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Sample records for actin polymerization state

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

    PubMed Central

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

    2010-01-01

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

  2. Amplification of actin polymerization forces

    PubMed Central

    Dmitrieff, Serge; Nédélec, François

    2016-01-01

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments. PMID:27002174

  3. Amplification of actin polymerization forces.

    PubMed

    Dmitrieff, Serge; Nédélec, François

    2016-03-28

    The actin cytoskeleton drives many essential processes in vivo, using molecular motors and actin assembly as force generators. We discuss here the propagation of forces caused by actin polymerization, highlighting simple configurations where the force developed by the network can exceed the sum of the polymerization forces from all filaments.

  4. Polymerization of actin by positively charged liposomes

    PubMed Central

    1988-01-01

    By cosedimentation, spectrofluorimetry, and electron microscopy, we have established that actin is induced to polymerize at low salt concentrations by positively charged liposomes. This polymerization occurs only at the surface of the liposomes, and thus monomers not in direct contact with the liposome remain monomeric. The integrity of the liposome membrane is necessary to maintain actin in its polymerized state since disruption of the liposome depolymerizes actin. Actin polymerized at the surface of the liposome is organized into two filamentous structures: sheets of parallel filaments in register and a netlike organization. Spectrofluorimetric analysis with the probe N- pyrenyl-iodoacetamide shows that actin is in the F conformation, at least in the environment of the probe. However, actin assembly induced by the liposome is not accompanied by full ATP hydrolysis as observed in vitro upon addition of salts. PMID:3360852

  5. 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

  6. Dynamics of Actin Cable Polymerization in Fission Yeast

    NASA Astrophysics Data System (ADS)

    Wang, Hui; Vavylonis, Dimitrios

    2008-03-01

    In fission yeast, formin for3p nucleates actin filament bundles (cables) at cell tips which contribute to polarized cell growth. Actin cables reach a steady state of dynamic turnover involving for3p-mediated actin polymerization at the barbed ends near the plasma membrane, retrograde flow of polymerized actin toward the cell center, and cable disassembly. Formin for3p associates with actin at the cable tip where it transiently polymerizes actin filaments and subsequently follows the retrograde actin cable flow (Martin and Chang, Curr. Biol. 16, 1161, 2006). Because of the small number of formin nucleators, the actin cable dynamics are subject to spatial and temporal fluctuations. We studied actin cable dynamics with simple analytical models and whole cell computational models which combine deterministic simulation of actin diffusion with stochastic simulation of formin reaction and diffusion. Our model successfully explains a large number of experimental observations, such as density of formin speckles and variance of actin cable density. The model predicts significant spatial gradient of actin and formin molecules in the cytoplasm, powered by the retrograde flow of actin cables.

  7. Actin cytoskeleton: putting a CAP on actin polymerization.

    PubMed

    Stevenson, V A; Theurkauf, W E

    2000-10-05

    Two recent studies have identified a Drosophila homolog of cyclase-associated protein (CAP) as a developmentally important negative regulator of actin polymerization that may also directly mediate signal transduction.

  8. Mechanism of actin polymerization in cellular ATP depletion.

    PubMed

    Atkinson, Simon J; Hosford, Melanie A; Molitoris, Bruce A

    2004-02-13

    Cellular ATP depletion in diverse cell types results in the net conversion of monomeric G-actin to polymeric F-actin and is an important aspect of cellular injury in tissue ischemia. We propose that this conversion results from altering the ratio of ATP-G-actin and ADP-G-actin, causing a net decrease in the concentration of thymosinactin complexes as a consequence of the differential affinity of thymosin beta4 for ATP- and ADP-G-actin. To test this hypothesis we examined the effect of ATP depletion induced by antimycin A and substrate depletion on actin polymerization, the nucleotide state of the monomer pool, and the association of actin monomers with thymosin and profilin in the kidney epithelial cell line LLC-PK1. ATP depletion for 30 min increased F-actin content to 145% of the levels under physiological conditions, accompanied by a corresponding decrease in G-actin content. Cytochalasin D treatment did not reduce F-actin formation during ATP depletion, indicating that it was predominantly not because of barbed end monomer addition. ATP-G-actin levels decreased rapidly during depletion, but there was no change in the concentration of ADP-G-actin monomers. The decrease in ATP-G-actin levels could be accounted for by dissociation of the thymosin-G-actin binary complex, resulting in a rise in the concentration of free thymosin beta4 from 4 to 11 microm. Increased detection of profilin-actin complexes during depletion indicated that profilin may participate in catalyzing nucleotide exchange during depletion. This mechanism provides a biochemical basis for the accumulation of F-actin aggregates in ischemic cells.

  9. 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.

  10. Neurite outgrowth is driven by actin polymerization even in the presence of actin polymerization inhibitors

    PubMed Central

    Chia, Jonathan X.; Efimova, Nadia; Svitkina, Tatyana M.

    2016-01-01

    Actin polymerization is a universal mechanism to drive plasma membrane protrusion in motile cells. One apparent exception to this rule is continuing or even accelerated outgrowth of neuronal processes in the presence of actin polymerization inhibitors. This fact, together with the key role of microtubule dynamics in neurite outgrowth, led to the concept that microtubules directly drive plasma membrane protrusion either in the course of polymerization or by motor-driven sliding. The possibility that unextinguished actin polymerization drives neurite outgrowth in the presence of actin drugs was not explored. We show that cultured hippocampal neurons treated with cytochalasin D or latrunculin B contained dense accumulations of branched actin filaments at ∼50% of neurite tips at all tested drug concentrations (1–10 μM). Actin polymerization is required for neurite outgrowth because only low concentrations of either inhibitor increased the length and/or number of neurites, whereas high concentrations inhibited neurite outgrowth. Of importance, neurites undergoing active elongation invariably contained a bright F-actin patch at the tip, whereas actin-depleted neurites never elongated, even though they still contained dynamic microtubules. Stabilization of microtubules by Taxol treatment did not stop elongation of cytochalasin–treated neurites. We conclude that actin polymerization is indispensable for neurite elongation. PMID:27682586

  11. Intracellular transport based on actin polymerization.

    PubMed

    Khaitlina, S Yu

    2014-09-01

    In addition to the intracellular transport of particles (cargo) along microtubules, there are in the cell two actin-based transport systems. In the actomyosin system the transport is driven by myosin, which moves the cargo along actin microfilaments. This transport requires the hydrolysis of ATP in the myosin molecule motor domain that induces conformational changes in the molecule resulting in the myosin movement along the actin filament. The other actin-based transport system of the cell does not involve myosin or other motor proteins. This system is based on a unidirectional actin polymerization, which depends on ATP hydrolysis in actin polymers and is initiated by proteins bound to the surface of transported particles. Obligatory components of the actin-based transport are proteins of the WASP/Scar family and a complex of Arp2/3 proteins. Moreover, the actin-based systems often contain dynamin and cortactin. It is known that a system of actin filaments formed on the surface of particles, the so-called "comet-like tail", is responsible for intracellular movements of pathogenic bacteria, micropinocytotic vesicles, clathrin-coated vesicles, and phagosomes. This movement is reproduced in a cell-free system containing extract of Xenopus oocytes. The formation of a comet-like structure capable of transporting vesicles from the plasma membrane into the cell depth has been studied in detail by high performance electron microscopy combined with electron tomography. A similar mechanism provides the movement of vesicles containing membrane rafts enriched with sphingolipids and cholesterol, changes in position of the nuclear spindle at meiosis, and other processes. This review will consider current ideas about actin polymerization and its regulation by actin-binding proteins and show how these mechanisms are realized in the intracellular actin-based vesicular transport system.

  12. 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.

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

    PubMed

    Skillman, Kristen M; Ma, Christopher I; Fremont, Daved H; Diraviyam, Karthikeyan; Cooper, John A; Sept, David; Sibley, L David

    2013-01-01

    Previous reports have indicated that parasite actins are short and inherently unstable, despite being required for motility. Here we re-examine the polymerization properties of actin in Toxoplasma gondii, unexpectedly finding that it exhibits isodesmic polymerization in contrast to the conventional nucleation-elongation process of all previously studied actins from both eukaryotes and bacteria. Polymerization kinetics of actin in T. gondii lacks both a lag phase and critical concentration, normally characteristic of actins. Unique among actins, the kinetics of assembly can be fit with a single set of rate constants for all subunit interactions, without need for separate nucleation and elongation rates. This isodesmic model accurately predicts the assembly, disassembly and the size distribution of actin filaments in T. gondii in vitro, providing a mechanistic explanation for actin dynamics in vivo. Our findings expand the repertoire of mechanisms by which actin polymerization is governed and offer clues about the evolution of self-assembling, stabilized protein polymers.

  14. Dynamics of Membranes Driven by Actin Polymerization

    PubMed Central

    Gov, Nir S.; Gopinathan, Ajay

    2006-01-01

    A motile cell, when stimulated, shows a dramatic increase in the activity of its membrane, manifested by the appearance of dynamic membrane structures such as lamellipodia, filopodia, and membrane ruffles. The external stimulus turns on membrane bound activators, like Cdc42 and PIP2, which cause increased branching and polymerization of the actin cytoskeleton in their vicinity leading to a local protrusive force on the membrane. The emergence of the complex membrane structures is a result of the coupling between the dynamics of the membrane, the activators, and the protrusive forces. We present a simple model that treats the dynamics of a membrane under the action of actin polymerization forces that depend on the local density of freely diffusing activators on the membrane. We show that, depending on the spontaneous membrane curvature associated with the activators, the resulting membrane motion can be wavelike, corresponding to membrane ruffling and actin waves, or unstable, indicating the tendency of filopodia to form. Our model also quantitatively explains a variety of related experimental observations and makes several testable predictions. PMID:16239328

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-03-01

    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.

  17. 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

  18. Effects of lithium ions on actin polymerization in the presence of magnesium ions.

    PubMed Central

    Colombo, R; Milzani, A; Contini, P; Dalle Donne, I

    1991-01-01

    In spite of the abundant literature, questions on the biological action of Li+ are far from being answered. In the present paper we demonstrate that modification of the salt composition of the medium for actin polymerization, by gradually replacing K+ with Li+, leads to a dose-related change in the time course of actin assembly. The presence of Li+ influences actin polymerization in vitro by enhancing nucleation and decreasing critical monomer concentration at steady state. Furthermore, Li+ stabilizes actin polymers mainly by lowering the absolute value of the dissociation rate constant (K-) and shifting (towards lower values of actin monomer concentrations) the range of G-actin concentrations in which filament-subunit flux can occur. The influence of Li+ on actin and tubulin polymerization in vitro suggests that cytoskeletal structures could be some of the cytoplasmic targets of this ion. Images Fig. 2. PMID:1848759

  19. Pattern Formation in Polymerizing Actin Flocks: Spirals, Spots, and Waves without Nonlinear Chemistry

    NASA Astrophysics Data System (ADS)

    Le Goff, T.; Liebchen, B.; Marenduzzo, D.

    2016-12-01

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

  20. The polymerization of actin. A study of the nucleation reaction.

    PubMed Central

    Grazi, E; Ferri, A; Cino, S

    1983-01-01

    We compared the properties of the nuclei that accumulate in 7.5 mM-KCl in ATP-G-actin solutions and of the oligomers that are formed by sonication of either G-actin or F-actin. We found that the ability of the above species to prime the polymerization of actin decays with different rates. The nuclei are stable in 7.5 mM-KCl (they decay with a rate constant of 1.5 X 10(-3) s -1 at pH 7.8 at 22 degrees C in the absence of KCl). The oligomers formed by sonication of either G-actin or F-actin, once the sonication is stopped, revert to simpler structures or evolve into F-actin, depending on the KCl concentration in which they are kept. In 10.5 mM-KCl at pH 7.8 at 22 degrees C their priming ability decays with a rate constant of 6 X 10(-3) s -1. We propose that the nuclei that form spontaneously in 7.5 mM-KCl are not directly susceptible to elongation. They must first be converted into activated nuclei, which exist in very low concentration at the steady state. The activated nuclei are directly susceptible to elongation, they have a short life and they decay rapidly into the ground state unless the elongation reaction occurs. Sonication displaces the steady-state concentration in favour of the activated state. PMID:6615456

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

    PubMed Central

    Miyata, H; Hotani, H

    1992-01-01

    Spherical giant liposomes that had encapsulated skeletal-muscle G-actin were made by swelling a dried lipid mixture of dimyristoyl phosphatidylcholine/cardiolipin, 1:1 (wt/wt), in a solution of G-actin/CaCl2 at 0 degree C. Polymerization of the encapsulated G-actin into actin filaments was achieved by raising the temperature to 30 degrees C. We observed the subsequent shape changes of the liposomes by dark-field and differential interference-contrast light microscopy. After approximately 40 min, which was required for completion of actin polymerization, two shapes of liposome were evident: dumbbell and disk. Elongation of the dumbbell-shaped liposomes was concomitant with actin polymerization. Polarization microscopy showed that actin filaments formed thick bundles in the liposomes and that these filaments lay contiguous to the periphery of the liposome. Localization of actin filaments in the liposomes was confirmed by observation of rhodamine phalloidin-conjugated actin filaments by fluorescence microscopy. Both dumbbell- and disk-shaped liposomes were rigid and kept their shapes as far as actin filaments were stabilized. In contrast, liposomes containing bovine serum albumin were fragile, and their shapes continually fluctuated from Brownian motion, indicating that the actin bundles served as mechanical support for the liposome shapes. Images PMID:1454846

  2. The effects of detergents on the polymerization properties of actin.

    PubMed

    Ujfalusi-Pozsonyi, Kinga; Hild, Gábor; Gróf, Pál; Gutay-Tóth, Zsuzsanna; Bacsó, Zsolt; Nyitrai, Miklós

    2010-05-01

    Effects of some detergents-most frequently used in membrane raft studies-on the polymerization properties of actin were examined under in vitro and in vivo conditions, for protein and cellular investigations, respectively. Under in vitro conditions the polymerization rates were measured with pyrene-labeled actin. We found that polymerization rate depended on the detergent concentration by following either biphasic characteristics or only decreasing tendency. The strongest effects were observed at relatively low detergent concentrations. SDS-PAGE electrophoresis and dynamic light-scattering measurements provided further evidences for the size distribution of actin filaments formed under the influence of detergents. Comparing the polymerization rates measured in the presence of different detergents to those obtained with various magnesium and KCl concentrations showed that detergents may influence the actin polymerization at three levels by modifying: (i) the monomer-monomer interaction, (ii) the local ionic strength, and (iii) the affinity of actin for various cations. In vivo studies on NIH 3T3MDR1 cells using TRITC-phalloidin detected fast depolymerization of large extent around the critical micellar concentrations of the detergents. We concluded that microdomain insolubility observed in the presence of detergents is hardly to be the result of the stabilization of the submembrane actin cytoskeleton merely; rather inter-lipid and lipid-protein interactions are also involved within the detergent-resistant membranes.

  3. Polymerization of actin does not regulate desensitization in human basophils

    PubMed Central

    MacGlashan, Donald; Vilariño, Natalia

    2009-01-01

    Previous studies have suggested that maintenance of IgE-mediated signaling results from regulation of the activity of signaling complexes by actin polymerization. This process is also hypothesized to be related to desensitization of basophils and mast cells. Recent studies demonstrated that any signaling process dependent on syk or PI-3K activity cannot be a mechanism of desensitization, and in this context, syk and PI-3K inhibitors were found to inhibit actin polymerization. Inhibitors of actin polymerization were tested for their effect on desensitization of human peripheral blood basophils. Latrunculin A, in particular, removed all resting and stimulated f-actin but did not inhibit desensitization. Cytochalasin D and latrunculin A also did not reverse the loss of syk phosphorylation that accompanies desensitization. These results demonstrate that desensitization mechanisms are not dependent on actin polymerization. In this context, it was also shown that progressive immobilization of FcεRI during aggregation was sensitive to syk or actin polymerization inhibition. Therefore, desensitization is also not dependent on receptor immobilization. These studies demonstrate that desensitization is not the result of two signaling pathways once considered relevant to down-regulation of IgE-mediated signaling. PMID:19150851

  4. Role of Actin Polymerization in Cell Locomotion: Molecules and Models

    PubMed Central

    Bearer, E. L.

    2015-01-01

    Actin filaments forming at the anterior margin of a migrating cell are essential for the formation of filopodia, lamellipodia, and pseudopodia, the “feet” that the cell extends before it. These structures in turn are required for cell locomotion. Yet the molecular nature of the “nucleator” that seeds the polymerization of actin at the leading edge is unknown. Recent advances, including video microscopy of actin dynamics, discovery of proteins unique to the leading edge such as ponticulin, the Mab 2E4 antigen, and ABP 120, and novel experimental models of actin polymerization such as the actin-based movements of intracellular parasites, promise to shed light on this problem in the near future. PMID:8323743

  5. Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadate.

    PubMed

    Ramos, Susana; Manuel, Miguel; Tiago, Teresa; Duarte, Rui; Martins, Jorge; Gutiérrez-Merino, Carlos; Moura, José J G; Aureliano, Manuel

    2006-11-01

    Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68+/-22 microM and 17+/-2 microM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2mM concentration of "metavanadate" solution that contains ortho and metavanadate species, as observed by combining kinetic with (51)V NMR spectroscopy studies. Although at 25 degrees C, decameric vanadate (10 microM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 microM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the "decavanadate" interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems.

  6. Nanotopography-induced symmetry-breaking and guidance of actin polymerization waves and cell migration

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    Many types of eukaryotic cells on a surfaces exhibit reaction diffusion-type waves of actin polymerization. Exposing migrating Dictyostelium discoideum cells to asymmetries at a length scale relevant to actin waves (300 nm) results in guidance of actin polymerization and of the migration of the cells themselves. Quantitative measurements of actin wave speed and direction distributions show that actin polymerization is preferentially localized to nanoridges and directed along the ridges, and that the velocity of guided actin polymerization waves decreases with decreasing ridge spacing. A stochastic growth model of actin polymerization dynamics reproduces these key observations. Supported by NSF-PoLS.

  7. 54Mn2+ as a tracer of the polymerization of actin. Intermediate oligomers condense to give F-actin.

    PubMed Central

    Grazi, E

    1984-01-01

    Mg2+, at submicromolar concentrations, is needed for the nucleation of actin [Maruyama (1981) J. Biol. Chem. 256, 1060-1062]. I show here that Mn2+ fulfils the same function. It binds to oligomers present in the ATP-G-actin solutions with a ratio of 2-3 Mn2+ ions per 100 actin monomers and with an association constant of 0.66 X 10(10) M-1 at pH 8.2 at 25 degrees C. The time course of the binding of Mn2+ to polymerizing actin is not affected by the initial concentration of the protein. Analysis of the distribution of the binding shows that, both in the large oligomeric species and in the polymers, 1 Mn2+ ion is bound for every 14-25 actin monomers, whereas in the smaller oligomeric species 1 Mn2+ ion is bound for every 4 actin monomers. The proposal is made that Mn2+ stabilizes actin nuclei and decreases the concentration of the monomers at the steady state. It is also proposed that, at least in some experimental conditions, the direct condensation of oligomers of intermediate length is an effective mechanism of F-actin formation. PMID:6508731

  8. Effect of temperature on the mechanism of actin polymerization.

    PubMed

    Zimmerle, C T; Frieden, C

    1986-10-21

    The rate of the Mg2+-induced polymerization of rabbit skeletal muscle G-actin has been measured as as function of temperature at pH 8 by using various concentrations of Mg2+, Ca2+, and G-actin. A polymerization mechanism similar to that proposed at this pH [Frieden, C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6513-6517] was found to fit the data from 10 to 35 degrees C. From the kinetic data, no evidence for actin filament fragmentation was found at any temperature. Dimer formation is the most temperature-sensitive step, with the ratio of forward and reverse rate constants changing 4 orders of magnitude from 10 to 35 degrees C. Over this temperature change, all other ratios of forward and reverse rate constants change 7-fold or less, and the critical concentration remains nearly constant. The reversible Mg2+-induced isomerization of G-actin monomer occurs to a greater extent with increasing temperature, measured either by using N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled actin or by simulation of the full-time course of the polymerization reaction. This is partially due to Mg2+ binding becoming tighter, and Ca2+ binding becoming weaker, with increasing temperature. Elongation rates from the filament-pointed end, determined by using actin nucleated by plasma gelsolin, show a temperature dependence slightly larger than that expected for a diffusion-limited reaction.

  9. Stoichiometry of Nck-dependent actin polymerization in living cells

    PubMed Central

    Ditlev, Jonathon A.; Michalski, Paul J.; Huber, Greg; Rivera, Gonzalo M.; Mohler, William A.

    2012-01-01

    Regulation of actin dynamics through the Nck/N-WASp (neural Wiskott–Aldrich syndrome protein)/Arp2/3 pathway is essential for organogenesis, cell invasiveness, and pathogen infection. Although many of the proteins involved in this pathway are known, the detailed mechanism by which it functions remains undetermined. To examine the signaling mechanism, we used a two-pronged strategy involving computational modeling and quantitative experimentation. We developed predictions for Nck-dependent actin polymerization using the Virtual Cell software system. In addition, we used antibody-induced aggregation of membrane-targeted Nck SH3 domains to test these predictions and to determine how the number of molecules in Nck aggregates and the density of aggregates affected localized actin polymerization in living cells. Our results indicate that the density of Nck molecules in aggregates is a critical determinant of actin polymerization. Furthermore, results from both computational simulations and experimentation support a model in which the Nck/N-WASp/Arp2/3 stoichiometry is 4:2:1. These results provide new insight into activities involving localized actin polymerization, including tumor cell invasion, microbial pathogenesis, and T cell activation. PMID:22613834

  10. 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. © 2015 by The American Society of Hematology.

  11. 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

  12. Sound attenuation of polymerizing actin reflects supramolecular structures: viscoelastic properties of actin gels modified by cytochalasin D, profilin and alpha-actinin.

    PubMed Central

    Wagner, O; Schüler, H; Hofmann, P; Langer, D; Dancker, P; Bereiter-Hahn, J

    2001-01-01

    Polymerization and depolymerization of cytoskeletal elements maintaining cytoplasmic stiffness are key factors in the control of cell crawling. Rheometry is a significant tool in determining the mechanical properties of the single elements in vitro. Viscoelasticity of gels formed by these polymers strongly depends on both the length and the associations of the filaments (e.g. entanglements, annealings and side-by-side associations). Ultrasound attenuation is related to viscosity, sound velocity and supramolecular structures in the sample. In combination with a small glass fibre (2 mm x 50 microm), serving as a viscosity sensor, an acoustic microscope was used to measure the elasticity and acoustic attenuation of actin solutions. Changes in acoustic attenuation of polymerizing actin by far exceed the values expected from calculations based on changes in viscosity and sound velocity. During the lag-phase of actin polymerization, attenuation slightly decreases, depending on actin concentration. After the half-maximum viscosity is accomplished and elasticity turns into steady state, attenuation distinctly rises. Changes in ultrasound attenuation depend on actin concentration, and they are modulated by the addition of alpha-actinin, cytochalasin D and profilin. Thus absorption and scattering of sound on the polymerization of actin is related to the packing density of the actin net, entanglements and the length of the actin filaments. Shortening of actin filaments by cytochalasin D was also confirmed by electron micrographs and falling-ball viscosimetry. In addition to viscosity and elasticity, the attenuation of sound proved to be a valuable parameter in characterizing actin polymerization and the supramolecular associations of F-actin. PMID:11311141

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

    PubMed Central

    1996-01-01

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

  14. Accelerated actin filament polymerization from microtubule plus-ends

    PubMed Central

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

    2016-01-01

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

  15. Effects of polymerization and nucleotide identity on the conformational dynamics of the bacterial actin homolog MreB.

    PubMed

    Colavin, Alexandre; Hsin, Jen; Huang, Kerwyn Casey

    2014-03-04

    The assembly of protein filaments drives many cellular processes, from nucleoid segregation, growth, and division in single cells to muscle contraction in animals. In eukaryotes, shape and motility are regulated through cycles of polymerization and depolymerization of actin cytoskeletal networks. In bacteria, the actin homolog MreB forms filaments that coordinate the cell-wall synthesis machinery to regulate rod-shaped growth and contribute to cellular stiffness through unknown mechanisms. Like actin, MreB is an ATPase and requires ATP to polymerize, and polymerization promotes nucleotide hydrolysis. However, it is unclear whether other similarities exist between MreB and actin because the two proteins share low sequence identity and have distinct cellular roles. Here, we use all-atom molecular dynamics simulations to reveal surprising parallels between MreB and actin structural dynamics. We observe that MreB exhibits actin-like polymerization-dependent structural changes, wherein polymerization induces flattening of MreB subunits, which restructures the nucleotide-binding pocket to favor hydrolysis. MreB filaments exhibited nucleotide-dependent intersubunit bending, with hydrolyzed polymers favoring a straighter conformation. We use steered simulations to demonstrate a coupling between intersubunit bending and the degree of flattening of each subunit, suggesting cooperative bending along a filament. Taken together, our results provide molecular-scale insight into the diversity of structural states of MreB and the relationships among polymerization, hydrolysis, and filament properties, which may be applicable to other members of the broad actin family.

  16. Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection*

    PubMed Central

    Gordón-Alonso, Mónica; Rocha-Perugini, Vera; Álvarez, Susana; Ursa, Ángeles; Izquierdo-Useros, Nuria; Martinez-Picado, Javier; Muñoz-Fernández, María A.; Sánchez-Madrid, Francisco

    2013-01-01

    HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4+ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1. PMID:23926103

  17. Actin Polymerization: An Event Regulated by Tyrosine Phosphorylation During Buffalo Sperm Capacitation.

    PubMed

    Naresh, S; Atreja, S K

    2015-12-01

    In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa.

  18. Adhesive F-actin Waves: A Novel Integrin-Mediated Adhesion Complex Coupled to Ventral Actin Polymerization

    PubMed Central

    Case, Lindsay B.; Waterman, Clare M.

    2011-01-01

    At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in “ventral F-actin waves” that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the extracellular matrix downstream of ventral F-actin waves in several mammalian cell lines as well as in primary mouse embryonic fibroblasts. These “adhesive F-actin waves” require a cycle of integrin engagement and disengagement to the extracellular matrix for their formation and propagation, and exhibit morphometry and a hierarchical assembly and disassembly mechanism distinct from other integrin-containing structures. After Arp2/3-mediated actin polymerization, zyxin and VASP are co-recruited to adhesive F-actin waves, followed by paxillin and vinculin, and finally talin and integrin. Adhesive F-actin waves thus represent a previously uncharacterized integrin-based adhesion complex associated with Arp2/3-mediated actin polymerization. PMID:22069459

  19. Propulsion and Comet-Tail Formation by Actin Polymerization

    NASA Astrophysics Data System (ADS)

    Katz, Jonathan; Carlsson, Anders

    2002-03-01

    We have developed and calculated a model of the propulsion of intracellular parasites by actin polymerization. A disc or sphere is uniformly coated with sites at which F-actin is bound and G-actin may intercalate. Intercalation displaces the sphere and polymer along the polymeric axis, in opposite directions, by amounts inversely proportional to their viscous drags. As the sphere is displaced and rotates the polymers are subject to drag in the flow field. We consider two cases: 1. The polymers remain perpendicular to the surface of the sphere, but are free to slide along it; 2. The polymers are hinged at fixed points of attachment. In each case fluctuations in displacement (resulting from stochastic choice of intercalation sites) grow as motion in one direction sweeps the polymers back, so further intercalation augments the directed motion. Numerical simulation shows that the second case leads to runaway (pinwheel) rotation and little translation, but that in the first case the sphere is effectively propelled after a latency time, and a comet tail is formed.

  20. Adhesion controls bacterial actin polymerization-based movement

    PubMed Central

    Soo, Frederick S.; Theriot, Julie A.

    2005-01-01

    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, Ea, over a wide range (8–21 kcal/mol), reminiscent of the “rate compensation effect” of classical catalytic reactions. Average Ea 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

  1. Early nucleation events in the polymerization of actin, probed by time-resolved small-angle x-ray scattering

    PubMed Central

    Oda, Toshiro; Aihara, Tomoki; Wakabayashi, Katsuzo

    2016-01-01

    Nucleators generating new F-actin filaments play important roles in cell activities. Detailed information concerning the events involved in nucleation of actin alone in vitro is fundamental to understanding these processes, but such information has been hard to come by. We addressed the early process of salt-induced polymerization of actin using the time-resolved synchrotron small-angle X-ray scattering (SAXS). Actin molecules in low salt solution maintain a monomeric state by an electrostatic repulsive force between molecules. On mixing with salts, the repulsive force was rapidly screened, causing an immediate formation of many of non-polymerizable dimers. SAXS kinetic analysis revealed that tetramerization gives the highest energetic barrier to further polymerization, and the major nucleation is the formation of helical tetramers. Filaments start to grow rapidly with the formation of pentamers. These findings suggest an acceleration mechanism of actin assembly by a variety of nucleators in cells. PMID:27775032

  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. Actin polymerization does not provide direct mechanical forces for vesicle fission during clathrin-mediated endocytosis.

