofilin to sever actin filaments to form cofilin/actin/fullerenol rods. Our findings suggest that fullerenol with structural activity binding disturbs actin filament structure, which may inhibit locomotion of cell or induce chronic side effects in to cells. PMID:27319217

  4. Alpha-herpesvirus infection induces the formation of nuclear actin filaments.

    PubMed

    Feierbach, Becket; Piccinotti, Silvia; Bisher, Margaret; Denk, Winfried; Enquist, Lynn W

    2006-08-01

    Herpesviruses are large double-stranded DNA viruses that replicate in the nuclei of infected cells. Spatial control of viral replication and assembly in the host nucleus is achieved by the establishment of nuclear compartments that serve to concentrate viral and host factors. How these compartments are established and maintained remains poorly understood. Pseudorabies virus (PRV) is an alpha-herpesvirus often used to study herpesvirus invasion and spread in the nervous system. Here, we report that PRV and herpes simplex virus type 1 infection of neurons results in formation of actin filaments in the nucleus. Filamentous actin is not found in the nucleus of uninfected cells. Nuclear actin filaments appear physically associated with the viral capsids, as shown by serial block-face scanning electron micropscopy and confocal microscopy. Using a green fluorescent protein-tagged viral capsid protein (VP26), we show that nuclear actin filaments form prior to capsid assembly and are required for the efficient formation of viral capsid assembly sites. We find that actin polymerization dynamics (e.g., treadmilling) are not necessary for the formation of these sites. Green fluorescent protein-VP26 foci co-localize with the actin motor myosin V, suggesting that viral capsids travel along nuclear actin filaments using myosin-based directed transport. Viral transcription, but not viral DNA replication, is required for actin filament formation. The finding that infection, by either PRV or herpes simplex virus type 1, results in formation of nuclear actin filaments in neurons, and that PRV infection of an epithelial cell line results in a similar phenotype is evidence that F-actin plays a conserved role in herpesvirus assembly. Our results suggest a mechanism by which assembly domains are organized within infected cells and provide insight into how the viral infectious cycle and host actin cytoskeleton are integrated to promote the infection process. PMID:16933992

  5. VASP Activation via the Gα13/RhoA/PKA Pathway Mediates Cucurbitacin-B-Induced Actin Aggregation and Cofilin-Actin Rod Formation

    PubMed Central

    Zhang, Yan-Ting; Xu, Li-Hui; Lu, Qun; Liu, Kun-Peng; Liu, Pei-Yan; Ji, Fang; Liu, Xiao-Ming; Ouyang, Dong-Yun; He, Xian-Hui

    2014-01-01

    Cucurbitacin B (CuB), a potent antineoplastic agent of cucurbitacin triterpenoids, induces rapid disruption of actin cytoskeleton and aberrant cell cycle inhibiting carcinogenesis. However, the underlying molecular mechanism of such anticancer effects remains incompletely understood. In this study, we showed that CuB treatment rapidly induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation (i.e. activation) at the Ser157 residue and generated VASP clumps which were co-localized with amorphous actin aggregates prior to the formation of highly-ordered cofilin-actin rods in melanoma cells. Knockdown of VASP or inhibition of VASP activation using PKA-specific inhibitor H89 suppressed CuB-induced VASP activation, actin aggregation and cofilin-actin rod formation. The VASP activation was mediated by cAMP-independent PKA activation as CuB decreased the levels of cAMP while MDL12330A, an inhibitor of adenylyl cyclase, had weak effect on VASP activation. Knockdown of either Gα13 or RhoA not only suppressed VASP activation, but also ameliorated CuB-induced actin aggregation and abrogated cofilin-actin rod formation. Collectively, our studies highlighted that the CuB-induced actin aggregation and cofilin-actin rod formation was mediated via the Gα13/RhoA/PKA/VASP pathway. PMID:24691407

  6. Cooperative and non-cooperative conformational changes of F-actin induced by cofilin

    SciTech Connect

    Aihara, Tomoki; Oda, Toshiro

    2013-05-31

    Highlights: •Mobility of MTSL attached to C374 in F-actin became high upon addition of cofilin. •Change of motility of MTSL attached to C374 with cofilin-binding was cooperative. •Mobility of MTSL attached to V43C in F-actin became high upon addition of cofilin. •Change of motility of MTSL attached to V43C with cofilin-binding was linear. -- Abstract: Cofilin is an actin-binding protein that promotes F-actin depolymerization. It is well-known that cofilin-coated F-actin is more twisted than naked F-actin, and that the protomer is more tilted. However, the means by which the local changes induced by the binding of individual cofilin proteins proceed to the global conformational changes of the whole F-actin molecule remain unknown. Here we investigated the cofilin-induced changes in several parts of F-actin, through site-directed spin-label electron paramagnetic resonance spectroscopy analyses of recombinant actins containing single reactive cysteines. We found that the global, cooperative conformational changes induced by cofilin-binding, which were detected by the spin-label attached to the Cys374 residue, occurred without the detachment of the D-loop in subdomain 2 from the neighboring protomer. The two processes of local and global changes do not necessarily proceed in sequence.

  7. Antibody against the Carboxyl Terminus of Intimin α Reduces Enteropathogenic Escherichia coli Adherence to Tissue Culture Cells and Subsequent Induction of Actin Polymerization

    PubMed Central

    Carvalho, Humberto M.; Teel, Louise D.; Kokai-Kun, John F.; O'Brien, Alison D.

    2005-01-01

    The C-terminal third of intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of intimin α. PMID:15784601

  8. Bending Flexibility of Actin Filaments during Motor-Induced Sliding

    PubMed Central

    Vikhorev, Petr G.; Vikhoreva, Natalia N.; Månsson, Alf

    2008-01-01

    Muscle contraction and other forms of cell motility occur as a result of cyclic interactions between myosin molecules and actin filaments. Force generation is generally attributed to ATP-driven structural changes in myosin, whereas a passive role is ascribed to actin. However, some results challenge this view, predicting structural changes in actin during motor activity, e.g., when the actin filaments slide on a myosin-coated surface in vitro. Here, we analyzed statistical properties of the sliding filament paths, allowing us to detect changes of this type. It is interesting to note that evidence for substantial structural changes that led to increased bending flexibility of the filaments was found in phalloidin-stabilized, but not in phalloidin-free, actin filaments. The results are in accordance with the idea that a high-flexibility structural state of actin is a prerequisite for force production, but not the idea that a low-to-high flexibility transition of the actin filament should be an important component of the force-generating step per se. Finally, our data challenge the general view that phalloidin-stabilized filaments behave as native actin filaments in their interaction with myosin. This has important implications, since phalloidin stabilization is a routine procedure in most studies of actomyosin function. PMID:18835897

  9. Evaluation of the sliding distance in shortening muscles and in polymerizing actin from Hill's force-velocity equation.

    PubMed

    Oplatka, Avraham

    2006-12-15

    The relationship derived earlier between the sliding distance, Deltal(m), and a/P(0), the characteristic parameter of Hill's force-velocity equation for muscle contraction, was re-formulated in order to get a more general relationship which can be applied also to other biological mechano-chemical energy converters: alpha x Deltal(m)=phi (0)(a/P(0))Deltal(m)=-Deltag where Deltag is the free energy change accompanying the hydrolysis of one ATP molecule while alpha and phi (0) are, respectively, the average forces developed by a myosin head-actin complex which are responsible for shortening and for isometric tension generation. These two molecular forces are different in magnitude and in nature and it is demonstrated that alpha , not phi (0), is the true contractile force. The values of alpha and of phi (0) have been calculated for three muscles. The equation has been successfully applied to actin polymerization-based motility. The value of Deltag in different muscles under different environmental conditions can be easily determined from this equation with the value of Deltal(m) derived experimentally. PMID:16904176

  10. Tau co-organizes dynamic microtubule and actin networks

    PubMed Central

    Elie, Auréliane; Prezel, Elea; Guérin, Christophe; Denarier, Eric; Ramirez-Rios, Sacnicte; Serre, Laurence; Andrieux, Annie; Fourest-Lieuvin, Anne; Blanchoin, Laurent; Arnal, Isabelle

    2015-01-01

    The crosstalk between microtubules and actin is essential for cellular functions. However, mechanisms underlying the microtubule-actin organization by cross-linkers remain largely unexplored. Here, we report that tau, a neuronal microtubule-associated protein, binds to microtubules and actin simultaneously, promoting in vitro co-organization and coupled growth of both networks. By developing an original assay to visualize concomitant microtubule and actin assembly, we show that tau can induce guided polymerization of actin filaments along microtubule tracks and growth of single microtubules along actin filament bundles. Importantly, tau mediates microtubule-actin co-alignment without changing polymer growth properties. Mutagenesis studies further reveal that at least two of the four tau repeated motifs, primarily identified as tubulin-binding sites, are required to connect microtubules and actin. Tau thus represents a molecular linker between microtubule and actin networks, enabling a coordination of the two cytoskeletons that might be essential in various neuronal contexts. PMID:25944224

  11. Formin-mediated actin polymerization cooperates with Mushroom body defect (Mud)–Dynein during Frizzled–Dishevelled spindle orientation

    PubMed Central

    Johnston, Christopher A.; Manning, Laurina; Lu, Michelle S.; Golub, Ognjen; Doe, Chris Q.; Prehoda, Kenneth E.

    2013-01-01

    Summary To position the mitotic spindle, cytoskeletal components must be coordinated to generate cortical forces on astral microtubules. Although the dynein motor is common to many spindle orientation systems, ‘accessory pathways’ are often also required. In this work, we identified an accessory spindle orientation pathway in Drosophila that functions with Dynein during planar cell polarity, downstream of the Frizzled (Fz) effector Dishevelled (Dsh). Dsh contains a PDZ ligand and a Dynein-recruiting DEP domain that are both required for spindle orientation. The Dsh PDZ ligand recruits Canoe/Afadin and ultimately leads to Rho GTPase signaling mediated through RhoGEF2. The formin Diaphanous (Dia) functions as the Rho effector in this pathway, inducing F-actin enrichment at sites of cortical Dsh. Chimeric protein experiments show that the Dia–actin accessory pathway can be replaced by an independent kinesin (Khc73) accessory pathway for Dsh-mediated spindle orientation. Our results define two ‘modular’ spindle orientation pathways and show an essential role for actin regulation in Dsh-mediated spindle orientation. PMID:23868974

  12. 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

  13. Dynamic in vivo analysis of drug induced actin cytoskeleton degradation by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Schnekenburger, Juergen; Bredebusch, Ilona; Langehanenberg, Patrik; Domschke, Wolfram; von Bally, Gert; Kemper, Björn

    2007-07-01

    The actin cytoskeleton mediates a variety of crucial cellular functions as migration, intracellular transport, exocytosis, endocytosis and force generation. The highly dynamic actin fibers are therefore targets for several drugs and toxins. However the study of actin interfering processes by standard microscopy techniques fails in the detailed resolution of dynamic spatial alterations required for a deeper understanding of toxic effects. Here we applied digital holographic microscopy in the online functional analysis of the actin cytoskeleton disrupting marine toxin Latrunculin B. SEM and fluorescence microscopy showed rapid Latrunculin B induced alterations in cell morphology and actin fiber degradation in pancreas tumor cells. The dynamic digital holographic in vivo analysis of the drug dependent cellular processes demonstrated differences in the actin cytoskeleton stability of highly differentiated and dedifferentiated pancreas tumor cell lines. The spatial resolution of the morphological alterations revealed unequal changes in cell morphology. While cells with a low metastatic potential showed Latrunculin B induced cell collapse within 4 h the metastatic tumor cells were increased in cell volume indicating Latrunculin B effects also on cell water content. These data demonstrate that marker free, non-destructive online analysis of cellular morphology and dynamic spatial processes in living cells by digital holography offers new insights in actin dependent cellular mechanisms. Digital holographic microscopy was shown to be a versatile tool in the screening of toxic drug effects and cancer cell biology.

  14. Electron Induced Fracture of Polymeric Materials

    NASA Astrophysics Data System (ADS)

    Klakken, Michael Lee

    The application of high energy electrons onto a polymeric sample is known to induce electronic excitations which cause many reactions including dissociation, bond scissions and chemical reactions. Dissociation and bond scission tend to "weaken" the material while the chemical reactions tend to "strengthen" the material. It is hypothesized that the introduction of energetic electrons onto a stressed sample causes a decrease in the effective bond energy of the polymers main chains. The effect of electron bombardment was studied on the following materials: polyisoprene, polybutadiene, polyethylene, BAMO/THF (an energetic elastomer), butyl rubber, Kapton-H and Teflon. The techniques used in the study are: (1) measurement of the mechanical response of a sample mounted in a tension mode due to the electron application, (2) measurement of the change in the tear energy of an elastic material due to the electron beam and (3) generating the observed responses using a molecular dynamics computer simulation method. It was found that the force required to cause crack propagation in a sample mounted in tension decreased when the applied electron current was increased. Periodic patterns were also observed on the fracture surfaces of many of the materials which indicates that both crosslinking and chain scissions occurred in the induced fracture process. The tear energy was also observed to change with the application of the electron beam. The tear energy of polybutadiene was found to first increase (i.e. predominantly crosslinking) and then decrease (i.e. predominantly chain scissions) with increasing current while the tear energy of butyl rubber was found to steadily decrease as the current was increased. The decrease of the butyl rubber was modeled using a kinetic rate process theory that gave results that agreed well with the data. A molecular dynamics computer program was also used to model the electron induced fracture event and the failure event itself. The results obtained

  15. The accessibility of etheno-nucleotides to collisional quenchers and the nucleotide cleft in G- and F-actin.

    PubMed Central

    Root, D. D.; Reisler, E.

    1992-01-01

    Recent publication of the atomic structure of G-actin (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C., 1990, Nature 347, 37-44) raises questions about how the conformation of actin changes upon its polymerization. In this work, the effects of various quenchers of etheno-nucleotides bound to G- and F-actin were examined in order to assess polymerization-related changes in the nucleotide phosphate site. The Mg(2+)-induced polymerization of actin quenched the fluorescence of the etheno-nucleotides by approximately 20% simultaneously with the increase in light scattering by actin. A conformational change at the nucleotide binding site was also indicated by greater accessibility of F-actin than G-actin to positively, negatively, and neutrally charged collisional quenchers. The difference in accessibility between G- and F-actin was greatest for I-, indicating that the environment of the etheno group is more positively charged in the polymerized form of actin. Based on calculations of the change in electric potential of the environment of the etheno group, specific polymerization-related movements of charged residues in the atomic structure of G-actin are suggested. The binding of S-1 to epsilon-ATP-G-actin increased the accessibility of the etheno group to I- even over that in Mg(2+)-polymerized actin. The quenching of the etheno group by nitromethane was, however, unaffected by the binding of S-1 to actin. Thus, the binding of S-1 induces conformational changes in the cleft region of actin that are different from those caused by Mg2+ polymerization of actin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1304380

  16. Biophysical characterization of cofilin-induced extension-torsion coupling in actin filaments.

    PubMed

    Kim, Jae In; Kwon, Junpyo; Baek, Inchul; Na, Sungsoo

    2016-06-14

    Cofilin makes the actin filament flexible and thermally unstable by disassembling the filament and inducing bending and torsional compliance. Actin monomers bound to cofilin are able to chemically and mechanically interact in response to external forces. In this study, we performed two molecular dynamics tensile tests for actin and cofilactin filaments under identical conditions. Surprisingly, cofilactin filaments were found to be twisted, generating shear stress caused by torsion. Additionally, analysis by plane stress assumption indicated that the extension-torsion coupling effect increases the amount of principal stress by 10%. Using elasticity and solid mechanics theories, our study elucidates the role of cofilin in the disassembly of actin filaments under tensile forces. PMID:27143106

  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. Pressure-induced Polymerization in Substituted Acetylenes

    NASA Astrophysics Data System (ADS)

    Chellappa, Raja; Dattelbaum, Dana; Sheffield, Stephen; Robbins, David

    2011-06-01

    A fundamental understanding of shock-induced chemical reactions in organics is still lacking and there are limited studies devoted to determining reaction mechanisms, evolution of bonding, and effect of functional group substitutions. The fast timescale of reactions occurring during shock compression create significant experimental challenges (diagnostics) to fully quantify the mechanisms involved. Static compression provides a complementary route to investigate the equilibrium phase space and metastable intermediates during high pressure chemistry, although at a much slower timescale. In this study, we present our results from our ongoing high pressure in situ synchrotron x-ray diffraction and vibrational spectroscopy experiments on substituted acetylenes: tert-butyl acetylene [TBA: (CH3)3 -C ≡CH] and ethynyl trimethylsilane [ETMS: (CH3)3 -Si ≡CH]. We observed that the onset pressure of chemical reactions (at room temperature) in these compounds is significantly higher in static compression (TBA: 11 GPa and ETMS: 26 GPa) when compared to shock input pressures (TBA: 6.1 GPa and ETMS: 6.6 GPa). The products were polymeric in nature, recovered to ambient conditions with little degradation and fully characterized using spectroscopy, calorimetry, and other techniques to identify reaction mechanisms. LDRD-DR (PI: Dana Dattelbaum)

  19. Cytoskeletal F-actin polymerization from cytosolic G-actin occurs in the phagocytosing immunocytes of arthropods (Limulus polyphemus and Gromphadorhina portentosa): does [cAMP]i play any role?

    PubMed

    Gupta, A P; Campenot, E S

    1996-09-01

    Phagocytosis is a major defense reaction in arthropods and is accomplished by two blood cells (hemocytes), the granulocyte (GRs) and plasmatocytes (PLs), collectively called immunocytes. Immunocytes (principally the GRs) from two arthropods, Limulus polyphemus (horseshoe crab) and Gromphadorhina portentosa (Madagascar hissing cockroach) effectively phagocytose fluorescein isothiocyanate (FITC)-conjugated fluoresbrite microspheres (FITC-FM) and chicken (Gallus domesticus) erythrocytes within 1 hr of incubation. Although actin polymerization and changes in intracellular cAMP ([cAMP]i) levels occur during the early stages of phagocytosis in vertebrates, these two phenomena have not been studied in arthropod immunocytes. Using the DNase I inhibition assay, we found a decrease in cytosolic G-actin and an increase in the cytoskeletal F-actin in the phagocytosing immunocytes; the total actin in both resting and phagocytosing immunocytes remained constant. These results showed an 86% increase in F-actin in G. portentosa immunocytes and a 29% increase in those of L. polyphemus after 1 hr of initial incubation with FITC-FM. As in some vertebrates, the role of [cAMP]i in the early stages of phagocytosis in these two animals- and perhaps in arthropods in general-is variable; although we detected some negligible amounts of [cAMP]i (0.10-0.80 pmol/cell at different time intervals) in L. polyphemus immunocytes, it was inconclusive whether those in G. portentosa also contained [cAMP]i. Even in L. polyphemus, the difference in the amounts of [cAMP]i in resting and phagocytosing cells was insignificant (P > 0.05). It was also inconclusive whether [Ca2+]i and/or [Mg2+]i play any roles in the early stages of phagocytosis in the two arthropods in this study. These results suggest that the two phenomena (F-actin polymerization and levels of [cAMP]i in arthropods) are basically similar to those in vertebrate neutrophils and macrophages, which suggests that certain immunological

  20. Actin-based modeling of a transcriptionally competent nuclear substructure induced by transcription inhibition

    SciTech Connect

    Wang, I-F.; Chang, H.-Y.; James Shen, C.-K. . E-mail: ckshen@ccvax.sinica.edu.tw

    2006-11-15

    During transcription inactivation, the nuclear bodies in the mammalian cells often undergo reorganization. In particular, the interchromatin granule clusters, or IGCs, become colocalized with RNA polymerase II (RNAP II) upon treatment with transcription inhibitors. This colocalization has also been observed in untreated but transcriptionally inactive cells. We report here that the reorganized IGC domains are unique substructure consisting of outer shells made of SC35, ERK2, SF2/ASF, and actin. The apparently hollow holes of these domains contain clusters of RNAP II, mostly phosphorylated, and the splicing regulator SMN. This class of complexes are also the sites where prominent transcription activities are detected once the inhibitors are removed. Furthermore, actin polymerization is required for reorganization of the IGCs. In connection with this, immunoprecipitation and immunostaining experiments showed that nuclear actin is associated with IGCs and the reorganized IGC domains. The study thus provides further evidence for the existence of an actin-based nuclear skeleton structure in association with the dynamic reorganization processes in the nucleus. Overall, our data suggest that mammalian cells have adapted to utilize the reorganized, uniquely shaped IGC domains as the temporary storage sites of RNAP II transcription machineries in response to certain transient states of transcription inactivation.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-10-01

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

  3. Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation

    PubMed Central

    Smith, Elenoe C.; Teixeira, Alexandra M.; Chen, Rachel C.; Wang, Lin; Gao, Yuan; Hahn, Katherine L.

    2013-01-01

    How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryoblast leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, promotes muscle, neuron, and megakaryocyte differentiation. In muscle cells, where MKL1 subcellular localization is one mechanism by which cells control SRF activity, MKL1 translocation from the cytoplasm to the nucleus in response to actin polymerization is critical for its function as a transcriptional regulator. MKL1 localization is cell-type specific; it is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types and is constitutively nuclear in neuronal cells. In the present study, we report that in megakaryocytes, subcellular localization and regulation of MKL1 is dependent on RhoA activity and actin organization. Induction of megakaryocytic differentiation of human erythroleukemia cells by 12-O-tetradecanoylphorbol-13-acetate and primary megakaryocytes by thrombopoietin promotes MKL1 nuclear localization. This MKL1 localization is blocked by drugs inhibiting RhoA activity or actin polymerization. We also show that nuclear-localized MKL1 activates the transcription of SRF target genes. This report broadens our knowledge of the molecular mechanisms regulating megakaryocyte differentiation. PMID:23243284

  4. Small GTPase Rab21 Mediates Fibronectin Induced Actin Reorganization in Entamoeba histolytica: Implications in Pathogen Invasion

    PubMed Central

    Emmanuel, Merlyn; Nakano, Yumiko Saito; Nozaki, Tomoyoshi; Datta, Sunando

    2015-01-01

    The protozoan parasite Entamoeba histolytica causes a wide spectrum of intestinal infections. In severe cases, the trophozoites can breach the mucosal barrier, invade the intestinal epithelium and travel via the portal circulation to the liver, where they cause hepatic abscesses, which can prove fatal if left untreated. The host Extra Cellular Matrix (ECM) plays a crucial role in amoebic invasion by triggering an array of cellular responses in the parasite, including induction of actin rich adhesion structures. Similar actin rich protrusive structures, known as ‘invadosomes’, promote chemotactic migration of the metastatic cancer cells and non-transformed cells by remodeling the ECM. Recent studies showed a central role for Rab GTPases, the master regulators of vesicular trafficking, in biogenesis of invadosomes. Here, we showed that fibronectin, a major host ECM component induced actin remodeling in the parasite in a Rab21 dependent manner. The focalized actin structures formed were reminiscent of the mammalian invadosomes. By using various approaches, such as immunofluorescence confocal microscopy and scanning electron microscopy, along with in vitro invasion assay and matrix degradation assay, we show that the fibronectin induced formation of amoebic actin dots depend on the nucleotide status of the GTPase. The ECM components, fibronectin and collagen type I, displayed differential control over the formation of actin dots, with fibronectin positively and collagen type I negatively modulating it. The cell surface adhesion molecule Gal/GalNAc complex was also found to impose additional regulation on this process, which might have implication in collagen type I mediated suppression of actin dots. PMID:25730114

  5. The regulation of actin polymerization in differentiating U937 cells correlates with increased membrane levels of the pertussis-toxin-sensitive G-protein Gi2.

    PubMed Central

    Sheth, B; Banks, P; Burton, D R; Monk, P N

    1991-01-01

    Undifferentiated U937 cells appear to lack a capacity of increase cellular F-actin. However, electropermeabilized cells gain the ability to respond in this way to a guanine nucleotide analogue, guanosine 5'-[gamma-thio]trisphosphate (GTP[S]) after 1 h of treatment with dibutyryl cyclic AMP (db-cAMP). The results reported here show that the levels of membrane association of the G-protein Gi2 alpha increase with a time course identical with that of the GTP[S]-sensitivity of electropermeabilized cells. These results suggest that Gi2 alpha may be involved in the signal-transduction pathway leading to actin polymerization in db-cAMP-differentiated U937 cells. PMID:1645523

  6. Direct interaction of Cucurbitacin E isolated from Alsomitra macrocarpa to actin filament

    PubMed Central

    Momma, Keiko; Masuzawa, Yuko; Nakai, Naomi; Chujo, Moeko; Murakami, Akira; Kioka, Noriyuki; Kiyama, Yasunori; Akita, Toru

    2007-01-01

    A methanol extract of Alsomitra macrocarpa leaves and branches induced a marked alteration of cell morphology in a human stellate cell line (LX-2). Similar morphologic alterations were observed in several other cell lines. Active compound was purified from the extract and determined to be cucurbitacin E (Cuc E). It has been known that Cuc E causes marked disruption of the actin cytoskeleton, supporting our observation, but how Cuc E altered the actin cytoskeleton has not been elucidated. By using the standard fluorescence assay using copolymerization and depolymerization of native and pyrene labelled actin, this study revealed that Cuc E interacted directly with actin consequently stabilizing the polymerized actin. When NIH-3T3 cells exogenously expressing YFP-labeled actin were treated with Cuc E, firstly the aggregation of globular actin and secondly the aggregation of actin including disrupted fibrous actin in the cells was observed. PMID:19002839

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

    PubMed

    Takaine, Masak; Numata, Osamu; Nakano, Kentaro

    2009-10-21

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

  8. Unprecedented inhibition of tubulin polymerization directed by gold nanoparticles inducing cell cycle arrest and apoptosis

    NASA Astrophysics Data System (ADS)

    Choudhury, Diptiman; Xavier, Paulrajpillai Lourdu; Chaudhari, Kamalesh; John, Robin; Dasgupta, Anjan Kumar; Pradeep, Thalappil; Chakrabarti, Gopal

    2013-05-01

    The effect of gold nanoparticles (AuNPs) on the polymerization of tubulin has not been examined till now. We report that interaction of weakly protected AuNPs with microtubules (MTs) could cause inhibition of polymerization and aggregation in the cell free system. We estimate that single citrate capped AuNPs could cause aggregation of ~105 tubulin heterodimers. Investigation of the nature of inhibition of polymerization and aggregation by Raman and Fourier transform-infrared (FTIR) spectroscopies indicated partial conformational changes of tubulin and microtubules, thus revealing that AuNP-induced conformational change is the driving force behind the observed phenomenon. Cell culture experiments were carried out to check whether this can happen inside a cell. Dark field microscopy (DFM) combined with hyperspectral imaging (HSI) along with flow cytometric (FC) and confocal laser scanning microscopic (CLSM) analyses suggested that AuNPs entered the cell, caused aggregation of the MTs of A549 cells, leading to cell cycle arrest at the G0/G1 phase and concomitant apoptosis. Further, Western blot analysis indicated the upregulation of mitochondrial apoptosis proteins such as Bax and p53, down regulation of Bcl-2 and cleavage of poly(ADP-ribose) polymerase (PARP) confirming mitochondrial apoptosis. Western blot run after cold-depolymerization revealed an increase in the aggregated insoluble intracellular tubulin while the control and actin did not aggregate, suggesting microtubule damage induced cell cycle arrest and apoptosis. The observed polymerization inhibition and cytotoxic effects were dependent on the size and concentration of the AuNPs used and also on the incubation time. As microtubules are important cellular structures and target for anti-cancer drugs, this first observation of nanoparticles-induced protein's conformational change-based aggregation of the tubulin-MT system is of high importance, and would be useful in the understanding of cancer therapeutics

  9. 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

  10. 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

  11. 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

  12. Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41.

    PubMed Central

    Kim, E; Miller, C J; Motoki, M; Seguro, K; Muhlrad, A; Reisler, E

    1996-01-01

    Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (S1) with the His-40-Gly-42 site in the 38-52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-S1 ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of K(m) and Vmax of the acto-S1 ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that S1 does not bind to the 40-42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-S1 binding in the absence of nucleotides, indicating that the 40-42 site does not contribute much to rigor acto-S1 binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by S1. It is proposed that these changes in the 38-52 loop on actin originate from S1 binding to other myosin recognition sites on actin. Images FIGURE 7 FIGURE 8 PMID:8785300

  13. 2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils.

    PubMed

    Conejeros, I; Velásquez, Z D; Carretta, M D; Alarcón, P; Hidalgo, M A; Burgos, R A

    2012-01-15

    2-Aminoethoxydiphenyl borate (2-APB) interferes with the Ca(2+) influx and reduces the ROS production, gelatinase secretion and CD11b expression in bovine neutrophils. Moreover, it has been suggested that inhibition of the Ca(2+) channel involved in the store operated Ca(2+) entry (SOCE) is a potential target for the development of new anti-inflammatory drugs in cattle, however it is unknown whether 2-APB affects neutrophil functions associated with the innate immune response. This study describes the effect of 2-APB, a putative SOCE inhibitor, on alkaline phosphatase activity a marker of secretory vesicles, CD63 a marker for azurophil granules, F-actin polymerization and in vitro chemotaxis in bovine neutrophils stimulated with platelet-activating factor (PAF). Also, we evaluated the effect of 2-APB in the phagocytic activity against Escherichia coli and Staphylococcus aureus bioparticles. We observed that doses of 2-APB ≥10 μM significantly reduced alkaline phosphatase activity and in vitro chemotaxis, whereas concentrations of 2-APB ≥50 μM reduced CD63 expression and F-actin polymerization. Finally, we observed that 2-APB did not affect the phagocytic activity in neutrophils incubated with E. coli and S. aureus bioparticles. We concluded that inhibition of Ca(2+) influx could be a useful strategy to reduce inflammatory process in cattle. PMID:22226550

  14. Polymeric nanocomposite proton exchange membranes prepared by radiation-induced polymerization for direct methanol fuel cell

    NASA Astrophysics Data System (ADS)

    Kim, Young-Seok; Seo, Kwang-Seok; Choi, Seong-Ho

    2016-01-01

    The vinyl group-modified montmorillonite clay (F-MMT), vinyl group-modified graphene oxide (F-GO), and vinyl group-modified multi-walled carbon nanotube (F-MWNT) were first prepared by ion exchange reaction of 1-[(4-ethylphenyl)methyl]-3-butyl-imidazolium chloride in order to use the materials for protection against methanol cross-over in direct methanol fuel cell (DMFC) membrane. Then polymeric nanocomposite membranes with F-MMT, F-GO, and F-MWNT were prepared by the solvent casting method after radiation-induced polymerization of vinyl monomers in water-methanol mixture solvents. The proton conductivity, water uptake, ion-exchange capacity, methanol permeability, and DMFC performance of the polymeric nanocomposite membranes with F-MMT, F-GO, and F-MWNT were evaluated.

  15. Actin Turnover-Mediated Gravity Response in Maize Root Apices

    PubMed Central

    Mancuso, Stefano; Barlow, Peter W; Volkmann, Dieter

    2006-01-01

    The dynamic actin cytoskeleton has been proposed to be linked to gravity sensing in plants but the mechanistic understanding of these processes remains unknown. We have performed detailed pharmacological analyses of the role of the dynamic actin cytoskeleton in gravibending of maize (Zea mays) root apices. Depolymerization of actin filaments with two drugs having different mode of their actions, cytochalasin D and latrunculin B, stimulated root gravibending. By contrast, drug-induced stimulation of actin polymerization and inhibition of actin turnover, using two different agents phalloidin and jasplakinolide, compromised the root gravibending. Importantly, all these actin drugs inhibited root growth to similar extents suggesting that high actin turnover is essential for the gravity-related growth responses rather than for the general growth process. Both latrunculin B and cytochalasin D treatments inhibited root growth but restored gravibending of the decapped root apices, indicating that there is a strong potential for effective actin-mediated gravity sensing outside the cap. This elusive gravity sensing outside the root cap is dependent not only on the high rate of actin turnover but also on weakening of myosin activities, as general inhibition of myosin ATPases induced stimulation of gravibending of the decapped root apices. Collectively, these data provide evidence for the actin turnover-mediated gravity sensing outside the root cap. PMID:19521476

  16. 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

  17. 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

  18. Photo-induced polymerization of polyaniline

    SciTech Connect

    Barros, R.A. de; Azevedo, W.M. de; Aguiar, F.M. de

    2003-03-15

    A straightforward route to prepare polyaniline is presented in which photons and metallic ions replace conventional oxidants to promote polymerization of aniline monomer. The photopolymerization methods yield a composite material that has been characterized by its UV, visible and Raman spectroscopic analysis and by scanning electron microscopy and X-ray microanalysis as well. Intriguing forms of silver wires embedded in polyaniline are observed, typically of 1 {mu}m wide and up to 100 {mu}m long. It is shown that the morphology of the resulting conducting polymer strongly depends on the excitation wavelength, while a globular morphology is found for the UV synthesis and a fibrillar one is found for visible light excitation.

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

    SciTech Connect

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

    2009-06-09

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

  20. Radiation-induced graft polymerization of amphiphilic monomers with different polymerization characteristics onto hydrophobic polysilane

    NASA Astrophysics Data System (ADS)

    Tanaka, Hidenori; Iwasaki, Isao; Kunai, Yuichiro; Sato, Nobuhiro; Matsuyama, Tomochika

    2011-08-01

    The structures of poly(methyl-n-propylsilane) (PMPrS) amphiphilically modified through γ-ray-induced graft polymerization were investigated with 1H NMR measurement. By the use of methyl methacrylate (MMA) or diethyl fumarate (DEF) as monomers for the graft polymerization, grafting yield rose with increasing total absorption dose and monomer concentrations, but decreased with increasing dose rate. This result means that grafting yield of modified PMPrS can be controlled by changing irradiation conditions. However, the number of PMMA or PDEF graft chains per PMPrS chain was estimated to be less than 1.0 by analysis of 1H NMR spectra, and this value was lower than that we had expected. To improve graft density, maleic anhydride (MAH), which is known as a non-homopolymerizable monomer in radical polymerization, was used as a monomer for grafting. As a result, high density grafting (one MAH unit for 4.2 silicon atoms) was attained. It demonstrates that the structure of γ-ray-modified polysilane strongly depends on the polymerization characteristics of grafted monomers.

  1. 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

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

    SciTech Connect

    Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki

    2013-11-29

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

  3. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

    SciTech Connect

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

    2010-06-04

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca{sup 2+}-dependent AMPK activation via calmodulin-dependent protein kinase kinase-{beta}(CaMKK{beta}), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKK{beta} inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  4. 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.

  5. Sulfonic acid catalysts prepared by radiation-induced graft polymerization

    SciTech Connect

    Mizota, Tomotoshi; Tsuneda, Satoshi; Saito, Kyoichi, Saito

    1994-09-01

    In this study, the authors prepared two variations of graft-type acid catalysts with different adjacent groups by radiation-induced graft polymerization (RIGP), and compared the hydrolytic activity of the resultant acid catalysts for methyl acetate with that of commercially available SO{sub 3}H-type ion-exchange beads with different degrees of cross-linking. 8 refs., 3 figs.

  6. Reaction-diffusion waves of reversible actin filament assembly drive cell oscillations and locomotion

    NASA Astrophysics Data System (ADS)

    Vicker, Michael G.

    Excitation waves of actin filament (F-actin) polymerization and depolymerization have been visualized in fixed and in living Dictyostelium cells by confocal and fluorescence resonance energy transfer (FRET) microscopy. F-actin waves generate supramolecular F-actin patterns, typical of chemical wave systems. Scroll waves distinguishable as sphere, ring and spiral patterns propagate up to several micrometres in diameter in a few seconds at wavefront speeds measured at up to 25 µm/min. These newly identified nonlinear F-actin dynamics drive eukaryotic cell locomotion. F-actin autowaves also induce oscillatory modi of temporally variable frequency and amplitude as cell surface projections, including pseudopodia and lamellipodia, which may traverse the cell surface as waves. F-actin waves may also govern a range of cell functions and behaviours, including phagocytosis, chemotaxis, cell surface receptor activity and biological rhythms.

  7. 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

  8. 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

  9. Self-Organized Gels in DNA/F-Actin Mixtures without Crosslinkers: Networks of Induced Nematic Domains with Tunable Density

    NASA Astrophysics Data System (ADS)

    Lai, Ghee Hwee; Butler, John C.; Zribi, Olena V.; Smalyukh, Ivan I.; Angelini, Thomas E.; Purdy, Kirstin R.; Golestanian, Ramin; Wong, Gerard C. L.