    PubMed

    Yao, Li-Hua; Rao, Yan; Bang, Chi; Kurilova, Svetlana; Varga, Kelly; Wang, Chun-Yang; Weller, Brandon D; Cho, Wonhwa; Cheng, Jun; Gong, Liang-Wei

    2013-10-02

    Actin polymerization is important for vesicle fission during clathrin-mediated endocytosis (CME), and it has been proposed that actin polymerization may promote vesicle fission during CME by providing direct mechanical forces. However, there is no direct evidence in support of this hypothesis. In the present study, the role of actin polymerization in vesicle fission was tested by analyzing the kinetics of the endocytic tubular membrane neck (the fission-pore) with cell-attached capacitance measurements to detect CME of single vesicles in a millisecond time resolution in mouse chromaffin cells. Inhibition in dynamin GTPase activity increased the fission-pore conductance (Gp), supporting the mechanical role of dynamin GTPase in vesicle fission. However, disruptions in actin polymerization did not alter the fission-pore conductance Gp, thus arguing against the force-generating role of actin polymerization in vesicle fission during CME. Similar to disruptions of actin polymerization, cholesterol depletion results in an increase in the fission-pore duration, indicating a role for cholesterol-dependent membrane reorganization in vesicle fission. Further experiments suggested that actin polymerization and cholesterol might function in vesicle fission during CME in the same pathway. Our results thus support a model in which actin polymerization promotes vesicle fission during CME by inducing cholesterol-dependent membrane reorganization.

  4. Effects of temperature on actin polymerized by Ca2+. Direct evidence of fragmentation.

    PubMed Central

    Grazi, E; Trombetta, G

    1985-01-01

    When the temperature is lowered from 20 to 4 degrees C, the specific viscosity of actin polymerized in the presence of either 4 mM-CaCl2 or 2 mM-MgCl2, but not of actin polymerized in the presence of 90 mM-KCl, is decreased by 50% in the absence of free ATP. Addition of ATP restores the viscosity of the actin polymerized by Mg2+, but not that of actin polymerized by Ca2+, to the original value. The effect of temperature on actin polymerized in the presence of Ca2+ is due to (a) polymer-into-monomer conversion, (b) latero-lateral aggregation of filaments, and (c) fragmentation of the filaments. Fragmentation, as demonstrated by fractional centrifugation and electron microscopy, was the most important of these. Images PMID:4084236

  5. 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

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

    SciTech Connect

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

    2008-08-27

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

  7. Profilin Regulates Apical Actin Polymerization to Control Polarized Pollen Tube Growth.

    PubMed

    Liu, Xiaonan; Qu, Xiaolu; Jiang, Yuxiang; Chang, Ming; Zhang, Ruihui; Wu, Youjun; Fu, Ying; Huang, Shanjin

    2015-12-07

    Pollen tube growth is an essential step during flowering plant reproduction, whose growth depends on a population of dynamic apical actin filaments. Apical actin filaments were thought to be involved in the regulation of vesicle fusion and targeting in the pollen tube. However, the molecular mechanisms that regulate the construction of apical actin structures in the pollen tube remain largely unclear. Here, we identify profilin as an important player in the regulation of actin polymerization at the apical membrane in the pollen tube. Downregulation of profilin decreased the amount of filamentous actin and induced disorganization of apical actin filaments, and reduced tip-directed vesicle transport and accumulation in the pollen tube. Direct visualization of actin dynamics revealed that the elongation of actin filaments originating at the apical membrane decreased in profilin mutant pollen tubes. Mutant profilin that is defective in binding poly-L-proline only partially rescues the actin polymerization defect in profilin mutant pollen tubes, although it fully rescues the actin turnover phenotype. We propose that profilin controls the construction of actin structures at the pollen tube tip, presumably by favoring formin-mediated actin polymerization at the apical membrane.

  8. A requirement for polymerized actin in DNA double-strand break repair.

    PubMed

    Andrin, Christi; McDonald, Darin; Attwood, Kathleen M; Rodrigue, Amélie; Ghosh, Sunita; Mirzayans, Razmik; Masson, Jean-Yves; Dellaire, Graham; Hendzel, Michael J

    2012-07-01

    Nuclear actin is involved in several nuclear processes from chromatin remodeling to transcription. Here we examined the requirement for actin polymerization in DNA double-strand break repair. Double-strand breaks are considered the most dangerous type of DNA lesion. Double-strand break repair consists of a complex set of events that are tightly regulated. Failure at any step can have catastrophic consequences such as genomic instability, oncogenesis or cell death. Many proteins involved in this repair process have been identified and their roles characterized. We discovered that some DNA double-strand break repair factors are capable of associating with polymeric actin in vitro and specifically, that purified Ku70/80 interacts with polymerized actin under these conditions. We find that the disruption of polymeric actin inhibits DNA double strand break repair both in vitro and in vivo. Introduction of nuclear targeted mutant actin that cannot polymerize, or the depolymerization of endogenous actin filaments by the addition of cytochalasin D, alters the retention of Ku80 at sites of DNA damage in live cells. Our results suggest that polymeric actin is required for proper DNA double-strand break repair and may function through the stabilization of the Ku heterodimer at the DNA damage site.

  9. Polymerization kinetics of ADP- and ADP-Pi-actin determined by fluorescence microscopy

    PubMed Central

    Fujiwara, Ikuko; Vavylonis, Dimitrios; Pollard, Thomas D.

    2007-01-01

    We used fluorescence microscopy to determine how polymerization of Mg-ADP-actin depends on the concentration of phosphate. From the dependence of the elongation rate on the actin concentration and direct observations of depolymerizing filaments, we measured the polymerization rate constants of ADP-actin and ADP-Pi-actin. Saturating phosphate reduces the critical concentration for polymerization of Mg-ADP-actin from 1.8 to 0.06 μM almost entirely by reducing the dissociation rate constants at both ends. Saturating phosphate increases the barbed end association rate constant of Mg-ADP-actin 15%, but this value is still threefold less than that of ATP-actin. Thus, ATP hydrolysis without phosphate dissociation must change the conformation of polymerized actin. Analysis of depolymerization experiments in the presence of phosphate suggests that phosphate dissociation near the terminal subunits is much faster than in the interior. Remarkably, 10 times more phosphate is required to slow the depolymerization of the pointed end than the barbed end, suggesting a weak affinity of phosphate near the pointed end. Our observations of single actin filaments provide clues about the origins of the difference in the critical concentration at the two ends of actin filaments in the presence of ATP. PMID:17517656

  10. Prolonged vasoconstriction of resistance arteries involves vascular smooth muscle actin polymerization leading to inward remodelling

    PubMed Central

    Staiculescu, Marius C.; Galiñanes, Edgar L.; Zhao, Guiling; Ulloa, Uri; Jin, Minshan; Beig, Mirza I.; Meininger, Gerald A.; Martinez-Lemus, Luis A.

    2013-01-01

    Aims Inward remodelling of the resistance vasculature is predictive of hypertension and life-threatening cardiovascular events. We hypothesize that the contractile mechanisms responsible for maintaining a reduced diameter over time in response to prolonged stimulation with vasoconstrictor agonists are in part responsible for the initial stages of the remodelling process. Here we investigated the role of vascular smooth muscle (VSM) actin polymerization on agonist-induced vasoconstriction and development of inward remodelling. Methods and results Experiments were conducted in Sprague–Dawley rat resistance vessels isolated from the cremaster and mesentery. Within blood vessels, actin dynamics of VSM were monitored by confocal microscopy after introduction of fluorescent actin monomers through electroporation and by differential centrifugation to probe globular (G) and filamentous (F) actin content. Results indicated that 4 h of agonist-dependent vasoconstriction induced inward remodelling and caused significant actin polymerization, elevating the F-/total-actin ratio. Inhibition of actin polymerization prevented vessels from maintaining prolonged vasoconstriction and developing inward remodelling. Activation of the small GTPases Rho/Rac/Cdc42 also increased the F-/total-actin ratio and induced inward remodelling, while inhibition of Rho kinase or Rac-1 prevented inward remodelling. Disruption of the actin cytoskeleton reversed the inward remodelling caused by prolonged vasoconstriction, but did not affect the passive diameter of freshly isolated vessels. Conclusion These results indicate that vasoconstriction-induced inward remodelling is in part caused by the polymerization of actin within VSM cells through activation of small GTPases. PMID:23417038

  11. Prolonged vasoconstriction of resistance arteries involves vascular smooth muscle actin polymerization leading to inward remodelling.

    PubMed

    Staiculescu, Marius C; Galiñanes, Edgar L; Zhao, Guiling; Ulloa, Uri; Jin, Minshan; Beig, Mirza I; Meininger, Gerald A; Martinez-Lemus, Luis A

    2013-06-01

    Inward remodelling of the resistance vasculature is predictive of hypertension and life-threatening cardiovascular events. We hypothesize that the contractile mechanisms responsible for maintaining a reduced diameter over time in response to prolonged stimulation with vasoconstrictor agonists are in part responsible for the initial stages of the remodelling process. Here we investigated the role of vascular smooth muscle (VSM) actin polymerization on agonist-induced vasoconstriction and development of inward remodelling. Experiments were conducted in Sprague-Dawley rat resistance vessels isolated from the cremaster and mesentery. Within blood vessels, actin dynamics of VSM were monitored by confocal microscopy after introduction of fluorescent actin monomers through electroporation and by differential centrifugation to probe globular (G) and filamentous (F) actin content. Results indicated that 4 h of agonist-dependent vasoconstriction induced inward remodelling and caused significant actin polymerization, elevating the F-/total-actin ratio. Inhibition of actin polymerization prevented vessels from maintaining prolonged vasoconstriction and developing inward remodelling. Activation of the small GTPases Rho/Rac/Cdc42 also increased the F-/total-actin ratio and induced inward remodelling, while inhibition of Rho kinase or Rac-1 prevented inward remodelling. Disruption of the actin cytoskeleton reversed the inward remodelling caused by prolonged vasoconstriction, but did not affect the passive diameter of freshly isolated vessels. These results indicate that vasoconstriction-induced inward remodelling is in part caused by the polymerization of actin within VSM cells through activation of small GTPases.

  12. 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.

  13. Regulation of microRNA expression in vascular smooth muscle by MRTF-A and actin polymerization.

    PubMed

    Alajbegovic, Azra; Turczyńska, Karolina M; Hien, Tran Thi; Cidad, Pilar; Swärd, Karl; Hellstrand, Per; Della Corte, Alessandro; Forte, Amalia; Albinsson, Sebastian

    2017-06-01

    The dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA. In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells. This analysis was performed using cardiovascular disease-focused miRNA arrays in both mouse and human cells. The potential clinical importance of actin polymerization in aortic aneurysm was evaluated using biopsies from mildly dilated human thoracic aorta in patients with stenotic tricuspid or bicuspid aortic valve. By integrating information from multiple qPCR based miRNA arrays we identified a group of five miRNAs (miR-1, miR-22, miR-143, miR-145 and miR-378a) that were sensitive to actin polymerization and MRTF-A overexpression in both mouse and human vascular smooth muscle. With the exception of miR-22, these miRNAs were also relatively enriched in striated and/or smooth muscle containing tissues. Actin polymerization was found to be dramatically reduced in the aorta from patients with mild aortic dilations. This was associated with a decrease in actin/MRTF-regulated miRNAs. In conclusion, the transcriptional co-activator MRTF-A and actin polymerization regulated a subset of miRNAs in vascular smooth muscle. Identification of novel miRNAs regulated by actin/MRTF-A may provide further insight into the mechanisms underlying

  14. Mechanism of Actin-Based Motility: A Dynamic State Diagram

    PubMed Central

    Bernheim-Groswasser, Anne; Prost, Jacques; Sykes, Cécile

    2005-01-01

    Cells move by a dynamical reorganization of their cytoskeleton, orchestrated by a cascade of biochemical reactions directed to the membrane. Designed objects or bacteria can hijack this machinery to undergo actin-based propulsion inside cells or in a cell-like medium. These objects can explore the dynamical regimes of actin-based propulsion, and display different regimes of motion, in a continuous or periodic fashion. We show that bead movement can switch from one regime to the other, by changing the size of the beads or the surface concentration of the protein activating actin polymerization. We experimentally obtain the state diagram of the bead dynamics, in which the transitions between the different regimes can be understood by a theoretical approach based on an elastic force opposing a friction force. Moreover, the experimental characteristics of the movement, such as the velocity and the characteristic times of the periodic movement, are predicted by our theoretical analysis. PMID:15923234

  15. Protein Kinase D Controls Actin Polymerization and Cell Motility through Phosphorylation of Cortactin*

    PubMed Central

    Eiseler, Tim; Hausser, Angelika; De Kimpe, Line; Van Lint, Johan; Pfizenmaier, Klaus

    2010-01-01

    We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein cortactin and the Arp actin polymerization machinery. PKD phosphorylates cortactin in vitro and in vivo at serine 298 thereby generating a 14-3-3 binding motif. In vitro, a phosphorylation-deficient cortactin-S298A protein accelerated VCA-Arp-cortactin-mediated synergistic actin polymerization and showed reduced F-actin binding, indicative of enhanced turnover of nucleation complexes. In vivo, cortactin co-localized with the nucleation promoting factor WAVE2, essential for lamellipodia extension, in the actin polymerization zone in Heregulin-treated MCF-7 cells. Using a 3-dye FRET-based approach we further demonstrate that WAVE2-Arp and cortactin prominently interact at these structures. Accordingly, cortactin-S298A significantly enhanced lamellipodia extension and directed cell migration. Our data thus unravel a previously unrecognized mechanism by which PKD controls cancer cell motility. PMID:20363754

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

    PubMed

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

    2000-12-22

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

  17. Directed actin polymerization is the driving force for epithelial cell-cell adhesion.

    PubMed

    Vasioukhin, V; Bauer, C; Yin, M; Fuchs, E

    2000-01-21

    We have found that epithelial cells engage in a process of cadherin-mediated intercellular adhesion that utilizes calcium and actin polymerization in unexpected ways. Calcium stimulates filopodia, which penetrate and embed into neighboring cells. E-cadherin complexes cluster at filopodia tips, generating a two-rowed zipper of embedded puncta. Opposing cell surfaces are clamped by desmosomes, while vinculin, zyxin, VASP, and Mena are recruited to adhesion zippers by a mechanism that requires alpha-catenin. Actin reorganizes and polymerizes to merge puncta into a single row and seal cell borders. In keratinocytes either null for alpha-catenin or blocked in VASP/Mena function, filopodia embed, but actin reorganization/polymerization is prevented, and membranes cannot seal. Taken together, a dynamic mechanism for intercellular adhesion is unveiled involving calcium-activated filopodia penetration and VASP/Mena-dependent actin reorganization/polymerization.

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

    PubMed

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

    2015-05-18

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

  19. Microtubules as Platforms for Assaying Actin Polymerization In Vivo

    PubMed Central

    Oelkers, J. Margit; Vinzenz, Marlene; Nemethova, Maria; Jacob, Sonja; Lai, Frank P. L.; Block, Jennifer; Szczodrak, Malgorzata; Kerkhoff, Eugen; Backert, Steffen; Schlüter, Kai; Stradal, Theresia E. B.; Small, J. Victor

    2011-01-01

    The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process. PMID:21603613

  20. Tubulin binding protein, CacyBP/SIP, induces actin polymerization and may link actin and tubulin cytoskeletons.

    PubMed

    Schneider, Gabriela; Nieznanski, Krzysztof; Jozwiak, Jolanta; Slomnicki, Lukasz P; Redowicz, Maria J; Filipek, Anna

    2010-11-01

    CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1-179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178-229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.

  1. The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation

    PubMed Central

    Deng, Su; Bothe, Ingo; Baylies, Mary K.

    2015-01-01

    The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease. PMID:26295716

  2. Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement.

    PubMed

    Disanza, A; Steffen, A; Hertzog, M; Frittoli, E; Rottner, K; Scita, G

    2005-05-01

    Dynamic assembly of actin filaments generates the forces supporting cell motility. Several recent biochemical and genetic studies have revealed a plethora of different actin binding proteins whose coordinated activity regulates the turnover of actin filaments, thus controlling a variety of actin-based processes, including cell migration. Additionally, emerging evidence is highlighting a scenario whereby the same basic set of actin regulatory proteins is also the convergent node of different signaling pathways emanating from extracellular stimuli, like those from receptor tyrosine kinases. Here, we will focus on the molecular mechanisms of how the machinery of actin polymerization functions and is regulated, in a signaling-dependent mode, to generate site-directed actin assembly leading to cell motility.

  3. 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

  4. Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils

    SciTech Connect

    Eberle, M.; Traynor-Kaplan, A.E.; Sklar, L.A.; Norgauer, J. )

    1990-10-05

    Stimulation of human neutrophils with the chemoattractant N-formyl peptide caused rapid polymerization of F-actin as detected by right angle light scatter and 7-nitrobenz-2-oxa-1,3-diazol (NBD)-phallacidin staining of F-actin. After labeling neutrophils with 32P, exposure to N-formyl peptide induced a fast decrease of phosphatidylinositol 4-bisphosphate (PIP)2, a slow increase of phosphatidic acid, and a rapid rise of phosphatidylinositol 4-trisphosphate (PIP3). Formation of PIP3 as well as actin polymerization was near maximal at 10 s after stimulation. Half-maximal response and PIP3 formation at early time points resulted from stimulation of neutrophils with 0.01 nM N-formyl peptide or occupation of about 200 receptors. Sustained elevation of PIP3, prolonged right angle light scatter response, and F-actin formation required higher concentrations of N-formyl peptide, occupation of thousands of receptors, and high binding rates. When ligand binding was interrupted with an antagonist, F-actin rapidly depolymerized, transient light scatter response recovered immediately, and elevated (32P)PIP3 levels decayed toward initial values. However, recovery of (32P)PIP2 was not influenced by the antagonist. Based on the parallel time courses and dose response of (32P) PIP3, the right angle light scatter response, and F-actin polymerization, PIP3 is more likely than PIP2 to be involved in modulation of actin polymerization and depolymerization in vivo.

  5. 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.

  6. Cooperative symmetry-breaking by actin polymerization in a model for cell motility.

    PubMed

    van Oudenaarden, A; Theriot, J A

    1999-12-01

    Polymerizing networks of actin filaments are capable of exerting significant mechanical forces, used by eukaryotic cells and their prokaryotic pathogens to change shape or to move. Here we show that small beads coated uniformly with a protein that catalyses actin polymerization are initially surrounded by symmetrical clouds of actin filaments. This symmetry is broken spontaneously, after which the beads undergo directional motion. We have developed a stochastic theory, in which each actin filament is modelled as an elastic brownian ratchet, that quantitatively accounts for the observed emergent symmetry-breaking behaviour. Symmetry-breaking can only occur for polymers that have a significant subunit off-rate, such as the biopolymers actin and tubulin.

  7. 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.

  8. 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.

  9. 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.

  10. Electrostatic balance between global repulsion and local attraction in reentrant polymerization of actin.

    PubMed

    Ohnuki, Jun; Yodogawa, Akira; Takano, Mitsunori

    2017-08-02

    Actin polymerization depends on the salt concentration, exhibiting a reentrant behavior: the polymerization is promoted by increasing KCl concentration up to 100 mM, and then depressed by further increase above 100 mM. We here investigated the physical mechanism of this reentrant behavior by calculating the polymerization energy, defined by the electrostatic energy change upon binding of an actin subunit to a filament, using an implicit solvent model based on the Poisson-Boltzmann (PB) equation. We found that the polymerization energy as a function of the salt concentration shows a non-monotonic reentrant-like behavior, with the minimum at about 100 mM (1:1 salt). By separately examining the salt concentration effect on the global electrostatic repulsion between the like-charged subunits and that on the local electrostatic attraction between the inter-subunit ionic-bond-forming residues in the filament, we clarified that the reentrant behavior is caused by the change in the balance between the two opposing electrostatic interactions. Our study showed that the non-specific nature of counterions, as described in the mean-field theory, plays an important role in the actin polymerization. We also discussed the endothermic nature of the actin polymerization and mentioned the effect of ATP hydrolysis on the G-F transformation, indicating that the electrostatic interaction is widely and intricately involved in the actin dynamics. © 2017 Wiley Periodicals, Inc.

  11. Directional Transport of a Bead Bound to Lamellipodial Surface Is Driven by Actin Polymerization

    PubMed Central

    Nobezawa, Daisuke; Ikeda, Sho-ichi; Wada, Eitaro; Nagano, Takashi

    2017-01-01

    The force driving the retrograde flow of actin cytoskeleton is important in the cellular activities involving cell movement (e.g., growth cone motility in axon guidance, wound healing, or cancer metastasis). However, relative importance of the forces generated by actin polymerization and myosin II in this process remains elusive. We have investigated the retrograde movement of the poly-d-lysine-coated bead attached with the optical trap to the edge of lamellipodium of Swiss 3T3 fibroblasts. The velocity of the attached bead drastically decreased by submicromolar concentration of cytochalasin D, latrunculin A, or jasplakinolide, indicating the involvement of actin turnover. On the other hand, the velocity decreased only slightly in the presence of 50 μM (−)-blebbistatin and Y-27632. Comparative fluorescence microscopy of the distribution of actin filaments and that of myosin II revealed that the inhibition of actin turnover by cytochalasin D, latrunculin A, or jasplakinolide greatly diminished the actin filament network. On the other hand, inhibition of myosin II activity by (−)-blebbistatin or Y-27632 little affected the actin network but diminished stress fibers. Based on these results, we conclude that the actin polymerization/depolymerization plays the major role in the retrograde movement, while the myosin II activity is involved in the maintenance of the dynamic turnover of actin in lamellipodium. PMID:28246604

  12. 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.

  13. 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

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

    PubMed

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

    2015-04-30

    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

  15. 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

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

    PubMed Central

    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

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

    PubMed

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

    2012-04-20

    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.

  18. Mena-GRASP65 interaction couples actin polymerization to Golgi ribbon linking.

    PubMed

    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.

  19. Actin Polymerization Driven Mitochondrial Transport in Mating S. cerevisiae by Fourier Imaging Correlation Spectroscopy

    NASA Astrophysics Data System (ADS)

    Senning, Eric; Marcus, Andrew

    2010-03-01

    The dynamic microenvironment of cells depends on macromolecular architecture, equilibrium fluctuations, and non-equilibrium forces generated by cytoskeletal proteins. We studied the influence of these factors on the motions of mitochondria in mating S. cerevisiae using Fourier imaging correlation spectroscopy (FICS). Our measurements provide detailed, length scale dependent information about the dynamic behavior of mitochondria. We investigate the influence of the actin cytoskeleton on mitochondrial motion, and make comparisons between conditions in which actin network assembly and disassembly is varied, either by using disruptive pharmacological agents, or mutations that alter the rates of actin polymerization. We find that non-equilibrium forces associated with actin polymerization lead to a 1.5-fold enhancement of the long-time mitochondrial diffusion coefficient, and a transient sub-diffusive temporal scaling of the mean-square displacement. Our results lend support to an existing model in which these forces are directly coupled to mitochondrial membrane surfaces.

  20. Analysis of Shape Dynamics and Actin Polymerization of Collectively Migrating Streams of Cells

    NASA Astrophysics Data System (ADS)

    Wang, Chenlu; Parent, Carole A.; Losert, Wolfgang

    We use Princiapl Component Analysis (PCA) to investigate cell-cell coupling during collective cell migration of Dictyostelium discoideun, and explore the underlying mechanisms that regulate the coupling. From PCA of the cell boundary motion obtained from time-lapse images of multicellular streams, we find that cells in streams exhibit more localized anterior protrusions than individually migrating cells. We also find that traveling protrusion waves along cell boundaries connect from cell to cell with high correlation. Further analysis of actin polymerization indicates that actin polymerization is significantly enhanced at the leading edge of cells at cell-cell contacts. The coupling of waves disappears when reducing F-actin polymerization with Latrunculin A.

  1. 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

  2. A human CXCL13-induced actin polymerization assay measured by fluorescence plate reader.

    PubMed

    Alley, Jennifer; Bloom, Laird; Kasaian, Marion; Gao, Huilan; Berstein, Gabriel; Clark, James D; Miao, Wenyan

    2010-02-01

    The chemokine receptor CXCR5 is predominantly expressed on mature B cells and follicular T-helper cells. CXCR5 and its ligand CXCL13 participate in ectopic germinal center formation at the inflammatory sites of multiple immune diseases such as rheumatoid arthritis, multiple sclerosis, and Sjogren's syndrome. Therefore, disrupting CXCL13-induced chemotaxis may be a fruitful approach for developing therapeutics in treating these diseases. Cells undergo cytoskeletal rearrangement prior to chemotaxis, and therefore actin polymerization can be used as a surrogate readout more proximal to chemokine receptor activation than chemotaxis. Conventionally, actin polymerization is measured by fluorescence microscopy or flow cytometry, which are either of low throughput or in need of special instruments. We developed a 96-well actin polymerization assay that can process 1,000 to 1,500 samples a day. This assay uses a standard laboratory fluorescence microplate reader as the detection instrument and was optimized for various experimental conditions such as cell density, actin filament staining reagent, staining buffer, and cell culture conditions. We demonstrate that this actin polymerization assay in 96-well format exhibits the expected pharmacology for human CXCR5 and is suitable as a primary functional assay to screen neutralizing scFv in crude bacterial peri-preps and a secondary assay for small compound collections.

  3. Antagonistic regulation of F-BAR protein assemblies controls actin polymerization during podosome formation.

    PubMed

    Tsujita, Kazuya; Kondo, Akihiro; Kurisu, Shusaku; Hasegawa, Junya; Itoh, Toshiki; Takenawa, Tadaomi

    2013-05-15

    FBP17, an F-BAR domain protein, has emerged as a crucial factor linking the plasma membrane to WASP-mediated actin polymerization. Although it is well established that FBP17 has a powerful self-polymerizing ability that promotes actin nucleation on membranes in vitro, knowledge of inhibitory factors that counteract this activity in vivo is limited. Here, we demonstrate that the assembly of FBP17 on the plasma membranes is antagonized by PSTPIP2, another F-BAR protein implicated in auto-inflammatory disorder. Knockdown of PSTPIP2 in macrophage promotes the assembly of FBP17 as well as subsequent actin nucleation at podosomes, resulting in an enhancement of matrix degradation. This phenotype is rescued by expression of PSTPIP2 in a manner dependent on its F-BAR domain. Time-lapse total internal reflection fluorescence (TIRF) microscopy observations reveal that the self-assembly of FBP17 at the podosomal membrane initiates actin polymerization, whereas the clustering of PSTPIP2 has an opposite effect. Biochemical analysis and live-cell imaging show that PSTPIP2 inhibits actin polymerization by competing with FBP17 for assembly at artificial as well as the plasma membrane. Interestingly, the assembly of FBP17 is dependent on WASP, and its dissociation by WASP inhibition strongly induces a self-organization of PSTPIP2 at podosomes. Thus, our data uncover a previously unappreciated antagonism between different F-BAR domain assemblies that determines the threshold of actin polymerization for the formation of functional podosomes and may explain how the absence of PSTPIP2 causes auto-inflammatory disorder.

  4. Ostrinia furnacalis integrin β1 may be involved in polymerization of actin to modulate spreading and encapsulation of plasmatocytes.

    PubMed

    Xu, Qiuyun; Yu, Xiaoqiang; Liu, Jia; Zhao, Huafu; Wang, Peng; Hu, Shengfeng; Chen, Jingya; Zhang, Wenqing; Hu, Jian

    2012-07-01

    Insect hemocytes must change their state from non-adhesive to adhesive when they spread on or encapsulate foreign invaders. Although integrin β has been reported to play an important role in hemocyte spreading and encapsulation in several insects, how it is involved in the encapsulation process is still unclear. Here we report that integrin β1 of Ostrinia furnacalis (Ofint β1) may modulate plasmatocyte spreading by regulating polymerization of F-actin and further affecting formation of capsules. In the Sephadex A-25 bead-injected larvae, hemocytes forming capsules expressed approximately ten times more Ofint β1 than hemocytes that are free in circulation in hemolymph. When the expression of Ofint β1 in hemocytes was inhibited by dsRNA of Ofint β1 (dsINT), polymerization of F-actin in hemocytes, especially in plasmatocytes, was significantly decreased, spreading of plasmatocytes was inhibited, and encapsulation rate of Sephadex beads was also significantly decreased. Furthermore, hemocytes formed individual aggregates on beads in the dsINT injected larvae, while hemocytes formed complete capsules surrounding the beads in the control larvae; and most of the hemocytes on the beads in the dsINT-injected larvae assumed round forms rather than spread forms. Based on these results, we speculate that integrins on cellular membranes may modulate hemocyte spreading by regulating polymerization of F-actin and further affecting encapsulation of foreign objects.