    2008-11-01

    We examine mixtures of DNA and filamentous actin (F-actin) as a model system of like-charged rigid rods and flexible chains. Confocal microscopy reveals the formation of elongated nematic F-actin domains reticulated via defect-free vertices into a network embedded in a mesh of random DNA. Synchrotron x-ray scattering results indicate that the DNA mesh squeezes the F-actin domains into a nematic state with an interactin spacing that decreases with increasing DNA concentration as dactin∝ρDNA-1/2. Interestingly, the system changes from a counterion-controlled regime to a depletion-controlled regime with added salt, with drastic consequences for the osmotic pressure induced phase behavior.

  10. Polymerization-Induced Phase Separation in Vinyl Ester Resins

    NASA Astrophysics Data System (ADS)

    Ganglani, Manisha; Torkelson, John; Carr, Stephen

    2000-03-01

    In certain multi-component, crosslinking polymer systems, phase separation is induced by polymerization, a process call polymerization-induced phase separation (PIPS) in which there exists a competition between reaction rate and phase separation rate. The final morphology and properties of a system that experiences PIPS depend on the outcome of this competition. Thus, by controlling these rates, it would be possible to control end properties. In fact, this theory has been applied for the creation of polymer-dispersed liquid crystals (PDLCs) where cure occurs via condensation reactions or via free radical polymerization initiated by UV light. This research examines PIPS in the vinyl ester (VE) resins, which are popular as matrix materials in polymer composites. Cure of the VE resins is more complicated because it uses initiators and therefore requires more time and offers less control than cure by photopolymerization. To better understand the PIPS process in the VE resins, this research separates the two competitive effects and examines each one in turn. Initial experiments use a model system to focus on the effect of PIPS in the absence of crosslinking.

  11. Mitochondrial Dysfunction, Disruption of F-Actin Polymerization, and Transcriptomic Alterations in Zebrafish Larvae Exposed to Trichloroethylene.

    PubMed

    Wirbisky, Sara E; Damayanti, Nur P; Mahapatra, Cecon T; Sepúlveda, Maria S; Irudayaraj, Joseph; Freeman, Jennifer L

    2016-02-15

    Trichloroethylene (TCE) is primarily used as an industrial degreasing agent and has been in use since the 1940s. TCE is released into the soil, surface, and groundwater. From an environmental and regulatory standpoint, more than half of Superfund hazardous waste sites on the National Priority List are contaminated with TCE. Occupational exposure to TCE occurs primarily via inhalation, while environmental TCE exposure also occurs through ingestion of contaminated drinking water. Current literature links TCE exposure to various adverse health effects including cardiovascular toxicity. Current studies aiming to address developmental cardiovascular toxicity utilized rodent and avian models, with the majority of studies using relatively higher parts per million (mg/L) doses. In this study, to further investigate developmental cardiotoxicity of TCE, zebrafish embryos were treated with 0, 10, 100, or 500 parts per billion (ppb; μg/L) TCE during embryogenesis and/or through early larval stages. After the appropriate exposure period, angiogenesis, F-actin, and mitochondrial function were assessed. A significant dose-response decrease in angiogenesis, F-actin, and mitochondrial function was observed. To further complement this data, a transcriptomic profile of zebrafish larvae was completed to identify gene alterations associated with the 10 ppb TCE exposure. Results from the transcriptomic data revealed that embryonic TCE exposure caused significant changes in genes associated with cardiovascular disease, cancer, and organismal injury and abnormalities with a number of targets in the FAK signaling pathway. Overall, results from our study support TCE as a developmental cardiovascular toxicant, provide molecular targets and pathways for investigation in future studies, and indicate a need for continued priority for environmental regulation. PMID:26745549

  12. α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

    PubMed Central

    Sousa, Vítor L.; Bellani, Serena; Giannandrea, Maila; Yousuf, Malikmohamed; Valtorta, Flavia; Meldolesi, Jacopo

    2009-01-01

    The function of α-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, α-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson's disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that α-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type α-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant α-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant α-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the α-synuclein gene, electroporation of wild-type α-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P α-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, α-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration. PMID:19553474

  13. Cytoarchitecture of Kirsten sarcoma virus-transformed rat kidney fibroblasts: butyrate-induced reorganization within the actin microfilament network.

    PubMed

    Ryan, M P; Higgins, P J

    1988-10-01

    Murine sarcoma virus-transformed rat fibroblasts (KNRK cells) undergo marked cytoarchitectural reorganization during in vitro exposure to sodium-n-butyrate (NaB) resulting in restoration of (1) a more typical fibroblastoid morphology, (2) proper cell-to-cell orientation, and (3) substratum adherence. Augmented cell spreading, involving greater than 90% of the population, was a function of culture density and time of exposure to NaB (2 mM final concentration). Induced cell spreading reflected a 2.5- to 3.0-fold increase in both total cellular actin content and deposition of actin into the detergent-resistant cytoskeleton. Cytoskeletal actin deposition in response to NaB was accompanied by the formation of occasionally dense, parallel alignments of F-actin-containing microfilaments and by a dramatic increase in the size and incidence of actin-enriched membrane ruffles. Long-term NaB-treated cells exhibited parallel orientations of microfilaments similar to those found in untransformed fibroblasts. Increased cytoskeletal actin occurred within 24 hr of NaB exposure, correlating with the initial reorganization of actin-containing microfilaments detected microscopically, and reflected concomitant 3-fold increases in cellular alpha-actinin and fibronectin content. In contrast, the amount of vimentin, tropomyosin, and tubulin in NaB-treated cells was significantly decreased. NaB-induced morphologic restructuring of sarcoma virus-transformed fibroblasts, thus, impacts on all three basic cytoskeletal systems. Selective increases, however, were evident in particular cytoskeletal proteins (actin, alpha-actinin, fibronectin) implicated in microfilament networking and cell spreading. PMID:2844835

  14. Reticulated Nanoporous Polymers by Controlled Polymerization-Induced Microphase Separation

    SciTech Connect

    Seo, Myungeun; Hillmyer, Marc A.

    2013-04-08

    Materials with percolating mesopores are attractive for applications such as catalysis, nanotemplating, and separations. Polymeric frameworks are particularly appealing because the chemical composition and the surface chemistry are readily tunable. We report on the preparation of robust nanoporous polymers with percolating pores in the 4- to 8-nanometer range from a microphase-separated bicontinuous precursor. We combined polymerization-induced phase separation with in situ block polymer formation from a mixture of multifunctional monomers and a chemically etchable polymer containing a terminal chain transfer agent. This marriage results in microphase separation of the mixture into continuous domains of the etchable polymer and the emergent cross-linked polymer. Precise control over pore size distribution and mechanical integrity renders these materials particularly suited for various advanced applications.

  15. Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells.

    PubMed

    Pfannes, Eva K B; Anielski, Alexander; Gerhardt, Matthias; Beta, Carsten

    2013-12-01

    Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system. PMID:24136144

  16. 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.

  17. MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

    PubMed Central

    Kobayashi, Miho; Nishita, Michiru; Mishima, Toshiaki; Ohashi, Kazumasa; Mizuno, Kensaku

    2006-01-01

    Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells. PMID:16456544

  18. BteA Secreted from the Bordetella bronchiseptica Type III Secetion System Induces Necrosis through an Actin Cytoskeleton Signaling Pathway and Inhibits Phagocytosis by Macrophages

    PubMed Central

    Kuwae, Asaomi; Momose, Fumitaka; Nagamatsu, Kanna; Suyama, Yasuharu; Abe, Akio

    2016-01-01

    BteA is one of the effectors secreted from the Bordetella bronchiseptica type III secretion system. It has been reported that BteA induces necrosis in mammalian cells; however, the roles of BteA during the infection process are largely unknown. In order to investigate the BteA functions, morphological changes of the cells infected with the wild-type B. bronchiseptica were examined by time-lapse microscopy. L2 cells, a rat lung epithelial cell line, spread at 1.6 hours after B. bronchiseptica infection. Membrane ruffles were observed at peripheral parts of infected cells during the cell spreading. BteA-dependent cytotoxicity and cell detachment were inhibited by addition of cytochalasin D, an actin polymerization inhibitor. Domain analyses of BteA suggested that two separate amino acid regions, 200–312 and 400–658, were required for the necrosis induction. In order to examine the intra/intermolecular interactions of BteA, the amino- and the carboxyl-terminal moieties were purified as recombinant proteins from Escherichia coli. The amino-terminal moiety of BteA appeared to interact with the carboxyl-terminal moiety in the pull-down assay in vitro. When we measured the amounts of bacteria phagocytosed by J774A.1, a macrophage-like cell line, the phagocytosed amounts of B. bronchiseptica strains that deliver BteA into the host cell cytoplasm were significantly lower than those of strains that lost the ability to translocate BteA into the host cell cytoplasm. These results suggest that B. bronchiseptica induce necrosis by exploiting the actin polymerization signaling pathway and inhibit macrophage phagocytosis. PMID:26828590

  19. Inhibition of FSS-induced actin cytoskeleton reorganization by silencing LIMK2 gene increases the mechanosensitivity of primary osteoblasts.

    PubMed

    Yang, Zhi; Tan, Shuyi; Shen, Yun; Chen, Rui; Wu, Changjing; Xu, Yajuan; Song, Zijun; Fu, Qiang

    2015-05-01

    Mechanical stimulation plays an important role in bone cell metabolic activity. However, bone cells lose their mechanosensitivity upon continuous mechanical stimulation (desensitization) and they can recover the sensitivity with insertion of appropriate rest period into the mechanical loading profiles. The concrete molecular mechanism behind the regulation of cell mechanosensitivity still remains unclear. As one kind of mechanosensitive cell to react to the mechanical stimulation, osteoblasts respond to fluid shear stress (FSS) with actin cytoskeleton reorganization, and the remodeling of actin cytoskeleton is closely associated with the alteration of cell mechanosensitivity. In order to find out whether inhibiting the actin cytoskeleton reorganization by silencing LIM-kinase 2 (LIMK2) gene would increase the mechanosensitivity of primary osteoblasts, we attenuated the formation of actin stress fiber under FSS in a more specific way: inhibiting the LIMK2 expression by RNA interference. We found that inhibition of LIMK2 expression by RNA interference attenuated the formation of FSS-induced actin stress fiber, and simultaneously maintained the integrity of actin cytoskeleton in primary osteoblasts. We confirmed that the decreased actin cytoskeleton reorganization in response to LIMK2 inhibition during FSS increased the mechanosensitivity of the osteoblasts, based on the increased c-Fos and COX-2 expression as well as the enhanced proliferative activity in response to FSS. These data suggest that osteoblasts can increase their mechanosensitivity under continuous mechanical stimulation by reducing the actin stress fiber formation through inhibiting the LIMK2 expression. This study provides us with a new and more specific method to regulate the osteoblast mechanosensitivity, and also a new therapeutic target to cure bone related diseases, which is of importance in maintaining bone mass and promoting osteogenesis. PMID:25549868

  20. 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

  1. 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

  2. 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.

  3. Unprecedented inhibition of tubulin polymerization directed by gold nanoparticles inducing cell cycle arrest and apoptosis

    NASA Astrophysics Data System (ADS)

    Choudhury, Diptiman; Xavier, Paulrajpillai Lourdu; Chaudhari, Kamalesh; John, Robin; Dasgupta, Anjan Kumar; Pradeep, Thalappil; Chakrabarti, Gopal

    2013-05-01

    The effect of gold nanoparticles (AuNPs) on the polymerization of tubulin has not been examined till now. We report that interaction of weakly protected AuNPs with microtubules (MTs) could cause inhibition of polymerization and aggregation in the cell free system. We estimate that single citrate capped AuNPs could cause aggregation of ~105 tubulin heterodimers. Investigation of the nature of inhibition of polymerization and aggregation by Raman and Fourier transform-infrared (FTIR) spectroscopies indicated partial conformational changes of tubulin and microtubules, thus revealing that AuNP-induced conformational change is the driving force behind the observed phenomenon. Cell culture experiments were carried out to check whether this can happen inside a cell. Dark field microscopy (DFM) combined with hyperspectral imaging (HSI) along with flow cytometric (FC) and confocal laser scanning microscopic (CLSM) analyses suggested that AuNPs entered the cell, caused aggregation of the MTs of A549 cells, leading to cell cycle arrest at the G0/G1 phase and concomitant apoptosis. Further, Western blot analysis indicated the upregulation of mitochondrial apoptosis proteins such as Bax and p53, down regulation of Bcl-2 and cleavage of poly(ADP-ribose) polymerase (PARP) confirming mitochondrial apoptosis. Western blot run after cold-depolymerization revealed an increase in the aggregated insoluble intracellular tubulin while the control and actin did not aggregate, suggesting microtubule damage induced cell cycle arrest and apoptosis. The observed polymerization inhibition and cytotoxic effects were dependent on the size and concentration of the AuNPs used and also on the incubation time. As microtubules are important cellular structures and target for anti-cancer drugs, this first observation of nanoparticles-induced protein's conformational change-based aggregation of the tubulin-MT system is of high importance, and would be useful in the understanding of cancer therapeutics

  4. A vital role for myosin-9 in puromycin aminonucleoside-induced podocyte injury by affecting actin cytoskeleton.

    PubMed

    Yuan, Yanggang; Zhao, Chuanyan; An, Xiaofei; Wu, Lin; Wang, Hui; Zhao, Min; Bai, Mi; Duan, Suyan; Zhang, Bo; Zhang, Aihua; Xing, Changying

    2016-06-01

    Podocyte injury is an early pathological change of many kidney diseases. In particular, the actin cytoskeleton plays an important role in maintaining the normal function of podocytes. Disruption of the actin cytoskeleton is a feature of podocyte injury in proteinuric nephropathies. Recent studies showed that myosin-9 was localized in the podocyte foot processes and was necessary in maintaining podocyte structural homeostasis. However, it is unclear whether myosin-9 maintains podocyte structure by affecting actin cytoskleton. Here, the role of myosin-9 in puromycin aminonucleoside (PAN)-induced podocyte injury was explored both in vitro and in vivo. In cultured mouse podocytes (MPC5), it was determined that PAN downregulated myosin-9 expression, disrupted the actin cytoskeleton and reduced the adhesion ability. Reduced myosin-9 expression by siRNA precipitated podocyte cytoskeletal damage and accelerated PAN-induced podocyte detachment. Overexpression of myosin-9 protected against PAN-induced podocyte detachment. Furthermore, administration of an antioxidant Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) inhibited PAN-induced podocyte cytoskeletal damage and podocyte detachment by restoring the expression of myosin-9. In the rat PAN nephropathy model, MnTBAP could also attenuate PAN-induced reduction of myosin-9 and podocyte loss. Taken together, these findings pinpointed that oxidative stress contributed to PAN-induced podocyte injury through the repression of a cytoskeletal protein myosin-9, which provided novel insights into a potential target for the treatment of podocyte injury-associated glomerulopathies. PMID:26902808

  5. 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 ...

  6. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-09-01

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

  9. Force-Driven Polymerization and Turgor-Induced Wall Expansion.

    PubMed

    Ali, Olivier; Traas, Jan

    2016-05-01

    While many molecular players involved in growth control have been identified in the past decades, it is often unknown how they mechanistically act to induce specific shape changes during development. Plant morphogenesis results from the turgor-induced yielding of the extracellular and load-bearing cell wall. Its mechanochemical equilibrium appears as a fundamental link between molecular growth regulation and the effective shape evolution of the tissue. We focus here on force-driven polymerization of the cell wall as a central process in growth control. We propose that mechanical forces facilitate the insertion of wall components, in particular pectins, a process that can be modulated through genetic regulation. We formalize this idea in a mathematical model, which we subsequently test with published experimental results. PMID:26895732

  10. 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

  11. The Disruption of the Cytoskeleton during Semaphorin 3A induced Growth Cone Collapse Correlates with Differences in Actin Organization and Associated Binding Proteins

    PubMed Central

    Brown, Jacquelyn A; Bridgman, Paul C

    2010-01-01

    Repulsive guidance cues induce growth cone collapse or collapse and retraction. Collapse results from disruption and loss of the actin cytoskeleton. Actin rich regions of growth cones contain binding proteins that influence filament organization, such as Arp2/3, cortactin, and fascin, but little is known about the role that these proteins play in collapse. Here we show that Semaphorin 3A (Sema 3A), which is repulsive to mouse dorsal root ganglion neurons, has unequal effects on actin binding proteins and their associated filaments. The immunofluorescence staining intensity of Arp-2 and cortactin decreases relative to total protein, while in unextracted growth cones fascin increases. Fascin and myosin IIB staining redistribute and show increased overlap. The degree of actin filament loss during collapse correlates with filament superstructures detected by rotary shadow electron microscopy. Collapse results in the loss of branched f-actin meshworks, while actin bundles are partially retained to varying degrees. Taken together with the known affects of Sema 3A on actin, this suggests a model for collapse that follows a sequence; depolymerization of actin meshworks followed by partial depolymerization of fascin associated actin bundles and their movement to the neurite to complete collapse. The relocated fascin associated actin bundles may provide the substrate for actomyosin contractions that produce retraction. PMID:19513995

  12. Industrialization of radiation-induced emulsion polymerization ----technological process and its advantages

    NASA Astrophysics Data System (ADS)

    Zhicheng, Zhang; Manwei, Zhang

    1993-07-01

    A technological process for industrialization of radiation induced emulsion polymerization was introduced briefly. A batch process rather than continuous one was adopted in the industrial-scale production. The advantages of radiation induced emulsion polymerization were described in comparison with chemical initiated process.

  13. Radiation-induced polymerization for the immobilization of penicillin acylase

    SciTech Connect

    Boccu, E.; Carenza, M.; Lora, S.; Palma, G.; Veronese, F.M.

    1987-06-01

    The immobilization of Escherichia coli penicillin acylase was investigated by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperature. A leak-proof composite that does not swell in water was obtained by adding the cross-linking agent trimethylolpropane trimethacrylate to the monomer-aqueous enzyme mixture. Penicillin acylase, which was immobilized with greater than 70% yield, possessed a higher Km value toward the substrate 6-nitro-3-phenylacetamidobenzoic acid than the free enzyme form (Km = 1.7 X 10(-5) and 1 X 10(-5) M, respectively). The structural stability of immobilized penicillin acylase, as assessed by heat, guanidinium chloride, and pH denaturation profiles, was very similar to that of the free-enzyme form, thus suggesting that penicillin acylase was entrapped in its native state into aqueous free spaces of the polymer matrix.

  14. 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

  15. Polymerization-Induced Self-Assembly of Block Copolymer Nano-objects via RAFT Aqueous Dispersion Polymerization

    PubMed Central

    2014-01-01

    In this Perspective, we discuss the recent development of polymerization-induced self-assembly mediated by reversible addition–fragmentation chain transfer (RAFT) aqueous dispersion polymerization. This approach has quickly become a powerful and versatile technique for the synthesis of a wide range of bespoke organic diblock copolymer nano-objects of controllable size, morphology, and surface functionality. Given its potential scalability, such environmentally-friendly formulations are expected to offer many potential applications, such as novel Pickering emulsifiers, efficient microencapsulation vehicles, and sterilizable thermo-responsive hydrogels for the cost-effective long-term storage of mammalian cells. PMID:24968281

  16. Polymerization-induced self-assembly of block copolymer nano-objects via RAFT aqueous dispersion polymerization.

    PubMed

    Warren, Nicholas J; Armes, Steven P

    2014-07-23

    In this Perspective, we discuss the recent development of polymerization-induced self-assembly mediated by reversible addition-fragmentation chain transfer (RAFT) aqueous dispersion polymerization. This approach has quickly become a powerful and versatile technique for the synthesis of a wide range of bespoke organic diblock copolymer nano-objects of controllable size, morphology, and surface functionality. Given its potential scalability, such environmentally-friendly formulations are expected to offer many potential applications, such as novel Pickering emulsifiers, efficient microencapsulation vehicles, and sterilizable thermo-responsive hydrogels for the cost-effective long-term storage of mammalian cells. PMID:24968281

  17. 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.

  18. Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking.

    PubMed

    Arnette, Christopher; Frye, Keyada; Kaverina, Irina

    2016-01-01

    The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms. PMID:26866809

  19. Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking

    PubMed Central

    Arnette, Christopher; Frye, Keyada; Kaverina, Irina

    2016-01-01

    The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms. PMID:26866809

  20. An insight into polymerization-induced self-assembly by dissipative particle dynamics simulation.

    PubMed

    Huang, Feng; Lv, Yisheng; Wang, Liquan; Xu, Pengxiang; Lin, Jiaping; Lin, Shaoliang

    2016-08-14

    Polymerization-induced self-assembly is a one-pot route to produce concentrated dispersions of block copolymer nano-objects. Herein, dissipative particle dynamics simulations with a reaction model were employed to investigate the behaviors of polymerization-induced self-assembly. The polymerization kinetics in the polymerization-induced self-assembly were analyzed by comparing with solution polymerization. It was found that the polymerization rate enhances in the initial stage and decreases in the later stage. In addition, the effects of polymerization rate, length of macromolecular initiators, and concentration on the aggregate morphologies and formation pathway were studied. The polymerization rate and the length of the macromolecular initiators are found to have a marked influence on the pathway of the aggregate formations and the final structures. Morphology diagrams were mapped correspondingly. A comparison between simulation results and experimental findings is also made and an agreement is shown. This work can enrich our knowledge about polymerization-induced self-assembly. PMID:27414465

  1. Symmetry breaking in actin gels - Implications for cellular motility

    NASA Astrophysics Data System (ADS)

    John, Karin; Peyla, Philippe; Misbah, Chaouqi

    2007-03-01

    The physical origin of cell motility is not fully understood. Recently minimal model systems have shown, that polymerizing actin itself can produce a motile force, without the help of motor proteins. Pathogens like Shigella or Listeria use actin to propel themselves forward in their host cell. The same process can be mimicked with polystyrene beads covered with the activating protein ActA, which reside in a solution containing actin monomers. ActA induces the growth of an actin gel at the bead surface. Initially the gel grows symmetrically around the bead until a critical size is reached. Subsequently one observes a symmetry breaking and the gel starts to grow asymmetrically around the bead developing a tail of actin at one side. This symmetry breaking is accompanied by a directed movement of the bead, with the actin tail trailing behind the bead. Force generation relies on the combination of two properties: growth and elasticity of the actin gel. We study this phenomenon theoretically within the framework of a linear elasticity theory and linear flux-force relationships for the evolution of an elastic gel around a hard sphere. Conditions for a parity symmetry breaking are identified analytically and illustrated numerically with the help of a phasefield model.

  2. 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

  3. 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

  4. Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

    PubMed

    Fujiwara, Takahiro K; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A; Watanabe, Yusuke; Umemura, Yasuhiro M; Murakoshi, Hideji; Suzuki, Kenichi G N; Nemoto, Yuri L; Morone, Nobuhiro; Kusumi, Akihiro

    2016-04-01

    The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed "hop diffusion") for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion. PMID:26864625

  5. Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane

    PubMed Central

    Fujiwara, Takahiro K.; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A.; Watanabe, Yusuke; Umemura, Yasuhiro M.; Murakoshi, Hideji; Suzuki, Kenichi G. N.; Nemoto, Yuri L.; Morone, Nobuhiro; Kusumi, Akihiro

    2016-01-01

    The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed “hop diffusion”) for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion. PMID:26864625

  6. Nonequilibrium-Driven Motion in Actin Networks: Comet Tails and Moving Beads

    NASA Astrophysics Data System (ADS)

    Burroughs, N. J.; Marenduzzo, D.

    2007-06-01

    We present 3D dynamic Monte-Carlo simulations of the growth of an actin network close to an obstacle coated with Wiskott-Aldrich syndrome protein (WASP), an inducer of actin branching. Our simulations incorporate both elasticity and relaxation of the actin tail, thus allowing for local network compression. Whilst steady state motility derives mainly from polymerization at the leading edge, nonthermal stored elastic energy and retrograde flow are observed in a thin slab of material close to the obstacle. We observe a crossover from steady to hopping bead motion as the branching rate is decreased.

  7. Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression

    PubMed Central

    Scheuring, David; Löfke, Christian; Krüger, Falco; Kittelmann, Maike; Eisa, Ahmed; Hughes, Louise; Smith, Richard S.; Hawes, Chris; Schumacher, Karin; Kleine-Vehn, Jürgen

    2016-01-01

    The cytoskeleton is an early attribute of cellular life, and its main components are composed of conserved proteins. The actin cytoskeleton has a direct impact on the control of cell size in animal cells, but its mechanistic contribution to cellular growth in plants remains largely elusive. Here, we reveal a role of actin in regulating cell size in plants. The actin cytoskeleton shows proximity to vacuoles, and the phytohormone auxin not only controls the organization of actin filaments but also impacts vacuolar morphogenesis in an actin-dependent manner. Pharmacological and genetic interference with the actin–myosin system abolishes the effect of auxin on vacuoles and thus disrupts its negative influence on cellular growth. SEM-based 3D nanometer-resolution imaging of the vacuoles revealed that auxin controls the constriction and luminal size of the vacuole. We show that this actin-dependent mechanism controls the relative vacuolar occupancy of the cell, thus suggesting an unanticipated mechanism for cytosol homeostasis during cellular growth. PMID:26715743

  8. Noise-induced cochlear F-actin depolymerization is mediated via ROCK2/p-ERM signaling

    PubMed Central

    Han, Yu; Wang, Xianren; Chen, Jun; Sha, Su-Hua

    2015-01-01

    Our previous work has suggested that traumatic noise activates Rho-GTPase pathways in cochlear outer hair cells (OHCs), resulting in cell death and noise-induced hearing loss (NIHL). In this study, we investigated Rho effectors, Rho-associated kinases (ROCKs), and the targets of ROCKs, the ezrin-radixin-moesin (ERM) proteins, in the regulation of the cochlear actin cytoskeleton using adult CBA/J mice under conditions of noise-induced temporary threshold shift (TTS) and permanent threshold shift (PTS) hearing loss, which result in changes to the F/G-actin ratio. The levels of cochlear ROCK2 and p-ERM decreased 1 h after either TTS- or PTS-noise exposure. In contrast, ROCK2 and p-ERM in OHCs decreased only after PTS-, not after TTS-noise exposure. Treatment with lysophosphatidic acid, an activator of the Rho pathway, resulted in significant reversal of the F/G-actin ratio changes caused by noise exposure and attenuated OHC death and NIHL. Conversely, the down-regulation of ROCK2 by pretreatment with ROCK2 siRNA reduced the expression of ROCK2 and p-ERM in OHCs, exacerbated TTS to PTS, and worsened OHC loss. Additionally, pretreatment with siRNA against radixin, an ERM protein, aggravated TTS to PTS. Our results indicate that a ROCK2-mediated ERM-phosphorylation signaling cascade modulates noise-induced hair cell loss and NIHL by targeting the cytoskeleton. PMID:25683353

  9. A semi-flexible model prediction for the polymerization force exerted by a living F-actin filament on a fixed wall

    NASA Astrophysics Data System (ADS)

    Pierleoni, Carlo; Ciccotti, Giovanni; Ryckaert, Jean-Paul

    2015-10-01

    We consider a single living semi-flexible filament with persistence length ℓp in chemical equilibrium with a solution of free monomers at fixed monomer chemical potential μ1 and fixed temperature T. While one end of the filament is chemically active with single monomer (de)polymerization steps, the other end is grafted normally to a rigid wall to mimic a rigid network from which the filament under consideration emerges. A second rigid wall, parallel to the grafting wall, is fixed at distance L < < ℓp from the filament seed. In supercritical conditions where monomer density ρ1 is higher than the critical density ρ1c, the filament tends to polymerize and impinges onto the second surface which, in suitable conditions (non-escaping filament regime), stops the filament growth. We first establish the grand-potential Ω(μ1, T, L) of this system treated as an ideal reactive mixture, and derive some general properties, in particular the filament size distribution and the force exerted by the living filament on the obstacle wall. We apply this formalism to the semi-flexible, living, discrete Wormlike chain model with step size d and persistence length ℓp, hitting a hard wall. Explicit properties require the computation of the mean force f ¯ i ( L ) exerted by the wall at L and associated potential f ¯ i ( L ) = - d W i ( L ) / d L on a filament of fixed size i. By original Monte-Carlo calculations for few filament lengths in a wide range of compression, we justify the use of the weak bending universal expressions of Gholami et al. [Phys. Rev. E 74, 041803 (2006)] over the whole non-escaping filament regime. For a filament of size i with contour length Lc = (i - 1) d, this universal form is rapidly growing from zero (non-compression state) to the buckling value f b ( L c , ℓ p ) = /π 2 k B T ℓ p 4 Lc 2 over a compression range much narrower than the size d of a monomer. Employing this universal form for living filaments, we find that the average force exerted

  10. [A study of quantitative dynamics of F-actin during oocyte maturation in the starfish Asterias amurensis].

    PubMed

    Lamash, N E; Eliseĭkina, M G

    2006-01-01

    We studied the actin cytoskeleton state in Asterias amurensis oocytes within 30 min after the 1-methyladenine-induced maturation until the germinal vesicle breakdown. The total amount of actin remained unchanged during oocyte maturation. In immature oocytes, the major part of actin is not a part of filaments, but in the presence of 1-methyladenine massive actin polymerization began already within 20 min. Electron immunocytochemistry methods demonstrated joint localization of actin and alpha-protein in the cytoplasm. They were redistributed from the cortex to the cytoplasm in the presence of 1-methyladenine. A possible involvement of actin cytoskeleton in transmembrane transduction of the hormonal signal at the postreceptor stages is discussed. PMID:17022441

  11. Cucumber Mosaic Virus Movement Protein Severs Actin Filaments to Increase the Plasmodesmal Size Exclusion Limit in Tobacco[W][OA

    PubMed Central

    Su, Shengzhong; Liu, Zhaohui; Chen, Cheng; Zhang, Yan; Wang, Xu; Zhu, Lei; Miao, Long; Wang, Xue-Chen; Yuan, Ming

    2010-01-01

    Plant viral movement proteins (MPs) enable viruses to pass through cell walls by increasing the size exclusion limit (SEL) of plasmodesmata (PD). Here, we report that the ability of Cucumber mosaic virus (CMV) MP to increase the SEL of the PD could be inhibited by treatment with the actin filament (F-actin)–stabilizing agent phalloidin but not by treatment with the F-actin–destabilizing agent latrunculin A. In vitro studies showed that CMV MP bound globular and F-actin, inhibited actin polymerization, severed F-actin, and participated in plus end capping of F-actin. Analyses of two CMV MP mutants, one with and one without F-actin severing activities, demonstrated that the F-actin severing ability was required to increase the PD SEL. Furthermore, the Tobacco mosaic virus MP also exhibited F-actin severing activity, and its ability to increase the PD SEL was inhibited by treatment with phalloidin. Our data provide evidence to support the hypothesis that F-actin severing is required for MP-induced increase in the SEL of PD. This may have broad implications in the study of the mechanisms of actin dynamics that regulate cell-to-cell transport of viral and endogenous proteins. PMID:20435906

  12. Two-photon induced polymerization of photo-driven microsensors

    NASA Astrophysics Data System (ADS)

    Lin, Chih-Lang; Wang, Irene; Bouriau, Michel; Casalegno, Roger; Andraud, Chantal; Baldeck, Patrice L.

    2004-10-01

    We report on the fabrication of photo-driven polymer microsensors for viscosimetry, velocimetry and micropump applications. They are readily made with a low-cost polymerization technique based on two-photon absorption. Microsensors are free-floating in the liquid to be characterized. A linearly-polarized optical tweezers is used to trap one sensor at the laser focal point and to generate the optical torque needed for local hydrodynamic measurements. Viscosity and velocity microsensors have slab shapes that align in the polarization direction. The local viscosity is deduced from the maximum rotation frequency generated by the rotating linear polarization, while the fluid velocity is obtained by measuring the maximum angle that equilibrates the optical torque and drag torque. Experimental results are in good agreement with theoretical calculations. The micropump is based on a micron-size Archimedes screw that rotates around its long axis when it is trapped at the focal point. The laser-induced rotation is due to the optical torque that is transferred by the laser scattering on the screw.

  13. Post-polymerization of urease-induced calcified, polymer hydrogels.

    PubMed

    Rauner, Nicolas; Buenger, Lea; Schuller, Stefanie; Tiller, Joerg C

    2015-01-01

    Urease-induced calcification is an innovative method to artificially produce highly filled CaCO3-based composite materials by intrinsic mineralization of hydrogels. The mechanical properties of these hybrid materials based on poly(2-hydroxyethylacrylate) cross-linked by triethylene glycol dimethacrylate are poor. Increasing the degree of calcification to up to 94 wt% improves the Young's moduli (YM) of the materials from some 40 MPa to more than 300 MPa. The introduction of calcium carbonate affine groups to the hydrogel matrix by copolymerizing acrylic acid and [2-(methacryloyloxy) ethyl]trimethylammonium chloride, respectively, does not increase the stiffness of the composites. A Young's modulus of more than 1 GPa is achieved by post-polymerization (PP) of the calcified hydrogels, which proves that the size of the contact area between the matrix and calcium carbonate crystals is the most crucial parameter for controlling the stiffness of hybrid materials. Switching from low Tg to high Tg hydrogel matrices (based on poly(N,N-dimethyl acrylamide)) results in a YM of up to 3.5 GPa after PP. PMID:25284027

  14. 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.

  15. Hydroxyapatite induces spontaneous polymerization of model self-etch dental adhesives.

    PubMed

    Zhang, Ying; Wu, Ningjing; Bai, Xinyan; Xu, Changqi; Liu, Yi; Wang, Yong

    2013-10-01

    The objective of this study is to report for the first time the spontaneous polymerization phenomenon of self-etch dental adhesives induced by hydroxylapatite (HAp). Model self-etch adhesives were prepared by using a monomer mixture of bis[2-(methacryloyloxy)ethyl] phosphate (2MP) with 2-hydroxyethyl methacrylate (HEMA). The initiator system consisted of camphorquinone (CQ, 0.022 mmol/g) and ethyl 4-dimethylaminobenzoate (4E, 0.022-0.088 mmol/g). HAp (2-8 wt.%) was added to the neat model adhesive. In a dark environment, the polymerization was monitored in-situ using ATR/FT-IR, and the mechanical properties of the polymerized adhesives were evaluated using nanoindentation technique. Results indicated that spontaneous polymerization was not observed in the absence of HAp. However, as different amounts of HAp were incorporated into the adhesives, spontaneous polymerization was induced. Higher HAp content led to higher degree of conversion (DC), higher rate of polymerization (RP) and shorter induction period (IP). In addition, higher 4E content also elevated DC and RP and reduced IP of the adhesives. Nanoindentation result suggested that the Young's modulus of the polymerized adhesives showed similar dependence on HAp and 4E contents. In summary, interaction with HAp could induce spontaneous polymerization of the model self-etch adhesives. This result provides important information for understanding the initiation mechanism of the self-etch adhesives, and may be of clinical significance to strengthen the adhesive/dentin interface based on the finding. PMID:23910263

  16. 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.

  17. Switching of actin-myosin motors by voltage-induced pH bias in vitro.

    PubMed

    Hatori, Kuniyuki; Iwase, Takahiro; Wada, Reito

    2016-08-01

    ATP-driven motor proteins, which function in cell motility and organelle transport, have potential applications as bio-inspired micro-devices; however, their control remains unsatisfactory. Here, we show rapid-velocity control of actin filaments interacting with myosin motors using voltage applied to Pt electrodes in an in vitro motility system, by which immediate increases and decreases in velocity were induced beside the cathode and anode, respectively. Indicator dye revealed pH changes after voltage application, and alternate voltage switching allowed actin filaments to cyclically alter their velocity in response to these changes. This principle provides a basis for on-demand control of not only motor proteins but also pH-sensitive events at a microscopic level. PMID:27210738

  18. Actin reorganization is required for the formation of polarized BCR signalosomes in response to both soluble and membrane-associated antigens1

    PubMed Central

    Liu, Chaohong; Miller, Heather; Orlowski, Gregory; Hang, Haiyin; Upadhyaya, Arpita

    2012-01-01

    B-cells encounter both soluble (sAg) and membrane-associated antigens (mAg) in the secondary lymphoid tissue, yet how the physical form of Ag modulates B-cell activation remains unclear. This study compares actin reorganization and its role in BCR signalosome formation in mAg- and sAg-stimulated B-cells. Both mAg and sAg induce F-actin accumulation and actin polymerization at BCR microclusters and at the outer rim of BCR central clusters, but the kinetics and magnitude of F-actin accumulation in mAg-stimulated B-cells are greater than those in sAg-stimulated B-cells. Accordingly, the actin regulatory factors, cofilin and gelsolin, are recruited to BCR clusters in both mAg- and sAg-stimulated B-cells but with different kinetics and patterns of cellular redistribution. Inhibition of actin reorganization by stabilizing F-actin inhibits BCR clustering and tyrosine phosphorylation induced by both forms of Ag. Depolymerization of F-actin leads to unpolarized microclustering of BCRs and tyrosine phosphorylation in BCR microclusters without mAg and sAg, but in much slower kinetics than those induced by Ag. Therefore, actin reorganization, mediated via both polymerization and depolymerization, is required for the formation of BCR signalosomes in response to both mAg and sAg. PMID:22387556

  19. Fibroblast growth factor (Fgf) 23 gene transcription depends on actin cytoskeleton reorganization.

    PubMed

    Fajol, Abul; Honisch, Sabina; Zhang, Bingbing; Schmidt, Sebastian; Alkahtani, Saad; Alarifi, Saud; Lang, Florian; Stournaras, Christos; Föller, Michael

    2016-03-01

    FGF23 regulates renal phosphate and vitamin D metabolism. Loss of FGF23 results in massive calcification and rapid aging. FGF23 production is stimulated by 1,25(OH)2 D3 and NFκB signaling. Here, we report that treatment of UMR106 osteoblast-like cells with 1,25(OH)2 D3 , inducing Fgf23 transcription, resulted in actin polymerization which was blocked by NFκB inhibitor wogonin. Interestingly, 1,25(OH)2 D3 -induced Fgf23 gene transcription was abolished by the actin microfilament-disrupting agent cytochalasin B, as well as by the inhibition of actin-regulating Rac1/PAK1 signaling. Our results provide strong evidence that actin redistribution regulated by the Rac1/PAK1 pathway participates in 1,25(OH)2 D3 -induced Fgf23 gene transcription. PMID:26878191

  20. Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function.

    PubMed

    Chan, Chun; Fan, Jun; Messer, Andrew E; Marston, Steve B; Iwamoto, Hiroyuki; Ochala, Julien

    2016-08-01

    In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomers are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. These phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients. PMID:27112274

  1. Effect of solvents on the radiation-induced polymerization of ethyl and isopropyl vinyl ethers

    SciTech Connect

    Hsieh, W.C.

    1981-01-01

    The effect of solvents on the radiation-induced cationic polymerization of ethyl and isopropyl vinyl ethers (EVE and IPVE, respectively) was investigated. EVE and IPVE polymerizations were carried out in bulk and in solution under superdry conditions in which polar impurities, especially water, have been reduced to negligible levels. This was accomplished by means of a sodium mirror technique using joint free baked out glass equipment and high vacuum. Plots of the monomer conversions and irradiation times were obtained for EVE and IPVE polymerizations in bulk and in benzene solution at constant monomer concentrations. The monomer concentration dependence of the polymerization rate was studied for EVE polymerization in bulk and in benzene, diethlyl ether, diglyme and methylene chloride, and for IPVE polymerization in bulk and in benzene. Solvent effect on the estimated propagating rate constants was examined for EVE and IPVE polymerization in bulk and in solution. The effect of temperature on the polymerization rate was also investigated for EVE polymerization in bulk ad in benzene, diethyl and diisopropyl ethers, methylene chloride and nitromethane, and for IPVE ploymerization in bulk and in benzene.

  2. The Pseudomonas syringae Type III Effector HopG1 Induces Actin Remodeling to Promote Symptom Development and Susceptibility during Infection1[OPEN

    PubMed Central

    Shimono, Masaki; Porter, Katie; Kvitko, Brian H.; Henty-Ridilla, Jessica; Creason, Allison; Chang, Jeff H.; Staiger, Christopher J.

    2016-01-01

    The plant cytoskeleton underpins the function of a multitude of cellular mechanisms, including those associated with developmental- and stress-associated signaling processes. In recent years, the actin cytoskeleton has been demonstrated to play a key role in plant immune signaling, including a recent demonstration that pathogens target actin filaments to block plant defense and immunity. Herein, we quantified spatial changes in host actin filament organization after infection with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), demonstrating that the type-III effector HopG1 is required for pathogen-induced changes to actin filament architecture and host disease symptom development during infection. Using a suite of pathogen effector deletion constructs, coupled with high-resolution microscopy, we found that deletion of hopG1 from Pst DC3000 resulted in a reduction in actin bundling and a concomitant increase in the density of filament arrays in Arabidopsis, both of which correlate with host disease symptom development. As a mechanism underpinning this activity, we further show that the HopG1 effector interacts with an Arabidopsis mitochondrial-localized kinesin motor protein. Kinesin mutant plants show reduced disease symptoms after pathogen infection, which can be complemented by actin-modifying agents. In total, our results support a model in which HopG1 induces changes in the organization of the actin cytoskeleton as part of its virulence function in promoting disease symptom development. PMID:27217495

  3. The Pseudomonas syringae Type III Effector HopG1 Induces Actin Remodeling to Promote Symptom Development and Susceptibility during Infection.

    PubMed

    Shimono, Masaki; Lu, Yi-Ju; Porter, Katie; Kvitko, Brian H; Henty-Ridilla, Jessica; Creason, Allison; He, Sheng Yang; Chang, Jeff H; Staiger, Christopher J; Day, Brad

    2016-07-01

    The plant cytoskeleton underpins the function of a multitude of cellular mechanisms, including those associated with developmental- and stress-associated signaling processes. In recent years, the actin cytoskeleton has been demonstrated to play a key role in plant immune signaling, including a recent demonstration that pathogens target actin filaments to block plant defense and immunity. Herein, we quantified spatial changes in host actin filament organization after infection with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), demonstrating that the type-III effector HopG1 is required for pathogen-induced changes to actin filament architecture and host disease symptom development during infection. Using a suite of pathogen effector deletion constructs, coupled with high-resolution microscopy, we found that deletion of hopG1 from Pst DC3000 resulted in a reduction in actin bundling and a concomitant increase in the density of filament arrays in Arabidopsis, both of which correlate with host disease symptom development. As a mechanism underpinning this activity, we further show that the HopG1 effector interacts with an Arabidopsis mitochondrial-localized kinesin motor protein. Kinesin mutant plants show reduced disease symptoms after pathogen infection, which can be complemented by actin-modifying agents. In total, our results support a model in which HopG1 induces changes in the organization of the actin cytoskeleton as part of its virulence function in promoting disease symptom development. PMID:27217495

  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. Phylogenetic Analysis Identifies Many Uncharacterized Actin-like Proteins (Alps) in Bacteria: Regulated Polymerization, Dynamic Instability, and Treadmilling in Alp7A

    PubMed Central

    Derman, Alan I.; Becker, Eric C.; Truong, Bao D.; Fujioka, Akina; Tucey, Timothy M.; Erb, Marcella L.; Patterson, Paula C.; Pogliano, Joe

    2010-01-01

    Summary Actin, one of the most abundant proteins in the eukaryotic cell, also has an abundance of relatives in the eukaryotic proteome. To date though, only five families of actins have been characterized in bacteria. We have conducted a phylogenetic search and uncovered more than 35 highly divergent families of actin-like proteins (Alps) in bacteria. Their genes are found primarily on phage genomes, on plasmids, and on integrating conjugative elements, and are likely to be involved in a variety of functions. We characterize three Alps and find that all form filaments in the cell. The filaments of Alp7A, a plasmid partitioning protein and one of the most divergent of the Alps, display dynamic instability and also treadmill. Alp7A requires other elements from the plasmid to assemble into dynamic polymers in the cell. Our findings suggest that most if not all of the Alps are indeed actin relatives, and that actin is very well represented in bacteria. PMID:19602153

  6. Synaptopodin-2 induces assembly of peripheral actin bundles and immature focal adhesions to promote lamellipodia formation and prostate cancer cell migration.

    PubMed

    Kai, FuiBoon; Fawcett, James P; Duncan, Roy

    2015-05-10

    Synaptopodin-2 (Synpo2), an actin-binding protein and invasive cancer biomarker, induces formation of complex stress fiber networks in the cell body and promotes PC3 prostate cancer cell migration in response to serum stimulation. The role of these actin networks in enhanced cancer cell migration is unknown. Using time-course analysis and live cell imaging of mock- and Synpo2-transduced PC3 cells, we now show that Synpo2 induces assembly of actin fibers near the cell periphery and Arp2/3-dependent lamellipodia formation. Lamellipodia formed in a non-directional manner or repeatedly changed direction, explaining the enhanced chemokinetic activity of PC3 cells in response to serum stimulation. Myosin contraction promotes retrograde flow of the Synpo2-associated actin filaments at the leading edge and their merger with actin networks in the cell body. Enhanced PC3 cell migration correlates with Synpo2-induced formation of lamellipodia and immature focal adhesions (FAs), but is not dependent on myosin contraction or FA maturation. The previously reported correlation between Synpo2-induced stress fiber assembly and enhanced PC3 cell migration therefore reflects the role of Synpo2 as a newly identified regulator of actin bundle formation and nascent FA assembly near the leading cell edge. PMID:25883213

  7. 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.

  8. 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

  9. 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

  10. Vacuole formation in mast cells responding to osmotic stress and to F-actin disassembly.

    PubMed

    Koffer, Anna; Williams, Mark; Johansen, Torben

    2002-01-01

    Fluorescent probes were used to visualize the morphology of membranes and of F-actin in rat peritoneal mast cells, exposed to hyperosmotic medium and consequently reversed to isotonicity. Hypertonicity induced cell shrinkage followed by a regulatory volume increase, and cell alkalinization that was sensitive to amiloride, an inhibitor of the Na(+)/H(+) exchanger (NHE), but not to Latrunculin B, an inhibitor of actin polymerization. Using Bodipy-Sphingomyelin, we have observed formation of vacuole-like dilations (VLDs), primarily at or close to the adhesion plane, following the reversal from hyper- to isotonic medium. VLD formation was not inhibited by Latrunculin B or by amiloride. Phalloidin staining has shown that actin filaments do not surround the vacuoles and latrunculin-induced depolymerization of actin has actually promoted vacuole formation, even in isotonic conditions. The results support the idea that a decrease in membrane tension promotes the internalization of the plasma membrane. PMID:12421579

  11. Electron-beam induced RAFT-graft polymerization of poly(acrylic acid) onto PVDF

    NASA Astrophysics Data System (ADS)

    Grasselli, M.; Betz, N.

    2005-07-01

    This paper explores for the first time the post-radiation-induced-graft polymerization on solid substrate using reversible addition-fragmentation transfer (RAFT) mechanism. Radiation-induced graft polymerization onto polymers is a potentially interesting technique to create easily new materials from highly resistant polymers, e.g. surface graft polymerization of acrylic acid (AA) onto poly(vinylidene difluoride) (PVDF) improves its surface properties without losing its excellent mechanical properties. As a consequence of the radical nature of the polymerization processes it is difficult to control molecular weight of grafted chains, and therefore design and standardize the properties of the final product. RAFT polymerization is a suitable method to obtain monodisperse polymers. The ability of the RAFT agents to control the polymer chain length could be an interesting approach to improve the grafted polymers obtained by post-radiation-induced-graft polymerization technique. In this way, graft polymerization of AA onto electron-beam irradiated α-PVDF was performed using trithiocarbonic acid bis(1-phenylethyl) ester as a RAFT agent to control the radical polymerization. We studied several grafting parameters such as solvent, monomer concentration and grafting time in order to achieve a poly(acrylic acid) (PAA) layer onto PVDF surface. Acetic acid was found to be the best solvent for many reasons, as to drive graft polymerization mainly to the polymer surface, complete solubility and stability of all reactants. Hydrolysis of PAA chains was also studied in order to remove the trithiocarbonate functionality from the grafted polymer. A mild chemical condition was achieved in order to have thiol groups that were detected onto the modified PVDF by specific enzymatic reaction.

  12. Characterization of the activities of actin-affecting drugs on tumor cell migration

    SciTech Connect

    Hayot, Caroline; Debeir, Olivier; Ham, Philippe van; Damme, Marc van; Kiss, Robert; Decaestecker, Christine . E-mail: cdecaes@ulb.ac.be

    2006-02-15

    Metastases kill 90% of cancer patients. It is thus a major challenge in cancer therapy to inhibit the spreading of tumor cells from primary tumor sites to those particular organs where metastases are likely to occur. Whereas the actin cytoskeleton is a key component involved in cell migration, agents targeting actin dynamics have been relatively poorly investigated. Consequently, valuable in vitro pharmacological tools are needed to selectively identify this type of agent. In response to the absence of any standardized process, the present work aims to develop a multi-assay strategy for screening actin-affecting drugs with anti-migratory potentials. To validate our approach, we used two cancer cell lines (MCF7 and A549) and three actin-affecting drugs (cytochalasin D, latrunculin A, and jasplakinolide). We quantified the effects of these drugs on the kinetics of actin polymerization in tubes (by means of spectrofluorimetry) and on the dynamics of actin cytoskeletons within whole cells (by means of fluorescence microscopy). Using quantitative videomicroscopy, we investigated the actual effects of the drugs on cell motility. Finally, the combined drug effects on cell motility and cell growth were evaluated by means of a scratch-wound assay. While our results showed concordant drug-induced effects on actin polymerization occurring in vitro in test tubes and within whole cells, the whole cell assay appeared more sensitive than the tube assay. The inhibition of actin polymerization induced by cytochalasin D was paralleled by a decrease in cell motility for both cell types. In the case of jasplakinolide, which induces actin polymerization, while it significantly enhanced the locomotion of the A549 cells, it significantly inhibited that of the MCF-7 ones. All these effects were confirmed by means of the scratch-wound assay except of the jasplakinolide-induced effects on MCF-7 cell motility. These later seemed compensated by an additional effect occurring during wound

  13. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.

    PubMed Central

    Fasano, A; Fiorentini, C; Donelli, G; Uzzau, S; Kaper, J B; Margaretten, K; Ding, X; Guandalini, S; Comstock, L; Goldblum, S E

    1995-01-01

    The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time- and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization. In IEC6 cells ZOT induced a peak increment of the PKC-alpha isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function. Images PMID:7635964

  14. The Biphasic Increase of PIP2 in the Fertilized Eggs of Starfish: New Roles in Actin Polymerization and Ca2+ Signaling

    PubMed Central

    Chun, Jong T.; Puppo, Agostina; Vasilev, Filip; Gragnaniello, Giovanni; Garante, Ezio; Santella, Luigia

    2010-01-01

    Background Fertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca2+. Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP3) and also regulates actin-binding proteins, PIP2 might be involved in these two processes. Methodology/Principal Findings In this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH) domain of PLC-δ1, which has specific binding affinity to PIP2, in combination with Ca2+ and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca2+ wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE). These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca2+ signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization. Conclusions/Significance Our results suggest that PIP2 plays comprehensive roles in shaping Ca2+ waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule exocytosis. PMID:21124897

  15. Berberine induces dedifferentiation by actin cytoskeleton reorganization via phosphoinositide 3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes.

    PubMed

    Yu, Seon-Mi; Cho, Hongsik; Kim, Gwang-Hoon; Chung, Ki-Wha; Seo, Sung-Yum; Kim, Song-Ja

    2016-04-01

    Osteoarthritis is a nonrheumatologic joint disease characterized by progressive degeneration of the cartilage extracellular matrix. Berberine (BBR) is an isoquinoline alkaloid used in traditional Chinese medicine, the majority of which is extracted from Huang Lian (Coptis chinensis). Although numerous studies have revealed the anticancer activity of BBR, its effects on normal cells, such as chondrocytes, and the molecular mechanisms underlying its actions remain elusive. Therefore, we examined the effects of BBR on rabbit articular chondrocytes, and the underlying molecular mechanisms, focusing on actin cytoskeletal reorganization. BBR induced dedifferentiation by inhibiting activation of phosphoinositide-3(PI3)-kinase/Akt and p38 kinase. Furthermore, inhibition of p38 kinase and PI3-kinase/Akt with SB203580 and LY294002, respectively, accelerated the BBR-induced dedifferentiation. BBR also caused actin cytoskeletal architecture reorganization and, therefore, we investigated if these effects were involved in the dedifferentiation. Disruption of the actin cytoskeleton by cytochalasin D reversed the BBR-induced dedifferentiation by activating PI3-kinase/Akt and p38 kinase. In contrast, the induction of actin filament aggregation by jasplakinolide accelerated the BBR-induced dedifferentiation via PI3-kinase/Akt inhibition and p38 kinase activation. Taken together, these data suggest that BBR strongly induces dedifferentiation, and actin cytoskeletal reorganization is a crucial requirement for this effect. Furthermore, the dedifferentiation activity of BBR appears to be mediated via PI3-kinase/Akt and p38 kinase pathways in rabbit articular chondrocytes. PMID:26851252

  16. Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    NASA Technical Reports Server (NTRS)

    Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1998-01-01

    Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

  17. 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

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

    PubMed Central

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

    2014-01-01

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

  19. 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

  20. β-Actin-binding Complementarity-determining Region 2 of Variable Heavy Chain from Monoclonal Antibody C7 Induces Apoptosis in Several Human Tumor Cells and Is Protective against Metastatic Melanoma*

    PubMed Central

    Arruda, Denise C.; Santos, Luana C. P.; Melo, Filipe M.; Pereira, Felipe V.; Figueiredo, Carlos R.; Matsuo, Alisson L.; Mortara, Renato A.; Juliano, Maria A.; Rodrigues, Elaine G.; Dobroff, Andrey S.; Polonelli, Luciano; Travassos, Luiz R.

    2012-01-01

    Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that β-actin is the receptor of C7H2 in the tumor cells. C7H2 induces β-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug. PMID:22334655

  1. Pressure-induced polymerization of P(CN){sub 3}

    SciTech Connect

    Gou, Huiyang E-mail: tstrobel@ciw.edu; Kim, Duck Young; Strobel, Timothy A. E-mail: tstrobel@ciw.edu; Yonke, Brendan L.; Epshteyn, Albert; Smith, Jesse S.

    2015-05-21

    Motivated to explore the formation of novel extended carbon-nitrogen solids via well-defined molecular precursor pathways, we studied the chemical reactivity of highly pure phosphorous tricyanide, P(CN){sub 3}, under conditions of high pressure at room temperature. Raman and infrared (IR) spectroscopic measurements reveal a series of phase transformations below 10 GPa, and several low-frequency vibrational modes are reported for the first time. Synchrotron powder X-ray diffraction measurements taken during compression show that molecular P(CN){sub 3} is highly compressible, with a bulk modulus of 10.0 ± 0.3 GPa, and polymerizes into an amorphous solid above ∼10.0 GPa. Raman and IR spectra, together with first-principles molecular-dynamics simulations, show that the amorphization transition is associated with polymerization of the cyanide groups into CN bonds with predominantly sp{sup 2} character, similar to known carbon nitrides, resulting in a novel phosphorous carbon nitride (PCN) polymeric phase, which is recoverable to ambient pressure.

  2. A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid.

    PubMed

    Au, Shelly; Wu, Wei; Zhou, Lixin; Theilmann, David A; Panté, Nelly

    2016-08-01

    The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin β superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle. PMID:27284005

  3. 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 ...

  4. 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 ...

  5. 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

  6. 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

  7. 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

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

    PubMed Central

    2016-01-01

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

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

    PubMed

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

    2003-05-01

    This report focuses on the identification of the molecular mechanisms of ethanol-induced in vitro angiogenesis. The manipulation of angiogenesis is an important therapeutic approach for the treatment of cancer, cardiovascular diseases, and chronic inflammation. Our results showed that ethanol stimulation altered the integrity of actin filaments and increased the formation of lamellipodia and filopodia in SVEC4-10 cells. Further experiments demonstrated that ethanol stimulation increased cell migration and invasion and induced in vitro angiogenesis in SVEC4-10 cells. Mechanistically, ethanol stimulation activated Cdc42 and produced H(2)O(2) a reactive oxygen species intermediate in SVEC4-10 cells. Measuring the time course of Cdc42 activation and H(2)O(2) production upon ethanol stimulation revealed that the Cdc42 activation and the increase of H(2)O(2) lasted more than 3 h, which indicates the mechanisms of the long duration effects of ethanol on the cells. Furthermore, either overexpression of a constitutive dominant negative Cdc42 or inhibition of H(2)O(2) production abrogated the effects of ethanol on SVEC4-10 cells, indicating that both the activation of Cdc42 and the production of H(2)O(2) are essential for the actions of ethanol. Interestingly, we also found that overexpression of a constitutive dominant positive Cdc42 itself was sufficient to produce H(2)O(2) and to induce in vitro angiogenesis. Taken together, our results suggest that ethanol stimulation can induce H(2)O(2) production through the activation of Cdc42, which results in reorganizing actin filaments and increasing cell motility and in vitro angiogenesis. PMID:12598535

  10. Antimicrobial fabric adsorbed iodine produced by radiation-induced graft polymerization

    NASA Astrophysics Data System (ADS)

    Aoki, Shoji; Fujiwara, Kunio; Sugo, Takanobu; Suzuki, Koichi

    2013-03-01

    Antimicrobial fabric was synthesized by radiation-induced graft polymerization of N-vinyl pyrrolidone onto polyolefine nonwoven fabric and subsequent adsorption of iodine. In response of the huge request for the antimicrobial material applied to face masks for swine flu in 2009, operation procedure of continuous radiation-induced graft polymerization apparatus was improved. The improved grafting production per week increased 3.8 times compared to the production by former operation procedure. Shipped antimicrobial fabric had reached 130,000 m2 from June until December, 2009.

  11. 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

  12. A biosensor for the protease TACE reveals actin damage induced TACE activation

    PubMed Central

    Chapnick, Douglas A.; Bunker, Eric; Liu, Xuedong

    2016-01-01

    Ligand shedding has gained increased attention as a major posttranslational modification mechanism used by cells to respond to diverse environmental conditions. The TACEadam17 protease is a critical mediator of such ligand shedding, regulating the maturation and release of an impressive range of extracellular substrates that drive diverse cellular responses. Exactly how this protease is itself activated remains unclear, in part due to the lack of available tools to measure TACE activity with temporal and spatial resolution in live cells. We have developed a FRET based biosensor for TACE activity (TSen), which is capable of reporting TACE activation kinetics in live cells with a high degree of specificity. TSen was used in combination with chemical biology to probe the dependence of various means of TACE activation on p38 and Erk kinase activities, as well as to identify a novel connection between actin cytoskeletal disruption and TACE activation. Such cytoskeletal disruption leads to rapid and robust TACE activation in some cell types and accumulation of TACE at the plasma membrane, allowing for increased cleavage of endogenous substrates. Our study highlights both the versatility of TSen as a tool to understand the mechanisms of TACE activation in live cells and the importance of actin cytoskeletal integrity as a modulator of TACE activity. PMID:25714465

  13. Pathogen-Mimicking Polymeric Nanoparticles based on Dopamine Polymerization as Vaccines Adjuvants Induce Robust Humoral and Cellular Immune Responses.

    PubMed

    Liu, Qi; Jia, Jilei; Yang, Tingyuan; Fan, Qingze; Wang, Lianyan; Ma, Guanghui

    2016-04-01

    Aiming to enhance the immunogenicity of subunit vaccines, a novel antigen delivery and adjuvant system based on dopamine polymerization on the surface of poly(D,L-lactic-glycolic-acid) nanoparticles (NPs) with multiple mechanisms of immunity enhancement is developed. The mussel-inspired biomimetic polydopamine (pD) not only serves as a coating to NPs but also functionalizes NP surfaces. The method is facile and mild including simple incubation of the preformed NPs in the weak alkaline dopamine solution, and incorporation of hepatitis B surface antigen and TLR9 agonist unmethylated cytosine-guanine (CpG) motif with the pD surface. The as-constructed NPs possess pathogen-mimicking manners owing to their size, shape, and surface molecular immune-activating properties given by CpG. The biocompatibility and biosafety of these pathogen-mimicking NPs are confirmed using bone marrow-derived dendritic cells. Pathogen-mimicking NPs hold great potential as vaccine delivery and adjuvant system due to their ability to: 1) enhance cytokine secretion and immune cell recruitment at the injection site; 2) significantly activate and maturate dendritic cells; 3) induce stronger humoral and cellular immune responses in vivo. Furthermore, this simple and versatile dopamine polymerization method can be applicable to endow NPs with characteristics to mimic pathogen structure and function, and manipulate NPs for the generation of efficacious vaccine adjuvants. PMID:26849717

  14. 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

  15. 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.

  16. Distribution of actin in etoposide-induced human leukemia cell line K-562 using fluorescence and immunoelectron microscopy technique.

    PubMed

    Grzanka, Alina; Grzanka, Dariusz

    2002-01-01

    Localization of actin was studied in erythroleukemic cell line K-562 after treatment with etoposide for 72 hours in a range of concentrations 0.02-200 microM/L. Actin was visualised by fluorescence microscopy and streptavidingold method. These findings indicate that changes in actin after treatment with etoposide were dose-dependent. Significant changes in the cellular distribution of F-actin in K-562 cells were obtained after treatment with 20 and 200 microM/L etoposide. In comparison with control cells, the number of the cells decreased and cells were larger especially at 200 microM/L. F-actin was diffusely distributed throughout the cell at 20 microM/L. Treatment of cells with 200 microM/L etoposide showed F-actin diffusely distributed throughout the cell with local actin assemblies and also at the cell periphery. Immunogold labelling of actin was observed in cells treated with all doses of etoposide and control cells. Labelling was found in the nucleus and also in the cytoplasm. At the ultrastructural level, cells treated with 200 microM/L etoposide showed protrusions at the surface, in which increase of actin was often observed. Etoposide causes changes in actin distribution of K-562 cells, and the changes in expression of actin were not only restricted to cell with features of apoptosis. PMID:12140866

  17. Thermally induced twin polymerization of 4H-1,3,2-benzodioxasilines.

    PubMed

    Kempe, Patrick; Löschner, Tina; Auer, Alexander A; Seifert, Andreas; Cox, Gerhard; Spange, Stefan

    2014-06-23

    The twin monomer 2,2'-spirobi[4H-1,3,2-benzodioxasiline] (1) can be polymerized to nanostructured SiO2/phenolic-resin composite material by thermally induced twin polymerization. Thermally induced twin polymerization represents a way to produce nanocomposites simply by thermal induction of twin monomers. Besides 1, the thermal reaction of several related salicylic (2-oxybenzylic) silicon molecules has been investigated. The thermal cleavage of the molecules is studied by using several trapping reagents (e.g., vinyl compounds). A significant occurrence of quinone methide adducts indicates that the thermal mechanism proceeds not only by a ring opening at the oxymethylene position, but also with the ortho-quinone methide as a central or alternative intermediate. This is supported by product analyses of thermally initialized reactions of 1 and its substituted analogues as well as by quantum chemical calculations. PMID:24849004

  18. Force Generation by Endocytic Actin Patches in Budding Yeast

    PubMed Central

    Carlsson, Anders E.; Bayly, Philip V.

    2014-01-01

    Membrane deformation during endocytosis in yeast is driven by local, templated assembly of a sequence of proteins including polymerized actin and curvature-generating coat proteins such as clathrin. Actin polymerization is required for successful endocytosis, but it is not known by what mechanisms actin polymerization generates the required pulling forces. To address this issue, we develop a simulation method in which the actin network at the protein patch is modeled as an active gel. The deformation of the gel is treated using a finite-element approach. We explore the effects and interplay of three different types of force driving invagination: 1), forces perpendicular to the membrane, generated by differences between actin polymerization rates at the edge of the patch and those at the center; 2), the inherent curvature of the coat-protein layer; and 3), forces parallel to the membrane that buckle the coat protein layer, generated by an actomyosin contractile ring. We find that with optimistic estimates for the stall stress of actin gel growth and the shear modulus of the actin gel, actin polymerization can generate almost enough force to overcome the turgor pressure. In combination with the other mechanisms, actin polymerization can the force over the critical value. PMID:24739159

  19. An unconventional form of actin in protozoan hemoflagellate, Leishmania.

    PubMed

    Kapoor, Prabodh; Sahasrabuddhe, Amogh A; Kumar, Ashutosh; Mitra, Kalyan; Siddiqi, Mohammad Imran; Gupta, Chhitar M

    2008-08-15

    Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs. PMID:18539603

  20. 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

  1. Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase–activating protein Gem-interacting protein

    PubMed Central

    Johnson, Jennifer L.; Monfregola, Jlenia; Napolitano, Gennaro; Kiosses, William B.; Catz, Sergio D.

    2012-01-01

    Cytoskeleton remodeling is important for the regulation of vesicular transport associated with exocytosis, but a direct association between granular secretory proteins and actin-remodeling molecules has not been shown, and this mechanism remains obscure. Using a proteomic approach, we identified the RhoA-GTPase–activating protein Gem-interacting protein (GMIP) as a factor that associates with the Rab27a effector JFC1 and modulates vesicular transport and exocytosis. GMIP down-regulation induced RhoA activation and actin polymerization. Importantly, GMIP-down-regulated cells showed impaired vesicular transport and exocytosis, while inhibition of the RhoA-signaling pathway induced actin depolymerization and facilitated exocytosis. We show that RhoA activity polarizes around JFC1-containing secretory granules, suggesting that it may control directionality of granule movement. Using quantitative live-cell microscopy, we show that JFC1-containing secretory organelles move in areas near the plasma membrane deprived of polymerized actin and that dynamic vesicles maintain an actin-free environment in their surroundings. Supporting a role for JFC1 in RhoA inactivation and actin remodeling during exocytosis, JFC1 knockout neutrophils showed increased RhoA activity, and azurophilic granules were unable to traverse cortical actin in cells lacking JFC1. We propose that during exocytosis, actin depolymerization commences near the secretory organelle, not the plasma membrane, and that secretory granules use a JFC1- and GMIP-dependent molecular mechanism to traverse cortical actin. PMID:22438581

  2. Chelation competition induced polymerization (CCIP): construction of integrated hollow polydopamine nanocontainers with tailorable functionalities.

    PubMed

    Xiang, Siyuan; Wang, Dandan; Zhang, Kai; Liu, Wendong; Wu, Ce; Meng, Qingnan; Sun, Hongchen; Yang, Bai

    2016-08-01

    A novel 'chelation competition induced polymerization' route was developed to construct hollow polydopamine nanocontainers with tailorable functionalities. The mechanism is systematically investigated and the nanocontainers constructed through this method show excellent chemo-thermo performance in vitro. This strategy is facile and is expected to be used for the construction of a series of hollow polymer nanostructures. PMID:27461771

  3. 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.

  4. 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

  5. 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

  6. Polymerization-Induced Self-Assembly of Galactose-Functionalized Biocompatible Diblock Copolymers for Intracellular Delivery

    PubMed Central

    2013-01-01

    Recent advances in polymer science are enabling substantial progress in nanobiotechnology, particularly in the design of new tools for enhanced understanding of cell biology and for smart drug delivery formulations. Herein, a range of novel galactosylated diblock copolymer nano-objects is prepared directly in concentrated aqueous solution via reversible addition–fragmentation chain transfer polymerization using polymerization-induced self-assembly. The resulting nanospheres, worm-like micelles, or vesicles interact in vitro with galectins as judged by a turbidity assay. In addition, galactosylated vesicles are highly biocompatible and allow intracellular delivery of an encapsulated molecular cargo. PMID:23941545

  7. Polymerization-induced self-assembly of galactose-functionalized biocompatible diblock copolymers for intracellular delivery.

    PubMed

    Ladmiral, Vincent; Semsarilar, Mona; Canton, Irene; Armes, Steven P

    2013-09-11

    Recent advances in polymer science are enabling substantial progress in nanobiotechnology, particularly in the design of new tools for enhanced understanding of cell biology and for smart drug delivery formulations. Herein, a range of novel galactosylated diblock copolymer nano-objects is prepared directly in concentrated aqueous solution via reversible addition-fragmentation chain transfer polymerization using polymerization-induced self-assembly. The resulting nanospheres, worm-like micelles, or vesicles interact in vitro with galectins as judged by a turbidity assay. In addition, galactosylated vesicles are highly biocompatible and allow intracellular delivery of an encapsulated molecular cargo. PMID:23941545

  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. PEGylated-thymoquinone-nanoparticle mediated retardation of breast cancer cell migration by deregulation of cytoskeletal actin polymerization through miR-34a.

    PubMed

    Bhattacharya, Saurav; Ahir, Manisha; Patra, Prasun; Mukherjee, Sudeshna; Ghosh, Swatilekha; Mazumdar, Minakshi; Chattopadhyay, Sreya; Das, Tanya; Chattopadhyay, Dhrubajyoti; Adhikary, Arghya

    2015-05-01

    Thymoquinone (TQ), a major active constituent of black seeds of Nigella sativa, has potential medical applications including spectrum of therapeutic properties against different cancers. However, little is known about their effect on breast cancer cell migration, which is the cause of over 90% of deaths worldwide. Herein, we have synthesized TQ-encapsulated nanoparticles using biodegradable, hydrophilic polymers like polyvinylpyrrolidone (PVP) and polyethyleneglycol (PEG) to overcome TQ's poor aqueous solubility, thermal and light sensitivity as well as consequently, minimal systemic bioavailability which can greatly improve the cancer treatment efficiency. Sizes of synthesized TQ-Nps were found to be below 50 nm and they were mostly spherical in shape with smooth surface texture. Estimation of the zeta potential also revealed that all the three TQ-Nps were negatively charged which also facilitated their cellular uptake. In the present investigation, we provide direct evidence that TQ-Nps showed more efficiency in killing cancer cells as well as proved to be less toxic to normal cells at a significantly lower dose than TQ. Interestingly, evaluation of the anti-migratory effect of the TQ-Nps, revealed that PEG4000-TQ-Nps showed much potent anti-migratory properties than the other types. Further studies indicated that PEG4000-TQ-Nps could significantly increase the expression of miR-34a through p53. Moreover, NPs mediated miR-34a up-regulation directly down-regulated Rac1 expression followed by actin depolymerisation thereby disrupting the actin cytoskeleton which leads to significant reduction in the lamellipodia and filopodia formation on cell surfaces thus retarding cell migration. Considering the biodegradability, non-toxicity and effectivity of PEG4000-TQ-Nps against cancer cell migration, TQ-Nps may provide new insights into specific therapeutic approach for cancer treatment. PMID:25771001

  10. Influence of matrix porosity on the immobilization of penicillin acylase by radiation-induced polymerization

    NASA Astrophysics Data System (ADS)

    Carenza, M.; Lora, S.; Palma, G.; Boccù, E.; Largajolli, R.; Veronese, F. M.

    Penicillin acylase was immobilized by low temperature radiation-induced polymerization into polymer matrices obtained from monomers of different hydrophilicities, at various ratios of monomer to enzyme solution and at different polymerization conversions. It was found that the penicillin acylase retention (60-85% of the starting enzyme) is independent of the monomer used in thepolymerization, of the polymerization conversion and of the porosity of the polymer matrix. On the other hand, the penicillin acylase retention strongly depends on the presence in the irradiation mixture of the hydrophobic crosslinking agent, trimethylolpropane trimethacrylate, even in low amounts. The data suggest that the enzyme is bound to the polymer matrix by hydrophobic interactions through crosslinking agent molecules.

  11. Rotavirus Infection of Cells in Culture Induces Activation of RhoA and Changes in the Actin and Tubulin Cytoskeleton

    PubMed Central

    Zambrano, Jose Luis; Sorondo, Orlando; Alcala, Ana; Vizzi, Esmeralda; Diaz, Yuleima; Ruiz, Marie Christine; Michelangeli, Fabian; Liprandi, Ferdinando; Ludert, Juan E.