  5. Actin Filament Polymerization Regulates Gliding Motility by Apicomplexan ParasitesV⃞

    PubMed Central

    Wetzel, D.M.; Håkansson, S.; Hu, K.; Roos, D.; Sibley, L.D.

    2003-01-01

    Host cell entry by Toxoplasma gondii depends critically on actin filaments in the parasite, yet paradoxically, its actin is almost exclusively monomeric. In contrast to the absence of stable filaments in conventional samples, rapid-freeze electron microscopy revealed that actin filaments were formed beneath the plasma membrane of gliding parasites. To investigate the role of actin filaments in motility, we treated parasites with the filament-stabilizing drug jasplakinolide (JAS) and monitored the distribution of actin in live and fixed cells using yellow fluorescent protein (YFP)-actin. JAS treatment caused YFP-actin to redistribute to the apical and posterior ends, where filaments formed a spiral pattern subtending the plasma membrane. Although previous studies have suggested that JAS induces rigor, videomicroscopy demonstrated that JAS treatment increased the rate of parasite gliding by approximately threefold, indicating that filaments are rate limiting for motility. However, JAS also frequently reversed the normal direction of motility, disrupting forward migration and cell entry. Consistent with this alteration, subcortical filaments in JAS-treated parasites occurred in tangled plaques as opposed to the straight, roughly parallel orientation observed in control cells. These studies reveal that precisely controlled polymerization of actin filaments imparts the correct timing, duration, and directionality of gliding motility in the Apicomplexa. PMID:12589042

  6. The polymerization of actin: Structural changes from small-angle neutron scattering

    NASA Astrophysics Data System (ADS)

    Norman, Alexander I.; Ivkov, Robert; Forbes, Jeffrey G.; Greer, Sandra C.

    2005-10-01

    We present a new analysis of small-angle neutron-scattering data from rabbit muscle actin in the course of the polymerization from G-actin to F-actin as a function of temperature. The data, from Ivkov et al. [J. Chem. Phys. 108, 5599 (1998)], were taken in D2O buffer with Ca2+ as the divalent cation on the G-actin in the presence of ATP and with KCl as the initiating salt. The new analysis of the data using modeling and the method of generalized indirect fourier transform (O. Glatter, GIFT, University of Graz, Austria, http://physchem.kfunigraz.ac.at/sm/) provide shapes and dimensions of the G-actin monomer and of the growing actin oligomer in solution as a function of temperature and salt concentration. This analysis indicates that the G-actin monomer, under the conditions given above, is a sphere 50-54Å in diameter as opposed to the oblate ellipsoid seen by x-ray crystallography. The F-actin dimensions are consistent with x-ray crystal structure determinations.

  7. 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

    2016-03-18

    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.

  8. Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes

    PubMed Central

    Bohdanowicz, Michal; Cosío, Gabriela; Backer, Jonathan M.

    2010-01-01

    Actin polymerization drives the extension of pseudopods that trap and engulf phagocytic targets. The polymerized actin subsequently dissociates as the phagocytic vacuole seals and detaches from the plasma membrane. We found that phagosomes formed by engagement of integrins that serve as complement receptors (CR3) undergo secondary waves of actin polymerization, leading to the formation of “comet tails” that propel the vacuoles inside the cells. Actin tail formation was accompanied by and required de novo formation of PI(3,4)P2 and PI(3,4,5)P3 on the phagosomal membrane by class I phosphoinositide 3-kinases (PI3Ks). Although the phosphatidylinositide phosphatase Inpp5B was recruited to nascent phagosomes, it rapidly detached from the membrane after phagosomes sealed. Detachment of Inpp5B required the formation of PI(3)P. Thus, class III PI3K activity was also required for the accumulation of PI(4,5)P2 and PI(3,4,5)P3 and for actin tail formation. These experiments reveal a new PI(3)P-sensitive pathway leading to PI(3,4)P2 and PI(3,4,5)P3 formation and signaling in endomembranes. PMID:21115805

  9. Propagating cell-membrane waves driven by curved activators of actin polymerization.

    PubMed

    Peleg, Barak; Disanza, Andrea; Scita, Giorgio; Gov, Nir

    2011-04-21

    Cells exhibit propagating membrane waves which involve the actin cytoskeleton. One type of such membranal waves are Circular Dorsal Ruffles (CDR) which are related to endocytosis and receptor internalization. Experimentally, CDRs have been associated with membrane bound activators of actin polymerization of concave shape. We present experimental evidence for the localization of convex membrane proteins in these structures, and their insensitivity to inhibition of myosin II contractility in immortalized mouse embryo fibroblasts cell cultures. These observations lead us to propose a theoretical model which explains the formation of these waves due to the interplay between complexes that contain activators of actin polymerization and membrane-bound curved proteins of both types of curvature (concave and convex). Our model predicts that the activity of both types of curved proteins is essential for sustaining propagating waves, which are abolished when one type of curved activator is removed. Within this model waves are initiated when the level of actin polymerization induced by the curved activators is higher than some threshold value, which allows the cell to control CDR formation. We demonstrate that the model can explain many features of CDRs, and give several testable predictions. This work demonstrates the importance of curved membrane proteins in organizing the actin cytoskeleton and cell shape.

  10. Propagating Cell-Membrane Waves Driven by Curved Activators of Actin Polymerization

    PubMed Central

    Peleg, Barak; Disanza, Andrea; Scita, Giorgio; Gov, Nir

    2011-01-01

    Cells exhibit propagating membrane waves which involve the actin cytoskeleton. One type of such membranal waves are Circular Dorsal Ruffles (CDR) which are related to endocytosis and receptor internalization. Experimentally, CDRs have been associated with membrane bound activators of actin polymerization of concave shape. We present experimental evidence for the localization of convex membrane proteins in these structures, and their insensitivity to inhibition of myosin II contractility in immortalized mouse embryo fibroblasts cell cultures. These observations lead us to propose a theoretical model which explains the formation of these waves due to the interplay between complexes that contain activators of actin polymerization and membrane-bound curved proteins of both types of curvature (concave and convex). Our model predicts that the activity of both types of curved proteins is essential for sustaining propagating waves, which are abolished when one type of curved activator is removed. Within this model waves are initiated when the level of actin polymerization induced by the curved activators is higher than some threshold value, which allows the cell to control CDR formation. We demonstrate that the model can explain many features of CDRs, and give several testable predictions. This work demonstrates the importance of curved membrane proteins in organizing the actin cytoskeleton and cell shape. PMID:21533032

  11. The control of actin nucleotide exchange by thymosin beta 4 and profilin. A potential regulatory mechanism for actin polymerization in cells.

    PubMed Central

    Goldschmidt-Clermont, P J; Furman, M I; Wachsstock, D; Safer, D; Nachmias, V T; Pollard, T D

    1992-01-01

    We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover. Images PMID:1330091

  12. Guanine nucleotide-induced polymerization of actin in electropermeabilized human neutrophils

    PubMed Central

    1989-01-01

    The effects of exogenous guanine nucleotides on the polymerization of actin in human neutrophils were tested in an electropermeabilized cell preparation. Close to 40% permeabilization was achieved with a single electric discharge as measured by nucleic acid staining with ethidium bromide or propidium iodide with minimal (less than 2%) release of the cytoplasmic marker lactate dehydrogenase. In addition, electropermeabilized neutrophils retained their capacity to produce superoxide anions and to sustain a polymerization of actin in response to surface-receptor dependent stimuli such as chemotactic factors. Electropermeabilization produced a rapid and transient permeabilization that allowed the entry of guanine nucleotides into the cells. GTP and, to a larger extent, its nonhydrolyzable analog guanosine 5'-O-2- thiotriphosphate (GTP[S]), induced a time- and concentration-dependent polymerization of actin, as determined by increased staining with 7- nitrobenz-2-oxa-1,3-diazolylphallacidin. The effects of the aforementioned guanine nucleotides were antagonized by GDP[S], but were insensitive to pertussis toxin. Cholera toxin potentiated to a small degree the amount of actin polymerization induced by GTP[S]. These results provided direct evidence for the involvement of GTP-binding proteins in the regulation of the organization of the cytoskeleton of neutrophils, an event that is of crucial importance to the performance of the defense-oriented functions of these cells. PMID:2768336

  13. 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

  14. Host actin polymerization tunes the cell division cycle of an intracellular pathogen.

    PubMed

    Siegrist, M Sloan; Aditham, Arjun K; Espaillat, Akbar; Cameron, Todd A; Whiteside, Sarah A; Cava, Felipe; Portnoy, Daniel A; Bertozzi, Carolyn R

    2015-04-28

    Growth and division are two of the most fundamental capabilities of a bacterial cell. While they are well described for model organisms growing in broth culture, very little is known about the cell division cycle of bacteria replicating in more complex environments. Using a D-alanine reporter strategy, we found that intracellular Listeria monocytogenes (Lm) spend a smaller proportion of their cell cycle dividing compared to Lm growing in broth culture. This alteration to the cell division cycle is independent of bacterial doubling time. Instead, polymerization of host-derived actin at the bacterial cell surface extends the non-dividing elongation period and compresses the division period. By decreasing the relative proportion of dividing Lm, actin polymerization biases the population toward cells with the highest propensity to form actin tails. Thus, there is a positive-feedback loop between the Lm cell division cycle and a physical interaction with the host cytoskeleton.

  15. Actin Polymerization Is Essential for Pollen Tube GrowthV⃞

    PubMed Central

    Vidali, Luis; McKenna, Sylvester T.; Hepler, Peter K.

    2001-01-01

    Actin microfilaments, which are prominent in pollen tubes, have been implicated in the growth process; however, their mechanism of action is not well understood. In the present work we have used profilin and DNAse I injections, as well as latrunculin B and cytochalasin D treatments, under quantitatively controlled conditions, to perturb actin microfilament structure and assembly in an attempt to answer this question. We found that a ∼50% increase in the total profilin pool was necessary to half-maximally inhibit pollen tube growth, whereas a ∼100% increase was necessary for half-maximal inhibition of cytoplasmic streaming. DNAse I showed a similar inhibitory activity but with a threefold more pronounced effect on growth than streaming. Latrunculin B, at only 1–4 nM in the growth medium, has a similar proportion of inhibition of growth over streaming to that of profilin. The fact that tip growth is more sensitive than streaming to the inhibitory substances and that there is no correlation between streaming and growth rates suggests that tip growth requires actin assembly in a process independent of cytoplasmic streaming. PMID:11514633

  16. 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

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

    PubMed

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

    2014-10-28

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

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

    PubMed

    Dovas, Athanassios; Gligorijevic, Bojana; Chen, Xiaoming; Entenberg, David; Condeelis, John; Cox, Dianne

    2011-02-14

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

  19. Changes in molar volume and heat capacity of actin upon polymerization.

    PubMed

    Quirion, F; Gicquaud, C

    1993-11-01

    We have used densimetry and microcalorimetry to measure the changes in molar volume and heat capacity of the actin molecule during Mg(2+)-induced polymerization. Molar volume is decreased by 720 ml/mol. This result is in contradiction with previous measurements by Ikkai and Ooi [(1966) Science 152, 1756-1757], and by Swezey and Somero [(1985) Biochemistry 24, 852-860]: both of these groups reported increases in actin volume during polymerization, of 391 ml/mol and 63 ml/mol respectively. We also observed a decrease in heat capacity of about 69.5 kJ.K-1.mol-1 during polymerization. This is in agreement with the concept of conformational fluctuation of proteins proposed by Lumry and Gregory [(1989) J.Mol. Liq. 42, 113-144]whereby either ligand binding by a protein or monomer-monomer interaction decreases the protein's conformational flexibility.

  20. Changes in molar volume and heat capacity of actin upon polymerization.

    PubMed Central

    Quirion, F; Gicquaud, C

    1993-01-01

    We have used densimetry and microcalorimetry to measure the changes in molar volume and heat capacity of the actin molecule during Mg(2+)-induced polymerization. Molar volume is decreased by 720 ml/mol. This result is in contradiction with previous measurements by Ikkai and Ooi [(1966) Science 152, 1756-1757], and by Swezey and Somero [(1985) Biochemistry 24, 852-860]: both of these groups reported increases in actin volume during polymerization, of 391 ml/mol and 63 ml/mol respectively. We also observed a decrease in heat capacity of about 69.5 kJ.K-1.mol-1 during polymerization. This is in agreement with the concept of conformational fluctuation of proteins proposed by Lumry and Gregory [(1989) J.Mol. Liq. 42, 113-144]whereby either ligand binding by a protein or monomer-monomer interaction decreases the protein's conformational flexibility. PMID:8240275

  1. 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

  2. Phosphoinositides and membrane curvature switch the mode of actin polymerization via selective recruitment of toca-1 and Snx9.

    PubMed

    Gallop, Jennifer L; Walrant, Astrid; Cantley, Lewis C; Kirschner, Marc W

    2013-04-30

    The membrane-cytosol interface is the major locus of control of actin polymerization. At this interface, phosphoinositides act as second messengers to recruit membrane-binding proteins. We show that curved membranes, but not flat ones, can use phosphatidylinositol 3-phosphate [PI(3)P] along with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to stimulate actin polymerization. In this case, actin polymerization requires the small GTPase cell cycle division 42 (Cdc42), the nucleation-promoting factor neural Wiskott-Aldrich syndrome protein (N-WASP) and the actin nucleator the actin-related protein (Arp) 2/3 complex. In liposomes containing PI(4,5)P2 as the sole phosphoinositide, actin polymerization requires transducer of Cdc42 activation-1 (toca-1). In the presence of phosphatidylinositol 3-phosphate, polymerization is both more efficient and independent of toca-1. Under these conditions, sorting nexin 9 (Snx9) can be implicated as a specific adaptor that replaces toca-1 to mobilize neural Wiskott-Aldrich syndrome protein and the Arp2/3 complex. This switch in phosphoinositide and adaptor specificity for actin polymerization from membranes has implications for how different types of actin structures are generated at precise times and locations in the cell.

  3. Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation

    PubMed Central

    Roa-Espitia, Ana L.; Hernández-Rendón, Eva R.; Baltiérrez-Hoyos, Rafael; Muñoz-Gotera, Rafaela J.; Cote-Vélez, Antonieta; Jiménez, Irma; González-Márquez, Humberto

    2016-01-01

    ABSTRACT Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by β1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca2+ dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton. PMID:27402964

  4. Inhibition of actin polymerization decreases osteogeneic differentiation of mesenchymal stem cells through p38 MAPK pathway

    PubMed Central

    2013-01-01

    Background Mesenchymal Stem Cells (MSC) are important candidates for therapeutic applications due to their ex vivo proliferation and differentiation capacity. MSC differentiation is controlled by both intrinsic and extrinsic factors and actin cytoskeleton plays a major role in the event. In the current study, we tried to understand the initial molecular mechanisms and pathways that regulate the differentiation of MSC into osteocytes or adipocytes. Results We observed that actin modification was important during differentiation and differentially regulated during adipogenesis and osteogenesis. Initial disruption of actin polymerization reduced further differentiation of MSC into osteocytes and osteogenic differentiation was accompanied by increase in ERK1/2 and p38 MAPK phosphorylation. However, only p38 MAPK phosphorylation was down regulated upon inhibition of actin polymerization which as accompanied by decreased CD49E expression. Conclusion Taken together, our results show that actin modification is a pre-requisite for MSC differentiation into osteocytes and adipocytes and osteogenic differentiation is regulated through p38 MAPK phosphorylation. Thus by modifying their cytoskeleton the differentiation potential of MSC could be controlled which might have important implications for tissue repair and regeneration. PMID:24070328

  5. Focal adhesion kinase is required for actin polymerization and remodeling of the cytoskeleton during sperm capacitation.

    PubMed

    Roa-Espitia, Ana L; Hernández-Rendón, Eva R; Baltiérrez-Hoyos, Rafael; Muñoz-Gotera, Rafaela J; Cote-Vélez, Antonieta; Jiménez, Irma; González-Márquez, Humberto; Hernández-González, Enrique O

    2016-09-15

    Several focal adhesion proteins are known to cooperate with integrins to link the extracellular matrix to the actin cytoskeleton; as a result, many intracellular signaling pathways are activated and several focal adhesion complexes are formed. However, how these proteins function in mammalian spermatozoa remains unknown. We confirm the presence of focal adhesion proteins in guinea pig spermatozoa, and we explore their role during capacitation and the acrosome reaction, and their relationship with the actin cytoskeleton. Our results suggest the presence of a focal adhesion complex formed by β1-integrin, focal adhesion kinase (FAK), paxillin, vinculin, talin, and α-actinin in the acrosomal region. Inhibition of FAK during capacitation affected the protein tyrosine phosphorylation associated with capacitation that occurs within the first few minutes of capacitation, which caused the acrosome reaction to become increasingly Ca(2+) dependent and inhibited the polymerization of actin. The integration of vinculin and talin into the complex, and the activation of FAK and paxillin during capacitation, suggests that the complex assembles at this time. We identify that vinculin and α-actinin increase their interaction with F-actin while it remodels during capacitation, and that during capacitation focal adhesion complexes are structured. FAK contributes to acrosome integrity, likely by regulating the polymerization and the remodeling of the actin cytoskeleton.

  6. Actin polymerization-dependent activation of Cas-L promotes immunological synapse stability

    PubMed Central

    Santos, Luís C; Blair, David A; Kumari, Sudha; Cammer, Michael; Iskratsch, Thomas; Herbin, Olivier; Alexandropoulos, Konstantina; Dustin, Michael L; Sheetz, Michael P

    2016-01-01

    The immunological synapse formed between a T-cell and an antigen-presenting cell is important for cell–cell communication during T-cell-mediated immune responses. Immunological synapse formation begins with stimulation of the T-cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization-dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte-specific Crk-associated substrate (Cas-L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas-L is phosphorylated at TCR microclusters in an actin polymerization-dependent fashion. Furthermore, Cas-L participates in a positive feedback loop leading to amplification of Ca2+ signaling, inside–out integrin activation, and actomyosin contraction. We propose a new role for Cas-L in T-cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin-dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T-cell-mediated immune responses. PMID:27359298

  7. Expression of type I collagen and tenascin C is regulated by actin polymerization through MRTF in dedifferentiated chondrocytes.

    PubMed

    Parreno, Justin; Raju, Sneha; Niaki, Mortah Nabavi; Andrejevic, Katarina; Jiang, Amy; Delve, Elizabeth; Kandel, Rita

    2014-10-16

    This study examined actin regulation of fibroblast matrix genes in dedifferentiated chondrocytes. We demonstrated that dedifferentiated chondrocytes exhibit increased actin polymerization, nuclear localization of myocardin related transcription factor (MRTF), increased type I collagen (col1) and tenascin C (Tnc) gene expression, and decreased Sox9 gene expression. Induction of actin depolymerization by latrunculin treatment or cell rounding, reduced MRTF nuclear localization, repressed col1 and Tnc expression, and increased Sox9 gene expression in dedifferentiated chondrocytes. Treatment of passaged chondrocytes with MRTF inhibitor repressed col1 and Tnc expression, but did not affect Sox9 expression. Our results show that actin polymerization regulates fibroblast matrix gene expression through MRTF in passaged chondrocytes.

  8. Role of actin polymerization in bending of the early heart tube.

    PubMed

    Latacha, Kimberly S; Rémond, Mathieu C; Ramasubramanian, Ashok; Chen, Amy Y; Elson, Elliot L; Taber, Larry A

    2005-08-01

    During cardiac c-looping, the heart transforms from a straight tube into a c-shaped tube, presenting the first evidence of left-right asymmetry in the embryo. C-looping consists of two primary deformation components: ventral bending and dextral rotation. This study examines the role of actin polymerization in bending of the heart tube. Exposure of stage 9-11 chick embryos to low concentrations of the actin polymerization inhibitors cytochalasin D (5 nM-2.0 microM) and latrunculin A (LA; 25 nM-2.0 microM) suppressed looping in a stage- and concentration-dependent manner in both whole embryos and isolated hearts. Local exposure of either the dorsal or ventral sides of isolated hearts to LA also inhibited looping, but less than global exposure, indicating that both sides contribute to the bending mechanism. Taken together, these data suggest that ongoing actin polymerization is required for the bending component of cardiac c-looping, and we speculate that polymerization-driven myocardial cell shape changes cause this deformation. (c) 2005 Wiley-Liss, Inc.

  9. Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization.

    PubMed

    Hubberstey, Andrew V; Mottillo, Emilio P

    2002-04-01

    Many extracellular signals elicit changes in the actin cytoskeleton, which are mediated through an array of signaling proteins and pathways. One family of proteins that plays a role in regulating actin remodeling in response to cellular signals are the cyclase-associated proteins (CAPs). CAPs are highly conserved monomeric actin binding proteins present in a wide range of organisms including yeast, fly, plants, and mammals. The original CAP was isolated as a component of the Saccharomyces cerevisiae adenylyl cyclase complex that serves as an effector of Ras during nutritional signaling. CAPs are multifunctional molecules that contain domains involved in actin binding, adenylyl cyclase association in yeast, SH3 binding, and oligomerization. Genetic studies in yeast have implicated CAPs in vesicle trafficking and endocytosis. CAPs play a developmental role in multicellular organisms, and studies of Drosophila have illuminated the importance of the actin cytoskeleton during eye development and in establishing oocyte polarity. This review will highlight the critical structural and functional domains of CAPs, describe recent studies that have implied important roles for these proteins in linking cell signaling with actin polymerization, and highlight their roles in vesicle trafficking and development.

  10. A WASp-VASP complex regulates actin polymerization at the plasma membrane.

    PubMed

    Castellano, F; Le Clainche, C; Patin, D; Carlier, M F; Chavrier, P

    2001-10-15

    Proteins of the Wiskott-Aldrich syndrome and Ena/VASP families both play essential functions in the regulation of actin dynamics at the cell leading edge. However, possibilities of functional interplay between members of these two families have not been addressed. Here we show that, in hemopoietic cells, recruitment of the C-terminal VCA (Verprolin homology, Cofilin homology, Acidic) domain of WASp at the plasma membrane by a ligand technique using rapamycin as an intermediate is not sufficient to elicit efficient Arp2/3 complex-mediated actin polymerization. Other domains of WASp, in particular the proline-rich domain, are required for the formation of actin-rich structures. An in vitro analysis demonstrates that the proline-rich domain of WASp binds VASP with an affinity of approximately 10(6) M(-1). In addition, WASp and VASP both accumulate in actin-rich phagocytic cups. Finally, in a reconstituted motility medium, VASP enhances actin-based propulsion of WASp-coated beads in a fashion reminiscent of its effect on Listeria movement. We propose that VASP and WASp cooperation is essential in stimulating actin assembly and membrane protrusion at the leading edge.

  11. 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

  12. Inhibition of actin polymerization prevents cocaine-induced changes in spine morphology in the nucleus accumbens.

    PubMed

    Toda, Shigenobu; Shen, Haowei; Kalivas, Peter W

    2010-11-01

    Withdrawal from daily cocaine administration causes an increase in actin cycling and increases spine head diameter in medium spiny neurons from the core of the nucleus accumbens. In order to determine if these two effects of cocaine are mechanistically linked, after 3 weeks of withdrawal from 1 week of daily cocaine treatments, we microinjected latrunculin into the accumbens to inhibit actin polymerization and prevent actin cycling. In cocaine-treated animals, latrunculin-reduced dendritic spine density and decreased the levels of F-actin and PSD-95 in postsynaptic density subfractions. In contrast, latrunculin did not affect spine density or protein levels in saline-treated subjects. Cocaine withdrawn animals show an increase in spine head diameter 45 min after an acute injection of cocaine, and latrunculin abolished the ability of acute cocaine to increase spine head diameter and simultaneously inhibited the sensitized behavioral response. In contrast, latrunculin had no effect in control animals on the acute locomotor response to cocaine. Altogether, these data support previous findings that withdrawal from cocaine is associated with increased actin cycling, and that the increase in actin cycling contributes to cocaine-induced changes in spine morphology of medium spiny neurons in the accumbens core.

  13. Yap/Taz transcriptional activity is essential for vascular regression via Ctgf expression and actin polymerization

    PubMed Central

    Nagasawa-Masuda, Ayumi; Terai, Kenta

    2017-01-01

    Vascular regression is essential to remove redundant vessels during the formation of an efficient vascular network that can transport oxygen and nutrient to every corner of the body. However, no mechanism is known to explain how major blood vessels regress during development. Here we use the dorsal part of the caudal vein plexus (dCVP) in Zebrafish to investigate the mechanism of regression and discover a new role of Yap/Taz in vascular regression. During regression, Yap/Taz is activated by blood circulation in the endothelial cells. This leads to induction of Ctgf and actin polymerization. Interference with Yap/Taz activation decreased Ctgf production, which decreased actin polymerization and vascular regression. These results implicate a novel role of Yap/Taz in vascular regression. PMID:28369143

  14. Polycomb group protein ezh2 controls actin polymerization and cell signaling.

    PubMed

    Su, I-hsin; Dobenecker, Marc-Werner; Dickinson, Ephraim; Oser, Matthew; Basavaraj, Ashwin; Marqueron, Raphael; Viale, Agnes; Reinberg, Danny; Wülfing, Christoph; Tarakhovsky, Alexander

    2005-05-06

    Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. Here, we report the existence of the cytosolic Ezh2-containing methyltransferase complex and tie the function of this complex to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes.

  15. 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.

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

    PubMed

    Cook, Mandy; Bolkan, Bonnie J; Kretzschmar, Doris

    2014-01-01

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

  17. 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

  18. 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

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

    PubMed

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

    2015-11-05

    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.

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

    PubMed

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

    2014-03-01

    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. Together with earlier studies, these results reveal that multiple L. pneumophila effectors target components of the host cytoskeleton. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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

    PubMed Central

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

    2008-01-01

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

  2. The putative pocket protein binding site of Autographa californica nucleopolyhedrovirus BV/ODV-C42 is required for virus-induced nuclear actin polymerization.

    PubMed

    Li, Kun; Wang, Yun; Bai, Huimin; Wang, Qian; Song, Jianhua; Zhou, Yuan; Wu, Chunchen; Chen, Xinwen

    2010-08-01

    Nuclear filamentous actin (F-actin) is essential for nucleocapsid morphogenesis of lepidopteran nucleopolyhedroviruses. Previously, we had demonstrated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-C42 (C42) is involved in nuclear actin polymerization by recruiting P78/83, an AcMNPV orf9-encoded N-WASP homology protein that is capable of activating an actin-related-protein 2/3 (Arp2/3) complex to initiate actin polymerization, to the nucleus. To further investigate the role of C42 in virus-induced actin polymerization, the recombinant bacmid vAc(p78/83nls-gfp), with a c42 knockout, p78/83 tagged with a nuclear localization signal coding sequence, and egfp as a reporter gene under the control of the Pp10 promoter, was constructed and transfected to Sf9 cells. In the nuclei of vAc(p78/83nls-gfp)-transfected cells, polymerized F-actin filaments were absent, whereas other actin polymerization elements (i.e., P78/83, G-actin, and Arp2/3 complex) were present. This in vivo evidence indicated that C42 actively participates in the nuclear actin polymerization process as a key element, besides its role in recruiting P78/83 to the nucleus. In order to collect in vitro evidence for the participation of C42 in actin polymerization, an anti-C42 antibody was used to neutralize the viral nucleocapsid, which is capable of initiating actin polymerization in vitro. Both the kinetics of pyrene-actin polymerization and F-actin-specific staining by phalloidin indicated that anti-C42 can significantly attenuate the efficiency of F-actin formation compared to that with control antibodies. Furthermore, we have identified the putative pocket protein binding sequence (PPBS) on C42 that is essential for C42 to exert its function in nuclear actin polymerization.

  3. Cortactin mediates elevated shear stress-induced mucin hypersecretion via actin polymerization in human airway epithelial cells.