    2012-01-01

    Rotavirus infection induces an increase in [Ca2+]cyto, which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca2+ chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca2+ homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton. PMID:23082182

  12. Ligand-induced changes in the location of actin, myosin, 95K (alpha- actinin), and 120K protein in amebae of Dictyostelium discoideum

    PubMed Central

    1985-01-01

    In this study we investigated concanavalin A (Con A) induced changes in the locations of actin, myosin, 120K, and 95K (alpha-actinin) to determine the extent to which actin and myosin are reorganized during capping and the roles that 120K and 95K might play in this reorganization. We observed the location of each protein by indirect immunofluorescence using affinity purified antibodies. Four morphological states were distinguished in vegetative Dictyostelium amebae: ameboid cells before Con A binding, patched cells, capped cells, and ameboid cells with caps. The location of each protein was distinct in ameboid cells both before and after capping Actin and 120K were found in the cell cortex usually associated with surface projections, and myosin and 95K were diffusely distributed. Myosin was excluded from surface projections in ameboid cells. During patching, all four proteins were localized below Con A patches. During capping, actin, myosin, and 95K protein moved with the Con A patches into the cap whereas 120K protein was excluded from the cap. During the late stages of cap formation actin and myosin were progressively lost from the cap, and 120K became concentrated in new actin-filled projections that formed away from the cap. However, 95K remained tightly associated with the cap. Poisoning cells with sodium azide inhibited capping but not patching of ligand. In azide-poisoned cells, myosin and 95K did not co-patch with Con A, whereas copatching of 120K and actin with Con A occurred as usual. Our results support the hypothesis that capping is an actomyosin-mediated motile event that involves a sliding interaction between actin filaments, which are anchored through the membrane to ligand patches, and myosin in the cortex. They are also consistent with a role for 120K in the formation of surface projections by promoting growth and/or cross-linking of actin filaments within projections, and with a role for 95K in regulating actomyosin-mediated contractility, earlier

  13. Adenosine diphosphate-ribosylation of G-actin by botulinum C2 toxin increases endothelial permeability in vitro.

    PubMed Central

    Suttorp, N; Polley, M; Seybold, J; Schnittler, H; Seeger, W; Grimminger, F; Aktories, K

    1991-01-01

    The endothelial cytoskeleton is believed to play an important role in the regulation of endothelial permeability. We used botulinum C2 toxin to perturb cellular actin and determined its effect on the permeability of endothelial cell monolayers derived from porcine pulmonary arteries. The substrate for botulinum C2 toxin is nonmuscle monomeric actin which becomes ADP-ribosylated. This modified actin cannot participate in actin polymerization and, in addition, acts as a capping protein. Exposure of endothelial cell monolayers to botulinum C2 toxin resulted in a dose- (3-100 ng/ml) and time-dependent (30-120 min) increase in the hydraulic conductivity and decrease in the selectivity of the cell monolayers. The effects of C2 toxin were accompanied by a time- and dose-dependent increase in ADP-ribosylatin of G-actin. G-Actin content increased and F-actin content decreased time- and dose-dependently in C2 toxin-treated endothelial cells. Phalloidin which stabilizes filamentous actin prevented the effects of botulinum C2 toxin on endothelial permeability. Botulinum C2 toxin induced interendothelial gaps. The effects occurred in the absence of overt cell damage and were not reversible within 2 h. The data suggest that the endothelial microfilament system is important for the regulation of endothelial permeability. Images PMID:2022729

  14. Effect of ATP on actin filament stiffness.

    PubMed

    Janmey, P A; Hvidt, S; Oster, G F; Lamb, J; Stossel, T P; Hartwig, J H

    1990-09-01

    Actin is an adenine nucleotide-binding protein and an ATPase. The bound adenine nucleotide stabilizes the protein against denaturation and the ATPase activity, although not required for actin polymerization, affects the kinetics of this assembly Here we provide evidence for another effect of adenine nucleotides. We find that actin filaments made from ATP-containing monomers, the ATPase activity of which hydrolyses ATP to ADP following polymerization, are stiff rods, whereas filaments prepared from ADP-monomers are flexible. ATP exchanges with ADP in such filaments and stiffens them. Because both kinds of actin filaments contain mainly ADP, we suggest the alignment of actin monomers in filaments that have bound and hydrolysed ATP traps them conformationally and stores elastic energy. This energy would be available for release by actin-binding proteins that transduce force or sever actin filaments. These data support earlier proposals that actin is not merely a passive cable, but has an active mechanochemical role in cell function. PMID:2168523

  15. Proteomic profiling of microbial transglutaminase-induced polymerization of milk proteins.

    PubMed

    Hsieh, J F; Pan, P H

    2012-02-01

    Microbial transglutaminase (MTGase)-induced polymerization of individual milk proteins during incubation was investigated using a proteomics-based approach. The addition of MTGase (0.25-2.0 units/mL) caused the milk proteins to polymerize after a 3-h incubation period. Sodium dodecyl sulfate-PAGE analysis showed that the total intensities of the protein bands that corresponded to α(S)-casein, β-casein, and κ-casein decreased from 8,245.6, 6,677.2, and 586.6 arbitrary units to 1,911.7, 0.0, and 66.2 arbitrary units, respectively. Components with higher molecular weights were observed, and the intensity of these proteins increased after 3h of incubation. These results support that inter- or intramolecular crosslinking occurred in the casein proteins of MTGase-treated milk. Two-dimensional electrophoresis analysis indicated that isomers of β-casein, κ-casein, a fraction of serum albumin, α(S1)-casein, α(S2)-casein, β-lactoglobulin, and α-lactalbumin in the milk were polymerized following incubation with MTGase. In addition, MTGase-induced polymerization occurred earlier for β-casein and κ-casein isomers than for other milk proteins. PMID:22281322

  16. Gelsolin, a Protein That Caps the Barbed Ends and Severs Actin Filaments, Enhances the Actin-Based Motility of Listeria monocytogenes in Host Cells

    PubMed Central

    Laine, Roney O.; Phaneuf, Katherine L.; Cunningham, Casey C.; Kwiatkowski, David; Azuma, Toshi; Southwick, Frederick S.

    1998-01-01

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

  17. On the organization of self-assembled actin networks in giant vesicles

    NASA Astrophysics Data System (ADS)

    Limozin, L.; Bärmann, M.; Sackmann, E.

    2003-04-01

    We studied the formation of actin scaffolds in giant vesicles of dimyristoylphosphatidylcholine (DMPC). Polymerization of actin was induced at low ionic strength through ionophore-mediated influx of Mg^{2+} (2 mM). The spatial organization of the filamentous actin was visualized by confocal and epifluorescence microscopy as a function of the filaments length and membrane composition, by including various amounts of cholesterol or lipids with neutral and positively charged polyethyleneglycol headgroups (PEG lipopolymers). In vesicles of pure DMPC, the newly polymerized actin adsorbs to the membrane and forms a thin shell. In the presence of 2.5 mol% lipopolymers or of cholesterol at a molar fraction x=0.37, formation of a thin adsorbed film is impeded. A fuzzy cortex is predominantly formed in vesicles of diameter d smaller than the filament persistence length (dleq 15 μm) while for larger vesicles a homogeneous network formation is favoured in the bulk of the vesicle. The fuzzy-cortex formation is interpreted as a consequence of the reduction of the bending energy if the actin filaments accumulate close to the vesicle wall.

  18. Induced Secondary Structure and Polymorphism in an Intrinsically Disordered Structural Linker of the CNS: Solid-State NMR and FTIR Spectroscopy of Myelin Basic Protein Bound to Actin

    PubMed Central

    Ahmed, Mumdooh A.M.; Bamm, Vladimir V.; Shi, Lichi; Steiner-Mosonyi, Marta; Dawson, John F.; Brown, Leonid; Harauz, George; Ladizhansky, Vladimir

    2009-01-01

    Abstract The 18.5 kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that maintains the structural integrity of the myelin sheath of the central nervous system by conjoining the cytoplasmic leaflets of oligodendrocytes and by linking the myelin membrane to the underlying cytoskeleton whose assembly it strongly promotes. It is a multifunctional, intrinsically disordered protein that behaves primarily as a structural stabilizer, but with elements of a transient or induced secondary structure that represent binding sites for calmodulin or SH3-domain-containing proteins, inter alia. In this study we used solid-state NMR (SSNMR) and Fourier transform infrared (FTIR) spectroscopy to study the conformation of 18.5 kDa MBP in association with actin microfilaments and bundles. FTIR spectroscopy of fully 13C,15N-labeled MBP complexed with unlabeled F-actin showed induced folding of both protein partners, viz., some increase in β-sheet content in actin, and increases in both α-helix and β-sheet content in MBP, albeit with considerable extended structure remaining. Solid-state NMR spectroscopy revealed that MBP in MBP-actin assemblies is structurally heterogeneous but gains ordered secondary structure elements (both α-helical and β-sheet), particularly in the terminal fragments and in a central immunodominant epitope. The overall conformational polymorphism of MBP is consistent with its in vivo roles as both a linker (membranes and cytoskeleton) and a putative signaling hub. PMID:19134474

  19. Neutrophil actin dysfunction is a genetic disorder associated with partial impairment of neutrophil actin assembly in three family members.

    PubMed Central

    Southwick, F S; Dabiri, G A; Stossel, T P

    1988-01-01

    A male infant with a severe neutrophil motility disorder and poorly polymerizable actin in PMN extracts was reported over a decade ago to have neutrophil actin dysfunction (NAD) (1974. N. Engl. J. Med. 291:1093-1099). Polymerized actin (F-actin) content of fixed and permeabilized intact neutrophils from the father, mother, and sister of the NAD index case have been measured using nitrobenzoxadiazole-phallacidin, a fluorescent compound which binds specifically to actin filaments. F-actin content of unstimulated PMN from all three family members was significantly lower than unstimulated control PMN (mean 23.6 +/- 0.4 SEM fluorescent units vs. 32.6 +/- 0.6 for controls). After stimulation with the chemotactic peptide FMLP, maximal F-actin content of NAD family member PMN was below that of controls (52.7 +/- 1.3 vs. 72.6 +/- 1.8). F-actin content of detergent insoluble cytoskeletons after stimulation with FMLP was also significantly lower in PMN from NAD family members as compared with controls (21 +/- 6% vs. 73 +/- 8%). PMN extracts from the father and mother, when treated with 0.6 M KCl, polymerized half as much actin as controls. Whereas diisopropylfluorophosphate treatment of normal PMN decreased actin polymerizability in cell extracts, this treatment increased the assembly of actin in parental PMN extract. Addition of purified actin to NAD extracts failed to reveal an abnormal actin polymerization inhibitory activity, and no obvious structural defect in actin purified from the father's PMNs was noted by HPLC and two dimensional thin layer chromatography of tryptic digests. The present studies of actin assembly in intact PMNs confirm that NAD is associated with a true defect in PMN actin assembly and is a genetic disorder that is recessively inherited. Images PMID:3183050

  20. Mechanics of biomimetic systems propelled by actin comet tails

    NASA Astrophysics Data System (ADS)

    Kang, Hyeran; Tambe, Dhananjay; Shenoy, Vivek; Tang, Jay

    2009-03-01

    The motility of intracellular bacterial pathogens such as Listeria monocytogenes is driven by filamentous actin comet tails in a variety of trajectories. Here, we present the in vitro study on the actin-based movements using spherical beads of different sizes coated with VCA protein, a partial domain of N-Wasp, in platelet extracts. Long term two-dimensional trajectories of the spherical beads motility show characteristic difference than those observed for bacteria, which have both elongated shape and asymmetric expression of the polymerization inducing enzyme. The trajectories also vary sensitively with the bead size and shape. These results provide a useful test to our new analytical model including the rotation of the bead relative to the tail.

  1. Low dose latrunculin-A inhibits dexamethasone-induced changes in the actin cytoskeleton and alters extracellular matrix protein expression in cultured human trabecular meshwork cells.

    PubMed

    Liu, X; Wu, Z; Sheibani, N; Brandt, C R; Polansky, J R; Kaufman, P L

    2003-08-01

    We determined the effects of a low dose of the actin-disrupting agent latrunculin (LAT)-A on dexamethasone (DEX)-induced changes in actin organization, focal adhesions, and production of extracellular matrix proteins in cultured human trabecular meshwork (HTM) cells. HTM cells were cultured to a highly confluent stage with stable endothelium-like morphology and incubated with 0.1 or 0.2 microM DEX and/or 0.1 microM LAT-A. Changes in the actin cytoskeleton and vinculin-containing focal contacts were evaluated by immunofluorescence microscopy. Expression of thrombospondin-1 (TSP1) and fibronectin (FN) in HTM cells was evaluated by Western blot analysis. The results showed that DEX induced morphological changes and actin reorganization in HTM cells. The cells partly recovered after DEX withdrawal, but the addition of low dose LAT-A hastened the recovery. In addition, DEX failed to induce changes when co-incubated with LAT-A for at least 4 weeks, and for at least 2 weeks when cells were pre-treated with LAT-A for 2 weeks. HTM cells treated with 0.1 microM LAT-A only for 5 days showed mild disorganization of the actin cytoskeleton and focal adhesions, which persisted during the 4 weeks of treatment. DEX stimulated production of FN in HTM cells independent of LAT-A treatment. LAT-A and, to a lesser extent, DEX inhibited production of TSP1 by HTM cells. Although LAT-A is not a DEX receptor antagonist, it is able to prevent the effects of DEX on the actin cytoskeleton in cultured HTM cells at a dose subthreshold for increasing outflow facility in monkeys. This suggests that LAT-A at low doses may be useful in treating steroid and other glaucomas. TSP1 may be an important target of LAT-A in HTM cells and modulation of TSP may influence the actin cytoskeleton of the trabecular meshwork (TM), and consequently, intraocular pressure. PMID:12873448

  2. Polymorphism of Cross-Linked Actin Networks in Giant Vesicles

    NASA Astrophysics Data System (ADS)

    Limozin, Laurent; Sackmann, Erich

    2002-09-01

    Actin networks cross-linked by natural linkers α-actinin and filamin are generated in giant vesicles by polymerization through ionophore-mediated influx of Mg2+. α-actinin induces the formation of randomly linked networks at 25 °C which transform at <15 °C into spiderweblike gels or ringlike bundles depending on the vesicle size. Muscle filamin forms ringlike structures under all experimental conditions which can supercoil by subsequent Mg2+ addition. The polymorphism is rationalized in terms of recent models of bivalent ion coupled semiflexible polyelectrolytes and by considering the topology of the linkers.

  3. Site-selective nanoscale-polymerization of pyrrole on gold nanoparticles via plasmon induced charge separation

    NASA Astrophysics Data System (ADS)

    Takahashi, Y.; Furukawa, Y.; Ishida, T.; Yamada, S.

    2016-04-01

    We proposed a nanoscale oxidative polymerization method which enables site-selective deposition on the surface of gold nanoparticles (AuNPs) combined with TiO2 by using plasmon induced charge separation (PICS) under visible-to-near infrared (IR) light irradiation. The method also revealed that the anodic site of PICS was located at the surface of AuNPs.We proposed a nanoscale oxidative polymerization method which enables site-selective deposition on the surface of gold nanoparticles (AuNPs) combined with TiO2 by using plasmon induced charge separation (PICS) under visible-to-near infrared (IR) light irradiation. The method also revealed that the anodic site of PICS was located at the surface of AuNPs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01531j

  4. The Effect of Hydrophile Topology in RAFT-Mediated Polymerization-Induced Self-Assembly.

    PubMed

    Lesage de la Haye, Jennifer; Zhang, Xuewei; Chaduc, Isabelle; Brunel, Fabrice; Lansalot, Muriel; D'Agosto, Franck

    2016-03-01

    Polymerization-induced self-assembly (PISA) was employed to compare the self-assembly of different amphiphilic block copolymers. They were obtained by emulsion polymerization of styrene in water using hydrophilic poly(N-acryloylmorpholine) (PNAM)-based macromolecular RAFT agents with different structures. An average of three poly (ethylene glycol acrylate) (PEGA) units were introduced either at the beginning, statistically, or at the end of a PNAM backbone, resulting in formation of nanometric vesicles and spheres from the two former macroRAFT architectures, and large vesicles from the latter. Compared to the spheres obtained with a pure PNAM macroRAFT agent, composite macroRAFT architectures promoted a dramatic morphological change. The change was induced by the presence of PEGA hydrophilic side-chains close to the hydrophobic polystyrene segment. PMID:26880016

  5. Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells

    PubMed Central

    Gao, Ying; Lui, Wing-yee; Lee, Will M.; Cheng, C. Yan

    2016-01-01

    Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES. PMID:27358069

  6. Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells.

    PubMed

    Gao, Ying; Lui, Wing-Yee; Lee, Will M; Cheng, C Yan

    2016-01-01

    Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES. PMID:27358069

  7. Surface Grafting via Photo-Induced Copper-Mediated Radical Polymerization at Extremely Low Catalyst Concentrations.

    PubMed

    Laun, Joachim; Vorobii, Mariia; de los Santos Pereira, Andres; Pop-Georgievski, Ognen; Trouillet, Vanessa; Welle, Alexander; Barner-Kowollik, Christopher; Rodriguez-Emmenegger, Cesar; Junkers, Thomas

    2015-09-01

    Surface-initiated photo-induced copper-mediated radical polymerization is employed to graft a wide range of polyacrylate brushes from silicon substrates at extremely low catalyst concentrations. This is the first time that the controlled nature of the reported process is demonstrated via block copolymer formation and re-initiation experiments. In addition to unmatched copper catalyst concentrations in the range of few ppb, film thicknesses up to almost 1 μm are achieved within only 1 h. PMID:26149622

  8. Spontaneous actin dynamics in contractile rings

    NASA Astrophysics Data System (ADS)

    Kruse, Karsten; Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Riveline, Daniel

    Networks of polymerizing actin filaments are known to be capable to self-organize into a variety of structures. For example, spontaneous actin polymerization waves have been observed in living cells in a number of circumstances, notably, in crawling neutrophils and slime molds. During later stages of cell division, they can also spontaneously form a contractile ring that will eventually cleave the cell into two daughter cells. We present a framework for describing networks of polymerizing actin filaments, where assembly is regulated by various proteins. It can also include the effects of molecular motors. We show that the molecular processes driven by these proteins can generate various structures that have been observed in contractile rings of fission yeast and mammalian cells. We discuss a possible functional role of each of these patterns. The work was supported by Agence Nationale de la Recherche, France, (ANR-10-LABX-0030-INRT) and by Deutsche Forschungsgemeinschaft through SFB1027.

  9. Activation of the cAMP Pathway Induces RACK1-Dependent Binding of β-Actin to BDNF Promoter

    PubMed Central

    Neasta, Jeremie; Fiorenza, Anna; He, Dao-Yao; Phamluong, Khanhky; Kiely, Patrick A.; Ron, Dorit

    2016-01-01

    RACK1 is a scaffolding protein that contributes to the specificity and propagation of several signaling cascades including the cAMP pathway. As such, RACK1 participates in numerous cellular functions ranging from cell migration and morphology to gene transcription. To obtain further insights on the mechanisms whereby RACK1 regulates cAMP-dependent processes, we set out to identify new binding partners of RACK1 during activation of the cAMP signaling using a proteomics strategy. We identified β-actin as a direct RACK1 binding partner and found that the association between β-actin and RACK1 is increased in response to the activation of the cAMP pathway. Furthermore, we show that cAMP-dependent increase in BDNF expression requires filamentous actin. We further report that β-actin associates with the BDNF promoter IV upon the activation of the cAMP pathway and present data to suggest that the association of β-actin with BDNF promoter IV is RACK1-dependent. Taken together, our data suggest that β-actin is a new RACK1 binding partner and that the RACK1 and β-actin association participate in the cAMP-dependent regulation of BDNF transcription. PMID:27505161

  10. 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.

  11. Non-Straub type actin from molluscan catch muscle.

    PubMed

    Shelud'ko, Nikolay S; Girich, Ulyana V; Lazarev, Stanislav S; Vyatchin, Ilya G

    2016-05-27

    We have developed a method of obtaining natural actin from smooth muscles of the bivalves on the example of the Сrenomytilus grayanus catch muscle. The muscles were previously rigorized to prevent a loss of thin filaments during homogenization and washings. Thin filaments were isolated with a low ionic strength solution in the presence of ATP and sodium pyrophosphate. Surface proteins of thin filaments-tropomyosin, troponin, calponin and some minor actin-binding proteins-were dissociated from actin filaments by increasing the ionic strength to 0.6 M KCL. Natural fibrillar actin obtained in that way depolymerizes easily in low ionic strength solutions commonly used for the extraction of Straub-type actin from acetone powder. Purification of natural actin was carried out by the polymerization-depolymerization cycle. The content of inactivated actin remaining in the supernatant is much less than at a similar purification of Straub-type actin. A comparative investigation was performed between the natural mussel actin and the Straub-type rabbit skeletal actin in terms of the key properties of actin: polymerization, activation of Mg-ATPase activity of myosin, and the electron-microscopic structure of actin polymers. PMID:27120462

  12. Simvastatin induces the apoptosis of normal vascular smooth muscle through the disruption of actin integrity via the impairment of RhoA/Rac-1 activity.

    PubMed

    Kang, Seojin; Kim, Keunyoung; Noh, Ji-Yoon; Jung, Yeryeon; Bae, Ok-Nam; Lim, Kyung-Min; Chung, Jin-Ho

    2016-08-30

    Statins, lipid-lowering agents for the prevention of atherosclerosis and fatal coronary heart diseases, have pleiotropic modalities on the function and physiology of vascular smooth muscle that include anti-contractile and pro-apoptotic effects. These effects were suggested to stem from the inhibition of small GTPase Rho A, but they are largely regarded as distinct and unrelated. Recently, we discovered that simvastatin causes both contractile dysfunction and apoptosis of vascular smooth muscle cells (VSMCs), reflecting that they may be closely related, yet their connecting link remains unexplained. Here, we elaborated the mechanism underlying simvastatin-induced apoptosis of normal VSMCs in connection with contractile dysfunction. Repeated oral administration of simvastatin to rats in vivo resulted in contractile dysfunction and apoptosis of vascular smooth muscle, of which pattern was well reproduced in rat VSMCs in vitro. Of note, contractile dysfunction and apoptosis occurred in concerted manners both in vivo and in vitro in the aspects of time course and dose of exposure. In rat VSMCs, simvastatin impaired the activation of small GTPases, RhoA along with Rac-1, which resulted in the disruption of actin integrity, a pivotal factor both for the generation of contractile force and survival of VSMCs. In line with the disruption of actin integrity, Bmf, a pro-apoptotic factor bound to intact actin, dissociated and translocated into mitochondria, which corresponded well with the dissipation of mitochondrial membrane potential, caspase-3 activation and ultimately apoptosis. These events were all rescued by an actin stabilisation agent, jasplakinolide as well as geranylgeraniol, indicating that damages of the actin integrity from disrupted activation of RhoA/Rac-1 lies at the center of simvastatin-induced contractile dysfunction and apoptosis in vascular smooth muscle. PMID:27306926

  13. 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

  14. EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin

    PubMed Central

    Mohan Kumar, Dipu; Lin, Mingqun; Xiong, Qingming; Webber, Mathew James; Kural, Comert

    2015-01-01

    ABSTRACT Obligate intracellular bacteria, such as Ehrlichia chaffeensis, perish unless they can enter eukaryotic cells. E. chaffeensis is the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. To infect cells, Ehrlichia uses the C terminus of the outer membrane invasin entry-triggering protein (EtpE) of Ehrlichia (EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drives Ehrlichia entry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibited Ehrlichia internalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impaired E. chaffeensis infection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibited Ehrlichia entry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulated in vitro actin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drives Ehrlichia entry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization. PMID:26530384

  15. The natural product cucurbitacin E inhibits depolymerization of actin filaments

    PubMed Central

    Sörensen, Pia M.; Iacob, Roxana E.; Fritzsche, Marco; Engen, John R.; Brieher, William M.; Charras, Guillaume; Eggert, Ulrike S.

    2012-01-01

    Although small molecule actin modulators have been widely used as research tools, only one cell permeable small molecule inhibitor of actin depolymerization (jasplakinolide) is commercially available. We report that the natural product cucurbitacin E inhibits actin depolymerization and show that its mechanism of action is different from jasplakinolide. In assays using pure fluorescently labeled actin, cucurbitacin E specifically affected depolymerization without affecting polymerization. It inhibited actin depolymerization at sub-stoichiometric concentrations up to 1:6 cucurbitacin:actin E. Cucurbitacin E specifically binds to filamentous actin (F-actin) forming a covalent bond at residue Cys257, but not to monomeric actin (G-actin). Based on its compatibility with phalloidin staining, we show that cucurbitacin E occupies a different binding site on actin filaments. Using loss of fluorescence after localized photoactivation, we found that cucurbitacin E inhibited actin depolymerization in live cells. Cucurbitacin E is a widely available plant-derived natural product, making it a useful tool to study actin dynamics in cells and actin-based processes such as cytokinesis. PMID:22724897

  16. Actin-dependent mechanisms in AMPA receptor trafficking

    PubMed Central

    Hanley, Jonathan G.

    2014-01-01

    The precise regulation of AMPA receptor (AMPAR) number and subtype at the synapse is crucial for the regulation of excitatory neurotransmission, synaptic plasticity and the consequent formation of appropriate neural circuits for learning and memory. AMPAR trafficking involves the dynamic processes of exocytosis, endocytosis and endosomal recycling, all of which involve the actin cytoskeleton. The actin cytoskeleton is highly dynamic and highly regulated by an abundance of actin-binding proteins and upstream signaling pathways that modulate actin polymerization and depolymerization. Actin dynamics generate forces that manipulate membranes in the process of vesicle biogenesis, and also for propelling vesicles through the cytoplasm to reach their destination. In addition, trafficking mechanisms exploit more stable aspects of the actin cytoskeleton by using actin-based motor proteins to traffic vesicular cargo along actin filaments. Numerous studies have shown that actin dynamics are critical for AMPAR localization and function. The identification of actin-binding proteins that physically interact with AMPAR subunits, and research into their mode of action is starting to shed light on the mechanisms involved. Such proteins either regulate actin dynamics to modulate mechanical forces exerted on AMPAR-containing membranes, or associate with actin filaments to target or transport AMPAR-containing vesicles to specific subcellular regions. In addition, actin-regulatory proteins that do not physically interact with AMPARs may influence AMPAR trafficking by regulating the local actin environment in the dendritic spine. PMID:25429259

  17. CDK5 Is Essential for Soluble Amyloid β-Induced Degradation of GKAP and Remodeling of the Synaptic Actin Cytoskeleton

    PubMed Central

    Roselli, Francesco; Livrea, Paolo; Almeida, Osborne F. X.

    2011-01-01

    The early stages of Alzheimer's disease are marked by synaptic dysfunction and loss. This process results from the disassembly and degradation of synaptic components, in particular of scaffolding proteins that compose the post-synaptic density (PSD), namely PSD95, Homer and Shank. Here we investigated in rat frontal cortex dissociated culture the mechanisms involved in the downregulation of GKAP (SAPAP1), which links the PSD95 complex to the Shank complex and cytoskeletal structures within the PSD. We show that Aβ causes the rapid loss of GKAP from synapses through a pathway that critically requires cdk5 activity, and is set in motion by NMDAR activity and Ca2+ influx. We show that GKAP is a direct substrate of cdk5 and that its phosphorylation results in polyubiquitination and proteasomal degradation of GKAP and remodeling (collapse) of the synaptic actin cytoskeleton; the latter effect is abolished in neurons expressing GKAP mutants that are resistant to phosphorylation by cdk5. Given that cdk5 also regulates degradation of PSD95, these results underscore the central position of cdk5 in mediating Aβ-induced PSD disassembly and synapse loss. PMID:21829588

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

    PubMed Central

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

    2016-01-01

    Summary During central nervous system development, oligodendrocytes wrap their plasma membrane around axons to generate multi-lamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/Cofilin1, which mediates high F-actin turnover rates, as essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading. PMID:26166299

  19. 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

  20. Regulation of the actin cytoskeleton in Helicobacter pylori-induced migration and invasive growth of gastric epithelial cells

    PubMed Central

    2011-01-01

    Dynamic rearrangement of the actin cytoskeleton is a significant hallmark of Helicobacter pylori (H. pylori) infected gastric epithelial cells leading to cell migration and invasive growth. Considering the cellular mechanisms, the type IV secretion system (T4SS) and the effector protein cytotoxin-associated gene A (CagA) of H. pylori are well-studied initiators of distinct signal transduction pathways in host cells targeting kinases, adaptor proteins, GTPases, actin binding and other proteins involved in the regulation of the actin lattice. In this review, we summarize recent findings of how H. pylori functionally interacts with the complex signaling network that controls the actin cytoskeleton of motile and invasive gastric epithelial cells. PMID:22044652

  1. Radiation-induced cationic polymerization of limonene oxide,. cap alpha. -pinene oxide, and. beta. -pinene oxide

    SciTech Connect

    Aikins, J.A.; Williams, F.

    1984-01-01

    After suitable drying, the subject monomers in the form of neat liquids undergo radiation-induced polymerization with no apparent side reactions and high conversions to precipitatable polymers of low molecular weight. A cationic mechanism is evidenced by the strongly retarding effect of tri-n-propylamine on the polymerization rate. At 25/sup 0/C, limonene oxide gives the highest polymerization rates, an average conversion of 36% per Mrad being obtained in comparison with values of 5.7 and 7.3% per Mrad for the ..cap alpha..-pinene and ..beta..-pinene oxides, respectively. Similarly, the average anti DP/sub n/ decreases from 11.8 for the limonene oxide polymer to 5.6 and 4.0 for the ..cap alpha..-pinene oxide and ..beta..-pinene oxide polymers, respectively. A high frequency of chain transfer to monomer is indicated in each case by the fact that the kinetic chain lengths are estimated to be on the order of a hundred times larger than the anti DP/sub n/ values. Structural characterization of the limonene oxide polymer by /sup 1/H and /sup 13/C NMR spectroscopy provides conclusive evidence that the polymerization proceeds by the opening of the epoxide ring to yield a 1,2-trans polyether. Similar NMR studies on the polymers formed from the ..cap alpha..-pinene and ..beta..-pinene oxides show that in the polymerization of these monomers, the opening of the epoxide ring is generally accompanied by the concomitant ring opening of the cyclobutane ring structure to yield a gem-dimethyl group in the main chain. The detection of isopropenyl end groups in the pinene oxide polymers is also consistent with this mode of propagation being followed by chain (proton) transfer to monomer.

  2. Sirtuin1 Maintains Actin Cytoskeleton by Deacetylation of Cortactin in Injured Podocytes

    PubMed Central

    Motonishi, Shuta; Wada, Takehiko; Ishimoto, Yu; Ohse, Takamoto; Matsusaka, Taiji; Kubota, Naoto; Shimizu, Akira; Kadowaki, Takashi; Tobe, Kazuyuki

    2015-01-01

    Recent studies have highlighted the renoprotective effect of sirtuin1 (SIRT1), a deacetylase that contributes to cellular regulation. However, the pathophysiologic role of SIRT1 in podocytes remains unclear. Here, we investigated the function of SIRT1 in podocytes. We first established podocyte-specific Sirt1 knockout (SIRT1pod−/−) mice. We then induced glomerular disease by nephrotoxic serum injection. The increase in urinary albumin excretion and BUN and the severity of glomerular injury were all significantly greater in SIRT1pod−/− mice than in wild-type mice. Western blot analysis and immunofluorescence showed a significant decrease in podocyte-specific proteins in SIRT1pod−/− mice, and electron microscopy showed marked exacerbation of podocyte injury, including actin cytoskeleton derangement in SIRT1pod−/− mice compared with wild-type mice. Protamine sulfate-induced podocyte injury was also exacerbated by podocyte-specific SIRT1 deficiency. In vitro, actin cytoskeleton derangement in H2O2-treated podocytes became prominent when the cells were pretreated with SIRT1 inhibitors. Conversely, this H2O2-induced derangement was ameliorated by SIRT1 activation. Furthermore, SIRT1 activation deacetylated the actin-binding and -polymerizing protein cortactin in the nucleus and facilitated deacetylated cortactin localization in the cytoplasm. Cortactin knockdown or inhibition of the nuclear export of cortactin induced actin cytoskeleton derangement and dissociation of cortactin from F-actin, suggesting the necessity of cytoplasmic cortactin for maintenance of the actin cytoskeleton. Taken together, these findings indicate that SIRT1 protects podocytes and prevents glomerular injury by deacetylating cortactin and thereby, maintaining actin cytoskeleton integrity. PMID:25424328

  3. A magnetically actuated cellular strain assessment tool for quantitative analysis of strain induced cellular reorientation and actin alignment

    NASA Astrophysics Data System (ADS)

    Khademolhosseini, F.; Liu, C.-C.; Lim, C. J.; Chiao, M.

    2016-08-01

    Commercially available cell strain tools, such as pneumatically actuated elastomer substrates, require special culture plates, pumps, and incubator setups. In this work, we present a magnetically actuated cellular strain assessment tool (MACSAT) that can be implemented using off-the-shelf components and conventional incubators. We determine the strain field on the MACSAT elastomer substrate using numerical models and experimental measurements and show that a specific region of the elastomer substrate undergoes a quasi-uniaxial 2D stretch, and that cells confined to this region of the MACSAT elastomer substrate undergo tensile, compressive, or zero axial strain depending on their angle of orientation. Using the MACSAT to apply cyclic strain on endothelial cells, we demonstrate that actin filaments within the cells reorient away from the stretching direction, towards the directions of minimum axial strain. We show that the final actin orientation angles in strained cells are spread over a region of compressive axial strain, confirming previous findings on the existence of a varied pre-tension in the actin filaments of the cytoskeleton. We also demonstrate that strained cells exhibit distinctly different values of actin alignment coherency compared to unstrained cells and therefore propose that this parameter, i.e., the coherency of actin alignment, can be used as a new readout to determine the occurrence/extent of actin alignment in cell strain experiments. The tools and methods demonstrated in this study are simple and accessible and can be easily replicated by other researchers to study the strain response of other adherent cells.

  4. Controlling and understanding the polymerization shrinkage-induced stresses in light-cured composites.

    PubMed

    Suh, B I

    1999-01-01

    The shrinkage of composites induces stress in the final restoration when the composite is bonded to the tooth surface. The amount of stress can be controlled by the method of pulse-delay cure used. The development of new composites has changed the energy requirements for obtaining polymerization. The total energy required for optimal polymerization has been reduced and the rate at which the energy is delivered can have a strong effect on the final properties of the restoration. Newer composites may employ different photoinitiators, making the spectral emission from the curing light an important factor for properties of the final restoration. It is proposed that labels for composites carry: (1) indications for the total energy required; (2) specification of the pulse-delay cure if applicable; and (3) the required spectral bandwidth of the curing lamp light emission. PMID:11908394

  5. Light-induced surface graft polymerizations initiated by an anthraquinone dye on cotton fibers.

    PubMed

    Zhuo, Jingyuan; Sun, Gang

    2014-11-01

    Anthraquinone and its derivatives could serve as photo-sensitizers and generate radicals and reactive oxygen species in polymers under exposure of UVA or day light. Such a property was utilized in development of novel light-induced surface radical graft polymerizations on cotton fibers that were dyed with an anthraquinone derivative, 2-ethylanthraquinone. Several functional monomers were directly grafted onto the dyed cotton fibers upon UVA exposure. The chemical and morphological structures and thermal properties of the grafted fibers were confirmed and characterized by Fourier transform infrared spectrometer (FTIR), scanning electron microscope (SEM) and thermal gravimetric analysis (TGA). Reaction conditions including concentrations of the photosensitizer, the amount of monomers, as well as UVA irradiation time could influence grafting efficiencies. More interestingly, the surface graft polymerization did not significantly change the light active functions of the agent, evidenced by the light-active antimicrobial functions of the grafted fibers. PMID:25129730

  6. Thermally induced release from polymeric microparticles with liquid core: the mechanism.

    PubMed

    Latnikova, Alexandra; Yildirim, Arda

    2015-03-14

    Herein we demonstrate how the volatility of a liquid can be manipulated by enclosing microdroplets of the liquid into thin polymeric shells. In this way, composite core-shell microparticles consisting of 80 wt% of a liquid core material and 20 wt% of a polymer can be made 150 °C more stable than the individual core component. The thermal stability of the composite microparticles is found to be determined by the boiling point of the core material and the average particle size, while the role of the particle shell thickness is much less relevant. Two mechanisms responsible for the release of the core material from the microparticles at elevated temperatures were resolved: (1) thermally induced degradation of the shell and (2) diffusion of the core material through the polymeric shell boosted by the increased inner pressure. PMID:25627164

  7. Scapinin, the Protein Phosphatase 1 Binding Protein, Enhances Cell Spreading and Motility by Interacting with the Actin Cytoskeleton

    PubMed Central

    Sagara, Junji; Arata, Toshiaki; Taniguchi, Shunichiro

    2009-01-01

    Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro. Next, we established a Hela cell line, where scapinin expression was induced by tetracycline. In these cells, expression of scapinin stimulated cell spreading and motility. Scapinin was colocalized with actin at the edge of spreading cells. To explore the roles of the RPEL-repeat and PP1-binding domains, we expressed wild-type and mutant scapinins as fusion proteins with green fluorescence protein (GFP) in Cos7 cells. Expression of GFP-scapinin (wild type) also stimulated cell spreading, but mutation in the RPEL-repeat domain abolished both the actin binding and the cell spreading activity. PP1-binding deficient mutants strongly induced cell retraction. Long and branched cytoplasmic processes were developed during the cell retraction. These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes. PMID:19158953

  8. Binding of WIP to Actin Is Essential for T Cell Actin Cytoskeleton Integrity and Tissue Homing

    PubMed Central

    Massaad, Michel J.; Oyoshi, Michiko K.; Kane, Jennifer; Koduru, Suresh; Alcaide, Pilar; Nakamura, Fumihiko; Ramesh, Narayanaswamy; Luscinskas, Francis W.; Hartwig, John

    2014-01-01

    The Wiskott-Aldrich syndrome protein (WASp) is important for actin polymerization in T cells and for their migration. WASp-interacting protein (WIP) binds to and stabilizes WASp and also interacts with actin. Cytoskeletal and functional defects are more severe in WIP−/− T cells, which lack WASp, than in WASp−/− T cells, suggesting that WIP interaction with actin may be important for T cell cytoskeletal integrity and function. We constructed mice that lack the actin-binding domain of WIP (WIPΔABD mice). WIPΔABD associated normally with WASp but not F-actin. T cells from WIPΔABD mice had normal WASp levels but decreased cellular F-actin content, a disorganized actin cytoskeleton, impaired chemotaxis, and defective homing to lymph nodes. WIPΔABD mice exhibited a T cell intrinsic defect in contact hypersensitivity and impaired responses to cutaneous challenge with protein antigen. Adoptively transferred antigen-specific CD4+ T cells from WIPΔABD mice had decreased homing to antigen-challenged skin of wild-type recipients. These findings show that WIP binding to actin, independently of its binding to WASp, is critical for the integrity of the actin cytoskeleton in T cells and for their migration into tissues. Disruption of WIP binding to actin could be of therapeutic value in T cell-driven inflammatory diseases. PMID:25246631

  9. The Tyrosine Kinase Activity of c-Src Regulates Actin Dynamics and Organization of Podosomes in Osteoclasts

    PubMed Central

    Destaing, Olivier; Sanjay, Archana; Itzstein, Cecile; Horne, William C.; Toomre, Derek

    2008-01-01

    Podosomes are dynamic actin-rich structures composed of a dense F-actin core surrounded by a cloud of more diffuse F-actin. Src performs one or more unique functions in osteoclasts (OCLs), and podosome belts and bone resorption are impaired in the absence of Src. Using Src−/− OCLs, we investigated the specific functions of Src in the organization and dynamics of podosomes. We found that podosome number and the podosome-associated actin cloud were decreased in Src−/− OCLs. Videomicroscopy and fluorescence recovery after photobleaching analysis revealed that the life span of Src−/− podosomes was increased fourfold and that the rate of actin flux in the core was decreased by 40%. Thus, Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes and in the actin cloud. Rescue of Src−/− OCLs with Src mutants showed that both the kinase activity and either the SH2 or the SH3 binding domain are required for Src to restore normal podosome organization and dynamics. Moreover, inhibition of Src family kinase activities in Src−/− OCLs by Src inhibitors or by expressing dominant-negative SrcK295M induced the formation of abnormal podosomes. Thus, Src is an essential regulator of podosome structure, dynamics and organization. PMID:17978100

  10. 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

  11. Distributed actin turnover in the lamellipodium and FRAP kinetics.

    PubMed

    Smith, Matthew B; Kiuchi, Tai; Watanabe, Naoki; Vavylonis, Dimitrios

    2013-01-01

    Studies of actin dynamics at the leading edge of motile cells with single-molecule speckle (SiMS) microscopy have shown a broad distribution of EGFP-actin speckle lifetimes and indicated actin polymerization and depolymerization over an extended region. Other experiments using FRAP with the same EGFP-actin as a probe have suggested, by contrast, that polymerization occurs exclusively at the leading edge. We performed FRAP experiments on XTC cells to compare SiMS to FRAP on the same cell type. We used speckle statistics obtained by SiMS to model the steady-state distribution and kinetics of actin in the lamellipodium. We demonstrate that a model with a single diffuse actin species is in good agreement with FRAP experiments. A model including two species of diffuse actin provides an even better agreement. The second species consists of slowly diffusing oligomers that associate to the F-actin network throughout the lamellipodium or break up into monomers after a characteristic time. Our work motivates studies to test the presence and composition of slowly diffusing actin species that may contribute to local remodeling of the actin network and increase the amount of soluble actin. PMID:23332077

  12. 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

  13. [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

  14. 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

  15. Reprint of: self-assembly of nanoparticles employing polymerization-induced phase separation.

    PubMed

    Williams, Roberto J J; Hoppe, Cristina E; Zucchi, Ileana A; Romeo, Hernán E; dell'Erba, Ignacio E; Gómez, María L; Puig, Julieta; Leonardi, Agustina B

    2015-06-01

    Nanoparticles (NPs) may be homogeneously dispersed in the precursors of a polymer (reactive solvent) by an adequate selection of their stabilizing ligands. However, the dispersion can become metastable or unstable in the course of polymerization. If this happens, NP-rich domains can be segregated by a process called polymerization-induced phase separation (PIPS). This occurs mainly due to the decrease in the entropic contribution of the reactive solvent to the free energy of mixing (increase in its average size) and, for a reactive solvent generating a cross-linked polymer, the additional contribution of the elastic energy in the post-gel stage. The extent of PIPS will depend on the competition between phase separation and polymerization rates. It can be completely avoided, limited to a local scale or conveyed to generate different types of NPs' aggregates such as crystalline platelets, self-assembled structures with a hierarchical order and partitioning at the interface, and bidimensional patterns of NPs at the film surface. The use of a third component in the initial formulation such as a linear polymer or a block copolymer, provides the possibility of generating an internal template for the preferential location and self-assembly of phase-separated NPs. Some illustrative examples of morphologies generated by PIPS in solutions of NPs in reactive solvents, are analyzed in this feature article. PMID:25736431

  16. Self-assembly of nanoparticles employing polymerization-induced phase separation.

    PubMed

    Williams, Roberto J J; Hoppe, Cristina E; Zucchi, Ileana A; Romeo, Hernán E; dell'Erba, Ignacio E; Gómez, María L; Puig, Julieta; Leonardi, Agustina B

    2014-10-01

    Nanoparticles (NPs) may be homogeneously dispersed in the precursors of a polymer (reactive solvent) by an adequate selection of their stabilizing ligands. However, the dispersion can become metastable or unstable in the course of polymerization. If this happens, NP-rich domains can be segregated by a process called polymerization-induced phase separation (PIPS). This occurs mainly due to the decrease in the entropic contribution of the reactive solvent to the free energy of mixing (increase in its average size) and, for a reactive solvent generating a cross-linked polymer, the additional contribution of the elastic energy in the post-gel stage. The extent of PIPS will depend on the competition between phase separation and polymerization rates. It can be completely avoided, limited to a local scale or conveyed to generate different types of NPs' aggregates such as crystalline platelets, self-assembled structures with a hierarchical order and partitioning at the interface, and bidimensional patterns of NPs at the film surface. The use of a third component in the initial formulation such as a linear polymer or a block copolymer, provides the possibility of generating an internal template for the preferential location and self-assembly of phase-separated NPs. Some illustrative examples of morphologies generated by PIPS in solutions of NPs in reactive solvents, are analyzed in this feature article. PMID:25014172

  17. Cuspal Displacement Induced by Bulk Fill Resin Composite Polymerization: Biomechanical Evaluation Using Fiber Bragg Grating Sensors

    PubMed Central

    Ramos, João; Alves, Sofia; Nogueira, Rogério

    2016-01-01

    Polymerization shrinkage is a major concern to the clinical success of direct composite resin restorations. The aim of this study was to compare the effect of polymerization shrinkage strain of two resin composites on cuspal movement based on the use of fiber Bragg grating (FBG) sensors. Twenty standardized Class II cavities prepared in upper third molars were allocated into two groups (n = 10). Restorations involved the bulk fill placement of conventional microhybrid resin composite (Esthet•X® HD, Dentsply DeTrey) (Group 1) or flowable “low-shrinkage” resin composite (SDR™, Dentsply DeTrey) (Group 2). Two FBG sensors were used per restoration for real-time measurement of cuspal linear deformation and temperature variation. Group comparisons were determined using ANCOVA (α = 0.05) considering temperature as the covariate. A statistically significant correlation between cuspal deflection, time, and material was observed (p < 0.01). Cuspal deflection reached 8.8 μm (0.23%) and 7.8 μm (0.20%) in Groups 1 and 2, respectively. When used with bulk fill technique, flowable resin composite SDR™ induced significantly less cuspal deflection than the conventional resin composite Esthet•X® HD (p = 0.015) and presented a smoother curve slope during the polymerization. FBG sensors appear to be a valid tool for accurate real-time monitoring of cuspal deformation. PMID:27190517

  18. Microfluidic Production of Semipermeable Microcapsules by Polymerization-Induced Phase Separation.

    PubMed

    Kim, Bomi; Jeon, Tae Yoon; Oh, You-Kwan; Kim, Shin-Hyun

    2015-06-01

    Semipermeable microcapsules are appealing for controlled release of drugs, study of cell-to-cell communication, and isolation of enzymes or artificial catalysts. Here, we report a microfluidic strategy for creating monodisperse microcapsules with size-selective permeability using polymerization-induced phase separation. Monodisperse water-in-oil-in-water (W/O/W) double-emulsion drops, whose ultrathin middle layer is composed of photocurable resin and inert oil, are generated in a capillary microfluidic device, and irradiated by UV light. Upon UV illumination, the monomers are photopolymerized, which leads to phase separation between the polymerized resin and the oil within the ultrathin shell. Subsequent dissolution of the oil leaves behind regular pores in the polymerized membrane that interconnect the interior and exterior of the microcapsules, thereby providing size-selective permeability. The degree of phase separation can be further tuned by adjusting the fraction of oil in the shell or the affinity of the oil to the monomers, thereby enabling the control of the cutoff value of permeation. High mechanical stability and chemical resistance of the microcapsules, as well as controllable permeability and high encapsulation efficiency, will provide new opportunity in a wide range of applications. PMID:26020458

  19. Cuspal Displacement Induced by Bulk Fill Resin Composite Polymerization: Biomechanical Evaluation Using Fiber Bragg Grating Sensors.

    PubMed

    Vinagre, Alexandra; Ramos, João; Alves, Sofia; Messias, Ana; Alberto, Nélia; Nogueira, Rogério

    2016-01-01

    Polymerization shrinkage is a major concern to the clinical success of direct composite resin restorations. The aim of this study was to compare the effect of polymerization shrinkage strain of two resin composites on cuspal movement based on the use of fiber Bragg grating (FBG) sensors. Twenty standardized Class II cavities prepared in upper third molars were allocated into two groups (n = 10). Restorations involved the bulk fill placement of conventional microhybrid resin composite (Esthet•X® HD, Dentsply DeTrey) (Group 1) or flowable "low-shrinkage" resin composite (SDR™, Dentsply DeTrey) (Group 2). Two FBG sensors were used per restoration for real-time measurement of cuspal linear deformation and temperature variation. Group comparisons were determined using ANCOVA (α = 0.05) considering temperature as the covariate. A statistically significant correlation between cuspal deflection, time, and material was observed (p < 0.01). Cuspal deflection reached 8.8 μm (0.23%) and 7.8 μm (0.20%) in Groups 1 and 2, respectively. When used with bulk fill technique, flowable resin composite SDR™ induced significantly less cuspal deflection than the conventional resin composite Esthet•X® HD (p = 0.015) and presented a smoother curve slope during the polymerization. FBG sensors appear to be a valid tool for accurate real-time monitoring of cuspal deformation. PMID:27190517

  20. Activation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid.

    PubMed

    Lee, Kwang Hee; Piao, Hai Lan; Kim, Ho-Youn; Choi, Sang Mi; Jiang, Fan; Hartung, Wolfram; Hwang, Ildoo; Kwak, June M; Lee, In-Jung; Hwang, Inhwan

    2006-09-22

    Abscisic acid (ABA) is a phytohormone critical for plant growth, development, and adaptation to various stress conditions. Plants have to adjust ABA levels constantly to respond to changing physiological and environmental conditions. To date, the mechanisms for fine-tuning ABA levels remain elusive. Here we report that AtBG1, a beta-glucosidase, hydrolyzes glucose-conjugated, biologically inactive ABA to produce active ABA. Loss of AtBG1 causes defective stomatal movement, early germination, abiotic stress-sensitive phenotypes, and lower ABA levels, whereas plants with ectopic AtBG1 accumulate higher ABA levels and display enhanced tolerance to abiotic stress. Dehydration rapidly induces polymerization of AtBG1, resulting in a 4-fold increase in enzymatic activity. Furthermore, diurnal increases in ABA levels are attributable to polymerization-mediated AtBG1 activation. We propose that the activation of inactive ABA pools by polymerized AtBG1 is a mechanism by which plants rapidly adjust ABA levels and respond to changing environmental cues. PMID:16990135

  1. β1 and β3 Integrins Cooperate to Induce Syndecan-4-Containing Cross-linked Actin Networks in Human Trabecular Meshwork Cells

    PubMed Central

    Filla, Mark S.; Woods, Anne; Kaufman, Paul L.; Peters, Donna M.

    2006-01-01

    Purpose To characterize the molecular composition of cross-linked actin networks (CLANs) and the regulation of their formation by integrins in normal human trabecular meshwork (TM) cells. CLANs have been observed in steroid-treated and glaucomatous TM cells and have been suggested to contribute to decreased outflow facility by altering the contractility of the TM. Methods Immunofluorescence microscopy was used to identify molecular components of CLANs and quantitate CLAN formation in HTM cells plated on coverslips coated with various extracellular matrix (ECM) proteins (fibronectin, types I and IV collagen, and vitronectin), vascular cell adhesion molecule (VCAM)-1, or activating antibodies against β1, β3, or α2β1 integrins. These integrin antibodies were also used as soluble ligands. Results CLAN vertices contained the actin-binding proteins α-actinin and filamin and the signaling molecules syndecan-4 and PIP2. CLANs lacked Arp3 and cortactin. CLAN formation was dependent on the ECM substrate and was significantly higher on fibronectin and VCAM-1 compared with vitronectin, types I or IV collagen. Adsorbed β1 integrin antibodies also induced CLANs, whereas adsorbed β3 or α2β1 integrin antibodies did not. Soluble β3 integrin antibodies, however, induced CLANs and actually enhanced CLAN formation in cells spread on fibronectin, VCAM-1, type I or type IV collagen, or β1 integrin antibodies. Conclusions CLANs are unique actin-branched networks whose formation can be regulated by β1 and β3 integrin signaling pathways. Thus, integrin-mediated signaling events can modulate the organization of the actin cytoskeleton in TM cells and hence could participate in regulating cytoskeletal events previously demonstrated to be involved in controlling outflow facility. PMID:16639003

  2. A magnetically actuated cellular strain assessment tool for quantitative analysis of strain induced cellular reorientation and actin alignment.

    PubMed

    Khademolhosseini, F; Liu, C-C; Lim, C J; Chiao, M

    2016-08-01

    Commercially available cell strain tools, such as pneumatically actuated elastomer substrates, require special culture plates, pumps, and incubator setups. In this work, we present a magnetically actuated cellular strain assessment tool (MACSAT) that can be implemented using off-the-shelf components and conventional incubators. We determine the strain field on the MACSAT elastomer substrate using numerical models and experimental measurements and show that a specific region of the elastomer substrate undergoes a quasi-uniaxial 2D stretch, and that cells confined to this region of the MACSAT elastomer substrate undergo tensile, compressive, or zero axial strain depending on their angle of orientation. Using the MACSAT to apply cyclic strain on endothelial cells, we demonstrate that actin filaments within the cells reorient away from the stretching direction, towards the directions of minimum axial strain. We show that the final actin orientation angles in strained cells are spread over a region of compressive axial strain, confirming previous findings on the existence of a varied pre-tension in the actin filaments of the cytoskeleton. We also demonstrate that strained cells exhibit distinctly different values of actin alignment coherency compared to unstrained cells and therefore propose that this parameter, i.e., the coherency of actin alignment, can be used as a new readout to determine the occurrence/extent of actin alignment in cell strain experiments. The tools and methods demonstrated in this study are simple and accessible and can be easily replicated by other researchers to study the strain response of other adherent cells. PMID:27587150

  3. A Critical Appraisal of RAFT-Mediated Polymerization-Induced Self-Assembly

    PubMed Central

    2016-01-01

    Recently, polymerization-induced self-assembly (PISA) has become widely recognized as a robust and efficient route to produce block copolymer nanoparticles of controlled size, morphology, and surface chemistry. Several reviews of this field have been published since 2012, but a substantial number of new papers have been published in the last three years. In this Perspective, we provide a critical appraisal of the various advantages offered by this approach, while also pointing out some of its current drawbacks. Promising future research directions as well as remaining technical challenges and unresolved problems are briefly highlighted. PMID:27019522

  4. Immobilization of yeast cells on hydrogel carriers obtained by radiation-induced polymerization

    NASA Astrophysics Data System (ADS)

    Xin, Lu Zhao; Carenza, Mario; Kaetsu, Isao; Kumakura, Minoru; Yoshida, Masaru; Fujimura, Takashi

    Polymer hydrogels were obtained by radiation-induced copolymerization at -78°C of aqueous solutions of acrylic and methacrylic esters. The matrices were characterized by equilibrium water content measurements, by optical microscopy observations and by scanning electron microscopy analysis. Yeast cells were immobilized on these hydrogels and the ethanol productivity by batch fermentation was determined. Matrix hydrophilicity and porosity were found to deeply influence the adhesion of yeast cells and, hence, the ethanol productivity. The latter as well as other physico-chemical properties were also affected by the presence of a crosslinking agent added in small amounts to the polymerizing mixture.

  5. Actin age orchestrates myosin-5 and myosin-6 run lengths.

    PubMed

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

    2015-08-01

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

  6. Structural Transitions of F-Actin:Espin Bundles

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin; Bartles, James; Wong, Gerard

    2006-03-01

    Espin is an actin bundling protein involved in the formation of the parallel bundles of filamentous actin in hair cell stereocilia. Mutations in espin are implicated in deafness phenotypes in mice and humans. We present measurements of the F-actin structures induced by wild type and by mutated espin obtained via small angle x-ray scattering and fluorescence microscopy. We found that wild type espin induced a paracrystalline hexagonal array of twisted F-actin, whereas the mutated espin only condensed the F-actin into a nematic-like phase. The possibility of coexisting nematic and bundled actin in mixtures containing both mutant and wild type espins was also investigated.

  7. New mechanism of γ-H2AX generation: Surfactant-induced actin disruption causes deoxyribonuclease I translocation to the nucleus and forms DNA double-strand breaks.

    PubMed

    Zhao, Xiaoxu; Yang, Gang; Toyooka, Tatsushi; Ibuki, Yuko

    2015-12-01

    We previously showed that nonionic surfactants, nonylphenol polyethoxylates (NPEOs), induced phosphorylation of histone H2AX, forming γ-H2AX. In this study, we analyzed the mechanism of γ-H2AX generation by an NPEO with 15 ethylene oxide units (NPEO(15)). In MCF-7 breast carcinoma cells, NPEO(15) treatment induced γ-H2AX in a dose-dependent manner. EDTA and ZnCl2, two inhibitors of deoxyribonuclease I (DNase I), inhibited both the γ-H2AX and DNA double-strand breaks induced by NPEO(15). NPEO(15) disrupted filamentous actin and released free DNase I as detected by cell fractionation analysis. Based on immunofluorescence staining of DNase I and monitoring DNase I-GFP localization, DNase I was translocated from the cytosol to the nucleus of cells after treatment with NPEO(15). This translocation did not occur with the common DNA damage inducers ultraviolet B irradiation and hydrogen peroxide. Other surfactants, Tween 20, Triton X-100 and Nonidet P-40, also generated γ-H2AX. These results show that γ-H2AX induction by surfactants including NPEOs, occurs via a new mechanism involving release of free DNase I with actin disruption. This mechanism is distinct from the process of γ-H2AX generation caused by direct chemically induced DNA damage. PMID:26653977

  8. Protein Interacting C-Kinase 1 Modulates Surface Expression of P2Y6 Purinoreceptor, Actin Polymerization and Phagocytosis in Microglia.

    PubMed

    Zhu, Jia; Wang, Zhen; Zhang, Nan; Ma, Jiao; Xu, Shui-Lin; Wang, Yin; Shen, Ying; Li, Yun-Hong

    2016-04-01

    Microglia clean up dead cells and debris through phagocytosis in the central nervous system. UDP-activated P2Y6 receptors (P2Y6Rs) induce the formation of phagocytic cup-like structure and P2Y6R expression is increased during the phagocytosis. However, it remains unclear how surface expression of P2Y6R is increased. PICK1 (protein interacting with C-kinase-1) interacts with various neurotransmitter receptors, transporters, and enzymes. We here report that PICK1 might interact with P2Y6R. Surface P2Y6R was reduced in microglia from PICK1-knockout mice and PICK1-knockdown BV2 cells, which was also confirmed by electrophysiological recordings, showing that P2Y6R-mediated current was increased by PICK1 overexpression but was reduced by PICK1-knockdown in BV2 microglia. Finally, PICK1 was sufficient to affect cytoskeletal aggregation and phagocytosis both in primary microglia and BV2 cells. These results indicate that PICK1 is an important regulator of P2Y6R expression and microglial phagocytosis. PMID:26566795

  9. Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement.

    PubMed

    Aldini, Giancarlo; Carini, Marina; Vistoli, Giulio; Shibata, Takahiro; Kusano, Yuri; Gamberoni, Luca; Dalle-Donne, Isabella; Milzani, Aldo; Uchida, Koji

    2007-03-13

    A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization. PMID:17297918

  10. A dynamic formin-dependent deep F-actin network in axons

    PubMed Central

    Ganguly, Archan; Tang, Yong; Wang, Lina; Ladt, Kelsey; Loi, Jonathan; Dargent, Bénédicte; Leterrier, Christophe

    2015-01-01

    Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles. PMID:26216902

  11. Tropomyosin diffusion over actin subunits facilitates thin filament assembly

    PubMed Central

    Fischer, Stefan; Rynkiewicz, Michael J.; Moore, Jeffrey R.; Lehman, William

    2016-01-01

    Coiled-coil tropomyosin binds to consecutive actin-subunits along actin-containing thin filaments. Tropomyosin molecules then polymerize head-to-tail to form cables that wrap helically around the filaments. Little is known about the assembly process that leads to continuous, gap-free tropomyosin cable formation. We propose that tropomyosin molecules diffuse over the actin-filament surface to connect head-to-tail to partners. This possibility is likely because (1) tropomyosin hovers loosely over the actin-filament, thus binding weakly to F-actin and (2) low energy-barriers provide tropomyosin freedom for 1D axial translation on F-actin. We consider that these unique features of the actin-tropomyosin interaction are the basis of tropomyosin cable formation. PMID:26798831

  12. F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

    PubMed Central

    Burger, Danielle; Fickentscher, Céline; de Moerloose, Philippe; Brandt, Karim J.

    2016-01-01

    NLRP3 and ASC are able to form a large multimeric complex called inflammasome in response to a number danger signals. The NLRP3 inflammasome is required for the activation of caspase-1 and subsequent maturation of pro-IL-1β into active IL-1β. Although the mechanisms regulating the formation and activity of NLRP3 inflammasome are yet not fully elucidated, data suggest that the assembly of NLRP3 inflammasome requires microtubules to induce the proximity of ASC and NLRP3. In this study we show that microfilaments (F-actin) inhibit NLRP3 inflammasome activity and interact with NLRP3 and ASC. We demonstrate that the inhibition depends on the actin polymerization state but not on the active polymerization process. In ATP- or nigericin-activated macrophages, our data further indicate that Flightless-I (FliI) and leucine-rich repeat FliI-interaction protein 2 (LRRFIP2) are required for the co-localization of NLRP3, ASC and F-actin. We also established that the ability of Ca2+ to accentuate the activity of NLRP3 inflammasome is abrogated in FliI- and LRRFIP2-knockdown macrophages, suggesting that Ca2+ signaling requires the presence of FliI and LRRFIP2. Accordingly, we observed that Ca2+/FliI-dependent severing of F-actin suppresses F-actin/FliI/LRRFIP2-dependent NLRP3 inflammasome inhibition leading to increase IL-1β production. Altogether, our results unveil a new function of F-actin in the regulation of NLRP3 inflammasome activity strengthening the importance of cytoskeleton in the regulation of inflammation. PMID:27431477

  13. 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

  14. Cucurbitacin IIa: a novel class of anti-cancer drug inducing non-reversible actin aggregation and inhibiting survivin independent of JAK2/STAT3 phosphorylation

    PubMed Central

    Boykin, C; Zhang, G; Chen, Y-H; Zhang, R-W; Fan, X-E; Yang, W-M; Lu, Q

    2011-01-01

    Background: Cucurbitacin (Cuc) and triterpene-derived natural products exhibit anti-cancer potential in addition to their conspicuous anti-bacterial and anti-inflammatory activity. Recently, inhibition of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling was shown to underlie the effects of Cuc family on inducing cell death in cancer. Method: We purified Cuc IIa, the active component from the medicinal plant Hemsleya amalils Diels, which shows different structural modifications from other Cuc derivatives. We investigated the mechanisms of its inhibitory effects on cancer cells in vitro and tumour growth in vivo. Results: Cuc IIa induced the irreversible clustering of filamentous actin and arrested cell cycle by the increases in G2/M populations. Cuc IIa resulted in the reduced phospho-Histone H3 and markedly increased cleavage of poly-(ADP-ribose) polymerase or PARP, immediate upstream of DNA breakdown as the result of caspase activation, consistent with mitotic blockage-induced cell death. However, unlike other Cuc members, Cuc IIa did not suppress JAK2/STAT3 phosphorylation or alter phosphorylation of mitogen-activated protein kinases. Instead, the expression of the cell cycle-regulated Inhibitor of Apoptosis Protein (IAP) survivin was reduced. Introducing oncoprotein δ-catenin, which increased survivin expression and suppressed small GTPase RhoA, reduced efficacy of Cuc IIa to induce cell death. Supporting the effects of Cuc IIa on actin cytoskeletal signaling, RhoA phosphorylation was reduced suggesting its increased activity. Conclusion: Cuc IIa is a novel class of anti-cancer drug in suppression of cancer cell expansion by disrupting the actin cytoskeleton and directing the cell to undergo PARP-mediated apoptosis through the inhibition of survivin downstream of JAK2/STAT3. PMID:21304528

  15. A myosin activator improves actin assembly and sarcomere function of human-induced pluripotent stem cell-derived cardiomyocytes with a troponin T point mutation.

    PubMed

    Broughton, K M; Li, J; Sarmah, E; Warren, C M; Lin, Y-H; Henze, M P; Sanchez-Freire, V; Solaro, R J; Russell, B

    2016-07-01

    We have investigated cardiac myocytes derived from human-induced pluripotent stem cells (iPSC-CMs) from two normal control and two family members expressing a mutant cardiac troponin T (cTnT-R173W) linked to dilated cardiomyopathy (DCM). cTnT is a regulatory protein of the sarcomeric thin filament. The loss of this basic charge, which is strategically located to control tension, has consequences leading to progressive DCM. iPSC-CMs serve as a valuable platform for understanding clinically relevant mutations in sarcomeric proteins; however, there are important questions to be addressed with regard to myocyte adaptation that we model here by plating iPSC-CMs on softer substrates (100 kPa) to create a more physiologic environment during recovery and maturation of iPSC-CMs after thawing from cryopreservation. During the first week of culture of the iPSC-CMs, we have determined structural and functional characteristics as well as actin assembly dynamics. Shortening, actin content, and actin assembly dynamics were depressed in CMs from the severely affected mutant at 1 wk of culture, but by 2 wk differences were less apparent. Sarcomeric troponin and myosin isoform composition were fetal/neonatal. Furthermore, the troponin complex, reconstituted with wild-type cTnT or recombinant cTnT-R173W, depressed the entry of cross-bridges into the force-generating state, which can be reversed by the myosin activator omecamtiv mecarbil. Therapeutic doses of this drug increased both contractility and the content of F-actin in the mutant iPSC-CMs. Collectively, our data suggest the use of a myosin activation reagent to restore function within patient-specific iPSC-CMs may aid in understanding and treating this familial DCM. PMID:27199119

  16. Mutant Profilin Suppresses Mutant Actin-dependent Mitochondrial Phenotype in Saccharomyces cerevisiae*

    PubMed Central

    Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema; Rubenstein, Peter A.

    2011-01-01

    In the Saccharomyces cerevisiae actin-profilin interface, Ala167 of the actin barbed end W-loop and His372 near the C terminus form a clamp around a profilin segment containing residue Arg81 and Tyr79. Modeling suggests that altering steric packing in this interface regulates actin activity. An actin A167E mutation could increase interface crowding and alter actin regulation, and A167E does cause growth defects and mitochondrial dysfunction. We assessed whether a profilin Y79S mutation with its decreased mass could compensate for actin A167E crowding and rescue the mutant phenotype. Y79S profilin alone caused no growth defect in WT actin cells under standard conditions in rich medium and rescued the mitochondrial phenotype resulting from both the A167E and H372R actin mutations in vivo consistent with our model. Rescue did not result from effects of profilin on actin nucleotide exchange or direct effects of profilin on actin polymerization. Polymerization of A167E actin was less stimulated by formin Bni1 FH1-FH2 fragment than was WT actin. Addition of WT profilin to mixtures of A167E actin and formin fragment significantly altered polymerization kinetics from hyperbolic to a decidedly more sigmoidal behavior. Substitution of Y79S profilin in this system produced A167E behavior nearly identical to that of WT actin. A167E actin caused more dynamic actin cable behavior in vivo than observed with WT actin. Introduction of Y79S restored cable movement to a more normal phenotype. Our studies implicate the importance of the actin-profilin interface for formin-dependent actin and point to the involvement of formin and profilin in the maintenance of mitochondrial integrity and function. PMID:21956104

  17. Facile synthesis of gradient mesoporous carbon monolith based on polymerization-induced phase separation

    NASA Astrophysics Data System (ADS)

    Xu, Shunjian; Luo, Yufeng; Zhong, Wei; Xiao, Zonghu; Luo, Yongping; Ou, Hui; Zhao, Xing-Zhong

    2014-06-01

    In this paper, a gradient mesoporous carbon (GMC) monolith derived from the mixtures of phenolic resin (PF) and ethylene glycol (EG) was prepared by a facile route based on polymerization-induced phase separation under temperature gradient (TG). A graded biphasic structure of PF-rich and EG-rich phases was first formed in preform under a TG, and then the preform was pyrolyzed to obtain the GMC monolith. The TG is mainly induced by the thermal resistance of the preferential phase separation layer at high temperature region. The pore structure of the monolith changes gradually along the TG direction. When the TG varies from 58°C to 29°C, the pore size, apparent porosity and specific surface area of the monolith range respectively from 18 nm to 83 nm, from 32% to 39% and from 140.5 m2/g to 515.3 m2/g. The gradient porous structure of the monolith is inherited from that of the preform, which depends on phase separation under TG in the resin mixtures. The pyrolysis mainly brings about the contraction of the pore size and wall thickness as well as the transformation of polymerized PF into glassy carbon.

  18. Adsorption and electron-induced polymerization of methyl methacrylate on Ru(1010)

    SciTech Connect

    Hedhili, M. N.; Yakshinskiy, B. V.; Wasielewski, R.; Ciszewski, A.; Madey, T. E.

    2008-05-07

    The adsorption and electron irradiation of methyl methacrylate (MMA) on a Ru(1010) surface have been studied using x-ray photoelectron spectroscopy (XPS), temperature programmed desorption (TPD), and low energy ion scattering. TPD analysis indicates that a monolayer of MMA chemisorbs and dissociates on the Ru(1010) surface. The reaction products observed upon heating include H{sub 2}, CO, CO{sub 2}, and a small amount of MMA. Physisorbed multilayers of MMA desorb at temperatures around 170 K. Electron irradiation of physisorbed MMA at 140 K leads to a modification of the MMA film: The XPS spectra show an increase in thermal stability of the film with retention of the MMA structure, and indicate that electron irradiation induces polymerization. An increase in the electron bombardment fluence induces a degradation of the formed polymerized species and leads to the accumulation of carbon on the Ru surface. These results are relevant to the accumulation of carbon on surfaces of Ru films that serve as capping layers on Mo/Si multilayer mirrors used in extreme ultraviolet lithography.

  19. Structural dynamics of an actin spring.

    PubMed

    Mahadevan, L; Riera, C S; Shin, Jennifer H

    2011-02-16

    Actin-based motility in cells is usually associated with either polymerization/depolymerization in the presence of cross-linkers or contractility in the presence of myosin motors. Here, we focus on a third distinct mechanism involving actin in motility, seen in the dynamics of an active actin spring that powers the acrosomal reaction of the horseshoe crab (Limulus polyphemus) sperm. During this process, a 60-μm bent and twisted bundle of cross-linked actin uncoils and becomes straight in a few seconds in the presence of Ca(2+). This straightening, which occurs at a constant velocity, allows the acrosome to forcefully penetrate the egg. Synthesizing ultrastructural information with the kinetics, energetics, and imaging of calcium binding allows us to construct a dynamical theory for this mechanochemical engine consistent with our experimental observations. It also illuminates the general mechanism by which energy may be stored in conformational changes and released cooperatively in ordered macromolecular assemblies. PMID:21320427

  20. The enteropathogenic E. coli effector EspH promotes actin pedestal formation and elongation via WASP-interacting protein (WIP)

    PubMed Central

    Wong, Alexander R. C.; Raymond, Benoit; Collins, James W.; Crepin, Valerie F.; Frankel, Gad

    2016-01-01

    Summary Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are diarrheagenic pathogens that colonize the gut mucosa via attaching-and-effacing lesion formation. EPEC and EHEC utilize a type III secretion system (T3SS) to translocate effector proteins that subvert host cell signalling to sustain colonization and multiplication. EspH, a T3SS effector that modulates actin dynamics, was implicated in the elongation of the EHEC actin pedestals. In this study we found that EspH is necessary for both efficient pedestal formation and pedestal elongation during EPEC infection. We report that EspH induces actin polymerization at the bacterial attachment sites independently of the Tir tyrosine residues Y474 and Y454, which are implicated in binding Nck and IRSp53/ITRKS respectively. Moreover, EspH promotes recruitment of neural Wiskott–Aldrich syndrome protein (N-WASP) and the Arp2/3 complex to the bacterial attachment site, in a mechanism involving the C-terminus of Tir and the WH1 domain of N-WASP. Dominant negative of WASP-interacting protein (WIP), which binds the N-WASP WH1 domain, diminished EspH-mediated actin polymerization. This study implicates WIP in EPEC-mediated actin polymerization and pedestal elongation and represents the first instance whereby N-WASP is efficiently recruited to the EPEC attachment sites independently of the Tir:Nck and Tir:IRTKS/IRSp53 pathways. Our study reveals the intricacies of Tir and EspH-mediated actin signalling pathways that comprise of distinct, convergent and synergistic signalling cascades. PMID:22372637

  1. Aggregation-Induced-Emissive Molecule Incorporated into Polymeric Nanoparticulate as FRET Donor for Observing Doxorubicin Delivery.

    PubMed

    Han, Xiongqi; Liu, De-E; Wang, Tieyan; Lu, Hongguang; Ma, Jianbiao; Chen, Qixian; Gao, Hui

    2015-10-28

    Tetraphenylethene (TPE) derivatives characterized with distinct aggregation-induced-emission, attempted to aggregate with doxorubicin (Dox) to formulate the interior compartment of polymeric nanoparticulate, served as fluorescence resonance energy transfer (FRET) donor to promote emission of acceptor Dox. Accordingly, this FRET formulation allowed identification of Dox in complexed form by detecting FRET. Important insight into the Dox releasing can be subsequently explored by extracting complexed Dox (FRET) from the overall Dox via direct single-photon excitation of Dox. Of note, functional catiomers were used to complex with FRET partners for a template formulation, which was verified to induce pH-responsive release in the targeted subcellular compartment. Hence, this well-defined multifunctional system entitles in situ observation of the drug releasing profile and insight on drug delivery journey from the tip of injection vein to the subcellular organelle of the targeted cells. PMID:26448180

  2. 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.

  3. Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1

    PubMed Central

    Grikscheit, Katharina; Frank, Tanja; Wang, Ying

    2015-01-01

    Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell–cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell–cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell–cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell–cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1. PMID:25963818

  4. Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1.