    PubMed

    Liu, Chunyi; Li, Qi; Zhou, Xiangdong; Kolosov, Victor P; Perelman, Juliy M

    2013-12-01

    Mucus hypersecretion is a remarkable pathophysiological manifestation in airway obstructive diseases. These diseases are usually accompanied with elevated shear stress due to bronchoconstriction. Previous studies have reported that shear stress induces mucin5AC (MUC5AC) secretion via actin polymerization in cultured nasal epithelial cells. Furthermore, it is well known that cortactin, an actin binding protein, is a central mediator of actin polymerization. Therefore, we hypothesized that cortactin participates in MUC5AC hypersecretion induced by elevated shear stress via actin polymerization in cultured human airway epithelial cells. Compared with the relevant control groups, Src phosphorylation, cortactin phosphorylation, actin polymerization and MUC5AC secretion were significantly increased after exposure to elevated shear stress. Similar effects were found when pretreating the cells with jasplakinolide, and transfecting with wild-type cortactin. However, these effects were significantly attenuated by pretreating with Src inhibitor, cytochalasin D or transfecting cells with the specific small interfering RNA of cortactin. Collectively, these results suggest that elevated shear stress induces MUC5AC hypersecretion via tyrosine-phosphorylated cortactin-associated actin polymerization in cultured human airway epithelial cells.

  4. 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.

  5. Absence of aryl hydrocarbon receptor alters CDC42 expression and prevents actin polymerization during capacitation.

    PubMed

    Angeles-Floriano, Tania; Roa-Espitia, Ana L; Baltiérrez-Hoyos, Rafael; Cordero-Martínez, Joaquin; Elizondo, Guillermo; Hernández-González, Enrique O

    2016-11-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the toxicity of a variety of environmental chemicals. The absence of this receptor causes serious reproductive complications. Ahr-knockout (Ahr-KO) male mice, for example, are considerably less fertile: Half of the few spermatozoa they produce exhibit morphological alterations, and those with typical morphology may have pathologic modifications. We therefore investigated the consequences of AHR loss on capacitation and the acrosome reaction, and asked if these effects are a consequence of changes to actin polymerization and the expression of Cdc42, which encodes Cell division control protein 42 (CDC42), a RHO protein that controls assembly of the actin cytoskeleton in somatic cells as well as during spermatogenesis. Nearly 50% of spermatozoa produced by Ahr-KO mice had alterations in the flagellum. Ahr-KO spermatozoa were frequently capacitated, but showed reduced spontaneous and progesterone-induced acrosome reaction-which is related to low CDC42 abundance and very limited actin polymerization during capacitation. Thus, the expression of CDC42 might be regulated by AHR, and both proteins are fundamental to the development of normal spermatozoa and the acrosome reaction. Mol. Reprod. Dev. 83: 1015-1026, 2016 © 2016 Wiley Periodicals, Inc.

  6. Actin polymerization as a key innate immune effector mechanism to control Salmonella infection.

    PubMed

    Man, Si Ming; Ekpenyong, Andrew; Tourlomousis, Panagiotis; Achouri, Sarra; Cammarota, Eugenia; Hughes, Katherine; Rizzo, Alessandro; Ng, Gilbert; Wright, John A; Cicuta, Pietro; Guck, Jochen R; Bryant, Clare E

    2014-12-09

    Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1β recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1β production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.

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

    PubMed

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

    2015-05-19

    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. We tracked spatial and temporal fluctuations in the molecular activities of individual moving cells to elucidate how extracellular signal-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 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. 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.

  8. Identification of a Distal GLUT4 Trafficking Event Controlled by Actin Polymerization

    PubMed Central

    Lopez, Jamie A.; Burchfield, James G.; Blair, Duncan H.; Mele, Katarina; Ng, Yvonne; Vallotton, Pascal

    2009-01-01

    The insulin-stimulated trafficking of GLUT4 to the plasma membrane in muscle and fat tissue constitutes a central process in blood glucose homeostasis. The tethering, docking, and fusion of GLUT4 vesicles with the plasma membrane (PM) represent the most distal steps in this pathway and have been recently shown to be key targets of insulin action. However, it remains unclear how insulin influences these processes to promote the insertion of the glucose transporter into the PM. In this study we have identified a previously uncharacterized role for cortical actin in the distal trafficking of GLUT4. Using high-frequency total internal reflection fluorescence microscopy (TIRFM) imaging, we show that insulin increases actin polymerization near the PM and that disruption of this process inhibited GLUT4 exocytosis. Using TIRFM in combination with probes that could distinguish between vesicle transport and fusion, we found that defective actin remodeling was accompanied by normal insulin-regulated accumulation of GLUT4 vesicles close to the PM, but the final exocytotic fusion step was impaired. These data clearly resolve multiple steps of the final stages of GLUT4 trafficking, demonstrating a crucial role for actin in the final stage of this process. PMID:19605560

  9. The effect of toxins on inorganic phosphate release during actin polymerization.

    PubMed

    Vig, Andrea; Ohmacht, Róbert; Jámbor, Eva; Bugyi, Beáta; Nyitrai, Miklós; Hild, Gábor

    2011-05-01

    During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P(i) release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P(i) release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P(i) from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P(i). The effect of both toxins can be sufficiently explained by local and mechanical blockade of P(i) dissociation.

  10. A new method for direct detection of the sites of actin polymerization in intact cells and its application to differentiated vascular smooth muscle.

    PubMed

    Kim, Hak Rim; Leavis, Paul C; Graceffa, Philip; Gallant, Cynthia; Morgan, Kathleen G

    2010-11-01

    Here we report and validate a new method, suitable broadly, for use in differentiated cells and tissues, for the direct visualization of actin polymerization under physiological conditions. We have designed and tested different versions of fluorescently labeled actin, reversibly attached to the protein transduction tag TAT, and have introduced this novel reagent into intact differentiated vascular smooth muscle cells (dVSMCs). A thiol-reactive version of the TAT peptide was synthesized by adding the amino acids glycine and cysteine to its NH(2)-terminus and forming a thionitrobenzoate adduct: viz. TAT-Cys-S-STNB. This peptide reacts readily with G-actin, and the complex is rapidly taken up by freshly enzymatically isolated dVSMC, as indicated by the fluorescence of a FITC tag on the TAT peptide. By comparing different versions of the construct, we determined that the optimal construct for biological applications is a nonfluorescently labeled TAT peptide conjugated to rhodamine-labeled actin. When TAT-Cys-S-STNB-tagged rhodamine actin (TSSAR) was added to live, freshly enzymatically isolated cells, we observed punctae of incorporated actin at the cortex of the cell. The punctae are indistinguishable from those we have previously reported to occur in the same cell type when rhodamine G-actin is added to permeabilized cells. Thus this new method allows the delivery of labeled G-actin into intact cells without disrupting the native state and will allow its further use to study the effect of physiological intracellular Ca(2+) concentration transients and signal transduction on actin dynamics in intact cells.

  11. act up controls actin polymerization to alter cell shape and restrict Hedgehog signaling in the Drosophila eye disc.

    PubMed

    Benlali, A; Draskovic, I; Hazelett, D J; Treisman, J E

    2000-04-28

    Cells in the morphogenetic furrow of the Drosophila eye disc undergo a striking shape change immediately prior to their neuronal differentiation. We have isolated mutations in a novel gene, act up (acu), that is required for this shape change. acu encodes a homolog of yeast cyclase-associated protein, which sequesters monomeric actin; we show that acu is required to prevent actin filament polymerization in the eye disc. In contrast, profilin promotes actin filament polymerization, acting epistatically to acu. However, both acu and profilin are required to prevent premature Hedgehog-induced photoreceptor differentiation ahead of the morphogenetic furrow. These findings suggest that dynamic changes in actin filaments alter cell shape to control the movement of signals that coordinate a wave of differentiation.

  12. Soluble TLT-1 modulates platelet-endothelial cell interactions and actin polymerization

    PubMed Central

    Morales, Jessica; Villa, Karina; Gattis, Jim; Castro, William; Colon, Katiria; Lubkowski, Jacek; Sanabria, Priscilla; Hunter, Robert; Washington, A. Valance

    2010-01-01

    TREM like transcript-1 (TLT-1) is a membrane protein receptor found in α-granules of platelets and megakaryocytes. Upon platelet activation TLT-1 is rapidly brought to the surface of platelets. Recently, we demonstrated that activated platelets release a soluble form of TLT-1 (sTLT-1) that is found in serum but not in the plasma of healthy individuals and can enhance platelet aggregation in vitro. Furthermore, evaluation of patients diagnosed with inflammatory diseases, such as sepsis, show that these patients have significantly elevated levels of sTLT-1 in their blood. Accordingly, mice deficient in TLT-1 are predisposed to bleeding in response to an inflammatory challenge; however the mechanism of TLT-1 function remains unknown. In this investigation we demonstrate an increase in the amount of platelets that adhere to endothelial cell monolayers in the presence of recombinant sTLT-1 (rsTLT-1). Additionally we present evidence that rsTLT-1 increases platelet adherence to glass slides by stimulating actin polymerization in platelets as determined by increased staining of rodamine phalloidin. These results suggest that during inflammation, sTLT-1 may mediate hemostasis by enhancing actin polymerization, resulting in increased platelet aggregation and adherence to the endothelium. PMID:20093931

  13. 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).

  14. Shwachman-Diamond syndrome neutrophils have altered chemoattractant-induced F-actin polymerization and polarization characteristics.

    PubMed

    Orelio, Claudia; Kuijpers, Taco W

    2009-03-01

    Shwachman-Diamond syndrome is a hereditary disorder characterized by pancreatic insufficiency and bone marrow failure. Most Shwachman-Diamond syndrome patients have mutations in the SBDS gene located at chromosome 7 and suffer from recurrent infections, due to neutropenia in combination with impaired neutrophil chemotaxis. Currently, the role of the actin cytoskeleton in Shwachman-Diamond syndrome neutrophils has not been investigated. Therefore, we performed immunofluorescence for SBDS and F-actin on human neutrophilic cells. Additionally, we examined in control neutrophils and cells from genetically defined Shwachman-Diamond syndrome patients F-actin polymerization and cytoskeletal polarization characteristics upon chemoattractant stimulation. These studies showed that SBDS and F-actin co-localize in neutrophilic cells and that F-actin polymerization and depolymerization characteristics are altered in Shwachman-Diamond syndrome neutrophils as compared to control neutrophils in response to both fMLP and C5a. Moreover, F-actin cytoskeletal polarization is delayed in Shwachman-Diamond syndrome neutrophils. Thus, Shwachman-Diamond syndrome neutrophils have aberrant chemoattractant-induced F-actin properties which might contribute to the impaired neutrophil chemotaxis.

  15. Disease causing mutations of troponin alter regulated actin state distributions.

    PubMed

    Chalovich, Joseph M

    2012-12-01

    Striated muscle contraction is regulated primarily through the action of tropomyosin and troponin that are bound to actin. Activation requires Ca(2+) binding to troponin and/or binding of high affinity myosin complexes to actin. Mutations within components of the regulatory complex may lead to familial cardiomyopathies and myopathies. In several cases examined, either physiological or pathological changes in troponin alter the distribution among states of actin-tropomyosin-troponin that differ in their abilities to stimulate myosin ATPase activity. These observations open possibilities for managing disorders of the troponin complex. Furthermore, analyses of mutant forms of troponin give insights into the regulation of striated muscle contraction.

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

    PubMed

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

    2013-11-08

    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.

  17. 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.

  18. Actin polymerization plays a significant role in asbestos-induced inflammasome activation in mesothelial cells in vitro.

    PubMed

    MacPherson, Maximilian; Westbom, Catherine; Kogan, Helen; Shukla, Arti

    2016-12-24

    Asbestos exposure leads to malignant mesothelioma (MM), a deadly neoplasm of mesothelial cells of various locations. Although there is no doubt about the role of asbestos in MM tumorigenesis, mechanisms are still not well explored. Recently, our group demonstrated that asbestos causes inflammasome priming and activation in mesothelial cells, which in part is dependent on oxidative stress. Our current study sheds light on yet another mechanism of inflammasome activation by asbestos. Here we show the role of actin polymerization in asbestos-induced activation of the nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome. Using human mesothelial cells, we first demonstrate that asbestos and carbon nanotubes induced caspase-1 activation and high-mobility group box 1, interleukin 1 beta and interleukin 18 secretion was blocked by Cytochalasin D (Cyto D) an actin polymerization inhibitor. Next, to understand the mechanism, we assessed whether phagocytosis of fibers by mesothelial cells is affected by actin polymerization inhibition. Transmission electron microscopy showed the inhibition of fiber uptake by mesothelial cells in the presence of Cyto D. Furthermore, localization of components of the inflammasome, apoptotic speck-like protein containing a CARD domain (ASC) and NLRP3, to the perinuclear space in mitochondria or endoplasmic reticulum in response to fiber exposure was also interrupted in the presence of Cyto D. Taken together, our studies suggest that actin polymerization plays important roles in inflammasome activation by fibers via regulation of phagocytosis and/or spatial localization of inflammasome components.

  19. Fronts and waves of actin polymerization in a bistability-based mechanism of circular dorsal ruffles

    NASA Astrophysics Data System (ADS)

    Bernitt, Erik; Döbereiner, Hans-Günther; Gov, Nir S.; Yochelis, Arik

    2017-06-01

    During macropinocytosis, cells remodel their morphologies for the uptake of extracellular matter. This endocytotic mechanism relies on the collapse and closure of precursory structures, which are propagating actin-based, ring-shaped vertical undulations at the dorsal (top) cell membrane, a.k.a. circular dorsal ruffles (CDRs). As such, CDRs are essential to a range of vital and pathogenic processes alike. Here we show, based on both experimental data and theoretical analysis, that CDRs are propagating fronts of actin polymerization in a bistable system. The theory relies on a novel mass-conserving reaction-diffusion model, which associates the expansion and contraction of waves to distinct counter-propagating front solutions. Moreover, the model predicts that under a change in parameters (for example, biochemical conditions) CDRs may be pinned and fluctuate near the cell boundary or exhibit complex spiral wave dynamics due to a wave instability. We observe both phenomena also in our experiments indicating the conditions for which macropinocytosis is suppressed.

  20. Differential regulation of cortactin and N-WASP-mediated actin polymerization by missing in metastasis (MIM) protein.

    PubMed

    Lin, Jinxiu; Liu, Jiali; Wang, Ying; Zhu, Jianwei; Zhou, Kang; Smith, Nicole; Zhan, Xi

    2005-03-17

    Missing in metastasis (MIM) gene encodes an actin binding protein that is expressed at low levels in a subset of malignant cell lines. MIM protein tagged by green fluorescent protein (GFP) colocalizes with cortactin, an Arp2/3 complex activator, and interacts directly with the SH3 domain of cortactin. Recombinant full-length MIM promotes markedly cortactin and Arp2/3 complex-mediated actin polymerization in an SH3 dependent manner. In contrast, MIM-CT, a short splicing variant of MIM, binds poorly to cortactin in vitro and is unable to enhance actin polymerization. Full-length MIM binds to G-actin with a similar affinity as N-WASP-VCA, a constitutively active form of N-WASP, and inhibits N-WASP-VCA-mediated actin polymerization as analysed in vitro. The significance of the association of MIM with cortactin and G-actin was evaluated in NIH3T3 cells expressing several MIM constructs. Overexpression of full-length wild-type MIM-GFP inhibited markedly the motility of NIH3T3 cells induced by PDGF and that of human vein umbilical endothelial cells induced by sphingosine 1 phosphate. However, an MIM mutant with deletion of the WH2 domain, which is responsible for G-actin binding, enhanced cell motility. The motility inhibition imposed by MIM was compromised in the cells overexpressing N-WASP. In contrast, deletion of an MIM proline-rich domain, which is required for an optimal binding to cortactin, substantiated the MIM-mediated inhibition of cell motility. These data imply that MIM regulates cell motility by modulating different Arp2/3 activators in a distinguished manner.

  1. State transitions of actin cortices in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Tan, Tzer Han; Keren, Kinneret; Mackintosh, Fred; Schmidt, Christoph; Fakhri, Nikta

    Most animal cells are enveloped by a thin layer of actin cortex which governs the cell mechanics. A functional cortex must be rigid to provide mechanical support while being flexible to allow for rapid restructuring events such as cell division. To satisfy these requirements, the actin cortex is highly dynamic with fast actin turnover and myosin-driven contractility. The regulatory mechanism responsible for the transition between a mechanically stable state and a restructuring state is not well understood. Here, we develop a technique to map the dynamics of reconstituted actin cortices in emulsion droplets using IR fluorescent single-walled carbon nanotubes (SWNTs). By increasing crosslinker concentration, we find that a homogeneous cortex transitions to an intermediate state with broken rotational symmetry and a globally contractile state which further breaks translational symmetry. We apply this new dynamic mapping technique to cortices of live starfish oocytes in various developmental stages. To identify the regulatory mechanism for steady state transitions, we subject the oocytes to actin and myosin disrupting drugs.

  2. F-actin structure destabilization and DNase I binding loop: fluctuations mutational cross-linking and electron microscopy analysis of loop states and effects on F-actin.

    PubMed

    Oztug Durer, Zeynep A; Diraviyam, Karthikeyan; Sept, David; Kudryashov, Dmitri S; Reisler, Emil

    2010-01-22

    The conformational dynamics of filamentous actin (F-actin) is essential for the regulation and functions of cellular actin networks. The main contribution to F-actin dynamics and its multiple conformational states arises from the mobility and flexibility of the DNase I binding loop (D-loop; residues 40-50) on subdomain 2. Therefore, we explored the structural constraints on D-loop plasticity at the F-actin interprotomer space by probing its dynamic interactions with the hydrophobic loop (H-loop), the C-terminus, and the W-loop via mutational disulfide cross-linking. To this end, residues of the D-loop were mutated to cysteines on yeast actin with a C374A background. These mutants showed no major changes in their polymerization and nucleotide exchange properties compared to wild-type actin. Copper-catalyzed disulfide cross-linking was investigated in equimolar copolymers of cysteine mutants from the D-loop with either wild-type (C374) actin or mutant S265C/C374A (on the H-loop) or mutant F169C/C374A (on the W-loop). Remarkably, all tested residues of the D-loop could be cross-linked to residues 374, 265, and 169 by disulfide bonds, demonstrating the plasticity of the interprotomer region. However, each cross-link resulted in different effects on the filament structure, as detected by electron microscopy and light-scattering measurements. Disulfide cross-linking in the longitudinal orientation produced mostly no visible changes in filament morphology, whereas the cross-linking of D-loop residues >45 to the H-loop, in the lateral direction, resulted in filament disruption and the presence of amorphous aggregates on electron microscopy images. A similar aggregation was also observed upon cross-linking the residues of the D-loop (>41) to residue 169. The effects of disulfide cross-links on F-actin stability were only partially accounted for by the simulations of current F-actin models. Thus, our results present evidence for the high level of conformational plasticity in

  3. Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization

    PubMed Central

    Grosheva, Inna; Haka, Abigail S.; Qin, Chunbo; Pierini, Lynda M.; Maxfield, Frederick R.

    2009-01-01

    Objective Interaction of macrophages with aggregated matrix-anchored lipoprotein deposits is an important initial step in atherogenesis. Aggregated lipoproteins require different cellular uptake processes than those used for endocytosis of monomeric lipoproteins. In this study, we tested the hypothesis that engagement of aggregated LDL (agLDL) by macrophages could lead to local increases in free cholesterol levels and that these increases in free cholesterol regulate signals that control cellular actin. Methods and Results AgLDL resides for prolonged periods in surface-connected compartments. While agLDL is still extracellular, we demonstrate that an increase in free cholesterol occurs at sites of contact between agLDL and cells due to hydrolysis of agLDL-derived cholesteryl ester. This increase in free cholesterol causes enhanced actin polymerization around the agLDL. Inhibition of cholesteryl ester hydrolysis results in decreased actin polymerization. Conclusions We describe a novel process that occurs during agLDL-macrophage interactions in which local release of free cholesterol causes local actin polymerization, promoting a pathologic positive feedback loop for increased catabolism of agLDL and eventual foam cell formation. PMID:19556523

  4. A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility

    PubMed Central

    Chang, Fanny S.; Han, Gil-Soo; Carman, George M.; Blumer, Kendall J.

    2005-01-01

    Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17ΔWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility. PMID:16216926

  5. A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility.

    PubMed

    Chang, Fanny S; Han, Gil-Soo; Carman, George M; Blumer, Kendall J

    2005-10-10

    Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

  6. Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.

    PubMed

    Grosheva, Inna; Haka, Abigail S; Qin, Chunbo; Pierini, Lynda M; Maxfield, Frederick R

    2009-10-01

    Interaction of macrophages with aggregated matrix-anchored lipoprotein deposits is an important initial step in atherogenesis. Aggregated lipoproteins require different cellular uptake processes than those used for endocytosis of monomeric lipoproteins. In this study, we tested the hypothesis that engagement of aggregated LDL (agLDL) by macrophages could lead to local increases in free cholesterol levels and that these increases in free cholesterol regulate signals that control cellular actin. AgLDL resides for prolonged periods in surface-connected compartments. Although agLDL is still extracellular, we demonstrate that an increase in free cholesterol occurs at sites of contact between agLDL and cells because of hydrolysis of agLDL-derived cholesteryl ester. This increase in free cholesterol causes enhanced actin polymerization around the agLDL. Inhibition of cholesteryl ester hydrolysis results in decreased actin polymerization. We describe a novel process that occurs during agLDL-macrophage interactions in which local release of free cholesterol causes local actin polymerization, promoting a pathological positive feedback loop for increased catabolism of agLDL and eventual foam cell formation.

  7. Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

    PubMed Central

    Damarla, Mahendra; Hasan, Emile; Boueiz, Adel; Le, Anne; Pae, Hyun Hae; Montouchet, Calypso; Kolb, Todd; Simms, Tiffany; Myers, Allen; Kayyali, Usamah S.; Gaestel, Matthias; Peng, Xinqi; Reddy, Sekhar P.; Damico, Rachel; Hassoun, Paul M.

    2009-01-01

    Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HVT MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling. PMID:19240800

  8. Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

    PubMed Central

    Martín-Villa, José Manuel; Benito-León, María; Martinez-Quiles, Narcisa

    2014-01-01

    Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization. PMID:24675776

  9. Crk adaptors negatively regulate actin polymerization in pedestals formed by enteropathogenic Escherichia coli (EPEC) by binding to Tir effector.

    PubMed

    Nieto-Pelegrin, Elvira; Meiler, Eugenia; Martín-Villa, José Manuel; Benito-León, María; Martinez-Quiles, Narcisa

    2014-03-01

    Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization.

  10. 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

    microfilament system to be restructured in a controlled manner via polymerization, depolymerization, severing, cross-linking, and anchorage. The structure the semicrystalline state of profilin:actin will challenge and validate current models of muscle contraction and cell motility. The methodology and theory under development will be easily extendable to other systems.

  11. 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

    microfilament system to be restructured in a controlled manner via polymerization, depolymerization, severing, cross-linking, and anchorage. The structure the semicrystalline state of profilin:actin will challenge and validate current models of muscle contraction and cell motility. The methodology and theory under development will be easily extendable to other systems.

  12. p21‐Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization

    PubMed Central

    Zhang, Wenwu; Huang, Youliang

    2016-01-01

    Key points In airway smooth muscle, tension development caused by a contractile stimulus requires phosphorylation of the 20 kDa myosin light chain (MLC), which activates crossbridge cycling and the polymerization of a pool of submembraneous actin.The p21‐activated kinases (Paks) can regulate the contractility of smooth muscle and non‐muscle cells, and there is evidence that this occurs through the regulation of MLC phosphorylation.We show that Pak has no effect on MLC phosphorylation during the contraction of airway smooth muscle, and that it regulates contraction by mediating actin polymerization.We find that Pak phosphorylates the adhesion junction protein, paxillin, on Ser273, which promotes the formation of a signalling complex that activates the small GTPase, cdc42, and the actin polymerization catalyst, neuronal Wiskott–Aldrich syndrome protein (N‐WASP).These studies demonstrate a novel role for Pak in regulating the contractility of smooth muscle by regulating actin polymerization. Abstract The p21‐activated kinases (Pak) can regulate contractility in smooth muscle and other cell and tissue types, but the mechanisms by which Paks regulate cell contractility are unclear. In airway smooth muscle, stimulus‐induced contraction requires phosphorylation of the 20 kDa light chain of myosin, which activates crossbridge cycling, as well as the polymerization of a small pool of actin. The role of Pak in airway smooth muscle contraction was evaluated by inhibiting acetylcholine (ACh)‐induced Pak activation through the expression of a kinase inactive mutant, Pak1 K299R, or by treating tissues with the Pak inhibitor, IPA3. Pak inhibition suppressed actin polymerization and contraction in response to ACh, but it did not affect myosin light chain phosphorylation. Pak activation induced paxillin phosphorylation on Ser273; the paxillin mutant, paxillin S273A, inhibited paxillin Ser273 phosphorylation and inhibited actin polymerization and contraction

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

    PubMed

    Vicker, M G

    2000-04-14

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

  14. Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization.

    PubMed

    Wakamatsu, Kairo; Fujimoto, Masaru; Nakazono, Mikio; Arimura, Shin-ichi; Tsutsumi, Nobuhiro

    2010-10-01

    Mitochondria in plant cells undergo fusion and fission frequently. Although the mechanisms and proteins of mitochondrial fusion are well known in yeast and mammalian cells, they remain poorly understood in plant cells. To clarify the physiological requirements for plant mitochondrial fusion, we investigated the fusion frequency of mitochondria in tobacco cultured cells using the photoconvertible fluorescent protein Kaede and some physiological inhibitors. The latter included two uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of mitochondrial ATP synthase, oligomycin, and an actin polymerization inhibitor, latrunculin B (Lat B). The frequency of mitochondrial fusion was clearly reduced by DNP, CCCP and oligomycin, but not by Lat B, although Lat B severely inhibited mitochondrial movement. Moreover, DNP, CCCP and oligomycin evidently lowered the cellular ATP levels. These results indicate that plant mitochondrial fusion depends on the cellular ATP level, but not on actin polymerization.

  15. Wound Closure in the Lamellipodia of Single Cells: Mediation by Actin Polymerization in the Absence of an Actomyosin Purse String

    PubMed Central

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

    2002-01-01

    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

  16. mTORC2 controls actin polymerization required for consolidation of long-term memory

    PubMed Central

    Huang, Wei; Zhu, Ping Jun; Zhang, Shixing; Zhou, Hongyi; Stoica, Loredana; Galiano, Mauricio; Krnjević, Krešimir; Roman, Gregg; Costa-Mattioli, Mauro

    2013-01-01

    A major goal of biomedical research has been the identification of molecular mechanisms that can enhance memory. Here we report a novel signaling pathway that regulates the conversion from short- to long-term memory. The mTOR complex 2 (mTORC2), which contains the key regulatory protein Rictor (Rapamycin-Insensitive Companion of mTOR), was discovered only recently, and little is known about its physiological role. We show that conditional deletion of rictor in the postnatal murine forebrain greatly reduces mTORC2 activity and selectively impairs both long-term memory (LTM) and the late (but not the early) phase of hippocampal long-term potentiation (LTP). Actin polymerization is reduced in the hippocampus of mTORC2-deficient mice and its restoration rescues both L-LTP and LTM. More importantly, a compound that selectively promotes mTORC2 activity converts early-LTP into late-LTP and enhances LTM. These findings indicate that mTORC2 could be a novel therapeutic target for the treatment of cognitive dysfunction. PMID:23455608

  17. 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.

  18. The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis.

    PubMed

    Hanna, Samer J; McCoy-Simandle, Kessler; Miskolci, Veronika; Guo, Peng; Cammer, Michael; Hodgson, Louis; Cox, Dianne

    2017-08-17

    Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed. Here we investigated the signaling pathways involved in TNT formation by macrophages using multiple imaging techniques including super-resolution microscopy (3D-SIM) and live-cell imaging including the use of FRET-based Rho GTPase biosensors. We found that formation of TNTs required the activity and differential localization of Cdc42 and Rac1. The downstream Rho GTPase effectors mediating actin polymerization through Arp2/3 nucleation, Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous 2 (WAVE2) proteins are also important, and both pathways act together during TNT biogenesis. Finally, TNT function as measured by transfer of cellular material between cells was reduced following depletion of a single factor demonstrating the importance of these factors in TNTs. Given that the characterization of TNT formation is still unclear in the field; this study provides new insights and would enhance the understanding of TNT formation towards investigating new markers.

  19. Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization.