    PubMed

    Grikscheit, Katharina; Frank, Tanja; Wang, Ying; Grosse, Robert

    2015-05-11

    Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell-cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell-cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell-cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell-cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1. PMID:25963818

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

    PubMed Central

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

    2015-01-01

    While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites. DOI: http://dx.doi.org/10.7554/eLife.11553.001 PMID:26609810

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

    PubMed

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

    2015-01-01

    While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites. PMID:26609810

  7. Arsenite-induced mitotic death involves stress response and is independent of tubulin polymerization

    SciTech Connect

    Taylor, B. Frazier; McNeely, Samuel C.; Miller, Heather L.; States, J. Christopher

    2008-07-15

    Arsenite, a known mitotic disruptor, causes cell cycle arrest and cell death at anaphase. The mechanism causing mitotic arrest is highly disputed. We compared arsenite to the spindle poisons nocodazole and paclitaxel. Immunofluorescence analysis of {alpha}-tubulin in interphase cells demonstrated that, while nocodazole and paclitaxel disrupt microtubule polymerization through destabilization and hyperpolymerization, respectively, microtubules in arsenite-treated cells remain comparable to untreated cells even at supra-therapeutic concentrations. Immunofluorescence analysis of {alpha}-tubulin in mitotic cells showed spindle formation in arsenite- and paclitaxel-treated cells but not in nocodazole-treated cells. Spindle formation in arsenite-treated cells appeared irregular and multi-polar. {gamma}-tubulin staining showed that cells treated with nocodazole and therapeutic concentrations of paclitaxel contained two centrosomes. In contrast, most arsenite-treated mitotic cells contained more than two centrosomes, similar to centrosome abnormalities induced by heat shock. Of the three drugs tested, only arsenite treatment increased expression of the inducible isoform of heat shock protein 70 (HSP70i). HSP70 and HSP90 proteins are intimately involved in centrosome regulation and mitotic spindle formation. HSP90 inhibitor 17-DMAG sensitized cells to arsenite treatment and increased arsenite-induced centrosome abnormalities. Combined treatment of 17-DMAG and arsenite resulted in a supra-additive effect on viability, mitotic arrest, and centrosome abnormalities. Thus, arsenite-induced abnormal centrosome amplification and subsequent mitotic arrest is independent of effects on tubulin polymerization and may be due to specific stresses that are protected against by HSP90 and HSP70.

  8. 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

  9. 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.

  10. Optogenetic control of PIP3: PIP3 is sufficient to induce the actin-based active part of growth cones and is regulated via endocytosis.

    PubMed

    Kakumoto, Toshiyuki; Nakata, Takao

    2013-01-01

    Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is highly regulated in a spatiotemporal manner and plays multiple roles in individual cells. However, the local dynamics and primary functions of PIP3 in developing neurons remain unclear because of a lack of techniques for manipulating PIP3 spatiotemporally. We addressed this issue by combining optogenetic control and observation of endogenous PIP3 signaling. Endogenous PIP3 was abundant in actin-rich structures such as growth cones and "waves", and PIP3-rich plasma membranes moved actively within growth cones. To study the role of PIP3 in developing neurons, we developed a PI3K photoswitch that can induce production of PIP3 at specific locations upon blue light exposure. We succeeded in producing PIP3 locally in mouse hippocampal neurons. Local PIP3 elevation at neurite tips did not induce neurite elongation, but it was sufficient to induce the formation of filopodia and lamellipodia. Interestingly, ectopic PIP3 elevation alone activated membranes to form actin-based structures whose behavior was similar to that of growth-cone-like "waves". We also found that endocytosis regulates effective PIP3 concentration at plasma membranes. These results revealed the local dynamics and primary functions of PIP3, providing fundamental information about PIP3 signaling in neurons. PMID:23951027

  11. Study of plasma-induced graft polymerization of stearyl methacrylate on cotton fabric substrates

    NASA Astrophysics Data System (ADS)

    Li, Yongqiang; Zhang, Yan; Zou, Chao; Shao, Jianzhong

    2015-12-01

    A simple and facile method to prepare the cotton fabric with hydrophobicity was described in the present work. In the one-step process, the cotton fabric pre-impregnated with the monomer solution of stearyl methacrylate (SMA) was placed in the plasma chamber and followed by glow discharge of the Helium low temperature plasma. The cotton fabrics before and after the plasma treatment were characterized by field emission scanning electron microscopy (FESEM), infrared spectroscopic analysis (FTIR), X-ray photoelectron spectroscopy (XPS) and thermogravimetric analysis (TGA), respectively. The wettability of the cotton fabrics was evaluated by contact angle measurement. Fabric Hand Values and mechanical properties were also measured in the experiment. The results showed that polymer films could be coated on the cotton fibers through the plasma induced grafting polymerization of SMA. The modified cotton fabrics exhibited an extraordinary hydrophobicity with a contact angle of 149° for a 5 μL water droplet and excellent thermal stability. The relative hand value and mechanical breaking strength of the cotton fabrics declined slightly after graft polymerization of SMA by the plasma.

  12. Radiation-induced cationic polymerization of limonene oxide,. cap alpha. -pinene oxide, and. beta. -pinene oxide

    SciTech Connect

    Aikins, J.A.; Williams, F.

    1985-01-01

    After suitable drying, the subject monomers in the form of neat liquids undergo radiation-induced polymerization with no apparent side reactions and high conversions to precipitatable polymers of low molecular weights. A high frequency of chain (proton) transfer to monomer is indicated by the fact that the kinetic chain lengths are estimated to be several hundred times larger than the range of DP/sub n/ values (12-4). Structural characterization of the limonene oxide polymer by /sup 1/H and /sup 13/C NMR spectroscopy provides conclusive evidence that the polymerization proceeds by the opening of the epoxide ring to yield a 1,2-trans polyether. Similar NMR studies on the polymers formed from the ..cap alpha..-pinene and ..beta..-pinene oxides show that the opening of the epoxide ring for these monomers is generally accompanied by the concomitant ring opening of the cyclobutane ring structure to yield a gem-di-methyl group in the main chain.

  13. Atrazine Molecular Imprinted Polymers: Comparative Analysis by Far-Infrared and Ultraviolet Induced Polymerization

    PubMed Central

    Chen, Jun; Bai, Lian-Yang; Liu, Kun-Feng; Liu, Run-Qiang; Zhang, Yu-Ping

    2014-01-01

    Atrazine molecular imprinted polymers (MIPs) were comparatively synthesized using identical polymer formulation by far-infrared (FIR) radiation and ultraviolet (UV)-induced polymerization, respectively. Equilibrium binding experiments were carried out with the prepared MIPs; the results showed that MIPuv possessed specific binding to atrazine compared with their MIPFIR radiation counterparts. Scatchard plot’s of both MIPs indicated that the affinities of the binding sites in MIPs are heterogeneous and can be approximated by two dissociation-constants corresponding to the high-and low-affinity binding sites. Moreover, several common pesticides including atrazine, cyromazine, metamitron, simazine, ametryn, terbutryn were tested to determine their specificity, similar imprinting factor (IF) and different selectivity index (SI) for both MIPs. Physical characterization of the polymers revealed that the different polymerization methods led to slight differences in polymer structures and performance by scanning electron microscope (SEM), Fourier transform infrared absorption (FT-IR), and mercury analyzer (MA). Finally, both MIPs were used as selective sorbents for solid phase extraction (SPE) of atrazine from lake water, followed by high performance liquid chromatography (HPLC) analysis. Compared with commercial C18 SPE sorbent (86.4%–94.8%), higher recoveries of atrazine in spiked lake water were obtained in the range of 90.1%–97.1% and 94.4%–101.9%, for both MIPs, respectively. PMID:24398982

  14. Atrazine molecular imprinted polymers: comparative analysis by far-infrared and ultraviolet induced polymerization.

    PubMed

    Chen, Jun; Bai, Lian-Yang; Liu, Kun-Feng; Liu, Run-Qiang; Zhang, Yu-Ping

    2014-01-01

    Atrazine molecular imprinted polymers (MIPs) were comparatively synthesized using identical polymer formulation by far-infrared (FIR) radiation and ultraviolet (UV)-induced polymerization, respectively. Equilibrium binding experiments were carried out with the prepared MIPs; the results showed that MIP(uv) possessed specific binding to atrazine compared with their MIP(FIR) radiation counterparts. Scatchard plot's of both MIPs indicated that the affinities of the binding sites in MIPs are heterogeneous and can be approximated by two dissociation-constants corresponding to the high- and low-affinity binding sites. Moreover, several common pesticides including atrazine, cyromazine, metamitron, simazine, ametryn, terbutryn were tested to determine their specificity, similar imprinting factor (IF) and different selectivity index (SI) for both MIPs. Physical characterization of the polymers revealed that the different polymerization methods led to slight differences in polymer structures and performance by scanning electron microscope (SEM), Fourier transform infrared absorption (FT-IR), and mercury analyzer (MA). Finally, both MIPs were used as selective sorbents for solid phase extraction (SPE) of atrazine from lake water, followed by high performance liquid chromatography (HPLC) analysis. Compared with commercial C18 SPE sorbent (86.4%-94.8%), higher recoveries of atrazine in spiked lake water were obtained in the range of 90.1%-97.1% and 94.4%-101.9%, for both MIPs, respectively. PMID:24398982

  15. Polymerization-induced spinodal decomposition of ethylene glycol∕phenolic resin solutions under electric fields.

    PubMed

    Zhang, Gang; Qiao, Guanjun

    2013-10-01

    Temporal evolution of polymerization-induced spinodal decomposition (PISD) under electric fields was investigated numerically in ethylene glycol∕phenolic resin solutions with different initial composition. A model composed of the nonlinear Cahn-Hilliard-Cook equation for spinodal decomposition and a rate equation for curing reaction was utilized to describe the PISD phenomenon. As initial composition varied, deformed droplet-like and aligned bi-continuous structures were observed in the presence of an electric field. Moreover, the anisotropic parameter (D), determined from the 2D-FFT power spectrum, was employed to quantitatively characterize the degree of morphology anisotropy. The value of D increased quickly in the early stage and then decreased in the intermediate stage of spinodal decomposition, which was attributed to the resistance of coarsening process to morphology deformation and the decline of electric stress caused by polymerization reaction. The results can also provide a guidance on how to control the morphology of monolithic porous polymer and carbon materials with anisotropic structures. PMID:24116581

  16. The use of radiation-induced graft polymerization for modification of polymer track membranes

    NASA Astrophysics Data System (ADS)

    Shtanko, N. I.; Kabanov, V. Ya.; Apel, P. Yu.; Yoshida, M.

    1999-05-01

    Track membranes (TM) made of poly(ethylene terephtalate) (PET) and polypropylene (PP) films have a number of peculiarities as compared with other ones. They have high mechanical strength at a low thickness, narrow pore size distribution, low content of extractables. However, TM have some disadvantages such as low chemical resistance in alkaline media (PET TM), the low water flow rate due to the hydrophobic nature of their surface. The use of radiation-induced graft polymerization makes it possible to improve the basic characteristics of TM. In this communication our results on the modification of PET and PP TM are presented. The modified membranes were prepared by radiation-induced graft polymerization from the liquid phase. Three methods of grafting were used: (a) the direct method in argon atmosphere; (b) the pre-irradiation of TM in air followed by grafting in argon atmosphere; (c) pre-irradiation in vacuum followed by grafting in vacuum without contacting oxygen. The aim of the work was to investigate some properties of TM modified by grafted poly(methylvinyl pyridine) (PMVP) and poly(N-isopropylacrylamide) (PNIPAAM). It was shown that the modification of TM with hydrophilic polymer results in the growth of the water flow rate. In the past few years many works have been devoted to the synthesis of new polymers - the so-called "intelligent" materials - such as PNIPAAM. However, it is very difficult to make thin membranes of this polymer. Recently, it has been proposed to manufacture composite membranes by grafting stimulus-responsive polymers onto TM. Following this principle, we prepared thermosensitive membranes by the radiation-induced graft polymerization of N-isopropylacrylamide (NIPAAM) onto PET TM. PET TM with the pore size of about 1 μm and pore density of 10 6 cm -2 were first inserted into a solution of NIPAAM containing inhibitor of homopolymerization (CuCl 2) and then exposed to the γ-rays from a 60Co source. The transport properties of the grafted

  17. Differential Incorporation of β-actin as A Component of RNA Polymerase II into Regulatory Regions of Stemness/Differentiation Genes in Retinoic Acid-Induced Differentiated Human Embryonic Carcinoma Cells

    PubMed Central

    Falahzadeh, Khadijeh; Shahhoseini, Maryam; Afsharian, Parvaneh

    2016-01-01

    Objective Nuclear actin is involved in transcription regulation by recruitment of histone modifiers and chromatin remodelers to the regulatory regions of active genes. In recent years, further attention has been focused on the role of actin as a nuclear protein in transcriptional processes. In the current study, the epigenetic role of nuclear actin on transcription regulation of two stemness (OCT4 and NANOG) and two differentiation) NESTIN and PAX6) marker genes was evaluated in a human embryonal carcinoma cell line (NT2) before and after differentiation induction. Materials and Methods In this experimental study, differentiation of embryonal cells was induced by retinoic acid (RA), and quantitative real-time polymerase chain reaction (PCR) was used to evaluate differential expression of marker genes before and 3 days after RA- induced differentiation. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR was then undertaken to monitor the incorporation of β-actin, as a functional component of RNA polymerase II, in the regulatory regions of marker genes. Results Data showed significant change in nuclear actin incorporation into the promoter regions of NESTIN and PAX6 after RA-induction. Conclusion We emphasize the dynamic functional role of nuclear actin in differentiation of embryonal cells and its role as a subunit of RNA polymerase II. PMID:27540526

  18. Bundling actin filaments from membranes: some novel players

    PubMed Central

    Thomas, Clément

    2012-01-01

    Progress in live-cell imaging of the cytoskeleton has significantly extended our knowledge about the organization and dynamics of actin filaments near the plasma membrane of plant cells. Noticeably, two populations of filamentous structures can be distinguished. On the one hand, fine actin filaments which exhibit an extremely dynamic behavior basically characterized by fast polymerization and prolific severing events, a process referred to as actin stochastic dynamics. On the other hand, thick actin bundles which are composed of several filaments and which are comparatively more stable although they constantly remodel as well. There is evidence that the actin cytoskeleton plays critical roles in trafficking and signaling at both the cell cortex and organelle periphery but the exact contribution of actin bundles remains unclear. A common view is that actin bundles provide the long-distance tracks used by myosin motors to deliver their cargo to growing regions and accordingly play a particularly important role in cell polarization. However, several studies support that actin bundles are more than simple passive highways and display multiple and dynamic roles in the regulation of many processes, such as cell elongation, polar auxin transport, stomatal and chloroplast movement, and defense against pathogens. The list of identified plant actin-bundling proteins is ever expanding, supporting that plant cells shape structurally and functionally different actin bundles. Here I review the most recently characterized actin-bundling proteins, with a particular focus on those potentially relevant to membrane trafficking and/or signaling. PMID:22936939

  19. Curvature and torsion in growing actin networks

    NASA Astrophysics Data System (ADS)

    Shaevitz, Joshua W.; Fletcher, Daniel A.

    2008-06-01

    Intracellular pathogens such as Listeria monocytogenes and Rickettsia rickettsii move within a host cell by polymerizing a comet-tail of actin fibers that ultimately pushes the cell forward. This dense network of cross-linked actin polymers typically exhibits a striking curvature that causes bacteria to move in gently looping paths. Theoretically, tail curvature has been linked to details of motility by considering force and torque balances from a finite number of polymerizing filaments. Here we track beads coated with a prokaryotic activator of actin polymerization in three dimensions to directly quantify the curvature and torsion of bead motility paths. We find that bead paths are more likely to have low rather than high curvature at any given time. Furthermore, path curvature changes very slowly in time, with an autocorrelation decay time of 200 s. Paths with a small radius of curvature, therefore, remain so for an extended period resulting in loops when confined to two dimensions. When allowed to explore a three-dimensional (3D) space, path loops are less evident. Finally, we quantify the torsion in the bead paths and show that beads do not exhibit a significant left- or right-handed bias to their motion in 3D. These results suggest that paths of actin-propelled objects may be attributed to slow changes in curvature, possibly associated with filament debranching, rather than a fixed torque.

  20. 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

  1. 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

  2. Characterization of Network Structure of Polyacrylamide Based Hydrogels Prepared By Radiation Induced Polymerization

    SciTech Connect

    Mahmudi, Naim; Sen, Murat; Gueven, Olgun; Rendevski, Stojan

    2007-04-23

    In this study network structure of polyacrylamide based hydrogels prepared by radiation induced polymerization has been investigated. Polyacrylamide based hydrogels in the rod form were prepared by copolymerization of acrylamide(AAm) with hydroxyl ethyl methacrylate(HEMA) and methyl acrylamide(MAAm) in the presence of cross-linking agent and water by gamma rays at ambient temperature. Molecular weight between cross-links and effective cross-link density of hydrogels were calculated from swelling as well as shear modulus data obtained from compression tests. The results have shown that simple compression analyses can be used for the determination of effective cross-link density of hydrogels without any need to some polymer-solvent based parameters as in the case of swelling based determinations. Diffusion of water into hydrogels was examined by analyzing water absorption kinetics and the effect of network, structure on the diffusion type and coefficient was discussed.

  3. Semi-interpenetrating polymer networks of poly(3-hydroxybutyrate) prepared by radiation-induced polymerization

    NASA Astrophysics Data System (ADS)

    Martellini, Flavia; Innocentini Mei, Lúcia H.; Lora, Silvano; Carenza, Mario

    2004-09-01

    Semi-interpenetrating polymer networks (IPNs) based on bacterial poly(3-hydroxy butyrate) with a hydrophilic monomer at different compositions were prepared by radiation-induced polymerization using γ-rays from a 60Co source with a total dose of 10-100 kGy. The swelling behaviour was determined by water content at equilibrium, while thermal properties and crystallinity were studied by differential scanning calorimetry. Extraction of the soluble part of PHB from the films at low and high temperature with water or chloroform as well as FTIR data indicate the occurrence of the crosslinking reaction in the hydrogels. The results show a water uptake increasing with the hydrophilic component until 25%.

  4. Graft polymerization using radiation-induced peroxides and application to textile dyeing

    NASA Astrophysics Data System (ADS)

    Enomoto, Ichiro; Katsumura, Yosuke; Kudo, Hisaaki; Soeda, Shin

    2011-02-01

    To improve the dyeing affinity of ultra high molecular weight polyethylene (UHMWPE) fiber, surface treatment by radiation-induced graft polymerization was performed. Methyl methacrylate (MMA), acrylic acid (AA) and styrene (St) were used as the monomers. The grafting yields as a function of storage time after irradiation were examined. Although the grafting yield of St after the sulfonation processing was quite low compared with those of MMA and AA, it was successfully dyed to a dark color with a cationic dye. Some acid dyes can dye the grafted fiber with AA. The acid dye is distributed to the amorphous domains of the AA grafted fiber. The dyeing concentration depended on the grafting yield, and the higher the grafting yield the darker the dye color.

  5. Unusual sintering behavior of porous chromatographic zirconia produced by polymerization-induced colloid aggregation

    SciTech Connect

    Lorenzano-Porras, C.F.; Reeder, D.H.; Annen, M.J.; Carr, P.W.; McCormick, A.V.

    1995-08-01

    The effects of sintering temperature and duration on the pore structure of chromatographic zirconia particles produced by the controlled polymerization-induced aggregation of 1,000 {angstrom} colloids are studied with an eye toward optimally strengthening the aggregates and eliminating small pores while preserving large pores. Nitrogen adsorption and mercury porosimetry are used to estimate the surface area, pore volume, and pore size distribution. Pulsed field gradient NMR measurements of solvent diffusion are used to estimate the diffusion tortuosity of the pore space. Initially of course, the pore volume and surface area decrease significantly, the decrease being more pronounced at higher temperatures. With prolonged sintering, the pore size, pore volume, and surface area change much more slowly, but the diffusion tortuosity seems to be minimized at a sintering temperature and time at which pores are allowed to redistribute so as to optimize large pores. The aggregates synthesized by this aggregation method apparently produce metastable large pores which are not easily collapsed.

  6. Identification of sucrose synthase as an actin-binding protein

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  7. 8-Modified-2′-Deoxyadenosine Analogues Induce Delayed Polymerization Arrest during HIV-1 Reverse Transcription

    PubMed Central

    Sleiman, Marwan; Smyth, Redmond; Ben Gaied, Nouha; Barhoum, Patrick; Laumond, Géraldine; Bec, Guillaume; Götte, Matthias; Mak, Johnson; Aubertin, Anne-Marie; Burger, Alain; Marquet, Roland

    2011-01-01

    The occurrence of resistant viruses to any of the anti-HIV-1 compounds used in the current therapies against AIDS underlies the urge for the development of new drug targets and/or new drugs acting through novel mechanisms. While all anti-HIV-1 nucleoside analogues in clinical use and in clinical trials rely on ribose modifications for activity, we designed nucleosides with a natural deoxyribose moiety and modifications of position 8 of the adenine base. Such modifications might induce a steric clash with helix αH in the thumb domain of the p66 subunit of HIV-1 RT at a distance from the catalytic site, causing delayed chain termination. Eleven new 2′-deoxyadenosine analogues modified on position 8 of the purine base were synthesized and tested in vitro and in cell-based assays. In this paper we demonstrate for the first time that chemical modifications on position 8 of 2′-deoxyadenosine induce delayed chain termination in vitro, and also inhibit DNA synthesis when incorporated in a DNA template strand. Furthermore, one of them had moderate anti-HIV-1 activity in cell-culture. Our results constitute a proof of concept indicating that modification on the base moiety of nucleosides can induce delayed polymerization arrest and inhibit HIV-1 replication. PMID:22087320

  8. Measuring F-actin properties in dendritic spines

    PubMed Central

    Koskinen, Mikko; Hotulainen, Pirta

    2014-01-01

    During the last decade, numerous studies have demonstrated that the actin cytoskeleton plays a pivotal role in the control of dendritic spine shape. Synaptic stimulation rapidly changes the actin dynamics and many actin regulators have been shown to play roles in neuron functionality. Accordingly, defects in the regulation of the actin cytoskeleton in neurons have been implicated in memory disorders. Due to the small size of spines, it is difficult to detect changes in the actin structures in dendritic spines by conventional light microscopy imaging. Instead, to know how tightly actin filaments are bundled together, and how fast the filaments turnover, we need to use advanced microscopy techniques, such as fluorescence recovery after photobleaching (FRAP), photoactivatable green fluorescent protein (PAGFP) fluorescence decay and fluorescence anisotropy. Fluorescence anisotropy, which measures the Förster resonance energy transfer (FRET) between two GFP fluorophores, has been proposed as a method to measure the level of actin polymerization. Here, we propose a novel idea that fluorescence anisotropy could be more suitable to study the level of actin filament bundling instead of actin polymerization. We validate the method in U2OS cell line where the actin structures can be clearly distinguished and apply to analyze how actin filament organization in dendritic spines changes during neuronal maturation. In addition to fluorescence anisotropy validation, we take a critical look at the properties and limitations of FRAP and PAGFP fluorescence decay methods and offer our proposals for the analysis methods for these approaches. These three methods complement each other, each providing additional information about actin dynamics and organization in dendritic spines. PMID:25140131

  9. Selenium nanoparticles induced membrane bio-mechanical property changes in MCF-7 cells by disturbing membrane molecules and F-actin.

    PubMed

    Pi, Jiang; Yang, Fen; Jin, Hua; Huang, Xun; Liu, Ruiying; Yang, Peihui; Cai, Jiye

    2013-12-01

    Selenium nanoparticles (Se NPs) have been served as promising materials for biomedical applications, especially for cancer treatment. The anti-cancer effects of Se NPs against cancer cells have been widely studied in recent years, but whether Se NPs can induce the changes of cell membrane bio-mechanical properties in cancer cells still remain unexplored. In this Letter, we prepared Se NPs for investigating the intracellular localization of Se NPs in MCF-7 cells and determined the effects of Se NPs on apoptosis and necrosis in MCF-7 cells. Especially, we reported for the first time about the effects of Se NPs on the bio-mechanical properties of cancer cells and found that Se NPs could remarkably decrease the adhesion force and Young's modulus of MCF-7 cells. To further understand the potential mechanisms about how Se NPs affect the bio-mechanical properties of MCF-7 cells, we also investigated the expression of CD44 molecules, the structure and the amounts of F-actin. The results indicated that the decreased adhesion force between AFM tip and cell membrane was partially due to the changes of membrane molecules induced by Se NPs, such as the down-regulation of trans-membrane CD44 molecules. Additionally, the decrease of Young's modulus of MCF-7 cells was due to the dis-organization and down-regulation of F-actin induced by Se NPs. These results collectively suggested that cell membrane was of vital importance in Se NPs induced toxicity in cancer cells, which could be served as a potential target for cancer treatment by Se NPs. PMID:24140445

  10. 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

  11. Epstein-Barr virus infection induces expression in B lymphocytes of a novel gene encoding an evolutionarily conserved 55-kilodalton actin-bundling protein.

    PubMed

    Mosialos, G; Yamashiro, S; Baughman, R W; Matsudaira, P; Vara, L; Matsumura, F; Kieff, E; Birkenbach, M

    1994-11-01

    A novel human mRNA whose expression is induced over 200-fold in B lymphocytes by latent Epstein-Barr virus (EBV) infection was reverse transcribed, cloned, and sequenced. The mRNA is predicted to encode a protein containing four peptides which precisely match amino acid sequences from a previously identified 55-kDa actin-bundling protein, p55. In vitro translation of the cDNA results in a 55-kDa protein which binds to actin filaments in the presence of purified p55 from HeLa cells. The p55 mRNA is undetectable in non-EBV-infected B- and T-cell lines or in a myelomonocytic cell line (U937). Newly infected primary human B lymphocytes, EBV-transformed B-cell lines, latently infected Burkitt tumor cells expressing EBNA2 and LMP1, a chronic myelogenous leukemia cell line (K562), and an osteosarcoma cell line (TK143) contain high levels of p55 mRNA or protein. In EBV-transformed B cells, p55 localizes to perinuclear cytoplasm and to cell surface processes that resemble filopodia. The p55 mRNA is detected at high levels in spleen and brain tissues, at moderate levels in lung and placenta tissues, and at low levels in skeletal muscle, liver, and tonsil tissues and is undetectable in heart, kidney, pancreas, and bone marrow tissues. Immunohistochemical staining of human brain tissue demonstrates p55 localization to the perinuclear cytoplasm and dendritic processes of many, but not all, types of cortical or cerebellar neurons, to glial cells, and to capillary endothelial cells. In cultured primary rat neurons, p55 is distributed throughout the perinuclear cytoplasm and in subcortical filamentous structures of dendrites and growth cones. p55 is highly evolutionarily conserved since it shows 40% amino acid sequence identity to the Drosophila singed gene product and 37% identity to fascin, an echinoderm actin-bundling protein. The evolutionary conservation of p55 and its lack of extensive homology to other actin-binding proteins suggest that p55 has specific microfilament

  12. Competition of two distinct actin networks for actin defines a bistable switch for cell polarization

    PubMed Central

    Lomakin, Alexis J.; Lee, Kun-Chun; Han, Sangyoon J.; Bui, D A.; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-01-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype upon relaxation of the actomyosin cytoskeleton. We find that myosin-II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. At low contractility regimes epithelial cells polarize in a front-back manner due to emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin-II from the front to the back of the cell, where the motor locally “locks” actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high contractility-driven cell motion is inefficient. PMID:26414403

  13. Study of fracture and stress-induced morphological instabilities in polymeric materials

    NASA Astrophysics Data System (ADS)

    Sabouri-Ghomi, Mohsen

    We study the phenomena of fracture in polymers at the molecular and continuum level. At a molecular level, we study the failure of polymer/polymer interfaces. Our main focus is on a specific mode of failure known as chain pull-out fracture, which is common to weak adhesive junctions, and polymer blends and mixtures. In the case of the interface between incompatible polymers, reinforcement is achieved by adding a block copolymer to the interface. We introduce a microscopic model based on Brownian dynamics to investigate the effect of the polymerization index N, of the block connector chain, on fracture toughness of such reinforced polymeric junctions. We consider the mushroom regime, where connector chains are grafted with low surface density, for the case of large pulling velocity. We find that for short chains the interface fracture toughness depends linearly on the polymerization index N of the connector chains, while for longer chains the dependence becomes N 3/2. We propose a scaling argument, based on the geometry of the initial configuration, that accounts for both short and long chains and the crossover between them. At the continuum level, we study the pattern selection mechanism of finger-like crack growth phenomena in gradient driven growth problems in general, and the structure of stress-induced morphological instabilities in crazing of polymer glasses in particular. We simulate solidification in a narrow channel through the use of a phase-field model with an adaptive grid. By tuning a dimensionless parameter, the Peclet number, we show a continuous crossover from a free dendrite at high Peclet numbers to anisotropic viscous fingering at low Peclet numbers. At low Peclet numbers we find good agreement between our results, theoretical predictions, and experiment, providing the first quantitative test of solvability theory for anisotropic viscous fingers. For high undercoolings, we find new phenomena, a solid forger which satisfies stability and

  14. 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

  15. DNA bending is induced by a transcription factor that interacts with the human c-FOS and alpha-actin promoters.

    PubMed Central

    Gustafson, T A; Taylor, A; Kedes, L

    1989-01-01

    Conserved sequence elements in the human cardiac and skeletal alpha-actin promoters that contain the CC(A + T-rich)6GG motif have been shown to regulate transcription of these genes. A similar sequence is found in the serum response element of the human c-FOS gene. In this study, we demonstrate that indistinguishable proteins bind to each of five CC(A + T-rich)6GG elements examined in the human cardiac and skeletal alpha-actin promoters and the c-FOS serum response element. Using electrophoretic techniques, we show that these factors induce a stable bend in the DNA upon binding, and the bend center is shown to coincide with the CC(A + T-rich)6GG element. In addition, the ability to bend DNA is retained by a small proteolytic fragment of the protein, suggesting that the DNA-binding domain of the protein is resistant to proteases and is sufficient to bend DNA. Images PMID:2494661

  16. Transgelin is a TGFβ-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization.

    PubMed

    Elsafadi, M; Manikandan, M; Dawud, R A; Alajez, N M; Hamam, R; Alfayez, M; Kassem, M; Aldahmash, A; Mahmood, A

    2016-01-01

    Regenerative medicine is a novel approach for treating conditions in which enhanced bone regeneration is required. We identified transgelin (TAGLN), a transforming growth factor beta (TGFβ)-inducible gene, as an upregulated gene during in vitro osteoblastic and adipocytic differentiation of human bone marrow-derived stromal (skeletal) stem cells (hMSC). siRNA-mediated gene silencing of TAGLN impaired lineage differentiation into osteoblasts and adipocytes but enhanced cell proliferation. Additional functional studies revealed that TAGLN deficiency impaired hMSC cell motility and in vitro transwell cell migration. On the other hand, TAGLN overexpression reduced hMSC cell proliferation, but enhanced cell migration, osteoblastic and adipocytic differentiation, and in vivo bone formation. In addition, deficiency or overexpression of TAGLN in hMSC was associated with significant changes in cellular and nuclear morphology and cytoplasmic organelle composition as demonstrated by high content imaging and transmission electron microscopy that revealed pronounced alterations in the distribution of the actin filament and changes in cytoskeletal organization. Molecular signature of TAGLN-deficient hMSC showed that several genes and genetic pathways associated with cell differentiation, including regulation of actin cytoskeleton and focal adhesion pathways, were downregulated. Our data demonstrate that TAGLN has a role in generating committed progenitor cells from undifferentiated hMSC by regulating cytoskeleton organization. Targeting TAGLN is a plausible approach to enrich for committed hMSC cells needed for regenerative medicine application. PMID:27490926

  17. Peroxidase induced oligo-tyrosine cross-links during polymerization of α-lactalbumin.

    PubMed

    Dhayal, Surender Kumar; Sforza, Stefano; Wierenga, Peter A; Gruppen, Harry

    2015-12-01

    Horseradish peroxidase (HRP) induced cross-linking of proteins has been reported to proceed through formation of di-tyrosine cross-links. In the case of low molar mass phenolic substrates, the enzymatic oxidation is reported to lead to polymerization of the phenols. The aim of this work was to investigate if during oxidative cross-linking of proteins oligo-tyrosine cross-links are formed in addition to dityrosine. To this end, α-lactalbumin (α-LA) was cross-linked using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The reaction products were acid hydrolysed, after which the cross-linked amino acids were investigated by LC-MS and MALDI-MS. To test the effect of the size of the substrate, the cross-linking reaction was also performed with L-tyrosine, N-acetyl L-tyrosinamide and angiotensin. These products were analyzed by LC-MS directly, as well as after acid hydrolysis. In the acid hydrolysates of all samples oligo-tyrosine (Yn, n=3-8) was found in addition to di-tyrosine (Y2). Two stages of cross-linking of α-LA were identified: a) 1-2 cross-links were formed per monomer until the monomers were converted into oligomers, and b) subsequent cross-linking of oligomers formed in the first stage to form nanoparticles containing 3-4 cross-links per monomer. The transition from first stage to the second stage coincided with the point where di-tyrosine started to decrease and more oligo-tyrosines were formed. In conclusion, extensive polymerization of α-LA using HRP via oligo-tyrosine cross-links is possible, as is the case for low molar mass tyrosine containing substrates. PMID:26282909

  18. Effect of solvents on the radiation-induced polymerization of ethyl and isopropyl vinyl ethers. [gamma rays

    SciTech Connect

    Hsieh, W.C.; Kubota, H.; Squire, D.R.; Stannett, V.

    1980-09-01

    The radiation-induced cationic polymerization of ethyl and isopropyl vinyl ethers was studied in a variety of solvents. The propagation rate constants were estimated and found to vary widely with the nature of the solvent. In particular, a good linear relationship existed between the logarithms of the rate constants and the reciprocal of the dielectric constants. The lowest rates were those of the highest dielectric constant solvents. These results have been interpreted in terms of the Laidler and Eyring theory of ion-molecule reactions. Isopropyl vinyl ether polymerizes much faster than ethyl vinyl ether, although the rates approach the same value at infinite dielectric constant. In contrast, free carbenium ion polymerizations initiated by stable carbenium ion salts in methylene chloride solution had similar values for both monomers. A comparison is made between the rate constants obtained with both methods of initiation.

  19. The Actin Cytoskeleton as a Therapeutic Target for the Prevention of Relapse to Methamphetamine Use.

    PubMed

    Young, Erica J; Briggs, Sherri B; Miller, Courtney A

    2015-01-01

    A high rate of relapse is a defining characteristic of substance use disorder for which few treatments are available. Exposure to environmental cues associated with previous drug use can elicit relapse by causing the involuntary retrieval of deeply engrained associative memories that trigger a strong motivation to seek out drugs. Our lab is focused on identifying and disrupting mechanisms that support these powerful consolidated memories, with the goal of developing therapeutics. A particularly promising mechanism is regulation of synaptic dynamics by actin polymerization within dendritic spines. Emerging evidence indicates that memory is supported by structural and functional plasticity dendritic spines, for which actin polymerization is critical, and that prior drug use increases both spine and actin dynamics. Indeed we have found that inhibiting amygdala (AMY) actin polymerization immediately or twenty-four hours prior to testing disrupted methamphetamine (METH)-associated memories, but not food reward or fear memories. Furthermore, METH training increased AMY spine density which was reversed by actin depolymerization treatment. Actin dynamics were also shifted to a more dynamic state by METH training. While promising, actin polymerization inhibitors are not a viable therapeutic, as a multitude of peripheral process (e.g. cardiac function) rely on dynamic actin. For this reason, we have shifted our focus upstream of actin polymerization to nonmuscle myosin II. We and others have demonstrated that myosin IIb imparts a mechanical force that triggers spine actin polymerization in response to synaptic stimulation. Similar to an actin depolymerizing compound, pre-test inhibition of myosin II ATPase activity in the AMY produced a rapid and lasting disruption of drug-seeking behavior. While many questions remain, these findings indicate that myosin II represents a potential therapeutic avenue to target the actin cytoskeleton and disrupt the powerful, extinction

  20. Laser-assisted high-pressure-induced polymerization of 2-(hydroxyethyl)methacrylate.

    PubMed

    Evlyukhin, E; Museur, L; Traore, M; Nikitin, S M; Zerr, A; Kanaev, A

    2015-02-26

    We report on a successful room-temperature polymerization of 2-(hydroxyethyl)methacrylate (HEMA) under high pressure. The polymerization is observed in a limited range of pressures 0.1 to 1.6 GPa without the use of any initiator. When the compressed sample is irradiated at 488 or 355 nm by a laser, the polymerization reaction rate is increased by a factor of 10 or 30, respectively. Moreover, the shift of the laser wavelength to the UV improves the polymerization yield of the recovered sample to 84%. The catalysis of the polymerization process by light results from a one-photon-assisted electron transfer to π* antibonding states of the monomer molecule. The observed polymerization is irreversible and almost complete, which makes this synthesis process suitable for applications. PMID:25632927

  1. 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

  2. Near-Infrared-Induced Heating of Confined Water in Polymeric Particles for Efficient Payload Release

    PubMed Central

    2015-01-01

    Near-infrared (NIR) light-triggered release from polymeric capsules could make a major impact on biological research by enabling remote and spatiotemporal control over the release of encapsulated cargo. The few existing mechanisms for NIR-triggered release have not been widely applied because they require custom synthesis of designer polymers, high-powered lasers to drive inefficient two-photon processes, and/or coencapsulation of bulky inorganic particles. In search of a simpler mechanism, we found that exposure to laser light resonant with the vibrational absorption of water (980 nm) in the NIR region can induce release of payloads encapsulated in particles made from inherently non-photo-responsive polymers. We hypothesize that confined water pockets present in hydrated polymer particles absorb electromagnetic energy and transfer it to the polymer matrix, inducing a thermal phase change. In this study, we show that this simple and highly universal strategy enables instantaneous and controlled release of payloads in aqueous environments as well as in living cells using both pulsed and continuous wavelength lasers without significant heating of the surrounding aqueous solution. PMID:24717072

  3. Colchicine-induced polyploidization depends on tubulin polymerization in c-metaphase cells.

    PubMed

    Caperta, A D; Delgado, M; Ressurreição, F; Meister, A; Jones, R N; Viegas, W; Houben, A

    2006-05-01

    The microtubule cytoskeleton plays a crucial role in the cell cycle and in mitosis. Colchicine is a microtubule-depolymerizing agent that has long been used to induce chromosome individualization in cells arrested at metaphase and also in the induction of polyploid plants. Although attempts have been made to explain the processes and mechanisms underlying polyploidy induction, the role of the cytoskeleton still remains largely unknown. Through immunodetection of alpha-tubulin, different concentrations (0.5 or 5 mM) of colchicine were found to produce opposite effects in the organization of the cytoskeleton in rye (Secale cereale L.). A low concentration (0.5 mM) induced depolymerization of the microtubular cytoskeleton in all phases of the cell cycle. In contrast, a high concentration (5 mM) was found to induce the polymerization of new tubulin-containing structures in c-metaphase cells. Furthermore, both treatments also showed contrasting effects in the induction of polyploid cells. Flow cytometric analysis and quantitative assessments of nucleolus-organizing regions revealed that only the high-concentration colchicine treatment was effective in the formation of polyploid cells. Our studies indicate that spindle disruption alone is insufficient for the induction of polyploid cells. The absence of any tubulin structures in plants treated with colchicine at the low concentration induced cell anomalies, such as the occurrence of nuclei with irregular shape and/or (additional) micronuclei, 12 h after recovery, pointing to a direct effect on cell viability. In contrast, the almost insignificant level of cell anomalies in the high-concentration treatment suggests that the presence of new tubulin-containing structures allows the reconstitution of 4C nuclei and their progression into the cell cycle. PMID:16520877

  4. Advances in light-induced polymerizations: I. Shadow cure in free radical photopolymerizations II. Experimental and modeling studies of photoinitiator systems for effective polymerizations with LEDs

    NASA Astrophysics Data System (ADS)

    Kitano, Hajime

    Photopolymerization has become the standard for many coating and printing applications that require rapid curing at room temperature due to its potential to reduce volatile organic compound (VOC) emissions while providing a means for efficient manufacturing processes. These advantages could be useful in a variety of emerging applications, such as anisotropic conductive films (ACF) if photopolymerization could extend into relatively narrow shadow regions which are not directly illuminated, and if visible wavelengths that are not absorbed by polyimide films could be used to trigger the reaction. The broad objectives of this research are i) to examine the factors that determine the attainable extent of shadow cure in free radical polymerizations, and ii) to develop initiator systems effective for polymerization using visible light and light emitting diode (LED) lamps. Project I: Shadow Cure in Free Radical Photopolymerizations. In this project, the extent of shadow cure in visible-light-induced free radical photopolymerization is investigated. A number of effective methods such as adding additives, utilizing a reflective stage, and increasing the light intensity are introduced. In addition, the use of fluorescent dyes in multi-component photoinitiator systems proved to be very effective for shadow cure because the fluorescent light emitted from the dye could irradiate the shadow region. When considering practical resins, mixtures of oligomers and monomers, the viscosity is the major barrier that must be overcome in order to achieve high conversion in the shadow regions using visible-light-induced multi-component photoinitiator systems. Hence, instead of using multi-component systems, a commercial visible-light-induced single-component photoinitiator is investigated. As a result, a high conversion in shadow regions of the viscous oligomer containing resin is achieved. Project II: Experimental and Modeling Studies of Photoinitiator Systems for Effective Polymerizations

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

    PubMed

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

    2016-10-01

    Host actin cytoskeleton polymerization has been shown to play an important role during Trypanosoma cruzi internalization into mammalian cell. The structure and dynamics of the actin cytoskeleton in cells are regulated by a vast number of actin-binding proteins. Here we aimed to verify the impact of AFAP-1L1, during invasion and multiplication of T. cruzi. Knocking-down AFAP-1L1 increased parasite cell invasion and intracellular multiplication. Thus, we have shown that the integrity of the machinery formed by AFAP-1L1 in actin cytoskeleton polymerization is important to hinder parasite infection. PMID:27349187

  6. Environmental toxicants perturb human Sertoli cell adhesive function via changes in F-actin organization mediated by actin regulatory proteins

    PubMed Central

    Xiao, Xiang; Mruk, Dolores D.; Tang, Elizabeth I.; Wong, Chris K.C.; Lee, Will M.; John, Constance M.; Turek, Paul J.; Silvestrini, Bruno; Cheng, C. Yan

    2014-01-01

    mislocalization of actin filament barbed end capping and bundling protein Eps8, and branched actin polymerization protein Arp3. Besides impeding actin dynamics, endocytic vesicle-mediated trafficking and the proper localization of actin regulatory proteins c-Src and annexin II in Sertoli cells were also affected. Results of statistical analysis demonstrate that these findings were not obtained by chance. LIMITATIONS, REASONS FOR CAUTION (i) This study was done in vitro and might not extrapolate to the in vivo state, (ii) conclusions are based on the use of Sertoli cell samples from three men and (iii) it is uncertain if the concentrations of toxicants used in the experiments are reached in vivo. WIDER IMPLICATIONS OF THE FINDINGS Human Sertoli cells cultured in vitro provide a robust model to monitor environmental toxicant-mediated disruption of Sertoli cell BTB function and to study the mechanism(s) of toxicant-induced testicular dysfunction. PMID:24532171

  7. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    NASA Technical Reports Server (NTRS)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  8. Preparation of polymer decorated graphene oxide by γ-ray induced graft polymerization

    NASA Astrophysics Data System (ADS)

    Zhang, Bowu; Zhang, Yujie; Peng, Cheng; Yu, Ming; Li, Linfan; Deng, Bo; Hu, Pengfei; Fan, Chunhai; Li, Jingye; Huang, Qing

    2012-02-01

    Herein, we report a facile approach to decorate graphene oxide (GO) sheets with poly(vinyl acetate) (PVAc) by γ-ray irradiation-induced graft polymerization. The content of PVAc in the obtained sample, i.e., PVAc grafted GO (GO-g-PVAc) is calculated by the loss weight in thermogravimetric analysis (TGA) curves. A GO-g-PVAc sample with a degree of grafting (DG) of 28.5% was well dispersed in common organic solvents and the dispersions obtained were extremely stable at room temperature without any aggregation, even after standing for 2 months. The excellent dispersibility and stability of GO-g-PVAc in common organic solvents are readily rationalized in terms of the full coverage of PVAc chains and solvated layer formation on graphene oxide sheets surface, which weakens the interlaminar attraction of GO sheets. This approach presents a facile route for the preparation of dispersible GO and shows great potential in the preparation of graphene-based composites by solution-processes.Herein, we report a facile approach to decorate graphene oxide (GO) sheets with poly(vinyl acetate) (PVAc) by γ-ray irradiation-induced graft polymerization. The content of PVAc in the obtained sample, i.e., PVAc grafted GO (GO-g-PVAc) is calculated by the loss weight in thermogravimetric analysis (TGA) curves. A GO-g-PVAc sample with a degree of grafting (DG) of 28.5% was well dispersed in common organic solvents and the dispersions obtained were extremely stable at room temperature without any aggregation, even after standing for 2 months. The excellent dispersibility and stability of GO-g-PVAc in common organic solvents are readily rationalized in terms of the full coverage of PVAc chains and solvated layer formation on graphene oxide sheets surface, which weakens the interlaminar attraction of GO sheets. This approach presents a facile route for the preparation of dispersible GO and shows great potential in the preparation of graphene-based composites by solution-processes. Electronic

  9. Upregulation of two actin genes and redistribution of actin during diapause and cold stress in the northern house mosquito, Culex pipiens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two actin genes cloned from Culex pipiens L. are upregulated during adult diapause. Though actins 1 and 2 were expressed throughout diapause, both genes were most highly expressed early in diapause. These changes in gene expression were accompanied by a conspicuous redistribution of polymerized acti...

  10. Shear flow induced nanostructure-micostructure transition in a polymeric bicontinuous microemulsion

    NASA Astrophysics Data System (ADS)

    Krishnan, Kasiraman; Bates, Frank S.; Lodge, Timothy P.; Burghardt, Wesley R.

    2001-03-01

    The effects of shear flow on a polymeric bicontinuous microemulsion have been studied using scattering, microscopy and rheology. The microemulsion consists of a ternary blend of poly(ethyl ethylene) (PEE), poly(dimethyl siloxane) (PDMS) and the diblock copolymer PEE-PDMS. Steady shear experiments reveal four regimes as a function of shear rate. At low shear rates (regime I), the bicontinuous nanostructure is unperturbed and the sample behaves as a Newtonian fluid. Shear thinning is observed at intermediate shear rates (regime II) concurrent with the development of anisotropy in the structure. Further increase of shear rate results in flow-induced phase separation; the stress is independent of shear rate in this regime III. Light scattering shows a streak-like pattern perpendicular to the flow direction while microscopy reveals a string-like, micron-sized morphology. Higher shear rates (regime IV) lead to binary blend-like behavior with the block copolymer playing no significant role; the stress increases strongly with the shear rate.

  11. Pressure-induced polymerization of carbon monoxide: disproportionation and synthesis of an energetic lactonic polymer

    SciTech Connect

    Evans, W J; Lipp, M J; Yoo, C; Herberg, J L; Maxwell, R S; Nicol, M F

    2005-10-04

    We have studied pressure-induced chemical reactions in carbon monoxide using both a diamond-anvil cell and a modified large volume press. Our spectroscopic data reveal that carbon monoxide disproportionates into molecular CO{sub 2} and a solid lactone-type polymer; photochemically above 3.2 GPa, thermochemically above 5 GPa at 300K, or at 3 GPa and {approx}2000K as achieved by laser heating. The solid product can be recovered at ambient conditions with a high degree of conversion, measured to be up to 95% of the original CO. Its fundamental chemical structure includes {beta}-lactone and conjugated C=C, which can be considered a severely modified polymeric carbon suboxide with open ladders and smaller five-membered rings. The polymer is metastable at ambient conditions, spontaneously liberating CO{sub 2} gases exothermically. We find that the recovered polymer has a high energy density, 1-8 KJ/g, and is very combustible. We estimate the density of recovered CO polymer to be at least 1.65 g/cm cm{sup 3}.

  12. Properties of triple shape memory composites prepared via polymerization-induced phase separation.

    PubMed

    Torbati, Amir H; Nejad, Hossein Birjandi; Ponce, Mileysa; Sutton, James P; Mather, Patrick T

    2014-05-01

    Research in the field of shape memory polymers has recently witnessed the introduction of increasing complexity of material response, including such phenomena as triple/multishape behavior, temperature memory, and reversible actuation. Ordinarily, such complexity in physical behaviour is achieved through comparable complexity in material composition and synthesis. Seeking to achieve a triple shape behaviour with a simple route to materials synthesis, we introduce here a method that utilizes polymerization induced phase separation (PIPS) to yield the requisite combination of microstructure and composition. Thus, two blends incorporating epoxy and poly(ε-caprolactone) were developed using commercially available reactants, one featuring a semicrystalline epoxy and the other featuring an amorphous epoxy. We show that both blends exhibited distinct transition temperatures and three modulus-temperature plateaus needed for triple shape behaviour. Despite these similarities, their physical character at room temperature is vastly different: the semicrystalline epoxy material is elastomeric and the amorphous epoxy material is highly stiff. Characterization of the triple shape behaviour revealed an ability of both systems to fix two separate deformations independently, one by PCL crystallization and a second one by epoxy crystallization or vitrification, and recover both programmed shapes separately upon heating. Given the simplicity of fabrication, we envision application as multi-shape coatings, adhesives, and films. PMID:24695693

  13. Anhydrous Proton Conducting Polymer Electrolyte Membranes via Polymerization-Induced Microphase Separation.

    PubMed

    Chopade, Sujay A; So, Soonyong; Hillmyer, Marc A; Lodge, Timothy P

    2016-03-01

    Solid-state polymer electrolyte membranes (PEMs) exhibiting high ionic conductivity coupled with mechanical robustness and high thermal stability are vital for the design of next-generation lithium-ion batteries and high-temperature fuel cells. We present the in situ preparation of nanostructured PEMs incorporating a protic ionic liquid (IL) into one of the domains of a microphase-separated block copolymer created via polymerization-induced microphase separation. This facile, one-pot synthetic strategy transforms a homogeneous liquid precursor consisting of a poly(ethylene oxide) (PEO) macro-chain-transfer agent, styrene and divinylbenzene monomers, and protic IL into a robust and transparent monolith. The resulting PEMs exhibit a bicontinuous morphology comprising PEO/protic IL conducting pathways and highly cross-linked polystyrene (PS) domains. The cross-linked PS mechanical scaffold imparts thermal and mechanical stability to the PEMs, with an elastic modulus approaching 10 MPa at 180 °C, without sacrificing the ionic conductivity of the system. Crucially, the long-range continuity of the PEO/protic IL conducting nanochannels results in an outstanding ionic conductivity of 14 mS/cm at 180 °C. We posit that proton conduction in the protic IL occurs via the vehicular mechanism and the PEMs exhibit an average proton transference number of 0.7. This approach is very promising for the development of high-temperature, robust PEMs with excellent proton conductivities. PMID:26927732

  14. Biomimetic phosphorylcholine polymer grafting from polydimethylsiloxane surface using photo-induced polymerization.

    PubMed

    Goda, Tatsuro; Konno, Tomohiro; Takai, Madoka; Moro, Toru; Ishihara, Kazuhiko

    2006-10-01

    The biomimetic synthetic phospholipid polymer containing a phosphorylcholine group, 2-methacryloyloxyethyl phosphorylcholine (MPC), has improved the surface property of biomaterials. Both hydrophilic and anti-biofouling surfaces were prepared on polydimethylsiloxane (PDMS) with MPC grafted by surface-initiated photo-induced radical polymerization. Benzophenone was used as the photoinitiator. The quantity of the adsorbed initiator on PDMS was determined by UV absorption and ellipsometry. The poly(MPC)-grafted PDMS surfaces were characterized by XPS, ATR-FTIR and static water contact angle (SCA) measurements. The SCA on PDMS decreased from 115 degrees to 25 degrees after the poly(MPC) grafting. The in vitro single protein adsorption on the poly(MPC)-grafted PDMS decreased 50-75% compared to the unmodified PDMS. The surface friction of the poly(MPC)-grafted PDMS was lower than the unmodified PDMS under wet conditions. The oxygen permeability of the poly(MPC)-grafted PDMS was as high as the unmodified PDMS. The tensile property of PDMS was maintained at about 90% of the ultimate stress and strain after the poly(MPC) grafting. The surface-modified PDMS is expected to be a novel medical elastomer which possesses an excellent surface hydrophilicity, anti-biofouling property, oxygen permeability and tensile property. PMID:16797692

  15. Synthesis of porous zirconia spheres for HPLC by polymerization-induced colloid aggregation (PICA)

    SciTech Connect

    Sun, L.; Annen, M.J.; Lorenzano-Porras, F.; Carr, P.W.; McCormick, A.V. )

    1994-03-15

    Porous, spherical zirconia particles with a narrow particle size distribution, which are useful as chromatographic packing materials for high performance liquid chromatography (HPLC), were synthesized by polymerization-induced colloid aggregation (PICA) first described by Iler and McQueston (US Patent 4,010,242, 1977.) and the effects of a number of crucial processing variables were examined. In this method, an aqueous zirconia sol consisting of 700 [angstrom] (mean diameter) particles is mixed with urea and formaldehyde polymer adsorbs onto the ZrO[sub 2] colloids, entraining the colloids in the precipitation of the polymer gel and thus alloying the colloids to aggregate. Features of the aggregation process are elucidated from responses of the process to variations in temperature, reaction mixture composition, and solvent polarity. Results suggest that the aggregation process resembles those reported for the bridging flocculation of colloids by adsorbed polymers. Porous zirconia particles obtained after polymer combustion and sintering of the aggregates are 3.5 [mu]m in diameter with a surface area of 13 m[sup 2]/g, a porosity of 29% and pores ranging from <50 to 350 [angstrom] in diameter. The particles are strong enough to withstand the packing of a HPLC column.

  16. High Modulus, High Conductivity Nanostructured Polymer Electrolyte Membranes via Polymerization-Induced Phase Separation

    NASA Astrophysics Data System (ADS)

    McIntosh, Lucas; Schulze, Morgan; Hillmyer, Marc; Lodge, Timothy

    2014-03-01

    Solvent-free, solid-state polymer electrolyte membranes (PEMs) will play a vital role in next-generation electrochemical devices such as Li-metal batteries and high- T fuel cells. The primary challenge is that these applications require PEMs with substantial mechanical robustness, as well as high ionic conductivity. The key to optimizing orthogonal macroscopic properties is to use a heterogeneous composite with well-defined nanoscopic morphology--specifically, long-range co-continuity of high modulus and ion transport domains, which has proven difficult to achieve in commonly-studied diblock copolymer-based electrolytes. We report a simple synthetic strategy to generate PEMs via polymerization-induced phase separation, where the delicate balance between controlled addition of styrene onto a poly(ethylene oxide) macro-chain transfer agent and simultaneous chemical crosslinking by divinylbenzene results in a disordered structure with domain size of order 10 nm. Crucially, both domains exhibit long-range continuity, which results in PEMs that are glassy solids (modulus ~ 1 GPa) owing to the isotropic network of stiff, crosslinked polystyrene, and are highly conductive (> 1 mS/cm at 70 °C) because ions migrate in channels of low Tg poly(ethylene oxide).

  17. Proton conducting, high modulus polymer electrolyte membranes by polymerization-induced microphase separation

    NASA Astrophysics Data System (ADS)

    Chopade, Sujay; Hillmyer, Marc; Lodge, Timothy

    Robust solid-state polymer electrolyte membranes (PEMs) are vital for designing next-generation lithium-ion batteries and high-temperature fuel cells. However, the performance of diblock polymer electrolytes is generally limited by poor mechanical stability and network defects in the conducting pathways. We present the in-situ preparation of robust cross-linked PEMs via polymerization-induced microphase separation, and incorporation of protic ionic liquid (IL) into one of the microphase separated domains. The facile design strategy involves a delicate balance between the controlled growth of polystyrene from a poly(ethylene oxide) macro-chain transfer agent (PEO-CTA) and simultaneous chemical cross-linking by divinylbenzene in the presence of IL. Small angle X-ray scattering and transmission electron microscopy confirmed the formation of a disordered structure with bicontinuous morphology and a characteristic domain size of order 20 nm. The long-range continuity of the PEO/protic IL conducting nanochannels and cross-linked polystyrene domains imparts high thermal and mechanical stability to the PEMs, with elastic modulus approaching 10 MPa and a high ionic conductivity of 15 mS/cm at 180 °C.

  18. 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

  19. 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

  20. 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

  1. Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs

    PubMed Central

    Zheng, Maozhong; Beck, Martina; Müller, Jens; Chen, Tong; Wang, Xiaohua; Wang, Feng; Wang, Qinli; Wang, Yuqing; Baluška, František; Logan, David C.; Šamaj, Jozef; Lin, Jinxing

    2009-01-01

    Background Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements. Methodology/Principal Findings We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria. Conclusions/Significance Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events. PMID:19536333

  2. 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

  3. 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.

  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. Actin purification from a gel of rat brain extracts.

    PubMed

    Levilliers, N; Peron-Renner, M; Coffe, G; Pudles, J

    1984-01-01

    Actin, 99% pure, has been recovered from rat brain with a high yield (greater than 15 mg/100 g brain). We have shown that: 1. a low ionic strength extract from rat brain tissue is capable of giving rise to a gel; 2. actin is the main gel component and its proportion is one order of magnitude higher than in the original extract; 3. actin can be isolated from this extract by a three-step procedure involving gelation, dissociation of the gel in 0.6 M KCl, followed by one or two depolymerization-polymerization cycles. PMID:6529588

  6. Towards the Structure Determination of a Modulated Protein Crystal: The Semicrystalline State of Profilin:Actin

    NASA Technical Reports Server (NTRS)

    Borgstahl, G.; Lovelace, J.; Snell, E. H.; Bellamy, H.

    2003-01-01

    One of the remaining challenges to structural biology is the solution of modulated structures. While small molecule crystallographers have championed this type of structure, to date, no modulated macromolecular structures have been determined. Modulation of the molecular structures within the crystal can produce satellite reflections or a superlattice of reflections in reciprocal space. We have developed the data collection methods and strategies that are needed to collect and analyze these data. If the macromolecule's crystal lattice is composed of physiologically relevant packing contacts, structural changes induced under physiological conditions can cause distortion relevant to the function and biophysical processes of the molecule making up the crystal. By careful measurement of the distortion, and the corresponding three-dimensional structure of the distorted molecule, we will visualize the motion and mechanism of the biological macromolecule(s). We have measured the modulated diffraction pattern produced by the semicrystalline state of profilin:actin crystals using highly parallel and highly monochromatic synchrotron radiation coupled with fine phi slicing (0.001-0.010 degrees) for structure determination. These crystals present these crystals present a unique opportunity to address an important question in structural biology. The modulation is believed to be due to the formation of actin helical filaments from the actin beta ribbon upon the pH-induced dissociation of profilin. To date, the filamentous state of actin has resisted crystallization and no detailed structures are available. The semicrystalline state profilin:actin crystals provides a unique opportunity to understand the many conformational states of actin. This knowledge is essential for understanding the dynamics underlying shape changes and motility of eukaryotic cells. Many essential processes, such as cytokinesis, phagocytosis, and cellular migration depend upon the capacity of the actin

  7. 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

  8. Pressure induced polymerization of acetylide anions in CaC2 and 107 fold enhancement of electrical conductivity

    DOE PAGESBeta

    Zheng, Haiyan; Wang, Lijuan; Li, Kuo; Yang, Youyou; Wang, Yajie; Wu, Jiajia; Dong, Xiao; Wang, Chun -Hai; Tulk, Christopher A.; Molaison, Jamie J.; et al

    2016-08-17

    Transformation between different types of carbon–carbon bonding in carbides often results in a dramatic change of physical and chemical properties. Under external pressure, unsaturated carbon atoms form new covalent bonds regardless of the electrostatic repulsion. It was predicted that calcium acetylide (also known as calcium carbide, CaC2) polymerizes to form calcium polyacetylide, calcium polyacenide and calcium graphenide under high pressure. In this work, the phase transitions of CaC2 under external pressure were systematically investigated, and the amorphous phase was studied in detail for the first time. Polycarbide anions like C66– are identified with gas chromatography-mass spectrometry and several other techniques,more » which evidences the pressure induced polymerization of the acetylide anions and suggests the existence of the polyacenide fragment. Additionally, the process of polymerization is accompanied with a 107 fold enhancement of the electrical conductivity. As a result, the polymerization of acetylide anions demonstrates that high pressure compression is a viable route to synthesize novel metal polycarbides and materials with extended carbon networks, while shedding light on the synthesis of more complicated metal organics.« less

  9. Simulation of the effect of confinement in actin ring formation

    NASA Astrophysics Data System (ADS)

    Adeli Koudehi, Maral; Vavylonis, Dimitrios; Haosu Tang Team; Dimitrios Vavylonis Team

    Actin filaments are vital for different network structures in living cells. During cytokinesis, they form a contractile ring containing myosin motor proteins and actin filament cross-linkers to separate one cell into two cells. Recent experimental studies have quantified the bundle, ring, and network structures that form when actin filaments polymerize in confined environments in vitro, in the presence of varying concentrations of cross-linkers. In this study, we performed numerical simulations to investigate the effect of actin spherical confinement and cross-linking in ring formation. We used a spring-bead model and Brownian dynamics to simulate semiflexible actin filaments that polymerize in a confining sphere with a rate proportional to the monomer concentration. Applying the model for different size of the confining spheres shows that the probability of ring formation decreases by increasing the radius (at fixed initial monomer concentration), in agreement with prior experimental data. We describe the effect of persistence length, orientation-dependent cross-linking, and initial actin monomer concentration. Simulations show that equilibrium configurations can be reached through zipping and unzipping of actin filaments in bundles and transient ring formation.

  10. Actin Dynamics in Growth Cone Motility and Navigation

    PubMed Central

    Gomez, Timothy M.; Letourneau, Paul C.

    2014-01-01

    Motile growth cones lead growing axons through developing tissues to synaptic targets. These behaviors depend on the organization and dynamics of actin filaments that fill the growth cone leading margin (peripheral (P-) domain). Actin filament organization in growth cones is regulated by actin-binding proteins that control all aspects of filament assembly, turnover, interactions with other filaments and cytoplasmic components, and participation in producing mechanical forces. Actin filament polymerization drives protrusion of sensory filopodia and lamellipodia, and actin filament connections to the plasma membrane link the filament network to adhesive contacts of filopodia and lamellipodia with other surfaces. These contacts stabilize protrusions and transduce mechanical forces generated by actomyosin activity into traction that pulls an elongating axon along the path towards its target. Adhesive ligands and extrinsic guidance cues bind growth cone receptors and trigger signaling activities involving Rho GTPases, kinases, phosphatases, cyclic nucleotides and [Ca++] fluxes. These signals regulate actin binding proteins to locally modulate actin polymerization, interactions and force transduction to steer the growth cone leading margin towards the sources of attractive cues and away from repellent guidance cues. PMID:24164353

  11. 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

  12. Phosphatidylinositol 5-phosphatase oculocerebrorenal syndrome of Lowe protein (OCRL) controls actin dynamics during early steps of Listeria monocytogenes infection.

    PubMed

    Kühbacher, Andreas; Dambournet, Daphné; Echard, Arnaud; Cossart, Pascale; Pizarro-Cerdá, Javier

    2012-04-13

    Listeria monocytogenes is a bacterial pathogen that induces its own entry into a broad range of mammalian cells through interaction of the bacterial surface protein InlB with the cellular receptor Met, promoting an actin polymerization/depolymerization process that leads to pathogen engulfment. Phosphatidylinositol bisphosphate (PI[4,5]P(2)) and trisphosphate (PI[3,4,5]P(3)) are two major phosphoinositide species that function as molecular scaffolds, recruiting cellular effectors that regulate actin dynamics during L. monocytogenes infection. Because the phosphatidylinositol 5'-phosphatase OCRL dephosphorylates PI(4,5)P(2) and to a lesser extent PI(3,4,5)P(3), we investigated whether this phosphatase modulates cell invasion by L. monocytogenes. Inactivation of OCRL by small interfering RNA (siRNA) leads to an increase in the internalization levels of L. monocytogenes in HeLa cells. Interestingly, OCRL depletion does not increase but rather decreases the surface expression of the receptor Met, suggesting that OCRL controls bacterial internalization by modulating signaling cascades downstream of Met. Immuno-fluorescence microscopy reveals that endogenous and overexpressed OCRL are present at L. monocytogenes invasion foci; live-cell imaging additionally shows that actin depolymerization coincides with EGFP-OCRL-a accumulation around invading bacteria. Together, these observations suggest that OCRL promotes actin depolymerization during L. monocytogenes infection; in agreement with this hypothesis, OCRL depletion leads to an increase in actin, PI(4,5)P(2), and PI(3,4,5)P(3) levels at bacterial internalization foci. Furthermore, in cells knocked down for OCRL, transfection of enzymatically active EGFP-OCRL-a (but not of a phosphatase-dead enzyme) decreases the levels of intracellular L. monocytogenes and of actin associated with invading bacteria. These results demonstrate that through its phosphatase activity, OCRL restricts L. monocytogenes invasion by modulating

  13. Phosphatidylinositol 5-Phosphatase Oculocerebrorenal Syndrome of Lowe Protein (OCRL) Controls Actin Dynamics during Early Steps of Listeria monocytogenes Infection*

    PubMed Central

    Kühbacher, Andreas; Dambournet, Daphné; Echard, Arnaud; Cossart, Pascale; Pizarro-Cerdá, Javier

    2012-01-01

    Listeria monocytogenes is a bacterial pathogen that induces its own entry into a broad range of mammalian cells through interaction of the bacterial surface protein InlB with the cellular receptor Met, promoting an actin polymerization/depolymerization process that leads to pathogen engulfment. Phosphatidylinositol bisphosphate (PI[4,5]P2) and trisphosphate (PI[3,4,5]P3) are two major phosphoinositide species that function as molecular scaffolds, recruiting cellular effectors that regulate actin dynamics during L. monocytogenes infection. Because the phosphatidylinositol 5′-phosphatase OCRL dephosphorylates PI(4,5)P2 and to a lesser extent PI(3,4,5)P3, we investigated whether this phosphatase modulates cell invasion by L. monocytogenes. Inactivation of OCRL by small interfering RNA (siRNA) leads to an increase in the internalization levels of L. monocytogenes in HeLa cells. Interestingly, OCRL depletion does not increase but rather decreases the surface expression of the receptor Met, suggesting that OCRL controls bacterial internalization by modulating signaling cascades downstream of Met. Immuno-fluorescence microscopy reveals that endogenous and overexpressed OCRL are present at L. monocytogenes invasion foci; live-cell imaging additionally shows that actin depolymerization coincides with EGFP-OCRL-a accumulation around invading bacteria. Together, these observations suggest that OCRL promotes actin depolymerization during L. monocytogenes infection; in agreement with this hypothesis, OCRL depletion leads to an increase in actin, PI(4,5)P2, and PI(3,4,5)P3 levels at bacterial internalization foci. Furthermore, in cells knocked down for OCRL, transfection of enzymatically active EGFP-OCRL-a (but not of a phosphatase-dead enzyme) decreases the levels of intracellular L. monocytogenes and of actin associated with invading bacteria. These results demonstrate that through its phosphatase activity, OCRL restricts L. monocytogenes invasion by modulating actin

  14. Cdc42 and p190RhoGAP activation by CCN2 regulates cell spreading and polarity and induces actin disassembly in migrating keratinocytes.

    PubMed

    Kiwanuka, Elizabeth; Lee, Cameron Cy; Hackl, Florian; Caterson, Edward J; Junker, Johan Pe; Gerdin, Bengt; Eriksson, Elof

    2016-06-01

    Cell migration requires spatiotemporal integration of signals that regulate cytoskeletal dynamics. In response to a migration-promoting agent, cells begin to polarise and extend protrusions in the direction of migration. These cytoskeletal rearrangements are orchestrated by a variety of proteins, including focal adhesion kinase (FAK) and the Rho family of GTPases. CCN2, also known as connective tissue growth factor, has emerged as a regulator of cell migration but the mechanism by which CCN2 regulates keratinocyte function is not well understood. In this article, we sought to elucidate the basic mechanism of CCN2-induced cell migration in human keratinocytes. Immunohistochemical staining was used to demonstrate that treatment with CCN2 induces a migratory phenotype through actin disassembly, spreading of lamellipodia and re-orientation of the Golgi. In vitro assays were used to show that CCN2-induced cell migration is dependent on FAK, RhoA and Cdc42, but independent of Rac1. CCN2-treated keratinocytes displayed increased Cdc42 activity and decreased RhoA activity up to 12 hours post-treatment, with upregulation of p190RhoGAP. An improved understanding of how CCN2 regulates cell migration may establish the foundation for future therapeutics in fibrotic and neoplastic diseases. PMID:25185742

  15. Frutalin, a galactose-binding lectin, induces chemotaxis and rearrangement of actin cytoskeleton in human neutrophils: involvement of tyrosine kinase and phosphoinositide 3-kinase.

    PubMed

    Brando-Lima, Aline C; Saldanha-Gama, Roberta F; Henriques, Maria das Graças M O; Monteiro-Moreira, Ana C O; Moreira, Renato A; Barja-Fidalgo, Christina

    2005-10-15

    Several lectin-like molecules have been shown as potent activators of leukocytes. Galactose-binding lectins are of special interest since they could interact with several endogenous molecules involved in the innate and specific immune responses. The effects of Frutalin (FTL), an alpha-D-galactose (Gal)-binding plant lectin, on the modulation of neutrophil (PMN) functions were investigated. FTL induced a dose-dependent PMN migration in mice pleural cavity. Moreover, FTL was also a potent direct chemotactic for human PMN, in vitro, and triggered oxidative burst in these cells. These effects were accompanied by a rearrangement of the actin cytoskeleton dynamic, activation of tyrosine kinase (TK) pathways, increase in focal adhesion kinase (FAK) phosphorylation, and its subsequent association to phosphoinositide3-kinase (PI3K). All those effects were inhibited in the presence of Gal, suggesting specific carbohydrate recognition for FTL effects. The activations of TK and PI3K pathways are essential events for FTL-induced chemotaxis, since inhibitors of these pathways, genistein and LY294002, inhibited neutrophil migration in vitro. The data indicate that sugar-protein interactions between a soluble lectin and galacto-components on neutrophil surface trigger the TK pathway, inducing FAK and PI3K activation, interfering with cell motility and oxidative response. PMID:16183388

  16. Nuclear and cytoplasmic actin in dinoflagellates.

    PubMed

    Soyer-Gobillard, M O; Ausseil, J; Géraud, M L

    1996-01-01

    Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the

  17. Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo {sup 1}H NMR and fluorescence spectroscopy

    SciTech Connect

    Wazawa, Tetsuichi; Sagawa, Takashi; Ogawa, Tsubasa; Morimoto, Nobuyuki; Kodama, Takao; Suzuki, Makoto

    2011-01-28

    Research highlights: {yields} Translationally hyper-mobile water has been detected around actin filaments. {yields} Translationally hyper-mobile water is formed upon polymerization of actin. {yields} Low water viscosity was found around F-actin using fluorescence anisotropy. {yields} Formation of hyper-mobile water may explain endothermic actin polymerization. -- Abstract: This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo {sup 1}H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by {approx}5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1 ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.

  18. Regulation of cellular actin architecture by S100A10.

    PubMed

    Jung, M Juliane; Murzik, Ulrike; Wehder, Liane; Hemmerich, Peter; Melle, Christian

    2010-04-15

    Actin structures are involved in several biological processes and the disruption of actin polymerisation induces impaired motility of eukaryotic cells. Different factors are involved in regulation and maintenance of the cytoskeletal actin architecture. Here we show that S100A10 participates in the particular organisation of actin filaments. Down-regulation of S100A10 by specific siRNA triggered a disorganisation of filamentous actin structures without a reduction of the total cellular actin concentration. In contrast, the formation of cytoskeleton structures containing tubulin was unhindered in S100A10 depleted cells. Interestingly, the cellular distribution of annexin A2, an interaction partner of S100A10, was unaffected in S100A10 depleted cells. Cells lacking S100A10 showed an impaired migration activity and were unable to close a scratched wound. Our data provide first insights of S100A10 function as a regulator of the filamentous actin network. PMID:20100475

  19. Modulation of the interaction between G-actin and thymosin beta 4 by the ATP/ADP ratio: possible implication in the regulation of actin dynamics.