    PubMed

    Akhter, Anwari; Caution, Kyle; Abu Khweek, Arwa; Tazi, Mia; Abdulrahman, Basant A; Abdelaziz, Dalia H A; Voss, Oliver H; Doseff, Andrea I; Hassan, Hoda; Azad, Abul K; Schlesinger, Larry S; Wewers, Mark D; Gavrilin, Mikhail A; Amer, Amal O

    2012-07-27

    Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.

  20. Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization

    PubMed Central

    Akhter, Anwari; Caution, Kyle; Khweek, Arwa Abu; Tazi, Mia; Abdulrahman, Basant A.; Abdelaziz, Dalia H.A.; Voss, Oliver H.; Doseff, Andrea I.; Hassan, Hoda; Azad, Abul K.; Schlesinger, Larry S.; Wewers, Mark D.; Gavrilin, Mikhail A.; Amer, Amal O.

    2012-01-01

    Summary Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins. Yet, its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and 5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila- vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing non-pathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo. PMID:22658523

  1. The Myosin IXb Motor Activity Targets the Myosin IXb RhoGAP Domain as Cargo to Sites of Actin Polymerization

    PubMed Central

    van den Boom, Frank; Düssmann, Heiko; Uhlenbrock, Katharina; Abouhamed, Marouan

    2007-01-01

    Myosin IXb (Myo9b) is a single-headed processive myosin that exhibits Rho GTPase-activating protein (RhoGAP) activity in its tail region. Using live cell imaging, we determined that Myo9b is recruited to extending lamellipodia, ruffles, and filopodia, the regions of active actin polymerization. A functional motor domain was both necessary and sufficient for targeting Myo9b to these regions. The head domains of class IX myosins comprise a large insertion in loop2. Deletion of the large Myo9b head loop 2 insertion abrogated the enrichment in extending lamellipodia and ruffles, but enhanced significantly the enrichment at the tips of filopodia and retraction fibers. The enrichment in the tips of filopodia and retraction fibers depended on four lysine residues C-terminal to the loop 2 insertion and the tail region. Fluorescence recovery after photobleaching and photoactivation experiments in lamellipodia revealed that the dynamics of Myo9b was comparable to that of actin. The exchange rates depended on the Myo9b motor region and motor activity, and they were also dependent on the turnover of F-actin. These results demonstrate that Myo9b functions as a motorized RhoGAP molecule in regions of actin polymerization and identify Myo9b head sequences important for in vivo motor properties. PMID:17314409

  2. 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

  3. Force-producing ADP state of myosin bound to actin.

    PubMed

    Wulf, Sarah F; Ropars, Virginie; Fujita-Becker, Setsuko; Oster, Marco; Hofhaus, Goetz; Trabuco, Leonardo G; Pylypenko, Olena; Sweeney, H Lee; Houdusse, Anne M; Schröder, Rasmus R

    2016-03-29

    Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V.

  4. A role for actin polymerization in persistent pulmonary hypertension of the newborn.

    PubMed

    Fediuk, Jena; Dakshinamurti, Shyamala

    2015-03-01

    Persistent pulmonary hypertension of the newborn (PPHN) is defined as the failure of normal pulmonary vascular relaxation at birth. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial myocytes, resulting in elevation of smooth muscle α-actin and γ-actin content in elastic and resistance pulmonary arteries in PPHN compared with age-matched controls. This review examines the original histological characterization of PPHN with attention to cytoskeletal structural remodeling and actin isoform abundance, reviews the existing evidence for understanding the biophysical and biochemical forces at play during neonatal circulatory transition, and specifically addresses the role of the cortical actin architecture, primarily identified as γ-actin, in the transduction of mechanical force in the hypoxic PPHN pulmonary circuit.

  5. Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization*

    PubMed Central

    Hien, Tran Thi; Turczyńska, Karolina M.; Dahan, Diana; Ekman, Mari; Grossi, Mario; Sjögren, Johan; Nilsson, Johan; Braun, Thomas; Boettger, Thomas; Garcia-Vaz, Eliana; Stenkula, Karin; Swärd, Karl; Gomez, Maria F.; Albinsson, Sebastian

    2016-01-01

    Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility. PMID:26683376

  6. 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

  7. The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes.

    PubMed

    Vicente-Manzanares, Miguel; Rey, Mercedes; Pérez-Martínez, Manuel; Yáñez-Mó, María; Sancho, David; Cabrero, José Román; Barreiro, Olga; de la Fuente, Hortensia; Itoh, Kazuyuki; Sánchez-Madrid, Francisco

    2003-07-15

    Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.

  8. 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.

  9. 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.

  10. Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation

    PubMed Central

    1995-01-01

    The phosphorylation of regulatory myosin light chains by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B. and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. USA. 87:16-20). We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2- to 2.5-fold within 5 min and remains elevated for at least 60 min. The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation. Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and

  11. Mechano-chemical energy transduction in biological systems. The effect of mechanical stimulation on the polymerization of actin: a kinetic study.

    PubMed Central

    Ferri, A; Grazi, E

    1982-01-01

    Mechanical stimulation (forced circulation in narrow tubing) accelerates as much as 10-fold the rate of polymerization of actin. The increase in the rate is proportional to the intensity of the stimulation for flow rates between 0 and 3 cm/s. This supports the hypothesis that a statistical factor (the orientation of the flowing particles) is influenced by the flow. Comparison of the kinetics of the polymerization of resting and of mechanically stimulated actin solutions shows that both the nucleation and the elongation steps are accelerated. It is thus concluded that flow orients not only the oligomeric structures but also the actin monomers. The elongation reaction, also in the flow-stimulated samples, occurs always by the addition of ATP--G-actin (or ATP-containing oligomers) and not by the fusion of ADP-containing oligomeric structures. PMID:7138502

  12. Correlation between ECM guidance and actin polymerization on osteogenic differentiation of human adipose-derived stem cells.

    PubMed

    Keller, Vivian; Deiwick, Andrea; Pflaum, Michael; Schlie-Wolter, Sabrina

    2016-10-01

    The correlation between extracellular matrix (ECM) components, cell shape, and stem cell guidance can shed light in understanding and mimicking the functionality of stem cell niches for various applications. This interplay on osteogenic guidance of human adipose-derived stem cells (hASCs) was focus of this study. Proliferation and osteogenic markers like alkaline phosphatase activity and calcium mineralization were slightly increased by the ECM components laminin (LA), collagen I (COL), and fibronectin (FIB); with control medium no differentiation occurred. ECM guided differentiation was rather dependent on osterix than on Runx2 pathway. FIB significantly enhanced cell elongation even in presence of actin polymerization blockers cytochalasin D (CytoD) and ROCK inhibitor Y-27632, which generally caused more rounded cells. Except for the COL surface, both inhibitors increased the extent of osterix, while the Runx2 pathway was more sensitive to the culture condition. Both inhibitors did not affect hASC proliferation. CytoD enabled osteogenic differentiation independently from the ECM, while it was rather blocked via Y-27632 treatment; on FIB the general highest extent of differentiation occurred. Taken together, the ECM effect on hASCs occurs indirectly and selectively via a dominant role of FIB: it sustains osteogenic differentiation in case of a tension-dependent control of actin polymerization.

  13. 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.

  14. Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts

    PubMed Central

    Kaipa, Balasankara Reddy; Shao, Huanjie; Schäfer, Gritt; Trinkewitz, Tatjana; Groth, Verena; Liu, Jianqi; Beck, Lothar; Bogdan, Sven; Abmayr, Susan M.; Önel, Susanne-Filiz

    2013-01-01

    Summary The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell–cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar– or Vrp1–WASp-dependent Arp2/3 activation. PMID:22992459

  15. Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts.

    PubMed

    Kaipa, Balasankara Reddy; Shao, Huanjie; Schäfer, Gritt; Trinkewitz, Tatjana; Groth, Verena; Liu, Jianqi; Beck, Lothar; Bogdan, Sven; Abmayr, Susan M; Önel, Susanne-Filiz

    2013-01-01

    The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

  16. Structured Functional Domains of Myelin Basic Protein: Cross Talk between Actin Polymerization and Ca2+-Dependent Calmodulin Interaction

    PubMed Central

    Bamm, Vladimir V.; De Avila, Miguel; Smith, Graham S.T.; Ahmed, Mumdooh A.M.; Harauz, George

    2011-01-01

    The 18.5-kDa myelin basic protein (MBP), the most abundant isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the sheath. Solution NMR spectroscopy and a hydrophobic moment analysis of MBP's amino-acid sequence have previously revealed three regions with high propensity to form strongly amphipathic α-helices. These regions, located in the central, N- and C-terminal parts of the protein, have been shown to play a role in the interactions of MBP with cytoskeletal proteins, Src homology 3-domain-containing proteins, Ca2+-activated calmodulin (Ca2+-CaM), and myelin-mimetic membrane bilayers. Here, we have further characterized the structure-function relationship of these three domains. We constructed three recombinant peptides derived from the 18.5-kDa murine MBP: (A22–K56), (S72–S107), and (S133–S159) (which are denoted α1, α2, and α3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, isothermal titration calorimetry, transmission electron microscopy, fluorimetry, and solution NMR spectroscopy and chemical shift index analysis) to characterize the interactions of these peptides with actin and Ca2+-CaM. Our results show that all three peptides can adopt α-helical structure inherently even in aqueous solution. Both α1- and α3-peptides showed strong binding with Ca2+-CaM, and both adopted an α-helical conformation upon interaction, but the binding of the α3-peptide appeared to be more dynamic. Only the α1-peptide exhibited actin polymerization and bundling activity, and the addition of Ca2+-CaM resulted in depolymerization of actin that had been polymerized by α1. The results of this study proved that there is an N-terminal binding domain in MBP for Ca2+-CaM (in addition to the primary site located in the C-terminus), and that it is sufficient for CaM-induced actin depolymerization. These three domains of MBP represent molecular recognition

  17. Pasteurella multocida toxin (PMT) activates RhoGTPases, induces actin polymerization and inhibits migration of human dendritic cells, but does not influence macropinocytosis.

    PubMed

    Blöcker, Dagmar; Berod, Luciana; Fluhr, Joachim W; Orth, Joachim; Idzko, Marco; Aktories, Klaus; Norgauer, Johannes

    2006-03-01

    Dendritic cells (DCs) are considered as one of the principal initiators of immune responses. In their immature state, they migrate into peripheral tissue in order to uptake antigen and to patrol for danger signals. Upon maturation, they acquire the ability to migrate to the lymph nodes and present the captured antigens to T cells in order to direct the development of specific immune responses. There is evidence that microbial compounds interfere with proper functions of DCs in order to block innate and specific immunity. Here we characterized the influence of Pasteurella multocida toxin (PMT) on monocyte-derived DCs. Using pull-down assays with recombinant rhotekin or p21-activated kinase, we demonstrated the activation of RhoGTPases by PMT in DCs. Moreover, PMT induced changes in DC morphology and actin polymerization, impaired chemotaxin-induced actin re-organization and inhibited their migration response. However, macropinocytosis was not influenced by PMT. In summary, these data indicate that PMT inhibits proper function of the motility machinery in DCs, which might limit the development of adaptive immune surveillance during infection with Pasteurella multocida.

  18. Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization.

    PubMed

    Hien, Tran Thi; Turczyńska, Karolina M; Dahan, Diana; Ekman, Mari; Grossi, Mario; Sjögren, Johan; Nilsson, Johan; Braun, Thomas; Boettger, Thomas; Garcia-Vaz, Eliana; Stenkula, Karin; Swärd, Karl; Gomez, Maria F; Albinsson, Sebastian

    2016-02-12

    Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

    PubMed Central

    Rao, Megha Vaman; Zaidel-Bar, Ronen

    2016-01-01

    Cadherin-mediated cell–cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell–cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell–cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell–cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair. PMID:27440924

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

    PubMed Central

    1984-01-01

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

  1. A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein

    PubMed Central

    1991-01-01

    The 25-kD inhibitor of actin polymerization (25-kD IAP), isolated from turkey smooth muscle (Miron, T., M. Wilchek, and B. Geiger, 1988. Eur. J. Biochem. 178:543-553), is shown here to be a low molecular mass heat shock protein (HSP). Direct sequence analysis of the purified protein, as well as cloning and sequencing of the respective cDNA, disclosed a high degree of homology (67% identity, 80% similarity) to the human 27- kD HSP. Southern blot of chicken genomic DNA disclosed one band, suggesting the presence of a single gene, and Northern blot analysis revealed abundant transcript of approximately 1 kb in gizzard and heart tissues and lower amounts in total 18-d chick embryo RNA and in cultured fibroblasts. Exposure of the latter cells to 45 degrees C resulted in over 15-fold increase in the apparent level of the 25-kD IAP protein, confirming that its expression is regulated by heat shock. Immunofluorescent microscopic localization indicated that after heat treatment, the levels of the 25-kD IAP were markedly increased and the protein was apparently associated with cytoplasmic granules. Heat shock also had a transient, yet prominent, effect on the microfilament system in cultured fibroblasts: stress fibers disintegrated within 10-15 min after incubation at 45 degrees C, yet upon further incubation at the elevated temperature, conspicuous actin bundles were apparently reformed. PMID:2071672

  2. Nuclear Actin in Development and Transcriptional Reprogramming.

    PubMed

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin's roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation.

  3. Bacterial nucleators: actin' on actin

    PubMed Central

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

    2015-01-01

    The actin cytoskeleton is a key target of numerous microbial pathogens, including protozoa, fungi, bacteria and viruses. In particular, bacterial pathogens produce and deliver virulence effector proteins that hijack actin dynamics to enable bacterial invasion of host cells, allow movement within the host cytosol, facilitate intercellular spread or block phagocytosis. Many of these effector proteins directly or indirectly target the major eukaryotic actin nucleator, the Arp2/3 complex, by either mimicking nucleation promoting factors or activating upstream small GTPases. In contrast, this review is focused on a recently identified class of effector proteins from Gram-negative bacteria that function as direct actin nucleators. These effector proteins mimic functional activities of formins, WH2-nucleators and Ena/VASP assembly promoting factors demonstrating that bacteria have coopted the complete set of eukaryotic actin assembly pathways. Structural and functional analyses of these nucleators have revealed several motifs and/or mechanistic activities that are shared with eukaryotic actin nucleators. However, functional effects of these proteins during infection extend beyond plain actin polymerization leading to interference with other host cell functions such as vesicle trafficking, cell cycle progression and cell death. Therefore, their use as model systems could not only help in the understanding of the mechanistic details of actin polymerization but also provide novel insights into the connection between actin dynamics and other cellular pathways. PMID:26416078

  4. Regulation of WASH-Dependent Actin Polymerization and Protein Trafficking by Ubiquitination

    PubMed Central

    Hao, Yi-Heng; Doyle, Jennifer M.; Ramanathan, Saumya; Gomez, Timothy S.; Jia, Da; Xu, Ming; Chen, Zhijian J.; Billadeau, Daniel D.; Rosen, Michael K.; Potts, Patrick Ryan

    2013-01-01

    SUMMARY Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a novel role for ubiquitination in this process. We find that the novel E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the Retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired Retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of Retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and Retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 and reveal novel aspects of retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex. PMID:23452853

  5. Regulation of actin polymerization and adhesion-dependent cell edge protrusion by the Abl-related gene (Arg) tyrosine kinase and N-WASp.

    PubMed

    Miller, Matthew M; Lapetina, Stefanie; MacGrath, Stacey M; Sfakianos, Mindan K; Pollard, Thomas D; Koleske, Anthony J

    2010-03-16

    Extracellular cues stimulate the Abl family nonreceptor tyrosine kinase Arg to promote actin-based cell edge protrusions. Several Arg-interacting proteins are potential links to the actin cytoskeleton, but exactly how Arg stimulates actin polymerization and cellular protrusion has not yet been fully elucidated. We used affinity purification to identify N-WASp as a novel binding partner of Arg. N-WASp activates the Arp2/3 complex and is an effector of Abl. We find that the Arg SH3 domain binds directly to N-WASp. Arg phosphorylates N-WASp on Y256, modestly increasing the affinity of Arg for N-WASp, an interaction that does not require the Arg SH2 domain. The Arg SH3 domain stimulates N-WASp-dependent actin polymerization in vitro, and Arg phosphorylation of N-WASp weakly stimulates this effect. Arg and N-WASp colocalize to adhesion-dependent cell edge protrusions in vivo. The cell edge protrusion defects of arg-/- fibroblasts can be complemented by re-expression of an Arg-yellow fluorescent protein (YFP) fusion, but not by an N-WASp binding-deficient Arg SH3 domain point mutant. These results suggest that Arg promotes actin-based protrusions in response to extracellular stimuli through phosphorylation of and physical interactions with N-WASp.

  6. Nuclear Actin in Development and Transcriptional Reprogramming

    PubMed Central

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin’s roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation. PMID:28326098

  7. Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent.

    PubMed

    Kim, Hak Rim; Gallant, Cynthia; Leavis, Paul C; Gunst, Susan J; Morgan, Kathleen G

    2008-09-01

    Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

  8. Actin and Actin-Binding Proteins.

    PubMed

    Pollard, Thomas D

    2016-08-01

    Organisms from all domains of life depend on filaments of the protein actin to provide structure and to support internal movements. Many eukaryotic cells use forces produced by actin polymerization for their motility, and myosin motor proteins use ATP hydrolysis to produce force on actin filaments. Actin polymerizes spontaneously, followed by hydrolysis of a bound adenosine triphosphate (ATP). Dissociation of the γ-phosphate prepares the polymer for disassembly. This review provides an overview of the properties of actin and shows how dozens of proteins control both the assembly and disassembly of actin filaments. These players catalyze nucleotide exchange on actin monomers, initiate polymerization, promote phosphate dissociation, cap the ends of polymers, cross-link filaments to each other and other cellular components, and sever filaments. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

  9. Regulation of glucose transport by ROCK1 differs from that of ROCK2 and is controlled by actin polymerization.

    PubMed

    Chun, Kwang-Hoon; Araki, Kazushi; Jee, Yuna; Lee, Dae-Ho; Oh, Byung-Chul; Huang, Hu; Park, Kyong Soo; Lee, Sam W; Zabolotny, Janice M; Kim, Young-Bum

    2012-04-01

    A role of Rho-associated coiled-coil-containing protein kinase (ROCK)1 in regulating whole-body glucose homeostasis has been reported. However, cell-autonomous effects of ROCK1 on insulin-dependent glucose transport in adipocytes and muscle cells have not been elucidated. To determine the specific role of ROCK1 in glucose transport directly, ROCK1 expression in 3T3-L1 adipocytes and L6 myoblasts was biologically modulated. Here, we show that small interfering RNA-mediated ROCK1 depletion decreased insulin-induced glucose transport in adipocytes and myoblasts, whereas adenovirus-mediated ROCK1 expression increased this in a dose-dependent manner, indicating that ROCK1 is permissive for glucose transport. Inhibition of ROCK1 also impaired glucose transporter 4 translocation in 3T3-L1 adipocytes. Importantly, the ED₅₀ of insulin for adipocyte glucose transport was reduced when ROCK1 was expressed, leading to hypersensitivity to insulin. These effects are dependent on actin cytoskeleton remodeling, because inhibitors of actin polymerization significantly decreased ROCK1's effect to promote insulin-stimulated glucose transport. Unlike ROCK2, ROCK1 binding to insulin receptor substrate (IRS)-1 was not detected by immunoprecipitation, although cell fractionation demonstrated both ROCK isoforms localize with IRS-1 in low-density microsomes. Moreover, insulin's ability to increase IRS-1 tyrosine 612 and serine 632/635 phosphorylation was attenuated by ROCK1 suppression. Replacing IRS-1 serine 632/635 with alanine reduced insulin-stimulated phosphatidylinositol 3-kinase activation and glucose transport in 3T3-L1 adipocytes, indicating that phosphorylation of these serine residues of IRS-1, which are substrates of the ROCK2 isoform in vitro, are crucial for maximal stimulation of glucose transport by insulin. Our studies identify ROCK1 as an important positive regulator of insulin action on glucose transport in adipocytes and muscle cells.

  10. Regulation of Glucose Transport by ROCK1 Differs from That of ROCK2 and Is Controlled by Actin Polymerization

    PubMed Central

    Chun, Kwang-Hoon; Araki, Kazushi; Jee, Yuna; Lee, Dae-Ho; Oh, Byung-Chul; Huang, Hu; Park, Kyong Soo; Lee, Sam W.; Zabolotny, Janice M.

    2012-01-01

    A role of Rho-associated coiled-coil-containing protein kinase (ROCK)1 in regulating whole-body glucose homeostasis has been reported. However, cell-autonomous effects of ROCK1 on insulin-dependent glucose transport in adipocytes and muscle cells have not been elucidated. To determine the specific role of ROCK1 in glucose transport directly, ROCK1 expression in 3T3-L1 adipocytes and L6 myoblasts was biologically modulated. Here, we show that small interfering RNA-mediated ROCK1 depletion decreased insulin-induced glucose transport in adipocytes and myoblasts, whereas adenovirus-mediated ROCK1 expression increased this in a dose-dependent manner, indicating that ROCK1 is permissive for glucose transport. Inhibition of ROCK1 also impaired glucose transporter 4 translocation in 3T3-L1 adipocytes. Importantly, the ED50 of insulin for adipocyte glucose transport was reduced when ROCK1 was expressed, leading to hypersensitivity to insulin. These effects are dependent on actin cytoskeleton remodeling, because inhibitors of actin polymerization significantly decreased ROCK1's effect to promote insulin-stimulated glucose transport. Unlike ROCK2, ROCK1 binding to insulin receptor substrate (IRS)-1 was not detected by immunoprecipitation, although cell fractionation demonstrated both ROCK isoforms localize with IRS-1 in low-density microsomes. Moreover, insulin's ability to increase IRS-1 tyrosine 612 and serine 632/635 phosphorylation was attenuated by ROCK1 suppression. Replacing IRS-1 serine 632/635 with alanine reduced insulin-stimulated phosphatidylinositol 3-kinase activation and glucose transport in 3T3-L1 adipocytes, indicating that phosphorylation of these serine residues of IRS-1, which are substrates of the ROCK2 isoform in vitro, are crucial for maximal stimulation of glucose transport by insulin. Our studies identify ROCK1 as an important positive regulator of insulin action on glucose transport in adipocytes and muscle cells. PMID:22355071

  11. Evolutionary conservation of the WASH complex, an actin polymerization machine involved in endosomal fission.

    PubMed

    Derivery, Emmanuel; Gautreau, Alexis

    2010-05-01

    WASH is the Arp2/3 activating protein that is localized at the surface of endosomes, where it induces the formation of branched actin networks. This activity of WASH favors, in collaboration with dynamin, the fission of transport intermediates from endosomes, and hence regulates endosomal trafficking of several cargos. We have purified a novel stable multiprotein complex containing WASH, the WASH complex, and we examine here the evolutionary conservation of its seven subunits across diverse eukaryotic phyla. This analysis supports the idea that the invention of the WASH complex has involved the incorporation of an independent complex, the CapZ alpha/beta heterodimer, forming the so-called Capping Protein (CP), as illustrated by the yeasts S. cerevisiae and S. pombe, which possess the CP heterodimer but no other subunits of the WASH complex. The alignements of the orthologous genes that we have generated give a view on the conservation of the different subunits and on their organization into domains. Moreover, we propose here a unique nomenclature for the different subunits to prevent future confusions in the field.

  12. Impairment of CCR6+ and CXCR3+ Th Cell Migration in HIV-1 Infection Is Rescued by Modulating Actin Polymerization

    PubMed Central

    Bernasconi, Enos; Speck, Roberto F.; Proietti, Michele; Sauermann, Ulrike; D’Agostino, Gianluca; Danelon, Gabriela; Rezzonico Jost, Tanja; Grassi, Fabio; Raeli, Lorenzo; Schöni-Affolter, Franziska; Stahl-Hennig, Christiane

    2017-01-01

    CD4+ T cell repopulation of the gut is rarely achieved in HIV-1–infected individuals who are receiving clinically effective antiretroviral therapy. Alterations in the integrity of the mucosal barrier have been indicated as a cause for chronic immune activation and disease progression. In this study, we present evidence that persistent immune activation causes impairment of lymphocytes to respond to chemotactic stimuli, thus preventing their trafficking from the blood stream to peripheral organs. CCR6+ and CXCR3+ Th cells accumulate in the blood of aviremic HIV-1–infected patients on long-term antiretroviral therapy, and their frequency in the circulation positively correlates to levels of soluble CD14 in plasma, a marker of chronic immune activation. Th cells show an impaired response to chemotactic stimuli both in humans and in the pathogenic model of SIV infection, and this defect is due to hyperactivation of cofilin and inefficient actin polymerization. Taking advantage of a murine model of chronic immune activation, we demonstrate that cytoskeleton remodeling, induced by okadaic acid, restores lymphocyte migration in response to chemokines, both in vitro and in vivo. This study calls for novel pharmacological approaches in those pathological conditions characterized by persistent immune activation and loss of trafficking of T cell subsets to niches that sustain their maturation and activities. PMID:27895171

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

    PubMed

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

    2012-03-27

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

  14. A Balance of Capping Protein and Profilin Functions Is Required to Regulate Actin Polymerization in Drosophila Bristle

    PubMed Central

    Hopmann, Roberta; Miller, Kathryn G.

    2003-01-01

    Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell. PMID:12529431

  15. 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.

  16. 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces rapid actin polymerization in HL-60 cells differentiated into macrophage-like cells.

    PubMed

    Gokoh, Maiko; Kishimoto, Seishi; Oka, Saori; Mori, Masahiro; Waku, Keizo; Ishima, Yoshio; Sugiura, Takayuki

    2005-03-15

    Delta9-Tetrahydrocannabinol, a major psychoactive constituent of marijuana, interacts with specific receptors, i.e. the cannabinoid receptors, thereby eliciting a variety of pharmacological responses. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, which is abundantly expressed in the nervous system, and the CB2 receptor, which is predominantly expressed in the immune system. Previously, we investigated in detail the structure-activity relationship of various cannabinoid receptor ligands and found that 2-AG (2-arachidonoylglycerol) is the most efficacious agonist. We have proposed that 2-AG is the true natural ligand for both the CB1 and CB2 receptors. Despite the potential physiological importance of 2-AG, not much information is available concerning its biological activities towards mammalian tissues and cells. In the present study, we examined the effect of 2-AG on morphology as well as the actin filament system in differentiated HL-60 cells, which express the CB2 receptor. We found that 2-AG induces rapid morphological changes such as the extension of pseudopods. We also found that it provokes a rapid actin polymerization in these cells. Actin polymerization induced by 2-AG was abolished when cells were treated with SR144528, a CB2 receptor antagonist, and pertussis toxin, suggesting that the response was mediated by the CB2 receptor and G(i/o). A phosphoinositide 3-kinase, Rho family small G-proteins and a tyrosine kinase were also suggested to be involved. Reorganization of the actin filament system is known to be indispensable for a variety of cellular events; it is possible that 2-AG plays physiologically essential roles in various inflammatory cells and immune-competent cells by inducing a rapid actin rearrangement.

  17. Co-transcriptional nuclear actin dynamics

    PubMed Central

    Percipalle, Piergiorgio

    2013-01-01

    Actin is a key player for nuclear structure and function regulating both chromosome organization and gene activity. In the cell nucleus actin interacts with many different proteins. Among these proteins several studies have identified classical nuclear factors involved in chromatin structure and function, transcription and RNA processing as well as proteins that are normally involved in controlling the actin cytoskeleton. These discoveries have raised the possibility that nuclear actin performs its multi task activities through tight interactions with different sets of proteins. This high degree of promiscuity in the spectrum of protein-to-protein interactions correlates well with the conformational plasticity of actin and the ability to undergo regulated changes in its polymerization states. Several of the factors involved in controlling head-to-tail actin polymerization have been shown to be in the nucleus where they seem to regulate gene activity. By focusing on the multiple tasks performed by actin and actin-binding proteins, possible models of how actin dynamics controls the different phases of the RNA polymerase II transcription cycle are being identified. PMID:23138849

  18. 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.

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

    PubMed

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

    2012-08-01

    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.

  20. 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

  1. 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

  2. Nckβ Adapter Regulates Actin Polymerization in NIH 3T3 Fibroblasts in Response to Platelet-Derived Growth Factor bb

    PubMed Central

    Chen, Min; She, Hongyun; Kim, Airie; Woodley, David T.; Li, Wei

    2000-01-01

    The SH3-SH3-SH3-SH2 adapter Nck represents a two-gene family that includes Nckα (Nck) and Nckβ (Grb4/Nck2), and it links receptor tyrosine kinases to intracellular signaling networks. The function of these mammalian Nck genes has not been established. We report here a specific role for Nckβ in platelet-derived growth factor (PDGF)-induced actin polymerization in NIH 3T3 cells. Overexpression of Nckβ but not Nckα blocks PDGF-stimulated membrane ruffling and formation of lamellipoda. Mutation in either the SH2 or the middle SH3 domain of Nckβ abolishes its interfering effect. Nckβ binds at Tyr-1009 in human PDGF receptor β (PDGFR-β) which is different from Nckα's binding site, Tyr-751, and does not compete with phosphatidylinositol-3 kinase for binding to PDGFR. Microinjection of an anti-Nckβ but not an anti-Nckα antibody inhibits PDGF-stimulated actin polymerization. Constitutively membrane-bound Nckβ but not Nckα blocks Rac1-L62-induced membrane ruffling and formation of lamellipodia, suggesting that Nckβ acts in parallel to or downstream of Rac1. This is the first report of Nckβ's role in receptor tyrosine kinase signaling to the actin cytoskeleton. PMID:11027258

  3. X-ray and cryo-EM structures of monomeric and filamentous actin-like protein MamK reveal changes associated with polymerization.

    PubMed

    Löwe, Jan; He, Shaoda; Scheres, Sjors H W; Savva, Christos G

    2016-11-22

    Magnetotactic bacteria produce iron-rich magnetic nanoparticles that are enclosed by membrane invaginations to form magnetosomes so they are able to sense and act upon Earth's magnetic field. In Magnetospirillum and other magnetotactic bacteria, to combine their magnetic moments, magnetosomes align along filaments formed by a bacterial actin homolog, MamK. Here, we present the crystal structure of a nonpolymerizing mutant of MamK from Magnetospirillum magneticum AMB-1 at 1.8-Å resolution, revealing its close similarity to actin and MreB. The crystals contain AMPPNP-bound monomeric MamK in two different conformations. To investigate conformational changes associated with polymerization, we used unmodified MamK protein and cryo-EM with helical 3D reconstruction in RELION to obtain a density map and a fully refined atomic model of MamK in filamentous form at 3.6-Å resolution. The filament is parallel (polar) double-helical, with a rise of 52.2 Å and a twist of 23.8°. As shown previously and unusually for actin-like filaments, the MamK subunits from each of the two strands are juxtaposed, creating an additional twofold axis along the filament. Compared with monomeric MamK, ADP-bound MamK in the filament undergoes a conformational change, rotating domains I and II against each other to further close the interdomain cleft between subdomains IB and IIB. The domain movement causes several loops to close around the nucleotide-binding pocket. Glu-143, a key residue for catalysis coordinating the magnesium ion, moves closer, presumably switching nucleotide hydrolysis upon polymerization-one of the hallmarks of cytomotive filaments of the actin type.

  4. Characterization of actin filament severing by actophorin from Acanthamoeba castellanii

    PubMed Central

    1991-01-01

    Actophorin is an abundant 15-kD actinbinding protein from Acanthamoeba that is thought to form a nonpolymerizable complex with actin monomers and also to reduce the viscosity of polymerized actin by severing filaments (Cooper et al., 1986. J. Biol. Chem. 261:477-485). Homologous proteins have been identified in sea urchin, chicken, and mammalian tissues. Chemical crosslinking produces a 1:1 covalent complex of actin and actophorin. Actophorin and profilin compete for crosslinking to actin monomers. The influence of actophorin on the steady-state actin polymer concentration gave a Kd of 0.2 microM for the complex of actophorin with actin monomers. Several new lines of evidence, including assays for actin filament ends by elongation rate and depolymerization rate, show that actophorin severs actin filaments both at steady state and during spontaneous polymerization. This is confirmed by direct observation in the light microscope and by showing that the effects of actophorin on the low shear viscosity of polymerized actin cannot be explained by monomer sequestration. The severing activity of actophorin is strongly inhibited by stoichiometric concentrations of phalloidin or millimolar concentrations of inorganic phosphate. PMID:1757465

  5. Percolation mechanism drives actin gels to the critically connected state

    NASA Astrophysics Data System (ADS)

    Lee, Chiu Fan; Pruessner, Gunnar

    2016-05-01

    Cell motility and tissue morphogenesis depend crucially on the dynamic remodeling of actomyosin networks. An actomyosin network consists of an actin polymer network connected by cross-linker proteins and motor protein myosins that generate internal stresses on the network. A recent discovery shows that for a range of experimental parameters, actomyosin networks contract to clusters with a power-law size distribution [J. Alvarado, Nat. Phys. 9, 591 (2013), 10.1038/nphys2715]. Here, we argue that actomyosin networks can exhibit a robust critical signature without fine-tuning because the dynamics of the system can be mapped onto a modified version of percolation with trapping (PT), which is known to show critical behavior belonging to the static percolation universality class without the need for fine-tuning of a control parameter. We further employ our PT model to generate experimentally testable predictions.

  6. The Proteome of BLOC-1 Genetic Defects Identifies the Arp2/3 Actin Polymerization Complex to Function Downstream of the Schizophrenia Susceptibility Factor Dysbindin at the Synapse

    PubMed Central

    Hartwig, Cortnie; Freeman, Amanda H.; Das, Ravi; Zlatic, Stephanie A.; Vistein, Rachel; Burch, Amelia; Carrot, Guillemette; Lewis, Arielle F.; Nelms, Sheldon; Dickman, Dion K.; Puthenveedu, Manojkumar A.; Cox, Daniel N.

    2016-01-01

    Proteome modifications downstream of monogenic or polygenic disorders have the potential to uncover novel molecular mechanisms participating in pathogenesis and/or extragenic modification of phenotypic expression. We tested this idea by determining the proteome sensitive to genetic defects in a locus encoding dysbindin, a protein required for synapse biology and implicated in schizophrenia risk. We applied quantitative mass spectrometry to identify proteins expressed in neuronal cells the abundance of which was altered after downregulation of the schizophrenia susceptibility factor dysbindin (Bloc1s8) or two other dysbindin-interacting polypeptides, which assemble into the octameric biogenesis of lysosome-related organelles complex 1 (BLOC-1). We found 491 proteins sensitive to dysbindin and BLOC-1 loss of function. Gene ontology of these 491 proteins singled out the actin cytoskeleton and the actin polymerization factor, the Arp2/3 complex, as top statistical molecular pathways contained within the BLOC-1-sensitive proteome. Subunits of the Arp2/3 complex were downregulated by BLOC-1 loss of function, thus affecting actin dynamics in early endosomes of BLOC-1-deficient cells. Furthermore, we demonstrated that Arp2/3, dysbindin, and subunits of the BLOC-1 complex biochemically and genetically interact, modulating Drosophila melanogaster synapse morphology and homeostatic synaptic plasticity. Our results indicate that ontologically prioritized proteomics identifies novel pathways that modify synaptic phenotypes associated with neurodevelopmental disorder gene defects. SIGNIFICANCE STATEMENT The mechanisms associated with schizophrenia are mostly unknown despite the increasing number of genetic loci identified that increase disease risk. We present an experimental strategy that impartially and comprehensively interrogates the proteome of neurons to identify effects of genetic mutations in a schizophrenia risk factor, dysbindin. We find that the expression of the

  7. The Proteome of BLOC-1 Genetic Defects Identifies the Arp2/3 Actin Polymerization Complex to Function Downstream of the Schizophrenia Susceptibility Factor Dysbindin at the Synapse.

    PubMed

    Gokhale, Avanti; Hartwig, Cortnie; Freeman, Amanda H; Das, Ravi; Zlatic, Stephanie A; Vistein, Rachel; Burch, Amelia; Carrot, Guillemette; Lewis, Arielle F; Nelms, Sheldon; Dickman, Dion K; Puthenveedu, Manojkumar A; Cox, Daniel N; Faundez, Victor

    2016-12-07

    Proteome modifications downstream of monogenic or polygenic disorders have the potential to uncover novel molecular mechanisms participating in pathogenesis and/or extragenic modification of phenotypic expression. We tested this idea by determining the proteome sensitive to genetic defects in a locus encoding dysbindin, a protein required for synapse biology and implicated in schizophrenia risk. We applied quantitative mass spectrometry to identify proteins expressed in neuronal cells the abundance of which was altered after downregulation of the schizophrenia susceptibility factor dysbindin (Bloc1s8) or two other dysbindin-interacting polypeptides, which assemble into the octameric biogenesis of lysosome-related organelles complex 1 (BLOC-1). We found 491 proteins sensitive to dysbindin and BLOC-1 loss of function. Gene ontology of these 491 proteins singled out the actin cytoskeleton and the actin polymerization factor, the Arp2/3 complex, as top statistical molecular pathways contained within the BLOC-1-sensitive proteome. Subunits of the Arp2/3 complex were downregulated by BLOC-1 loss of function, thus affecting actin dynamics in early endosomes of BLOC-1-deficient cells. Furthermore, we demonstrated that Arp2/3, dysbindin, and subunits of the BLOC-1 complex biochemically and genetically interact, modulating Drosophila melanogaster synapse morphology and homeostatic synaptic plasticity. Our results indicate that ontologically prioritized proteomics identifies novel pathways that modify synaptic phenotypes associated with neurodevelopmental disorder gene defects. The mechanisms associated with schizophrenia are mostly unknown despite the increasing number of genetic loci identified that increase disease risk. We present an experimental strategy that impartially and comprehensively interrogates the proteome of neurons to identify effects of genetic mutations in a schizophrenia risk factor, dysbindin. We find that the expression of the actin polymerization

  8. The Impact of the Current United States Guidelines on the Management of Actinic Keratosis

    PubMed Central

    2010-01-01

    Actinic keratosis is one of the most common diagnoses made by dermatologists. Many experts recommend treating all actinic keratoses because of their potential to progress to invasive squamous cell carcinoma. Physicians have a large armamentarium of actinic keratosis treatment modalities available to them, including destructive therapies, such as cryotherapy, curettage and electrodessication, chemical peels, photodynamic therapy, and topical therapies, including 5-fluorouracil, imiquimod, and diclofenac. In addition to standardized monotherapy regimens, combinations of two concomitant or sequential therapies and alternative topical dosing regimens have been studied in a number of clinical trials. Such therapeutic courses are used to maintain or enhance efficacy while improving tolerability, convenience, and/or patient adherence. This abundance of treatment options prompted development of several actinic keratosis management guidelines. Whereas two sets of treatment guidelines were published by European organizations within the past three years, the most recent United States-based guidelines for dermatologists were published by the American Academy of Dermatology in 1995. Because they are not up to date, the 1995 United States guidelines lack recent clinical developments and an evidence rating system and can no longer effectively guide practitioners. While there are benefits and potential limitations to developing an updated set of United States-based guidelines, there is a clearly defined need for a unified, comprehensive, evidence-based guideline approach to actinic keratosis treatment that balances the need to tailor long-term management of the disease to the needs of the individual patient. PMID:21103312

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

    PubMed

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

    2013-01-08

    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.

  10. 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

  11. Modification of Lys-237 on actin by 2,4-pentanedione. Alteration of the interaction of actin with tropomyosin.

    PubMed

    El-Saleh, S C; Thieret, R; Johnson, P; Potter, J D

    1984-09-10

    It has been possible to specifically label rabbit skeletal muscle actin at Lys-237 with 2,4-pentanedione, producing an enamine. This reaction can be reversed with hydroxylamine. The modification can be carried out with actin in either the G- or F-forms and does not affect polymerization-depolymerization. The modification does affect, however, the interaction of tropomyosin (Tm) with the modified F-actin. In the absence of Ca2+ and Mg2+ (mu = 0.12), Tm failed to bind to the modified F-actin whereas it did bind to unmodified F-actin (1 Tm:7 actins). Tm binding could be restored under these conditions by the addition of either troponin (Tn), Mg2+, or Mg2+ and Ca2+. Under certain conditions, Tm alone has been shown to inhibit actin-activated heavy meromyosin (HMM)-Mg2+-ATPase. This inhibition did not occur with the modified F-actin even though Tm was bound (approximately 1 Tm:7 actins). Even when Tn was added to this system (in the absence of Ca2+), no inhibition of ATPase could be observed. Thus, this modification appears to prevent F-actin X Tm from assuming the "blocking" inhibitory position (conformation). In addition, Tn appears to enhance the activation of heavy meromyosin-Mg2+-ATPase by the modified F-actin X Tm complex whether Ca2+ is present or not. This state may be analogous to the potentiated state (Murray, J. M., Knox, M. K., Trueblood, C. E., and Weber, A. (1982) Biochemistry 27, 906-915) seen with myosin subfragment 1-saturated actin at low ATP levels. Thus, using modified and unmodified F-actin, it is possible to produce three Tm X actin states: off (F-actin X Tm), on (modified F-actin X Tm), and "potentiated" (modified F-actin X Tm X Tn).

  12. 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.

  13. cAMP signaling by anthrax edema toxin induces transendothelial cell tunnels, which are resealed by MIM via Arp2/3-driven actin polymerization.

    PubMed

    Maddugoda, Madhavi P; Stefani, Caroline; Gonzalez-Rodriguez, David; Saarikangas, Juha; Torrino, Stéphanie; Janel, Sebastien; Munro, Patrick; Doye, Anne; Prodon, François; Aurrand-Lions, Michel; Goossens, Pierre L; Lafont, Frank; Bassereau, Patricia; Lappalainen, Pekka; Brochard, Françoise; Lemichez, Emmanuel

    2011-11-17

    RhoA-inhibitory bacterial toxins, such as Staphylococcus aureus EDIN toxin, induce large transendothelial cell macroaperture (TEM) tunnels that rupture the host endothelium barrier and promote bacterial dissemination. Host cells repair these tunnels by extending actin-rich membrane waves from the TEM edges. We reveal that cyclic-AMP signaling produced by Bacillus anthracis edema toxin (ET) also induces TEM formation, which correlates with increased vascular permeability. We show that ET-induced TEM formation resembles liquid dewetting, a physical process of nucleation and growth of holes within a thin liquid film. We also identify the cellular mechanisms of tunnel closure and reveal that the I-BAR domain protein Missing in Metastasis (MIM) senses de novo membrane curvature generated by the TEM, accumulates at the TEM edge, and triggers Arp2/3-dependent actin polymerization, which induces actin-rich membrane waves that close the TEM. Thus, the balance between ET-induced TEM formation and resealing likely determines the integrity of the host endothelium barrier.

  14. Prevention of RhoA activation and cofilin-mediated actin polymerization mediates the antihypertrophic effect of adenosine receptor agonists in angiotensin II- and endothelin-1-treated cardiomyocytes.

    PubMed

    Zeidan, Asad; Gan, Xiaohong Tracey; Thomas, Ashley; Karmazyn, Morris

    2014-01-01

    Adenosine receptor activation has been shown to be associated with diminution of cardiac hypertrophy and it has been suggested that endogenously produced adenosine may serve to blunt pro-hypertrophic processes. In the present study, we determined the effects of two pro-hypertrophic stimuli, angiotensin II (Ang II, 100 nM) and endothelin-1 (ET-1, 10 nM) on Ras homolog gene family, member A (RhoA)/Rho-associated, coiled-coil containing protein kinase (ROCK) activation in cultured neonatal rat ventricular myocytes and whether the latter serves as a target for the anti-hypertrophic effect of adenosine receptor activation. Both hypertrophic stimuli potently increased RhoA activity with peak activation occurring 15-30 min following agonist addition. These effects were associated with significantly increased phosphorylation (inactivation) of cofilin, a downstream mediator of RhoA, an increase in actin polymerization, and increased activation and nuclear import of p38 mitogen activated protein kinase. The ability of both Ang II and ET-1 to activate the RhoA pathway was completely prevented by the adenosine A1 receptor agonist N (6)-cyclopentyladenosine, the A2a receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine, the A3 receptor agonist N (6)-(3-iodobenzyl)adenosine-5'-methyluronamide as well as the nonspecific adenosine analog 2-chloro adenosine. All effects of specific receptor agonists were prevented by their respective receptor antagonists. Moreover, all adenosine agonists prevented either Ang II- or ET-1-induced hypertrophy, a property shared by the RhoA inhibitor Clostridium botulinum C3 exoenzyme, the ROCK inhibitor Y-27632 or the actin depolymerizing agent latrunculin B. Our study therefore demonstrates that both Ang II and ET-1 can activate the RhoA pathway and that prevention of the hypertrophic response to both agonists by adenosine receptor activation is mediated by prevention of RhoA stimulation and actin polymerization.

  15. X-ray and cryo-EM structures of monomeric and filamentous actin-like protein MamK reveal changes associated with polymerization

    PubMed Central

    He, Shaoda; Savva, Christos G.

    2016-01-01

    Magnetotactic bacteria produce iron-rich magnetic nanoparticles that are enclosed by membrane invaginations to form magnetosomes so they are able to sense and act upon Earth’s magnetic field. In Magnetospirillum and other magnetotactic bacteria, to combine their magnetic moments, magnetosomes align along filaments formed by a bacterial actin homolog, MamK. Here, we present the crystal structure of a nonpolymerizing mutant of MamK from Magnetospirillum magneticum AMB-1 at 1.8-Å resolution, revealing its close similarity to actin and MreB. The crystals contain AMPPNP-bound monomeric MamK in two different conformations. To investigate conformational changes associated with polymerization, we used unmodified MamK protein and cryo-EM with helical 3D reconstruction in RELION to obtain a density map and a fully refined atomic model of MamK in filamentous form at 3.6-Å resolution. The filament is parallel (polar) double-helical, with a rise of 52.2 Å and a twist of 23.8°. As shown previously and unusually for actin-like filaments, the MamK subunits from each of the two strands are juxtaposed, creating an additional twofold axis along the filament. Compared with monomeric MamK, ADP-bound MamK in the filament undergoes a conformational change, rotating domains I and II against each other to further close the interdomain cleft between subdomains IB and IIB. The domain movement causes several loops to close around the nucleotide-binding pocket. Glu-143, a key residue for catalysis coordinating the magnesium ion, moves closer, presumably switching nucleotide hydrolysis upon polymerization—one of the hallmarks of cytomotive filaments of the actin type. PMID:27821762

  16. Actinous enigma or enigmatic actin

    PubMed Central

    Povarova, Olga I; Uversky, Vladimir N; Kuznetsova, Irina M; Turoverov, Konstantin K

    2014-01-01

    Being the most abundant protein of the eukaryotic cell, actin continues to keep its secrets for more than 60 years. Everything about this protein, its structure, functions, and folding, is mysteriously counterintuitive, and this review represents an attempt to solve some of the riddles and conundrums commonly found in the field of actin research. In fact, actin is a promiscuous binder with a wide spectrum of biological activities. It can exist in at least three structural forms, globular, fibrillar, and inactive (G-, F-, and I-actin, respectively). G-actin represents a thermodynamically instable, quasi-stationary state, which is formed in vivo as a result of the energy-intensive, complex posttranslational folding events controlled and driven by cellular folding machinery. The G-actin structure is dependent on the ATP and Mg2+ binding (which in vitro is typically substituted by Ca2+) and protein is easily converted to the I-actin by the removal of metal ions and by action of various denaturing agents (pH, temperature, and chemical denaturants). I-actin cannot be converted back to the G-form. Foldable and “natively folded” forms of actin are always involved in interactions either with the specific protein partners, such as Hsp70 chaperone, prefoldin, and the CCT chaperonin during the actin folding in vivo or with Mg2+ and ATP as it takes place in the G-form. We emphasize that the solutions for the mysteries of actin multifunctionality, multistructurality, and trapped unfolding can be found in the quasi-stationary nature of this enigmatic protein, which clearly possesses many features attributed to both globular and intrinsically disordered proteins. PMID:28232879

  17. Membrane targeting of WAVE2 is not sufficient for WAVE2-dependent actin polymerization: a role for IRSp53 in mediating the interaction between Rac and WAVE2.

    PubMed

    Abou-Kheir, Wassim; Isaac, Beth; Yamaguchi, Hideki; Cox, Dianne

    2008-02-01

    Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous (WAVE) proteins play a major role in Rac-induced actin dynamics, but Rac does not bind directly to WAVE proteins. It has been proposed that either the insulin receptor substrate protein 53 (IRSp53) or a complex of proteins containing Abelson interactor protein 1 (Abi1) mediates the interaction of WAVE2 and Rac. Depletion of endogenous IRSp53 by RNA-mediated interference (RNAi) in a RAW/LR5 macrophage cell line resulted in a significant reduction of Rac1Q61L-induced surface ruffles and colony-stimulating factor 1 (CSF-1)-induced actin polymerization, protrusion and cell migration. However, IRSp53 was not essential for Fcgamma-R-mediated phagocytosis, formation of podosomes or for formation of Cdc42V12-induced filopodia. IRSp53 was found to be present in an immunoprecipitable complex with WAVE2 and Abi1 in a Rac1-activation-dependent manner in RAW/LR5 cells in vivo. Importantly, reduction of endogenous IRSp53 or expression of IRSp53 lacking the WAVE2-binding site (IRSp53DeltaSH3) resulted in a significant reduction in the association of Rac1 with WAVE2 and Abi1, indicating that the association of Rac1 with WAVE2 and Abi1 is IRSp53 dependent. While it has been proposed that WAVE2 activity is regulated by membrane recruitment, membrane targeting of WAVE2 in RAW/LR5 and Cos-7 cells did not induce actin polymerization or protrusion, suggesting that membrane recruitment was insufficient for regulation of WAVE2. Combined, these data suggest that IRSp53 links Rac1 to WAVE2 in vivo and its function is crucial for production of CSF-1-induced F-actin-rich protrusions and cell migration in macrophages. This study indicates that Rac1, along with IRSp53 and Abi1, is involved in a more complex and tight regulation of WAVE2 than one operating solely through membrane localization.

  18. The actin cytoskeleton as a therapeutic target for the prevention of relapse to methamphetamine use

    PubMed Central

    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

  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. [Physical-chemical properties of actin in different structural states. New ideas about its folding-unfolding pathways].

    PubMed

    Povarova, O I; Kuznetsova, I M; Turoverov, K K

    2005-01-01

    Results of actin folding-unfolding pathways examination and characterization of intermediate and misfolded states are summarized. Properties of microenvironments and peculiarities of location of tryptophan residues in protein are analysed in detail. This allowed to conclude that the main contribution to the bulk fluorescence of native protein is made by internal tryptophan residues Trp 340 and Trp 356, localized in hydrophobic regions, while tryptophan residues Trp 79 and Trp 86 are quenched. It has been shown that inactivated actin, previously regarded as an intermediate state between native and completely unfolded state of protein is in reality a misfolded aggregated state. The properties of actin in this state were characterized in detail. In particular, it is shown that inactivated actin is a monodisperse associate consisting of 15 monomer unit. Two earlier unknown intermediate states, which precede completely unfolding of protein macromolecule and formation of inactivated actin, were visualized. A new scheme of folding-unfolding processes was proposed. It is shown that the reason of anomalous effects, which are recorded for actin in solutions with small concentrations of GdnHCl, is a specific interaction of actin with a denaturant.

  1. Structural reorganization of parallel actin bundles by crosslinking proteins: Incommensurate states of twist

    NASA Astrophysics Data System (ADS)

    Shin, Homin; Grason, Gregory M.

    2010-11-01

    We construct a coarse-grained model of parallel actin bundles crosslinked by compact globular bundling proteins, such as fascin and espin, necessary components of filopodial and mechanosensory bundles. Consistent with structural observations of bundles, we find that the optimal geometry for crosslinking is overtwisted, requiring a coherent structural change of the helical geometry of the filaments. We study the linker-dependent thermodynamic transition of bundled actin filaments from their native state to the overtwisted state and map out the “twist-state” phase diagram in terms of the availability as well as the flexibility of crosslinker proteins. We predict that the transition from the uncrosslinked to fully crosslinked state is highly sensitive to linker flexibility: flexible crosslinking smoothly distorts the twist state of bundled filaments, while rigidly crosslinked bundles undergo a phase transition, rapidly overtwisting filaments over a narrow range of free crosslinker concentrations. Additionally, we predict a rich spectrum of intermediate structures, composed of alternating domains of sparsely bound (untwisted) and strongly bound (overtwisted) filaments. This model reveals that subtle differences in crosslinking agents themselves modify not only the detailed structure of parallel actin bundles, but also the thermodynamic pathway by which they form.

  2. AmpA protein functions by different mechanisms to influence early cell type specification and to modulate cell adhesion and actin polymerization in Dictyostelium discoideum.

    PubMed

    Cost, Hoa N; Noratel, Elizabeth F; Blumberg, Daphne D

    2013-01-01

    The Dictyostelium discoideum ampA gene encodes a multifunctional regulator protein that modulates cell-cell and cell-substrate adhesions and actin polymerization during growth and is necessary for correct cell type specification and patterning during development. Insertional inactivation of the ampA gene results in defects that define two distinct roles for the ampA gene during development. AmpA is necessary in a non-cell autonomous manner to prevent premature expression of a prespore gene marker. It is also necessary in a cell autonomous manner for the anterior like cells, which express the ampA gene, to migrate to the upper cup during culmination. It is also necessary to prevent excessive cell-cell agglutination when cells are developed in a submerged suspension culture. Here, we demonstrate that a supernatant source of AmpA protein, added extracellularly, can prevent the premature mis-expression of the prespore marker. Synthetic oligopeptides are used to identify the domain of the AmpA protein that is important for preventing cells from mis-expressing the prespore gene. We further demonstrate that a factor capable of inducing additional cells to express the prespore gene marker accumulates extracellularly in the absence of AmpA protein. While the secreted AmpA acts extracellularly to suppress prespore gene expression, the effects of AmpA on cell agglutination and on actin polymerization in growing cells are not due to an extracellular role of secreted AmpA protein. Rather, these effects appear to reflect a distinct cell autonomous role of the ampA gene. Finally, we show that secretion of AmpA protein is brought about by elevating the levels of expression of ampA so that the protein accumulates to an excessive level.

  3. Non-equilibrium steady states in supramolecular polymerization

    NASA Astrophysics Data System (ADS)

    Sorrenti, Alessandro; Leira-Iglesias, Jorge; Sato, Akihiro; Hermans, Thomas M.

    2017-06-01

    Living systems use fuel-driven supramolecular polymers such as actin to control important cell functions. Fuel molecules like ATP are used to control when and where such polymers should assemble and disassemble. The cell supplies fresh ATP to the cytosol and removes waste products to sustain steady states. Artificial fuel-driven polymers have been developed recently, but keeping them in sustained non-equilibrium steady states (NESS) has proven challenging. Here we show a supramolecular polymer that can be kept in NESS, inside a membrane reactor where ATP is added and waste removed continuously. Assembly and disassembly of our polymer is regulated by phosphorylation and dephosphorylation, respectively. Waste products lead to inhibition, causing the reaction cycle to stop. Inside the membrane reactor, however, waste can be removed leading to long-lived NESS conditions. We anticipate that our approach to obtain NESS can be applied to other stimuli-responsive materials to achieve more life-like behaviour.

  4. 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

  5. Nuclear role of WASp in gene transcription is uncoupled from its ARP2/3-dependent cytoplasmic role in actin polymerization.

    PubMed

    Sadhukhan, Sanjoy; Sarkar, Koustav; Taylor, Matthew; Candotti, Fabio; Vyas, Yatin M

    2014-07-01

    Defects in Wiskott-Aldrich Syndrome protein (WASp) underlie development of WAS, an X-linked immunodeficiency and autoimmunity disorder of childhood. Nucleation-promoting factors (NPFs) of the WASp family generate F-actin in the cytosol via the VCA (verprolin-homology, cofilin-homology, and acidic) domain and support RNA polymerase II-dependent transcription in the nucleus. Whether nuclear-WASp requires the integration of its actin-related protein (ARP)2/3-dependent cytoplasmic function to reprogram gene transcription, however, remains unresolved. Using the model of human TH cell differentiation, we find that WASp has a functional nuclear localizing and nuclear exit sequences, and accordingly, its effects on transcription are controlled mainly at the level of its nuclear entry and exit via the nuclear pore. Human WASp does not use its VCA-dependent, ARP2/3-driven, cytoplasmic effector mechanisms to support histone H3K4 methyltransferase activity in the nucleus of TH1-skewed cells. Accordingly, an isolated deficiency of nuclear-WASp is sufficient to impair the transcriptional reprogramming of TBX21 and IFNG promoters in TH1-skewed cells, whereas an isolated deficiency of cytosolic-WASp does not impair this process. In contrast, nuclear presence of WASp in TH2-skewed cells is small, and its loss does not impair transcriptional reprogramming of GATA3 and IL4 promoters. Our study unveils an ARP2/3:VCA-independent function of nuclear-WASp in TH1 gene activation that is uncoupled from its cytoplasmic role in actin polymerization.