    PubMed Central

    Carlier, M F; Jean, C; Rieger, K J; Lenfant, M; Pantaloni, D

    1993-01-01

    The interaction of G-actin with thymosin beta 4 (T beta 4), the major G-actin-sequestering protein in motile and proliferating cells, has been analyzed in vitro. T beta 4 is found to have a 50-fold higher affinity for MgATP-actin than for MgADP-actin. These results imply that in resting platelets and neutrophils, actin is sequestered by T beta 4 as MgATP-G-actin. Kinetic experiments and theoretical calculations demonstrate that this ATP/ADP dependence of T beta 4 affinity for G-actin can generate a mechanism of desequestration of G-actin by ADP, in the presence of physiological concentrations of T beta 4 (approximately 0.1 mM). The desequestration of G-actin by ADP is kinetically enhanced by profilin, which accelerates the dissociation of ATP from G-actin. Whether a local drop in the ATP/ADP ratio can allow local, transient desequestration and polymerization of actin either close to the plasma membrane, following platelet or neutrophil stimulation, or behind the Listeria bacterium in the host cell, while the surrounding cytoplasm contains sequestered ATP-G-actin, is an open issue raised by the present work. PMID:8506348

  20. Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    NASA Technical Reports Server (NTRS)

    Love, Felisha D.; Melhado, Caroline; Bosah, Francis; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1997-01-01

    A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

  1. Arabidopsis CROLIN1, a Novel Plant Actin-binding Protein, Functions in Cross-linking and Stabilizing Actin Filaments*

    PubMed Central

    Jia, Honglei; Li, Jisheng; Zhu, Jingen; Fan, Tingting; Qian, Dong; Zhou, Yuelong; Wang, Jiaojiao; Ren, Haiyun; Xiang, Yun; An, Lizhe

    2013-01-01

    Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which the higher order cytoskeleton is formed in plants remains unknown. In this study, we identified a novel actin-cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom. There are six isovariants of CROLIN in the Arabidopsis genome, with CROLIN1 specifically expressed in pollen. In vitro biochemical analyses showed that CROLIN1 is a novel actin-cross-linking protein with binding and stabilizing activities. Remarkably, CROLIN1 can cross-link actin bundles into actin networks. CROLIN1 loss of function induces pollen germination and pollen tube growth hypersensitive to latrunculin B. All of these results demonstrate that CROLIN1 may play an important role in stabilizing and remodeling actin filaments by binding to and cross-linking actin filaments. PMID:24072702

  2. F-actin Severing Facilitates Distinct Mechanisms of Stress Relaxation in the Actin Cytoskeleton

    NASA Astrophysics Data System (ADS)

    Kim, Taeyoon; Jung, Wonyeong; Murrell, Michael

    Rheological behaviors of actin cytoskeleton play an important role in physiological processes including cell migration and division. The actin cytoskeleton shows a wide variety of viscoelastic responses to external mechanical cues, such as strain-stiffening and stress relaxation. It has been hypothesized that the stress relaxation originates mainly from transient nature of cross-linkers that connect pairs of F-actins. By contrast, potential impacts of rich F-actin dynamics to the stress relaxation have been neglected in most previous studies. Here, using a computational model, we demonstrated that severing of F-actins induced by buckling during strain-stiffening can facilitate a very distinct mode of stress relaxation in the actin cytoskeleton from that induced by the transient cross-linkers. We also explored conditions where the severing-induced stress relaxation becomes prominent. This finding provides a more complete understanding of rheological behaviors of the actin cytoskeleton. We gratefully acknowledge the support of the National Science Foundation (1434013-CMMI and 1434095-CMMI).

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

    PubMed Central

    Vitriol, Eric A.; McMillen, Laura M.; Kapustina, Maryna; Gomez, Shawn M.; Vavylonis, Dimitrios; Zheng, James Q.

    2015-01-01

    Summary Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer binding protein thymosin β4 (Tβ4) for optimal leading edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it doesn’t interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions. PMID:25865895

  4. A gelatin-free model system for the study of the basic radiation-induced polymerization in PAG dosimeters

    NASA Astrophysics Data System (ADS)

    Babic, S.; Park, Y. S.; Schreiner, L. J.

    2004-01-01

    In this presentation we show results of investigations on gelatin-free dosimeters containing equal amounts of acrylamide and N,N'-methylene-bisacrylamide (named Aqueous Polyacrylamide, APA, dosimeters). The dosimeters were prepared with three different total monomer concentrations (2, 6, and 8% by weight). Nuclear magnetic resonance (NMR) spin-spin and spin-lattice proton relaxation measurements at 20 MHz, and gravimetric analyses performed on all three dosimeters, show a continuous degree of polymerization over the range of dose 0.5 - 25 Gy. The developed NMR model explains the relationship observed between the relaxation data and the amount of cross-linked polymer formed at each dose. This model may be extended with gelatin relaxation data to provide a fundamental understanding of radiation-induced polymerization in the conventional PAG dosimeters.

  5. 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.

  6. Modification of linear prepolymers to tailor heterogeneous network formation through photo-initiated Polymerization-Induced Phase Separation

    PubMed Central

    Szczepanski, Caroline R.; Stansbury, Jeffrey W.

    2015-01-01

    Polymerization-induced phase separation (PIPS) was studied in ambient photopolymerizations of triethylene glycol dimethacrylate (TEGDMA) modified by poly(methyl methacrylate) (PMMA). The molecular weight of PMMA and the rate of network formation (through incident UV-irradiation) were varied to influence both the promotion of phase separation through increases in overall free energy, as well as the extent to which phase development occurs during polymerization through diffusion prior to network gelation. The overall free energy of the polymerizing system increases with PMMA molecular weight, such that PIPS is promoted thermodynamically at low loading levels (5 wt%) of a higher molecular weight PMMA (120 kDa), while a higher loading level (20 wt%) is needed to induce PIPS with lower PMMA molecular weight (11 kDa), and phase separation was not promoted at any loading level tested of the lowest molecular weight PMMA (1 kDa). Due to these differences in overall free energy, systems modified by PMMA (11 kDa) underwent phase separation via Nucleation and Growth, and systems modified by PMMA (120 kDa), followed the Spinodal Decomposition mechanism. Despite differences in phase structure, all materials form a continuous phase rich in TEGDMA homopolymer. At high irradiation intensity (Io=20mW/cm2), the rate of network formation prohibited significant phase separation, even when thermodynamically preferred. A staged curing approach, which utilizes low intensity irradiation (Io=300µW/cm2) for the first ~50% of reaction to allow phase separation via diffusion, followed by a high intensity flood-cure to achieve a high degree of conversion, was employed to form phase-separated networks with reduced polymerization stress yet equivalent final conversion and modulus. PMID:26190865

  7. A polar-localized iron-binding protein determines the polar targeting of Burkholderia BimA autotransporter and actin tail formation.

    PubMed

    Lu, Qiuhe; Xu, Yue; Yao, Qing; Niu, Miao; Shao, Feng

    2015-03-01

    Intracellular bacterial pathogens including Shigella, Listeria, Mycobacteria, Rickettsia and Burkholderia spp. deploy a specialized surface protein onto one pole of the bacteria to induce filamentous actin tail formation for directional movement within host cytosol. The mechanism underlying polar targeting of the actin tail proteins is unknown. Here we perform a transposon screen in Burkholderia thailandensis and identify a conserved bimC that is required for actin tail formation mediated by BimA from B. thailandensis and its closely related pathogenic species B. pseudomallei and B. mallei. bimC is located upstream of bimA in the same operon. Loss of bimC results in even distribution of BimA on the outer membrane surface, where actin polymerization still occurs. BimC is targeted to the same bacterial pole independently of BimA. BimC confers polar targeting of BimA prior to BimA translocation across bacterial inner membrane. BimC is an iron-binding protein, requiring a four-cysteine cluster at the carboxyl terminus. Mutation of the cysteine cluster disrupts BimC polar localization. Truncation analyses identify the transmembrane domain in BimA being responsible for its polar targeting. Consistently, BimC can interact with BimA transmembrane domain in an iron binding-dependent manner. Our study uncovers a new mechanism that determines the polar distribution of bacteria-induced actin tail in infected host cells. PMID:25293534

  8. Actin dynamics and the evolution of the memory trace.

    PubMed

    Rudy, Jerry W

    2015-09-24

    The goal of this essay is to link the regulation of actin dynamics to the idea that the synaptic changes that support long-term potentiation and memory evolve in temporally overlapping stages-generation, stabilization, and consolidation. Different cellular/molecular processes operate at each stage to change the spine cytoarchitecture and, in doing so, alter its function. Calcium-dependent processes that degrade the actin cytoskeleton network promote a rapid insertion of AMPA receptors into the post synaptic density, which increases a spine's capacity to express a potentiated response to glutamate. Other post-translation events then begin to stabilize and expand the actin cytoskeleton by increasing the filament actin content of the spine and reorganizing it to be resistant to depolymerizing events. Disrupting actin polymerization during this stabilization period is a terminal event-the actin cytoskeleton shrinks and potentiated synapses de-potentiate and memories are lost. Late-arriving, new proteins may consolidate changes in the actin cytoskeleton. However, to do so requires a stabilized actin cytoskeleton. The now enlarged spine has properties that enable it to capture other newly transcribed mRNAs or their protein products and thus enable the synaptic changes that support LTP and memory to be consolidated and maintained. This article is part of a Special Issue entitled SI: Brain and Memory. PMID:25498985

  9. Highly efficient and selective pressure-assisted photon-induced polymerization of styrene

    NASA Astrophysics Data System (ADS)

    Guan, Jiwen; Song, Yang

    2016-06-01

    The polymerization process of condensed styrene to produce polystyrene as an industrially important polymeric material was investigated using a novel approach by combining external compression with ultraviolet radiation. The reaction evolution was monitored as a function of time and the reaction products were characterized by in situ Fourier transform infrared spectroscopy. By optimizing the loading pressures, we observed highly efficient and selective production of polystyrene of different tacticities. Specifically, at relatively low loading pressures, infrared spectra suggest that styrene monomers transform to amorphous atactic polystyrene (APS) with minor crystalline isotactic polystyrene. In contrast, APS was found to be the sole product when polymerization occurs at relatively higher loading pressures. The time-dependent reaction profiles allow the examination of the polymerization kinetics by analyzing the rate constant and activation volume as a function of pressure. As a result, an optimized pressure condition, which allows a barrierless reaction to proceed, was identified and attributed to the very desirable reaction yield and kinetics. Finally, the photoinitiated reaction mechanism and the growth geometry of the polymer chains were investigated from the energy diagram of styrene and by the topology analysis of the crystal styrene. This study shows strong promise to produce functional polymeric materials in a highly efficient and controlled manner.

  10. Highly efficient and selective pressure-assisted photon-induced polymerization of styrene.

    PubMed

    Guan, Jiwen; Song, Yang

    2016-06-01

    The polymerization process of condensed styrene to produce polystyrene as an industrially important polymeric material was investigated using a novel approach by combining external compression with ultraviolet radiation. The reaction evolution was monitored as a function of time and the reaction products were characterized by in situ Fourier transform infrared spectroscopy. By optimizing the loading pressures, we observed highly efficient and selective production of polystyrene of different tacticities. Specifically, at relatively low loading pressures, infrared spectra suggest that styrene monomers transform to amorphous atactic polystyrene (APS) with minor crystalline isotactic polystyrene. In contrast, APS was found to be the sole product when polymerization occurs at relatively higher loading pressures. The time-dependent reaction profiles allow the examination of the polymerization kinetics by analyzing the rate constant and activation volume as a function of pressure. As a result, an optimized pressure condition, which allows a barrierless reaction to proceed, was identified and attributed to the very desirable reaction yield and kinetics. Finally, the photoinitiated reaction mechanism and the growth geometry of the polymer chains were investigated from the energy diagram of styrene and by the topology analysis of the crystal styrene. This study shows strong promise to produce functional polymeric materials in a highly efficient and controlled manner. PMID:27276967

  11. Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.

    PubMed Central

    Li, G; Rungger-Brändle, E; Just, I; Jonas, J C; Aktories, K; Wollheim, C B

    1994-01-01

    To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of

  12. Polymerized-Type I Collagen Induces Upregulation of Foxp3-Expressing CD4 Regulatory T Cells and Downregulation of IL-17-Producing CD4+ T Cells (Th17) Cells in Collagen-Induced Arthritis

    PubMed Central

    Furuzawa-Carballeda, Janette; Macip-Rodríguez, Perla; Galindo-Feria, Angeles S.; Cruz-Robles, David; Soto-Abraham, Virgina; Escobar-Hernández, Sergio; Aguilar, Diana; Alpizar-Rodríguez, Deshiré; Férez-Blando, Karen; Llorente, Luis

    2012-01-01

    Previous studies showed that polymerized-type I collagen (polymerized collagen) exhibits potent immunoregulatory properties. This work evaluated the effect of intramuscular administration of polymerized collagen in early and established collagen-induced arthritis (CIA) in mice and analyzed changes in Th subsets following therapy. Incidence of CIA was of 100% in mice challenged with type II collagen. Clinimorphometric analysis showed a downregulation of inflammation after administration of all treatments (P < 0.05). Histological analysis showed that the CIA-mice group had extensive bone erosion, pannus and severe focal inflammatory infiltrates. In contrast, there was a remarkable reduction in the severity of arthritis in mice under polymerized collagen, methotrexate or methotrexate/polymerized collagen treatment. Polymerized Collagen but not methotrexate induced tissue joint regeneration. Polymerized Collagen and methotrexate/polymerized collagen but not methotrexate alone induces downregulation of CD4+/IL17A+ T cells and upregulation of Tregs and CD4+/IFN-γ+ T cells. Thus, Polymerized Collagen could be an effective therapeutic agent in early and established rheumatoid arthritis by exerting downregulation of autoimmune inflammation. PMID:22028728

  13. 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.

  14. Length regulation of mechanosensitive stereocilia depends on very slow actin dynamics and filament-severing proteins.

    PubMed

    Narayanan, Praveena; Chatterton, Paul; Ikeda, Akihiro; Ikeda, Sakae; Corey, David P; Ervasti, James M; Perrin, Benjamin J

    2015-01-01

    Auditory sensory hair cells depend on stereocilia with precisely regulated lengths to detect sound. Since stereocilia are primarily composed of crosslinked, parallel actin filaments, regulated actin dynamics are essential for controlling stereocilia length. Here we assessed stereocilia actin turnover by monitoring incorporation of inducibly expressed β-actin-GFP in adult mouse hair cells in vivo and by directly measuring β-actin-GFP turnover in explants. Stereocilia actin incorporation is remarkably slow and restricted to filament barbed ends in a small tip compartment, with minimal accumulation in the rest of the actin core. Shorter rows of stereocilia, which have mechanically gated ion channels, show more variable actin turnover than the tallest stereocilia, which lack channels. Finally, the proteins ADF and AIP1, which both mediate actin filament severing, contribute to stereocilia length maintenance. Altogether, the data support a model whereby stereocilia actin cores are largely static, with dynamic regulation at the tips to maintain a critical length. PMID:25897778

  15. Radiation-induced reduction-polymerization route for the synthesis of PEDOT conducting polymers

    NASA Astrophysics Data System (ADS)

    Cui, Zhenpeng; Coletta, Cecilia; Rebois, Rolando; Baiz, Sarah; Gervais, Matthieu; Goubard, Fabrice; Aubert, Pierre-Henri; Dazzi, Alexandre; Remita, Samy

    2016-02-01

    Synthesis of conducting poly(3,4-ethylenedioxythiophene), PEDOT, is achieved through an original reduction-polymerization route: γ-radiolysis of aqueous solutions containing EDOT monomers under N2 atmosphere. According to UV-vis absorption spectrophotometry and ATR-FTIR spectroscopy, reduction of EDOT is initiated by hydrated electrons produced by water radiolysis and leads to PEDOT polymers through coupling reactions. The morphology of PEDOT is characterized by Cryo- TEM microscopy in aqueous solution and by SEM after deposition. In an original way, high resolution AFM microscopy, coupled with infrared nanospectroscopy, is used to probe the local chemical composition of PEDOT nanostructures. The results demonstrate that spherical self-assembled PEDOT nanostructures are formed. TGA analysis and four point probe measurements demonstrate that thermal stability and electrical conductivity of PEDOT polymers obtained by the present original reduction-polymerization method are close to those of PEDOT we previously prepared by radiolysis according to an oxidation-polymerization route.

  16. Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts.

    PubMed

    Kacena, Melissa A; Eleniste, Pierre P; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E; Mayo, Lindsey D; Bruzzaniti, Angela

    2012-05-18

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  17. Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts*

    PubMed Central

    Kacena, Melissa A.; Eleniste, Pierre P.; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E.; Mayo, Lindsey D.; Bruzzaniti, Angela

    2012-01-01

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  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. Contact potential induced enhancement of magnetization in polyaniline coated nanomagnetic iron oxides by plasma polymerization

    NASA Astrophysics Data System (ADS)

    Sethulakshmi, N.; Sooraj, V.; Sajeev, U. S.; Nair, Swapna S.; Narayanan, T. N.; Joy, Lija K.; Joy, P. A.; Ajayan, P. M.; Anantharaman, M. R.

    2013-10-01

    The present work derives motivation from the so called surface/interfacial magnetism in core shell structures and commercial samples of Fe3O4 and γ Fe2O3 with sizes ranging from 20 to 30 nm were coated with polyaniline using plasma polymerization and studied. The High Resolution Transmission Electron Microscopy images indicate a core shell structure after polyaniline coating and exhibited an increase in saturation magnetization by 2 emu/g. For confirmation, plasma polymerization was performed on maghemite nanoparticles which also exhibited an increase in saturation magnetization. This enhanced magnetization is rather surprising and the reason is found to be an interfacial phenomenon resulting from a contact potential.

  20. 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

  1. A new approach to network heterogeneity: Polymerization Induced Phase Separation in photo-initiated, free-radical methacrylic systems.

    PubMed

    Szczepanski, Caroline R; Pfeifer, Carmem S; Stansbury, Jeffrey W

    2012-09-28

    Non-reactive, thermoplastic prepolymers (poly- methyl, ethyl and butyl methacrylate) were added to a model homopolymer matrix composed of triethylene glycol dimethacrylate (TEGDMA) to form heterogeneous networks via polymerization induced phase separation (PIPS). PIPS creates networks with distinct phase structure that can partially compensate for volumetric shrinkage during polymerization through localized internal volume expansion. This investigation utilizes purely photo-initiated, free-radical systems, broadening the scope of applications for PIPS since these processing conditions have not been studied previously.The introduction of prepolymer into TEGDMA monomer resulted in stable, homogeneous monomer formulations, most of which underwent PIPS upon photo-irradiation, creating heterogeneous networks. During polymerization the presence of prepolymer enhanced autoacceleration, allowing for a more extensive ambient cure of the material. Phase separation, as characterized by dynamic changes in sample turbidity, was monitored simultaneously with monomer conversion and either preceded or was coincident with network gelation. Dynamic mechanical analysis shows a broadening of the tan delta peak and secondary peak formation, characteristic of phase-separated materials, indicating one phase rich in prepolymer and another depleted form upon phase separation. In certain cases, PIPS leads to an enhanced physical reduction of volumetric shrinkage, which is attractive for many applications including dental composite materials. PMID:23109733

  2. Rational synthesis of low-polydispersity block copolymer vesicles in concentrated solution via polymerization-induced self-assembly.

    PubMed

    Gonzato, Carlo; Semsarilar, Mona; Jones, Elizabeth R; Li, Feng; Krooshof, Gerard J P; Wyman, Paul; Mykhaylyk, Oleksandr O; Tuinier, Remco; Armes, Steven P

    2014-08-01

    Block copolymer self-assembly is normally conducted via post-polymerization processing at high dilution. In the case of block copolymer vesicles (or "polymersomes"), this approach normally leads to relatively broad size distributions, which is problematic for many potential applications. Herein we report the rational synthesis of low-polydispersity diblock copolymer vesicles in concentrated solution via polymerization-induced self-assembly using reversible addition-fragmentation chain transfer (RAFT) polymerization of benzyl methacrylate. Our strategy utilizes a binary mixture of a relatively long and a relatively short poly(methacrylic acid) stabilizer block, which become preferentially expressed at the outer and inner poly(benzyl methacrylate) membrane surface, respectively. Dynamic light scattering was utilized to construct phase diagrams to identify suitable conditions for the synthesis of relatively small, low-polydispersity vesicles. Small-angle X-ray scattering (SAXS) was used to verify that this binary mixture approach produced vesicles with significantly narrower size distributions compared to conventional vesicles prepared using a single (short) stabilizer block. Calculations performed using self-consistent mean field theory (SCMFT) account for the preferred self-assembled structures of the block copolymer binary mixtures and are in reasonable agreement with experiment. Finally, both SAXS and SCMFT indicate a significant degree of solvent plasticization for the membrane-forming poly(benzyl methacrylate) chains. PMID:25026466

  3. The yin-yang of dendrite morphology: unity of actin and microtubules.

    PubMed

    Georges, Penelope C; Hadzimichalis, Norell M; Sweet, Eric S; Firestein, Bonnie L

    2008-12-01

    Actin and microtubules (MT) are targets of numerous molecular pathways that control neurite outgrowth. To generate a neuronal protrusion, coordinated structural changes of the actin and MT cytoskeletons must occur. Neurite formation occurs when actin filaments (F-actin) are destabilized, filopodia are extended, and MTs invade filopodia. This process results in either axon or dendrite formation. Axonal branching involves interplay between F-actin and MTs, with F-actin and MTs influencing polymerization, stabilization, and maintenance of each other. Our knowledge of the mechanisms regulating development of the axon, however, far eclipses our understanding of dendritic development and branching. The two classes of neurites, while fundamentally similar in their ability to elongate and branch, dramatically differ in growth rate, orientation of polarized MT bundles, and mechanisms that initiate branching. In this review, we focus on how F-actin, MTs, and proteins that link the two cytoskeletons coordinate to specifically initiate dendritic events. PMID:18987787

  4. Whole Cell Model of Actin Diffusion and Reaction based on Single Molecule Speckle Microscopy Measurements

    NASA Astrophysics Data System (ADS)

    McMillen, Laura; Vavylonis, Dimitrios; Vavylonis Group Team

    It is debated whether transport of actin across the cell by diffusion alone is sufficiently fast to account for the rapid reorganization of actin filaments at the leading edge of motile cells. In order to investigate this question, we created a 3D model of the whole cell that includes reaction and diffusion of actin using a particle Monte Carlo method. For the lamellipodium of the simulated cell we use the model by Smith et al. Biophys. J 104:247 (2013), which includes two diffuse pools of actin, one which is slowly diffusing and the other which diffuses more quickly, as well as a pool of filamentous actin undergoing retrograde flow towards the cell center. We adjusted this model to fit a circular geometry around the whole cell. We also consider actin in the cell center which is either diffusing or in stationary filamentous form, representing cortical actin or actin in stress fibers. The local rates of polymerization and the lifetime distributions of polymerized actin were estimated from single molecule speckle microscopy experiments by the group of N. Watanabe. With this model we are able to simulate prior experiments that monitored the redistribution of actin after photoactivation or fluorescence recovery after photobleaching in various parts of the cell. We find that transport by diffusion is sufficient to fit these data, without the need for an active transport mechanism, however significant concentration gradients may develop at steady state.

  5. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators

    PubMed Central

    Dopie, Joseph; Rajakylä, Eeva K.; Joensuu, Merja S.; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K.

    2015-01-01

    ABSTRACT Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes. PMID:26021350

  6. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators.

    PubMed

    Dopie, Joseph; Rajakylä, Eeva K; Joensuu, Merja S; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K

    2015-07-01

    Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes. PMID:26021350

  7. Acrosomal reaction of the Thyone sperm. III. The relationship between actin assembly and water influx during the extension of the acrosomal process.

    PubMed

    Tilney, L G; Inoué, S

    1985-04-01

    In an attempt to investigate the role of water influx in the extension of the acrosomal process of Thyone sperm, we induced the acrosomal reaction in sea water whose osmolarity varied from 50 to 150% of that of sea water. (a) Video sequences of the elongation of the acrosomal processes were made; plots of the length of the acrosomal process as a function of (time)1/2 produced a straight line except at the beginning of elongation and at the end in both hypotonic and hypertonic sea water (up to 1.33 times the osmolarity of sea water), although the rate of elongation was fastest in hypotonic sea water and was progressively slower as the tonicity was raised. (b) Close examination of the video sequences revealed that regardless of the tonicity of the sea water, the morphology of the acrosomal processes were similar. (c) From thin sections of fixed sperm, the amount of actin polymerization that takes place is roughly coupled to the length of the acrosomal process formed so that sperm with short processes only polymerize a portion of the actin that must be present in those sperm. From these facts we conclude that the influx of water and the release of actin monomers from their storage form in the profilactin (so that these monomers can polymerize) are coupled. The exact role of water influx, why it occurs, and whether it could contribute to the extension of the acrosomal process by a hydrostatic pressure mechanism is discussed. PMID:3920226

  8. Synthesis and biological evaluation of arylcinnamide linked combretastatin-A4 hybrids as tubulin polymerization inhibitors and apoptosis inducing agents.

    PubMed

    Kamal, Ahmed; Bajee, Shaik; Lakshma Nayak, Vadithe; Venkata Subba Rao, Ayinampudi; Nagaraju, Burri; Ratna Reddy, Challa; Jeevak Sopanrao, Kapure; Alarifi, Abdullah

    2016-06-15

    A series of new molecules have been designed based on a hybridization approach by combining the arylcinnamide and combretastatin pharmacophores. These were synthesized and evaluated for their cytotoxic activity, effect on inhibition of tubulin polymerization and apoptosis inducing ability. Most of the conjugates exhibited significant cytotoxic activity against some representative human cancer cell lines and two of the conjugates 6i and 6p displayed potent cytotoxicity with GI50 values of 56nM and 31nM respectively against the human breast cancer cell line (MCF-7). SAR studies revealed that 3,4-substitution on the phenyl ring of the cinnamide moiety is beneficial for enhanced cytotoxicity. Moreover, G2/M cell cycle arrest was induced by these conjugates (6i and 6p) apart from tubulin polymerization inhibition (IC50 of 1.97μM and 1.05μM respectively). Further, mitochondrial membrane potential, Annexin V-FITC and caspase-9 activation assays suggested that these conjugates induce cell death by apoptosis. Docking studies revealed that these conjugates interact and bind at the colchicine binding site of the tubulin. PMID:27161282

  9. Band structure of cavity-type hypersonic phononic crystals fabricated by femtosecond laser-induced two-photon polymerization

    NASA Astrophysics Data System (ADS)

    Rakhymzhanov, A. M.; Gueddida, A.; Alonso-Redondo, E.; Utegulov, Z. N.; Perevoznik, D.; Kurselis, K.; Chichkov, B. N.; El Boudouti, E. H.; Djafari-Rouhani, B.; Fytas, G.

    2016-05-01

    The phononic band diagram of a periodic square structure fabricated by femtosecond laser pulse-induced two photon polymerization is recorded by Brillouin light scattering (BLS) at hypersonic (GHz) frequencies and computed by finite element method. The theoretical calculations along the two main symmetry directions quantitatively capture the band diagrams of the air- and liquid-filled structure and moreover represent the BLS intensities. The theory helps identify the observed modes, reveals the origin of the observed bandgaps at the Brillouin zone boundaries, and unravels direction dependent effective medium behavior.

  10. 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.

  11. Cortical actin regulation modulates vascular contractility and compliance in veins

    PubMed Central

    Saphirstein, Robert J; Gao, Yuan Z; Lin, Qian Qian; Morgan, Kathleen G

    2015-01-01

    Abstract The literature on arterial mechanics is extensive, but far less is known about mechanisms controlling mechanical properties of veins. We use here a multi-scale approach to identify subcellular sources of venous stiffness. Portal vein tissue displays a severalfold decrease in passive stiffness compared to aortic tissues. The α-adrenergic agonist phenylephrine (PE) increased tissue stress and stiffness, both attenuated by cytochalasin D (CytoD) and PP2, inhibitors of actin polymerization and Src activity, respectively. We quantify, for the first time, cortical cellular stiffness in freshly isolated contractile vascular smooth muscle cells using magnetic microneedle technology. Cortical stiffness is significantly increased by PE and CytoD inhibits this increase but, surprisingly, PP2 does not. No detectable change in focal adhesion size, measured by immunofluorescence of FAK and zyxin, accompanies the PE-induced changes in cortical stiffness. Probing with phospho-specific antibodies confirmed activation of FAK/Src and ERK pathways and caldesmon phosphorylation. Thus, venous tissue stiffness is regulated both at the level of the smooth muscle cell cortex, via cortical actin polymerization, and by downstream smooth muscle effectors of Src/ERK signalling pathways. These findings identify novel potential molecular targets for the modulation of venous capacitance and venous return in health and disease. Key points Most cardiovascular research focuses on arterial mechanisms of disease, largely ignoring venous mechanisms. Here we examine ex vivo venous stiffness, spanning tissue to molecular levels, using biomechanics and magnetic microneedle technology, and show for the first time that venous stiffness is regulated by a molecular actin switch within the vascular smooth muscle cell in the wall of the vein. This switch connects the contractile apparatus within the cell to adhesion structures and facilitates stiffening of the vessel wall, regulating blood flow return

  12. 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

  13. Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction*

    PubMed Central

    Dalghi, Marianela G.; Fernández, Marisa M.; Ferreira-Gomes, Mariela; Mangialavori, Irene C.; Malchiodi, Emilio L.; Strehler, Emanuel E.; Rossi, Juan Pablo F. C.

    2013-01-01

    As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca2+ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca2+-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca2+-ATPase activity was related to an increase in the apparent affinity for Ca2+ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca2+ homeostasis. PMID:23803603

  14. 2E4 (Kaptin): A novel actin-associated protein from human blood platelets found in lamellipodia and the tips of the stereocilia of the inner ear

    PubMed Central

    Bearer, Elaine L.; Abraham, Manoj T.

    2010-01-01

    Actin – 2E4/kaptin – platelet activation – stereocilia – sensory epithelium Platelet activation, crucial for hemostasis, requires actin polymerization, yet the molecular mechanisms by which localized actin polymerization is mediated are not clear. Here we report the characterization of a novel actin-binding protein. 2E4, originally isolated from human blood platelets and likely to be involved in the actin rearrangements occurring during activation. 2E4 binds to filamentous (F)-actin by F-actin affinity chromatography and is eluted from F-actin affinity columns and extracted from cells with ATP. Its presence at the leading edge of platelets spread on glass and in the lamellipodia of motile fibroblasts suggests a role in actin dynamics. Using localization to obtain clues about function, we stained the sensory epithelium of the embryonic inner car to determine whether 2E4 is at the barbed end of actin filaments during their elongation. Indeed, 2E4 was present at the tips of the elongating stereocilium. 2E4 is novel by DNA sequence and has no identifiable structural motifs. Its unusual amino acid sequence, its ATP-sensitive actin association and its location at sites of actin polymerization in cells suggest 2E4 plays a unique role in the actin rearrangements that accompany platelet activation and stereocilia formation. PMID:10099934

  15. Inducing local T cell apoptosis with anti-Fas-functionalized polymeric coatings fabricated via surface-initiated photopolymerizations

    PubMed Central

    Hume, Patrick S.; Anseth, Kristi S.

    2010-01-01

    Cell encapsulation has long been investigated as a means to achieve transplant immunoprotection as it creates a physical barrier between allograft tissue and host immune cells. Encapsulation with passive barrier materials alone, however, is generally insufficient to protect donor tissue from rejection, because small cytotoxic molecules produced by activated T cells can diffuse readily into the capsule and mediate allograft death. As a means to provide bioactive protection for polymeric encapsulation devices, we investigated a functionalized polymeric coating that mimics a natural T cell regulation pathway. T cells are regulated in vivo via Fas, a well-known ‘death receptor,’ whereby effector cells express Fas ligand and elicit T cell apoptosis upon binding the Fas receptor on a T cell surface. Anti-Fas antibodies are capable of replicating this effect and induce T cell apoptosis in solution. Here, an iniferter-based living radical polymerization was utilized to fabricate surface-anchored polymer chains containing poly(ethylene glycol) with covalently-incorporated pendant anti-Fas antibody. Using this reaction mechanism, we demonstrate fabrication conditions that yield surface densities in excess of 1.5 ng/cm2 of incorporated therapeutic, as detected by ELISA. Additionally, we show that coatings containing anti-Fas antibody induced significant T cell apoptosis, 21±2 % of cells, after 24 hours. Finally, the incorporation of a T cell adhesion ligand, intracellular adhesion molecule-1, along with anti-Fas antibody, yielded even higher levels of apoptosis, 34±1% of T cells, compared to either signal alone. PMID:20138358

  16. Molecular mechanisms underlying the force-dependent regulation of actin-to-ECM linkage at the focal adhesions.

    PubMed

    Hirata, Hiroaki; Sokabe, Masahiro; Lim, Chwee Teck

    2014-01-01

    The linkage of the actin cytoskeleton to extracellular matrices (ECMs) at focal adhesions provides a physical path for cells to exert traction forces on substrates during cellular processes such as migration and morphogenesis. Mechanical strength of the actin-to-ECM linkage increases in response to forces loaded at this linkage. This is achieved by local accumulations of actin filaments, as well as linker proteins connecting actins to integrins, at force-bearing adhesion sites, which leads to an increase in the number of molecular bonds between the actin cytoskeleton- and ECM-bound integrins. Zyxin-dependent actin polymerization and filamin-mediated actin bundling are seemingly involved in the force-dependent actin accumulation. Each actin-integrin link is primarily mediated by the linker protein talin, which is strengthened by another linker protein vinculin connecting the actin filaments to talin in a force-dependent manner. This eliminates slippage between the actin cytoskeleton and talin (clutch mechanism), thus playing a crucial role in creating cell membrane protrusions mediated by actin polymerization. Finally, each integrin-ECM bond is also strengthened when a force is loaded on it, which ensures force transmission at focal adhesions, contributing to stable cell-substrate adhesion in cell migration. PMID:25081617

  17. Changes in F-actin organization induced by hard metal particle exposure in rat pulmonary epithelial cells using laser scanning confocal microscopy.

    PubMed

    Antonini, J M; Starks, K; Roberts, J R; Millecchia, L; Yang, H M; Rao, K M

    2000-01-01

    Chronic inhalation of hard metal (WC-Co) particles causes alveolitis and the eventual development of pulmonary fibrosis. The initial inflammatory response includes a change in the alveolar epithelial cell-capillary barrier, which has been shown to be regulated by the state of assembly and organization of the actin cytoskeletal network. The objective of this study was to evaluate the effect WC-Co particles have on F-actin organization of lung epithelial cells in an in vitro culture system. Rat lung epithelial (L2) cells were exposed to 5, 25, and 100 microg/mL of WC-Co particles, as well as the individual components (Co and WC) of the hard metal mixture particles for 24 h. The effect on F-actin organization was visualized by laser scanning confocal microscopy (LSCM) following Bodipy-Phallacidin staining. Minimal changes in the F-actin microfilaments of L2 cells were observed by LSCM after exposure to WC and WC-Co at 5 and 25 microg/mL, while at 100 microg/mL, there was a noticeable disruption in the uniform distribution of L2 cell F-actin microfilaments. After exposure to Co, a dose-dependent change in the F-actin organization of the L2 cells was observed. Little change in F-actin assembly was observed after treatment with 5 microg/mL of Co (the concentration equivalent to the 5% amount of Co commonly present in 100 microg/mL of the WC-Co sample mixture). However, at 100 microg/mL of Co, the microfilaments aggregated into homogeneous masses within the cells, and a significant loss in the organization of L2 F-actin was observed. These dramatic alterations in F-actin organization seen after exposure to the higher doses of Co were attributed to an increase in L2 cell injury as measured by lactate dehydrogenase and trypan blue exclusion. We conclude the pulmonary response evoked in the lung by inhalation of high levels of WC-Co particles is unlikely due to alterations in the F-actin microfilaments of lung-epithelial cells. PMID:10900403

  18. Competition for actin between two distinct F-actin networks defines a bistable switch for cell polarization.

    PubMed

    Lomakin, Alexis J; Lee, Kun-Chun; Han, Sangyoon J; Bui, Duyen A; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-11-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype after relaxation of the actomyosin cytoskeleton. We find that myosin II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. Under low-contractility regimes, epithelial cells polarize in a front-back manner owing to the emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin II from the front to the back of the cell, where the motor locally 'locks' actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high-contractility-driven cell motion is inefficient. PMID:26414403

  19. Cell culture on polymers prepared by radiation-induced polymerization of various glass-forming monomers.

    PubMed

    Yoshii, F; Kaetsu, I

    1983-04-01

    The growth of cells on polymers prepared by the radiation polymerization of monomethacrylate and dimethacrylate was investigated. Cell growth was affected greatly by such properties of the polymers as water content, wettability, and porosity. Growth was promoted remarkably by rinsing the polymers with warm water at 60-70 degrees C and by irradiation of polymers with an electron beam. Cell growth decreased with increasing oxyethylene length (n) in the polymerized dimethacrylate of same series, CH2C(CH3)CO(OC