  6. Thymoquinone decreases F-actin polymerization and the proliferation of human multiple myeloma cells by suppressing STAT3 phosphorylation and Bcl2/Bcl-XL expression

    PubMed Central

    2011-01-01

    Background Thymoquinone (TQ), the major active component of the medicinal herb Nigella sativa Linn., has been described as a chemopreventive and chemotherapeutic compound. Methods In this study, we investigated the effect of TQ on survival, actin cytoskeletal reorganization, proliferation and signal transduction in multiple myeloma (MM) cells. Results We found that TQ induces growth arrest in both MDN and XG2 cells in a dose- and time-dependent manner. TQ also inhibited CXC ligand-12 (CXCL-12)-mediated actin polymerization and cellular proliferation, as shown by flow cytometry. The signal transducer and activator of transcription (STAT) and B-cell lymphoma-2 (Bcl-2) signaling pathways may play important roles in the malignant transformation of a number of human malignancies. The constitutive activation of the STAT3 and Bcl-2 pathways is frequently observed in several cancer cell lines, including MM cells. Using flow cytometry, we found that TQ markedly decreased STAT3 phosphorylation and Bcl-2 and Bcl-XL expression without modulating STAT5 phosphorylation in MM cells. Using western blotting, we confirmed the inhibitory effect of TQ on STAT3 phosphorylation and Bcl-2 and Bcl-XL expression. Conclusions Taken together, our data suggests that TQ could potentially be applied toward the treatment of MM and other malignancies. PMID:22177381

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

    PubMed

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

    2015-10-14

    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 f̄i(L) exerted by the wall at L and associated potential f̄i(L)=-dWi(L)/dL 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 fb(Lc,ℓp)=π(2)kBTℓp4Lc (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 by a living filament on a wall at

  8. Imaging Modulated Reflections from a Semi-Crystalline State of Profilin:Actin Crystals

    NASA Technical Reports Server (NTRS)

    Lovelace, J.; Bellamy, H.; Snell, E. H.; Borgstahl, G.

    2003-01-01

    Commensurate and incommensurate modulation in protein crystals remain terra incognita for crystallographers. While small molecule crystallographers have successfully wrestled with this type of structure, no modulated macromolecular structures have been determined to date. In this work, methods and strategies have been developed to collect and analyze data from modulated macromolecular crystals. Preliminary data using these methods are presented for a semi-crystalline state of profilin:actin.

  9. Imaging Modulated Reflections from a Semi-Crystalline State of Profilin:Actin Crystals

    NASA Technical Reports Server (NTRS)

    Lovelace, J.; Bellamy, H.; Snell, E. H.; Borgstahl, G.

    2003-01-01

    Commensurate and incommensurate modulation in protein crystals remain terra incognita for crystallographers. While small molecule crystallographers have successfully wrestled with this type of structure, no modulated macromolecular structures have been determined to date. In this work, methods and strategies have been developed to collect and analyze data from modulated macromolecular crystals. Preliminary data using these methods are presented for a semi-crystalline state of profilin:actin.

  10. Cross-linking study on skeletal muscle actin: properties of suberimidate-treated actin.

    PubMed

    Ohara, O; Takahashi, S; Ooi, T; Fujiyoshi, Y

    1982-06-01

    Cross-linking experiments were performed on muscle skeletal actin, using imidoesters of various chain lengths. Chemical analyses on all products except one (derived from succinimidate) show evidence of the presence of intramolecular cross-links in the molecule. The detailed properties of suberimidate-treated actin (SA) are as follows: SA contains nearly 1 mol of intramolecular cross-link per mol of actin and less than 15% of intermolecularly cross-linked products. Even at a low salt concentration, SA is polymeric, exchanges slowly its bound nucleotide with free nucleotides in solution, and shows an F-actin-type CD spectrum. Electron micrographs of SA reveal that SA exists actually as fibrous polymers in solutions of low ionic strength, although the fibers seem to be less rigid than those at high salt concentration. The F-form of SA at a high salt concentration is indistinguishable from intact F-actin. SA can bind heavy meromyosin and activate the ATPase of heavy meromyosin as observed for intact F-actin. Tropomyosin binds SA only at a high salt concentration. These results show that SA possesses the properties of F-actin even in media of low salt concentration, which are favorable for depolymerization of F-actin. Thus, we may infer that the conformation of SA is frozen in the F-state of actin by the introduction of intramolecular cross-links in the protein.

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

    PubMed

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

    2016-09-15

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

  12. 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.

  13. 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

  14. Interactive, Computer-Assisted Tracking of Speckle Trajectories in Fluorescence Microscopy: Application to Actin Polymerization and Membrane Fusion

    PubMed Central

    Smith, Matthew B.; Karatekin, Erdem; Gohlke, Andrea; Mizuno, Hiroaki; Watanabe, Naoki; Vavylonis, Dimitrios

    2011-01-01

    Analysis of particle trajectories in images obtained by fluorescence microscopy reveals biophysical properties such as diffusion coefficient or rates of association and dissociation. Particle tracking and lifetime measurement is often limited by noise, large mobilities, image inhomogeneities, and path crossings. We present Speckle TrackerJ, a tool that addresses some of these challenges using computer-assisted techniques for finding positions and tracking particles in different situations. A dynamic user interface assists in the creation, editing, and refining of particle tracks. The following are results from application of this program: 1), Tracking single molecule diffusion in simulated images. The shape of the diffusing marker on the image changes from speckle to cloud, depending on the relationship of the diffusion coefficient to the camera exposure time. We use these images to illustrate the range of diffusion coefficients that can be measured. 2), We used the program to measure the diffusion coefficient of capping proteins in the lamellipodium. We found values ∼0.5 μm2/s, suggesting capping protein association with protein complexes or the membrane. 3), We demonstrate efficient measuring of appearance and disappearance of EGFP-actin speckles within the lamellipodium of motile cells that indicate actin monomer incorporation into the actin filament network. 4), We marked appearance and disappearance events of fluorescently labeled vesicles to supported lipid bilayers and tracked single lipids from the fused vesicle on the bilayer. This is the first time, to our knowledge, that vesicle fusion has been detected with single molecule sensitivity and the program allowed us to perform a quantitative analysis. 5), By discriminating between undocking and fusion events, dwell times for vesicle fusion after vesicle docking to membranes can be measured. PMID:21961607

  15. Dehydroepiandrosterone sulfate and allopregnanolone directly stimulate catecholamine production via induction of tyrosine hydroxylase and secretion by affecting actin polymerization.

    PubMed

    Charalampopoulos, I; Dermitzaki, E; Vardouli, L; Tsatsanis, C; Stournaras, C; Margioris, A N; Gravanis, A

    2005-08-01

    Adrenal cortical cells of zona reticularis produce the neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester dehydroepiandrosterone sulfate (DHEAS), and allopregnanolone (ALLO). An interaction between zona reticularis and adrenal medulla has been postulated based on their close proximity and their interwoven borders. The aim of this paper was to examine in vitro the possible paracrine effects of these steroids on catecholamine production from adrenomedullary chromaffin cells, using an established in vitro model of chromaffin cells, the PC12 rat pheochromocytoma cell line. We have found the following: 1) DHEA, DHEAS, and ALLO increased acutely (peak effect between 10-30 min) and dose-dependently (EC50 in the nanomolar range) catecholamine levels (norepinephrine and dopamine). 2) It appears that the acute effect of these steroids involved actin depolymerization/actin filament disassembly, a fast-response cellular system regulating trafficking of catecholamine vesicles. Specifically, 10(-6) m phallacidin, an actin filament stabilizer, completely prevented steroid-induced catecholamine secretion. 3) DHEAS and ALLO, but not DHEA, also affected catecholamine synthesis. Indeed, DHEAS and ALLO increased catecholamine levels at 24 h, an effect blocked by L-2-methyl-3-(-4-hydroxyphenyl)alanine and 3-(hydrazinomethyl)phenol hydrochloride, inhibitors of tyrosine hydroxylase and L-aromatic amino acid decarboxylase, respectively, suggesting that this effect involved catecholamine synthesis. The latter hypothesis was confirmed by finding that DHEAS and ALLO increased both the mRNA and protein levels of tyrosine hydroxylase. In conclusion, our findings suggest that neuroactive steroids exert a direct tonic effect on adrenal catecholamine synthesis and secretion. These data associate the adrenomedullary malfunction observed in old age and neuroactive steroids.

  16. Effects of polychlorinated biphenyls, hexachlorocyclohexanes, and mercury on human neutrophil apoptosis, actin cytoskelton, and oxidative state

    USGS Publications Warehouse

    Sweet, L.I.; Passino-Reader, D. R.; Meier, P.G.; Omann, G.M.

    2006-01-01

    Apoptosis, or programmed cell death, has been proposed as a biomarker for environmental contaminant effects. In this work, we test the hypothesis that in vitro assays of apoptosis are sensitive indicators of immunological effects of polychlorinated biphenyls, hexachlorocyclohexanes, and mercury on human neutrophils. Apoptosis, necrosis, and viability as well as the related indicators F-actin levels, and active thiol state were measured in purified human neutrophils after treatment with contaminants. Effective concentrations observed were 0.3 μM (60 μg/L) mercury, 750 μg/L Aroclor 1254, and 50 μM (14,500 μg/L) hexachlorocylcohexanes. Concentrations of contaminants that induced apoptosis also decreased cellular F-actin levels. Active thiols were altered by mercury, but not organochlorines. Comparison of these data with levels of contaminants reported to be threats to human health indicate neutrophil apoptosis is a sensitive indicator of mercury toxicity.

  17. Why is Actin Patchy?

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2009-03-01

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

  18. The myosin start-of-power stroke state and how actin binding drives the power stroke.

    PubMed

    Preller, Matthias; Holmes, Kenneth C

    2013-10-01

    We propose that on binding to actin at the start of the power stroke the myosin cross-bridge takes on the rigor configuration at the actin interface. Starting from the prepower stroke state, this can be achieved by a small movement (16° rotation) of the lower 50K domain without twisting the central β-sheet or opening switch-1 or switch-2. The movement of the lower 50K domain puts a strain on the W-helix. This strain tries to twist the β-sheet, which could drive the power stroke. This would provide a coupling between actin binding and the execution of the power stroke. During the power stroke the β-sheet twists, moving the P-loop away from switch-2, which opens the nucleotide binding pocket and separates ADP from Pi . The power stroke is different from the recovery stroke because the upper and lower 50K domains are tethered in the rigor configuration. Copyright © 2013 Wiley Periodicals, Inc.

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

    PubMed Central

    Serebryannyy, Leonid A.; Parilla, Megan; Cruz, Christina M.; Laster, Kyle; Kudryashov, Dmitri; Kosak, Steven T.

    2016-01-01

    ABSTRACT Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin–RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II. PMID:27505898

  20. 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.

  1. Novel actin depolymerizing macrolide aplyronine A.

    PubMed

    Saito, S; Watabe, S; Ozaki, H; Kigoshi, H; Yamada, K; Fusetani, N; Karaki, H

    1996-09-01

    Aplyronine A is a macrolide isolated from Aplysia kurodai. By monitoring fluorescent intensity of pyrenyl-actin, it was found that aplyronine A inhibited both the velocity and the degree of actin polymerization. Aplyronine A also quickly depolymerized F-actin. The kinetics of depolymerization suggest that aplyronine A severs F-actin. The relationship between the concentration of total actin and F-actin at different concentrations of aplyronine A suggests that aplyronine A forms a 1:1 complex with G-actin. From these results, it is concluded that aplyronine A inhibits actin polymerization and depolymerizes F-actin by nibbling. Comparison of the chemical structure of aplyronine A and another actin-depolymerizing macrolide, mycalolide B, suggests that the side-chain but not the macrolide ring of aplyronine A may account for its actin binding and severing activity.

  2. 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.

  3. Structural dynamics of F-actin: II. Cooperativity in structural transitions.

    PubMed

    Orlova, A; Prochniewicz, E; Egelman, E H

    1995-02-03

    A large body of biochemical evidence suggests that the F-actin filament can have internal cooperativity. We have observed large cooperative effects on the low-resolution structure of actin filaments under three very different conditions. First, when G-Ca(2+)-actin is polymerized by both Mg2+ and KCl, filaments may be found in two different populations, with two discrete positions seen for subdomain 2. When G-Ca2+ actin is polymerized by only Mg2+, a single F-Mg(2+)-actin population is seen. The structural data suggest that an entire filament exists with subdomain 2 in one state or the other when there is a heterogenous mixture of Mg2+ and Ca(2+)-actin. Second, when actin filaments are nucleated from gelsolin there is a conformational change that can be observed throughout the filament that is consistent with a large shift in the actin C terminus. There must be a large cooperative propagation of this effect throughout the filament from the nucleation point. Third, we have used phalloidin to stabilize F-actin in which two C-terminal residues have been proteolytically removed by trypsin. It has been shown biochemically that this stabilization occurs at substoichiometric amounts of phalloidin. Phalloidin, at either a 1:1 or a 1:20 molar ratio with actin, restores the connectivity between the long-pitch helical strands. F-actin's internal cooperativity will have large implications in vivo, particularly in muscle.

  4. State of actin cytoskeleton and development of slow-frozen and vitrified rabbit pronuclear zygotes.

    PubMed

    Kulíková, Barbora; Jiménez-Trigos, Estrella; Makarevich, Alexander V; Chrenek, Peter; Vicente, José S; Marco-Jiménez, Francisco

    2016-02-01

    This study was focused on the effect of cryopreservation on the state of actin cytoskeleton and development of rabbit pronuclear zygotes. Zygotes were collected from superovulated females and immediately used for 1) slow-freezing in a solution containing 1.5 M 1,2-propanediol and 0.2 M sucrose, or 2) vitrification in a solution containing 42.0% (v/v) of ethylene glycol, 18.0% (w/v) of dextran and 0.3 M sucrose as cryoprotectants. After thawing or warming, respectively, zygotes were evaluated for 1) actin distribution, 2) in vitro or 3) in vivo development to blastocyst. Comparing actin filaments distribution, a significantly higher number of vitrified zygotes with actin distributed in cell border was observed (55 ± 7.7 vs. 74 ± 6.1% for slow-frozen vs. vitrified, respectively). After 24 and 72 h of in vitro development, significant differences in the cleavage and morula rate among the groups were observed (9 ± 2.4 and 3 ± 1.3 vs. 44 ± 3.0 and 28 ± 2.7% for slow-frozen vs. vitrified, respectively). None of the slow-frozen zygotes reached the blastocyst stage, in contrast to the vitrified counterparts (11 ± 1.9%). Under in vivo culture conditions, a significant difference in blastocyst rate was observed between vitrified and fresh embryos (6 ± 1.5 vs. 35 ± 4.4% respectively). Our results showed that alterations in actin cytoskeleton and deteriorated development are more evident in slow-frozen than vitrified pronuclear zygotes. Vitrification method seems to be a more effective option for rabbit zygotes cryopreservation, although pronuclear zygotes manipulation per se resulted in a notable decrease in embryo development. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. 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

  6. 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

    2016-03-15

    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.

  7. Fragmentation Is Crucial for the Steady-State Dynamics of Actin Filaments

    PubMed Central

    Schmoller, Kurt M.; Niedermayer, Thomas; Zensen, Carla; Wurm, Christine; Bausch, Andreas R.

    2011-01-01

    Despite the recognition that actin filaments are important for numerous cellular processes, and decades of investigation, the dynamics of in vitro actin filaments are still not completely understood. Here, we follow the time evolution of the length distribution of labeled actin reporter filaments in an unlabeled F-actin solution via fluorescence microscopy. Whereas treadmilling and diffusive length fluctuations cannot account for the observed dynamics, our results suggest that at low salt conditions, spontaneous fragmentation is crucial. PMID:21843470

  8. Interaction of Phalloidin with Actin

    PubMed Central

    Lengsfeld, Anneliese M.; Löw, Irmentraut; Wieland, Theodor; Dancker, Peter; Hasselbach, Wilhelm

    1974-01-01

    Phalloidin, a toxic bicyclic peptide of rapid action from the toadstool, Amanita phalloides, gives rise to polymerization of G-actin to filamentous structures (Ph-actin) in a medium of low ionic strength. Ph-actin closely resembles the microfilaments found in liver membrane fractions (Ph-filaments) after in vivo or in vitro poisoning. Both phalloidin induced filaments are resistant to 0.6 M KI in contrast to F-actin, and become decorated by heavy meromyosin. After preincubation with cytochalasin B significantly fewer actin filaments are observed. Images PMID:4368830

  9. 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.

  10. The impact of the current United States guidelines on the management of actinic keratosis: is it time for an update?

    PubMed

    Martin, George

    2010-11-01

    Actinic keratosis is one of the most common diagnoses made by dermatologists. Many experts recommend treating all actinic keratoses because of their potential to progress to invasive squamous cell carcinoma. Physicians have a large armamentarium of actinic keratosis treatment modalities available to them, including destructive therapies, such as cryotherapy, curettage and electrodessication, chemical peels, photodynamic therapy, and topical therapies, including 5-fluorouracil, imiquimod, and diclofenac. In addition to standardized monotherapy regimens, combinations of two concomitant or sequential therapies and alternative topical dosing regimens have been studied in a number of clinical trials. Such therapeutic courses are used to maintain or enhance efficacy while improving tolerability, convenience, and/or patient adherence. This abundance of treatment options prompted development of several actinic keratosis management guidelines. Whereas two sets of treatment guidelines were published by European organizations within the past three years, the most recent United States-based guidelines for dermatologists were published by the American Academy of Dermatology in 1995. Because they are not up to date, the 1995 United States guidelines lack recent clinical developments and an evidence rating system and can no longer effectively guide practitioners. While there are benefits and potential limitations to developing an updated set of United States-based guidelines, there is a clearly defined need for a unified, comprehensive, evidence-based guideline approach to actinic keratosis treatment that balances the need to tailor long-term management of the disease to the needs of the individual patient.

  11. Control of actin-based motility through localized actin binding

    PubMed Central

    Banigan, Edward J.; Lee, Kun-Chun; Liu, Andrea J.

    2014-01-01

    A wide variety of cell biological and biomimetic systems use actin polymerization to drive motility. It has been suggested that an object such as a bacterium can propel itself by self-assembling a high concentration of actin behind it if it is repelled by actin. However, it is also known that it is essential for the moving object to bind actin. Therefore, a key question is how the actin tail can propel an object when it both binds and repels the object. We present a physically consistent Brownian dynamics model for actin-based motility that includes the minimal components of the dendritic nucleation model and allows for both attractive and repulsive interactions between actin and a moveable disk. We find that the concentration gradient of filamentous actin generated by polymerization is sufficient to propel the object, even with moderately strong binding interactions. Additionally, actin binding can act as a biophysical cap, and may directly control motility through modulation of network growth. Overall, this mechanism is robust in that it can drive motility against a load up to a stall pressure that depends on the Young’s modulus of the actin network and can explain several aspects of actin-based motility. PMID:24225232

  12. β- and γ-Actins in the nucleus of human melanoma A375 cells.

    PubMed

    Migocka-Patrzałek, Marta; Makowiecka, Aleksandra; Nowak, Dorota; Mazur, Antonina J; Hofmann, Wilma A; Malicka-Błaszkiewicz, Maria

    2015-11-01

    Actin is a highly conserved protein that is expressed in all eukaryotic cells and has essential functions in the cytoplasm and the nucleus. Nuclear actin is involved in transcription by all three RNA polymerases, chromatin remodelling, RNA processing, intranuclear transport, nuclear export and in maintenance of the nuclear architecture. The nuclear actin level and polymerization state are important factors regulating nuclear processes such as transcription. Our study shows that, in contrast to the cytoplasm, the majority of endogenous nuclear actin is unpolymerized in human melanoma A375 cells. Most mammalian cells express the two non-muscle β- and γ-actin isoforms that differ in only four amino acids. Despite their sequence similarity, studies analysing the cytoplasmic functions of these isoforms demonstrated that β- and γ-actins show differences in localization and function. However, little is known about the involvement of the individual actin isoforms in nuclear processes. Here, we used the human melanoma A375 cell line to analyse actin isoforms in regard to their nuclear localization. We show that both β- and γ-non-muscle actin isoforms are present in nuclei of these cells. Immunolocalization studies demonstrate that both isoforms co-localize with RNA polymerase II and hnRNP U. However, we observe differences in the ratio of cytoplasmic to nuclear actin distribution between the isoforms. We show that β-actin has a significantly higher nucleus-to-cytoplasm ratio than γ-actin.

  13. Coordinated integrin activation by actin-dependent force during T-cell migration.

    PubMed

    Nordenfelt, Pontus; Elliott, Hunter L; Springer, Timothy A

    2016-10-10

    For a cell to move forward it must convert chemical energy into mechanical propulsion. Force produced by actin polymerization can generate traction across the plasma membrane by transmission through integrins to their ligands. However, the role this force plays in integrin activation is unknown. Here we show that integrin activity and cytoskeletal dynamics are reciprocally linked, where actin-dependent force itself appears to regulate integrin activity. We generated fluorescent tension-sensing constructs of integrin αLβ2 (LFA-1) to visualize intramolecular tension during cell migration. Using quantitative imaging of migrating T cells, we correlate tension in the αL or β2 subunit with cell and actin dynamics. We find that actin engagement produces tension within the β2 subunit to induce and stabilize an active integrin conformational state and that this requires intact talin and kindlin motifs. This supports a general mechanism where localized actin polymerization can coordinate activation of the complex machinery required for cell migration.

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

    PubMed

    Blotnick, Edna; Sol, Asaf; Muhlrad, Andras

    2017-01-01

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

  15. Fluorescence of fluorescein attached to myosin SH1 distinguishes the rigor state from the actin-myosin-nucleotide state.

    PubMed

    Ando, T

    1984-01-17

    It has been found that the fluorescence intensity of 5-(iodoacetamido)fluorescein (5-IAF) attached to the SH1 of myosin subfragment 1 (S-1) increases 3-fold on formation of the rigor complex. On adding Mg2+-ADP, light scattering indicates no dissociation, but the fluorescence increment disappears. Thus, this fluorescence signal can distinguish the rigor state from other states, especially from ternary complexes such as actin-myosin-nucleotide. We demonstrate that by using this signal we can measure spectroscopically several kinetic parameters of acto-S-1-nucleotide interaction: In the presence of 20 mM KCl, 2 mM MgSO4, and 10 mM TES (pH 7.5) at 22 degrees C, Mg2+-ADP binds to acto-S-1(5)* (S-1(5) denotes 5-IAF-labeled S-1) with a Ka = 2 X 10(6) M-1, and Mg2+-PPi binds to acto-S-1(5)* with two apparent affinities, Ka = 8 X 10(4) M-1 and 1.4 X 10(3) M-1; the association rates of Mg2+-ADP and Mg2+-ATP for acto-S-1(5)* are 10(7) s-1 M-1 and 4 X 10(6) s-1 M-1, respectively, and the dissociation rate of Mg2+-ADP for acto-S-1(5)* is 5 s-1. In contrast to the fluorescence intensity of the dye, the lifetime and the absorbance are essentially unaffected by complex formation with F-actin or nucleotides. Therefore, we conclude that there must be a static quencher such as Trp, Tyr, or Met in the neighborhood of the attached dye and that the contact between dye and quencher is modulated by actin-induced or nucleotide-induced conformational changes in S-1.

  16. Plasma membrane calcium ATPase activity is regulated by actin oligomers through direct interaction.

    PubMed

    Dalghi, Marianela G; Fernández, Marisa M; Ferreira-Gomes, Mariela; Mangialavori, Irene C; Malchiodi, Emilio L; Strehler, Emanuel E; Rossi, Juan Pablo F C

    2013-08-09

    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 Ca(2+) with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]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 Ca(2+)-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca(2+)-ATPase activity was related to an increase in the apparent affinity for Ca(2+) 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 Ca(2+) homeostasis.

  17. 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

  18. Labeling F-actin barbed ends with rhodamine-actin in permeabilized neuronal growth cones.

    PubMed

    Marsick, Bonnie M; Letourneau, Paul C

    2011-03-17

    The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant. Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells. When rhodamine-actin is added at a concentration above the critical concentration

  19. Effects of recombinant baculovirus AcMNPV-BmK IT on the formation of early cables and nuclear polymerization of actin in Sf9 cells.

    PubMed

    Fu, Yuejun; Lin, Taotao; Liang, Aihua; Hu, Fengyun

    2016-05-01

    Autographa californica nuclearpoly hedrosis virus (AcMNPV) is one of the most important baculoviridae. However, the application of AcMNPV as a biocontrol agent has been limited. Previously, we engineered Buthus martensii Karsch insect toxin (BmK IT) gene into the genome of AcMNPV. The bioassay data indicated that the recombinant baculovirus AcMNPV-BmK IT significantly enhanced the anti-insect efficacy of the virus. The actin cytoskeleton is the major component beneath the surface of eukaryotic cells. In this report, the effects of AcMNPV-BmK IT on the formation of early cables of actin and nuclear filamentous-actin (F-actin) were studied. The results indicated that these baculovirus induced rearrangement of the actin cytoskeleton of host cells during infection and actin might participate in the transportation of baculovirus from cytoplasm to the nuclei. AcMNPV-BmK IT delayed the formation of early cables of actin and nuclear F-actin and accelerated the clearance of actin in the nuclei.

  20. Excited-state Raman spectroscopy with and without actinic excitation: S{sub 1} Raman spectra of trans-azobenzene

    SciTech Connect

    Dobryakov, A. L.; Quick, M.; Ioffe, I. N.; Granovsky, A. A.; Ernsting, N. P.; Kovalenko, S. A.

    2014-05-14

    We show that femtosecond stimulated Raman spectroscopy can record excited-state spectra in the absence of actinic excitation, if the Raman pump is in resonance with an electronic transition. The approach is illustrated by recording S{sub 1} and S{sub 0} spectra of trans-azobenzene in n-hexane. The S{sub 1} spectra were also measured conventionally, upon nπ* (S{sub 0} → S{sub 1}) actinic excitation. The results are discussed and compared to earlier reports.

  1. Synthetic mimetics of actin-binding macrolides: rational design of actin-targeted drugs.

    PubMed

    Perrins, Richard D; Cecere, Giuseppe; Paterson, Ian; Marriott, Gerard

    2008-03-01

    Actin polymerization and dynamics are involved in a wide range of cellular processes such as cell division and migration of tumor cells. At sites of cell lysis, such as those occurring during a stroke or inflammatory lung diseases, actin is released into the serum where it polymerizes, leading to problems with clot dissolution and sputum viscosity. Therefore, drugs that target these actin-mediated processes may provide one mechanism to treat these conditions. Marine-organism-derived macrolides, such as reidispongiolide A, can bind to, sever, and inhibit polymerization of actin. Our studies show that the function of these complex macrolides resides in their tail region, whereas the head group stabilizes the actin-drug complex. Synthetic compounds derived from this tail region could therefore be used as a mimetic of the natural product, providing a range of designer compounds to treat actin-associated diseases or as probes to study actin polymerization.

  2. Load Adaptation of Lamellipodial Actin Networks.

    PubMed

    Mueller, Jan; Szep, Gregory; Nemethova, Maria; de Vries, Ingrid; Lieber, Arnon D; Winkler, Christoph; Kruse, Karsten; Small, J Victor; Schmeiser, Christian; Keren, Kinneret; Hauschild, Robert; Sixt, Michael

    2017-09-21

    Actin filaments polymerizing against membranes power endocytosis, vesicular traffic, and cell motility. In vitro reconstitution studies suggest that the structure and the dynamics of actin networks respond to mechanical forces. We demonstrate that lamellipodial actin of migrating cells responds to mechanical load when membrane tension is modulated. In a steady state, migrating cell filaments assume the canonical dendritic geometry, defined by Arp2/3-generated 70° branch points. Increased tension triggers a dense network with a broadened range of angles, whereas decreased tension causes a shift to a sparse configuration dominated by filaments growing perpendicularly to the plasma membrane. We show that these responses emerge from the geometry of branched actin: when load per filament decreases, elongation speed increases and perpendicular filaments gradually outcompete others because they polymerize the shortest distance to the membrane, where they are protected from capping. This network-intrinsic geometrical adaptation mechanism tunes protrusive force in response to mechanical load. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Actin Dynamics: From Nanoscale to Microscale

    PubMed Central

    Carlsson, Anders E.

    2010-01-01

    The dynamic nature of actin in cells manifests itself in many ways: Polymerization near the cell edge is balanced by depolymerization in the interior, externally induced actin polymerization is followed by depolymerization, and spontaneous oscillations of 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 rate of actin incorporation at the leading edge of a moving cell is roughly consistent with existing theories, and the factors determining the characteristic time of actin polymerization are fairly well understood. However, our understanding of actin disassembly is limited, in particular the interplay between severing and depolymerization and the role of specific combinations of proteins in implementing disassembly events. The origins of cell-edge oscillations, and their possible relation to actin waves, are a fruitful area of future research. PMID:20462375

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

    PubMed Central

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

    2004-01-01

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

  5. Reversible Membrane Pearling in Live Cells upon Destruction of the Actin Cortex

    PubMed Central

    Heinrich, Doris; Ecke, Mary; Jasnin, Marion; Engel, Ulrike; Gerisch, Günther

    2014-01-01

    Membrane pearling in live cells is observed when the plasma membrane is depleted of its support, the cortical actin network. Upon efficient depolymerization of actin, pearls of variable size are formed, which are connected by nanotubes of ∼40 nm diameter. We show that formation of the membrane tubes and their transition into chains of pearls do not require external tension, and that they neither depend on microtubule-based molecular motors nor pressure generated by myosin-II. Pearling thus differs from blebbing. The pearling state is stable as long as actin is prevented from polymerizing. When polymerization is restored, the pearls are retracted into the cell, indicating continuity of the membrane. Our data suggest that the alternation of pearls and strings is an energetically favored state of the unsupported plasma membrane, and that one of the functions of the actin cortex is to prevent the membrane from spontaneously assuming this configuration. PMID:24606932

  6. Identification of another actin-related protein (Arp) 2/3 complex binding site in neural Wiskott-Aldrich syndrome protein (N-WASP) that complements actin polymerization induced by the Arp2/3 complex activating (VCA) domain of N-WASP.

    PubMed

    Suetsugu, S; Miki, H; Takenawa, T

    2001-08-31

    Neural Wiskott-Aldrich syndrome protein (N-WASP) is an essential regulator of actin cytoskeleton formation via its association with the actin-related protein (Arp) 2/3 complex. It is believed that the C-terminal Arp2/3 complex-activating domain (verprolin homology, cofilin homology, and acidic (VCA) or C-terminal region of WASP family proteins domain) of N-WASP is usually kept masked (autoinhibition) but is opened upon cooperative binding of upstream regulators such as Cdc42 and phosphatidylinositol 4,5-bisphosphate (PIP2). However, the mechanisms of autoinhibition and association with Arp2/3 complex are still unclear. We focused on the acidic region of N-WASP because it is thought to interact with Arp2/3 complex and may be involved in autoinhibition. Partial deletion of acidic residues from the VCA portion alone greatly reduced actin polymerization activity, demonstrating that the acidic region contributes to Arp2/3 complex-mediated actin polymerization. Surprisingly, the same partial deletion of the acidic region in full-length N-WASP led to constitutive activity comparable with the activity seen with the VCA portion. Therefore, the acidic region in full-length N-WASP plays an indispensable role in the formation of the autoinhibited structure. This mutant contains WASP-homology (WH) 1 domain with weak affinity to the Arp2/3 complex, leading to activity in the absence of part of the acidic region. Furthermore, the actin comet formed by the DeltaWH1 mutant of N-WASP was much smaller than that of wild-type N-WASP. Partial deletion of acidic residues did not affect actin comet size, indicating the importance of the WH1 domain in actin structure formation. Collectively, the acidic region of N-WASP plays an essential role in Arp2/3 complex activation as well as in the formation of the autoinhibited structure, whereas the WH1 domain complements the activation of the Arp2/3 complex achieved through the VCA portion.

  7. Differential Effects of G- and F-Actin on the Plasma Membrane Calcium Pump Activity

    PubMed Central

    Vanagas, Laura; de La Fuente, María Candelaria; Dalghi, Marianela; Ferreira-Gomes, Mariela; Rossi, Rolando C.; Strehler, Emanuel E.; Rossi, Juan P. F. C.

    2014-01-01

    We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641–1644, 2007). Results show evidences for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca2+ extrusion through the membrane. Our results provide further evidence of the activation–inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts. PMID:23152090

  8. Cofilin-mediated actin dynamics promotes actin bundle formation during Drosophila bristle development

    PubMed Central

    Wu, Jing; Wang, Heng; Guo, Xuan; Chen, Jiong

    2016-01-01

    The actin bundle is an array of linear actin filaments cross-linked by actin-bundling proteins, but its assembly and dynamics are not as well understood as those of the branched actin network. Here we used the Drosophila bristle as a model system to study actin bundle formation. We found that cofilin, a major actin disassembly factor of the branched actin network, promotes the formation and positioning of actin bundles in the developing bristles. Loss of function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate the positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated. PMID:27385345

  9. 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.

  10. Regulation of the actin cycle in vivo by actin filament severing

    PubMed Central

    McGrath, James L.; Osborn, Eric A.; Tardy, Yanik S.; Dewey, C. Forbes; Hartwig, John H.

    2000-01-01

    Cycling of actin subunits between monomeric and filamentous phases is essential for cell crawling behavior. We investigated actin filament turnover rates, length, number, barbed end exposure, and binding of cofilin in bovine arterial endothelial cells moving at different speeds depending on their position in a confluent monolayer. Fast-translocating cells near the wound edge have short filament lifetimes compared with turnover values that proportionately increase in slower moving cells situated at increasing distances from the wound border. Contrasted with slow cells exhibiting slow actin filament turnover speeds, fast cells have less polymerized actin, shorter actin filaments, more free barbed ends, and less actin-associated cofilin. Cultured primary fibroblasts manifest identical relationships between speed and actin turnover as the endothelial cells, and fast fibroblasts expressing gelsolin have higher actin turnover rates than slow fibroblasts that lack this actin-severing protein. These results implicate actin filament severing as an important control mechanism for actin cycling in cells. PMID:10823888

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

    PubMed

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

    2000-06-01

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

  12. Ca2+ regulation of gelsolin activity: binding and severing of F-actin.

    PubMed Central

    Kinosian, H J; Newman, J; Lincoln, B; Selden, L A; Gershman, L C; Estes, J E

    1998-01-01

    Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow. PMID:9826630

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  16. Actinic keratosis

    MedlinePlus

    Solar keratosis; Sun-induced skin changes - keratosis; Keratosis - actinic (solar); Skin lesion - actinic keratosis ... likely to develop it if you: Have fair skin, blue or green eyes, or blond or red ...

  17. Actin: Structure, Function, Dynamics, and Interactions with Bacterial Toxins.

    PubMed

    Kühn, Sonja; Mannherz, Hans Georg

    Actin is one of the most abundant proteins in any eukaryotic cell and an indispensable component of the cytoskeleton. In mammalian organisms, six highly conserved actin isoforms can be distinguished, which differ by only a few amino acids. In non-muscle cells, actin polymerizes into actin filaments that form actin structures essential for cell shape stabilization, and participates in a number of motile activities like intracellular vesicle transport, cytokinesis, and also cell locomotion. Here, we describe the structure of monomeric and polymeric actin, the polymerization kinetics, and its regulation by actin-binding proteins. Probably due to its conserved nature and abundance, actin and its regulating factors have emerged as prefered targets of bacterial toxins and effectors, which subvert the host actin cytoskeleton to serve bacterial needs.

  18. A change in actin conformation associated with filament instability after Pi release.

    PubMed

    Belmont, L D; Orlova, A; Drubin, D G; Egelman, E H

    1999-01-05

    The ability of actin to both polymerize into filaments and to depolymerize permits the rapid rearrangements of actin structures that are essential for actin's function in most cellular processes. Filament polarity and dynamic properties are conferred by the hydrolysis of ATP on actin filaments. Release of inorganic phosphate (Pi) from filaments after ATP hydrolysis promotes depolymerization. We identify a yeast actin mutation, Val-159 to Asn, which uncouples Pi release from the conformational change that results in filament destabilization. Three-dimensional reconstructions of electron micrographs reveal a conformational difference between ADP-Pi filaments and ADP filaments and show that ADP V159N filaments resemble ADP-Pi wild-type filaments. Crystal structures of mammalian beta-actin in which the nucleotide binding cleft is in the "open" and "closed" states can be used to model actin filaments in the ADP and ADP-Pi conformations, respectively. We propose that these two conformations of G-actin may be related to two functional states of F-actin.

  19. Soft Listeria: actin-based propulsion of liquid drops.

    PubMed

    Boukellal, Hakim; Campás, Otger; Joanny, Jean-François; Prost, Jacques; Sykes, Cécile

    2004-06-01

    We study the motion of oil drops propelled by actin polymerization in cell extracts. Drops deform and acquire a pearlike shape under the action of the elastic stresses exerted by the actin comet, a tail of cross-linked actin filaments. We solve this free boundary problem and calculate the drop shape taking into account the elasticity of the actin gel and the variation of the polymerization velocity with normal stress. The pressure balance on the liquid drop imposes a zero propulsive force if gradients in surface tension or internal pressure are not taken into account. Quantitative parameters of actin polymerization are obtained by fitting theory to experiment.

  20. From dense monomer salt crystals to CO2 selective microporous polyimides via solid-state polymerization.

    PubMed

    Unterlass, Miriam M; Emmerling, Franziska; Antonietti, Markus; Weber, Jens

    2014-01-14

    Fully aromatic polyimides are synthesized via solid-state polymerization of the corresponding monomer salts. The crystal structure of salts shows strong hydrogen bonding of the reactive groups and thereby paves the way for solid-state transformations. The polycondensation yields copies of the initial salt crystallite habits, accompanied by the development of a porosity especially suited for CO2.

  1. 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.

  2. 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.

  3. Dynamic interaction between actin and nesprin2 maintain the cell nucleus in a prestressed state

    NASA Astrophysics Data System (ADS)

    Kumar, Abhishek; Shivashankar, G. V.

    2016-12-01

    Mechanical coupling between the nucleus and the cytoskeleton is indispensable for direct force transduction from the extra cellular matrix (ECM) to the chromatin. Although this physical coupling has been shown to be crucial for nuclear positioning and its function, the quantification of nuclear-cytoskeleton interaction has been lacking. In this paper, using various quantitative fluorescence spectroscopy techniques, we investigate the nature of this connection. High-resolution 3D imaging shows that nesprin2G forms short linear structures along actin stress fibers (ASFs) in the apical region of the nucleus. Fluorescence recovery after photobleaching (FRAP) revealed that the alignment of nesprin2G becomes heterogeneous when cell shape is engineered from elongated rectangular shape to square using micropatterned substrates. Further, fluorescence cross-correlation spectroscopy (FCCS) revealed that actin interacts transiently with outer nuclear membrane protein nesprin2G with a time scale of 12 ms. In addition, fluorescence resonance energy transfer (FRET) experiments show that the apical ASFs and nesprin2G are in close physical proximity. This interaction is spatially heterogeneous with high FRET along the ASFs. Lastly, we show that the disruption of actin to nuclear connection by over-expression of Dominant Negative Klarsicht, ANC-1, Syne Homology (DNKASH) leads to an increase in nuclear height. These results not only reveal the characteristics of actin-nesprin2G interaction and its significance in regulating nuclear morphology, but also validate the utility of quantitative fluorescence techniques in deciphering physical connections that are essential for mechanotransduction.

  4. S-NO-actin: S-nitrosylation kinetics and the effect on isolated vascular smooth muscle.

    PubMed

    Dalle-Donne, I; Milzani, A; Giustarini, D; Di Simplicio, P; Colombo, R; Rossi, R

    2000-02-01

    We describe the modification of reactive actin sulfhydryls by S-nitrosoglutathione. Kinetics of S-nitrosylation and denitrosylation suggest that only one cysteine of actin is involved in the reactions. By using the bifunctional sulfhydryl cross-linking reagent N,N'-1,4-phenylenebismaleimide and the monofunctional reagent N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine, we identified this residue as Cys374. The time course of filament formation followed by high-shear viscosity changes revealed that S-nitrosylated G-actin polymerizes less efficiently than native monomers. The observed decrease in specific viscosity at steady state is due mainly to a marked inhibition of filament end-to-end annealing and, partially, to a reduction in F-actin concentration. Finally, S-nitrosylated actin acts as nitric oxide donor showing a fast, potent vasodilating activity at unusually low concentrations, being comparable with that of low molecular weight nitrosothiols.

  5. Actin-Based Feedback Circuits in Cell Migration and Endocytosis

    NASA Astrophysics Data System (ADS)

    Wang, Xinxin

    In this thesis, we study the switch and pulse functions of actin during two important cellular processes, cell migration and endocytosis. Actin is an abundant protein that can polymerize to form a dendritic network. The actin network can exert force to push or bend the cell membrane. During cell migration, the actin network behaves like a switch, assembling mostly at one end or at the other end. The end with the majority of the actin network is the leading edge, following which the cell can persistently move in the same direction. The other end, with the minority of the actin network, is the trailing edge, which is dragged by the cell as it moves forward. When subjected to large fluctuations or external stimuli, the leading edge and the trailing edge can interchange and change the direction of motion, like a motion switch. Our model of the actin network in a cell reveals that mechanical force is crucial for forming the motion switch. We find a transition from single state symmetric behavior to switch behavior, when tuning parameters such as the force. The model is studied by both stochastic simulations, and a set of rate equations that are consistent with the simulations. Endocytosis is a process by which cells engulf extracellular substances and recycle the cell membrane. In yeast cells, the actin network is transiently needed to overcome the pressure difference across the cell membrane caused by turgor pressure. The actin network behaves like a pulse, which assembles and then disassembles within about 30 seconds. Using a stochastic model, we reproduce the pulse behaviors of the actin network and one of its regulatory proteins, Las17. The model matches green fluorescence protein (GFP) experiments for wild-type cells. The model also predicts some phenotypes that modify or diminish the pulse behavior. The phenotypes are verified with both experiments performed at Washington University and with other groups' experiments. We find that several feedback mechanisms are

  6. 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

  7. State-dependent diffusion of actin-depolymerizing factor/cofilin underlies the enlargement and shrinkage of dendritic spines

    PubMed Central

    Noguchi, Jun; Hayama, Tatsuya; Watanabe, Satoshi; Ucar, Hasan; Yagishita, Sho; Takahashi, Noriko; Kasai, Haruo

    2016-01-01

    Dendritic spines are the postsynaptic sites of most excitatory synapses in the brain, and spine enlargement and shrinkage give rise to long-term potentiation and depression of synapses, respectively. Because spine structural plasticity is accompanied by remodeling of actin scaffolds, we hypothesized that the filamentous actin regulatory protein cofilin plays a crucial role in this process. Here we investigated the diffusional properties of cofilin, the actin-severing and depolymerizing actions of which are activated by dephosphorylation. Cofilin diffusion was measured using fluorescently labeled cofilin fusion proteins and two-photon imaging. We show that cofilins are highly diffusible along dendrites in the resting state. However, during spine enlargement, wild-type cofilin and a phosphomimetic cofilin mutant remain confined to the stimulated spine, whereas a nonphosphorylatable mutant does not. Moreover, inhibition of cofilin phosphorylation with a competitive peptide disables spine enlargement, suggesting that phosphorylated-cofilin accumulation is a key regulator of enlargement, which is localized to individual spines. Conversely, spine shrinkage spreads to neighboring spines, even though triggered by weaker stimuli than enlargement. Diffusion of exogenous cofilin injected into a pyramidal neuron soma causes spine shrinkage and reduced PSD95 in spines, suggesting that diffusion of dephosphorylated endogenous cofilin underlies the spreading of spine shrinkage and long-term depression. PMID:27595610

  8. 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

  9. Reconstituted polymeric materials derived from post-consumer waste, industrial scrap and virgin resins made by solid state shear pulverization

    DOEpatents

    Khait, Klementina

    2001-01-30

    A method of making polymeric particulates wherein polymeric scrap material, virgin polymeric material and mixtures thereof are supplied to intermeshing extruder screws which are rotated to transport the polymeric material along their length and subject the polymeric material to solid state shear pulverization and in-situ polymer compatibilization, if two or more incompatible polymers are present. Uniform pulverized particulates are produced without addition of a compatibilizing agent. The pulverized particulates are directly melt processable (as powder feedstock) and surprisingly yield a substantially homogeneous light color product.

  10. Reconstituted polymeric materials derived from post-consumer waste, industrial scrap and virgin resins made by solid state pulverization

    DOEpatents

    Khait, Klementina

    1998-09-29

    A method of making polymeric particulates wherein polymeric scrap material, virgin polymeric material and mixtures thereof are supplied to intermeshing extruder screws which are rotated to transport the polymeric material along their length and subject the polymeric material to solid state shear pulverization and in-situ polymer compatibilization, if two or more incompatible polymers are present. Uniform pulverized particulates are produced without addition of a compatibilizing agent. The pulverized particulates are directly melt processable (as powder feedstock) and surprisingly yield a substantially homogeneous light color product.

  11. Reconstituted Polymeric Materials Derived From Post-Consumer Waste, Industrial Scrap And Virgin Resins Made By Solid State Shear Pulverizat

    DOEpatents

    Khait, Klementina

    2005-02-01

    A method of making polymeric particulates wherein polymeric scrap material, virgin polymeric material and mixtures thereof are supplied to intermeshing extruder screws which are rotated to transport the polymeric material along their length and subject the polymeric material to solid state shear pulverization and in-situ polymer compatibilization, if two or more incompatible polymers are present. Uniform pulverized particulates are produced without addition of a compatibilizing agent. The pulverized particulates are directly melt processable (as powder feedstock) and surprisingly yield a substantially homogeneous light color product.

  12. Reconstituted polymeric materials derived from post-consumer waste, industrial scrap and virgin resins made by solid state pulverization

    DOEpatents

    Khait, K.

    1998-09-29

    A method of making polymeric particulates is described wherein polymeric scrap material, virgin polymeric material and mixtures thereof are supplied to intermeshing extruder screws which are rotated to transport the polymeric material along their length and subject the polymeric material to solid state shear pulverization and in-situ polymer compatibilization, if two or more incompatible polymers are present. Uniform pulverized particulates are produced without addition of a compatible agent. The pulverized particulates are directly melt processable (as powder feedstock) and surprisingly yield a substantially homogeneous light color product. 29 figs.

  13. Dye Photodestruction in a Solid-State Dye Laser with a Polymeric Gain Medium

    NASA Astrophysics Data System (ADS)

    Popov, Sergei

    1998-09-01

    The process of dye photodestruction in a solid-state dye laser is studied, and implemented is a polymeric gain medium doped with a strongly concentrated dye. The behavior of the conversion efficiency in the polymeric gain medium pumped with different laser-pulse repetition rates and the process of dye photobleaching are analyzed. The contribution of the heating of the host material into the dye molecules deactivation is discussed. The negative effect of high dye concentration on the dye stability under a high pump repetition rate is reported and analyzed for the first time to my knowledge. A comparison of the present results with recently published data demonstrates the major role of photodestruction, rather than direct thermodestruction, in the dye stability of the solid-state gain medium. The role of additives with low molecular weights in the polymeric matrix, for increasing the stability of the gain material, is discussed.

  14. Dye photodestruction in a solid-state dye laser with a polymeric gain medium.

    PubMed

    Popov, S

    1998-09-20

    The process of dye photodestruction in a solid-state dye laser is studied, and implemented is a polymeric gain medium doped with a strongly concentrated dye. The behavior of the conversion efficiency in the polymeric gain medium pumped with different laser-pulse repetition rates and the process of dye photobleaching are analyzed. The contribution of the heating of the host material into the dye molecules' deactivation is discussed. The negative effect of high dye concentration on the dye stability under a high pump repetition rate is reported and analyzed for the first time to my knowledge. A comparison of the present results with recently published data demonstrates the major role of photodestruction, rather than direct thermodestruction, in the dye stability of the solid-state gain medium. The role of additives with low molecular weights in the polymeric matrix, for increasing the stability of the gain material, is discussed.

  15. 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.

  16. Crowded surfaces change annealing dynamics of actin filaments.

    PubMed

    Popp, David; Yamamoto, Akihiro; Maéda, Yuichiro

    2007-04-27

    Changes in cell shape that occur in many cellular processes are thought to arise from polymerization of actin filaments near the cell membrane. End-to-end annealing of actin filaments is believed to play only a minor role in this process, as annealing in solution was shown to be a slow process, which is not typical for a bimolecular reaction, its rate constant decreasing over time, being inversely proportional to the filament length. Furthermore, in vitro studies on f-actin solutions were found to display an exponential steady-state length distribution. In the cell, many physiologically important parameters, such as mechanical strength or viscoelastic response are a direct function of the physical properties of the underlying actin cytoskeleton, such as actin filament length distribution and dynamics. How the underlying physical parameters of the actin cytoskeleton may be influenced by the cell surface or molecular crowding remains poorly understood. Using total internal reflection fluorescence (TIRF) microscopy we reinvestigated actin end-to-end annealing in vitro in a more realistic environment. We studied the process near a hydrophilic surface together with crowding agents, in order to mimic the physiological media near the cell membrane, which has substantial amounts of macromolecules present. We find that actin end-to-end annealing changes in three ways near a crowded hydrophilic surface as compared to solution. First the annealing rate becomes a factor of 20 faster than in solution. Second the rate of annealing becomes typical of a bimolecular reaction, shows no length dependence and is basically just a function of the square of the concentration of ends. Lastly the length distribution is Gaussian throughout the entire annealing process. This implicates that dynamic rearrangement of actin filaments by annealing near the leading edge of the cell, could change physical parameters like the mechanical response and contribute significantly to cell motility.

  17. Bacterial Actins and Their Interactors.

    PubMed

    Gayathri, Pananghat

    2017-01-01

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

  18. Dendritic Actin Nucleation Causes Traveling Waves and Patches

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2010-03-01

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

  19. Polycation induced actin bundles.

    PubMed

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

    2011-04-01

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

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

    PubMed

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

    1984-05-01

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

  1. 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

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

    PubMed

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

    2011-04-12

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

  3. Toxofilin, a Novel Actin-binding Protein from Toxoplasma gondii, Sequesters Actin Monomers and Caps Actin Filaments

    PubMed Central

    Poupel, Olivier; Boleti, Haralabia; Axisa, Sophie; Couture-Tosi, Evelyne; Tardieux, Isabelle

    2000-01-01

    Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end. PMID:10637313

  4. Control of actin filament length by phosphorylation of fragmin-actin complex

    PubMed Central

    1990-01-01

    Fragmin is a Ca2(+)-sensitive F-actin-severing protein purified from a slime mold, Physarum polycephalum (Hasegawa, T., S. Takahashi, H. Hayashi, and S. Hatano. 1980. Biochemistry. 19:2677-2683). It binds to G-actin to form a 1:1 fragmin/actin complex in the presence of micromolar free Ca2+. The complex nucleates actin polymerization and caps the barbed end of the short F-actin (Sugino, H., and S. Hatano. 1982. Cell Motil. 2:457-470). Subsequent removal of Ca2+, however, hardly dissociates the complex. This complex nucleates actin polymerization and caps the F-actin regardless of Ca2+ concentration. Here we report that this activity of fragmin-actin complex can be abolished by phosphorylation of actin of the complex. When crude extract from Physarum plasmodium was incubated with 5 mM ATP and 1 mM EGTA, the activities of the complex decreased to a great extent. The inactivation of the complex in the crude extract was not observed in the presence of Ca2+. In addition, the activities of the complex inactivated in the crude extract were restored under conditions suitable for phosphatase reactions. We purified factors that inactivated fragmin-actin complex from the crude extract. These factors phosphorylated actin of the complex, and the activities of the complex decreased with an increased level of phosphorylation of the complex. These factors, termed actin kinase, also inactivated the complex that capped the barbed end of short F-actin, leading to elongation of the short F-actin to long F-actin. Thus the length of F-actin can be controlled by phosphorylation of fragmin-actin complex by actin kinase. PMID:2202733

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

    PubMed Central

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

    2011-01-01

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

  6. Conformational polymorphs and solid-state polymerization of 9-(1,3-butadiynyl)carbazole derivatives

    NASA Astrophysics Data System (ADS)

    Tabata, Hideyuki; Kuwamoto, Kazunori; Okuno, Tsunehisa

    2016-02-01

    The novel diacetylenes, 9-(5-(4-nitrophenoxy)penta-1,3-diyn-1-yl)-9H-carbazole (1) and 4-((5-(9H-carbazol-9-yl)penta-2,4-diyn-1-yl)oxy)benzonitrile (2), were prepared and characterized by crystallographic analyses. Compound 1 gave two conformational polymorphs, 1-(I) and 1-(II), whose differences were concluded to originate in intermolecular interactions among nitrophenyl groups. Crystal 1-(I) and 2 had suitable molecular arrangements for solid-state polymerization and polymerized by thermal annealing to give crystalline polydiacetylenes (PDAs). While an arrangement of 1-(II) was unsuitable for the polymerization. The PDAs showed broad absorption from UV to near IR region (ca. 900 nm), suggesting effective expansion of π-conjugated system by carbazolyl groups.

  7. [Actinic keratoses].

    PubMed

    Babilas, P; Landthaler, M; Szeimies, R-M

    2003-06-01

    Actinic keratoses are defined as proliferation of cytologically atypical keratinocytes in the zone of epidermal-dermal junction in photodamaged skin. In the northern hemisphere the prevalence of actinic keratoses ranges depending on different epidemiological studies from 11% to 25% for people aged 40 or older. The main cause of actinic keratoses is exposure to UVB radiation in sunlight UVB radiation induces mutations in the telomerase gene and in the tumor suppressor gene P53, which can also be detected in invasive squamous cell carcinoma. The only histological parameter to distinguish between actinic keratoses and SCC is the level of invasiveness. The risk for actinic keratoses to develop into SCC is about 16% over lo years. For this reason and because of the high prevalence of actinic keratoses, it has been suggested to replace the term,, actinic keratosis K with intraepidermal squamous cell carcinoma' to better characterize the lesion. In the following review recent aspects of pathogenesis and therapy of actinic keratoses are discussed.

  8. Density of conformon states in a disordered polymeric crystal

    NASA Astrophysics Data System (ADS)

    Klinskikh, A. F.

    1999-03-01

    Following Volkenstein's idea the density of conformon states (rho) (E) is investigated. Using the path-integral method in conditions of the strong conformational disorder the numerical account (rho) (E) is carried out. The results of account (rho) (E) under the new formula are discussed.

  9. Structural Differences Explain Diverse Functions of Plasmodium Actins

    PubMed Central

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

    2014-01-01

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

  10. Crystal structure of a nuclear actin ternary complex

    PubMed Central

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

    2016-01-01

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

  11. Raman Study of Solid State Reactions in Polymeric Materials.

    DTIC Science & Technology

    1981-08-01

    and dynamics of the conducting complex: ( Benzophenone )9 (KI)2 .17 CHCl3" Some preliminary work was done on this novel group of conducting materials...the conducting complex: ( Benzophenone )9 (KI)2 17 CHCl3. Some preliminary work was done on this novel group of conducting materials. (e) Phonon...investigate photochemistry in solid state of two systems which are prototypes of two broad classes of compounds. The first reaction is the photodimerization

  12. Thermal unfolding and aggregation of actin.

    PubMed

    Levitsky, Dmitrii I; Pivovarova, Anastasiya V; Mikhailova, Valeria V; Nikolaeva, Olga P

    2008-09-01

    Actin is one of the most abundant proteins in nature. It is found in all eukaryotes and plays a fundamental role in many diverse and dynamic cellular processes. Also, actin is one of the most ubiquitous proteins because actin-like proteins have recently been identified in bacteria. Actin filament (F-actin) is a highly dynamic structure that can exist in different conformational states, and transitions between these states may be important in cytoskeletal dynamics and cell motility. These transitions can be modulated by various factors causing the stabilization or destabilization of actin filaments. In this review, we look at actin stabilization and destabilization as expressed by changes in the thermal stability of actin; specifically, we summarize and analyze the existing data on the thermal unfolding of actin as measured by differential scanning calorimetry. We also analyze in vitro data on the heat-induced aggregation of actin, the process that normally accompanies actin thermal denaturation. In this respect, we focus on the effects of small heat shock proteins, which can prevent the aggregation of thermally denatured actin with no effect on actin thermal unfolding. As a result, we have proposed a mechanism describing the thermal denaturation and aggregation of F-actin. This mechanism explains some of the special features of the thermal unfolding of actin filaments, including the effects of their stabilization and destabilization; it can also explain how small heat shock proteins protect the actin cytoskeleton from damage caused by the accumulation of large insoluble aggregates under heat shock conditions.

  13. Actin binding proteins and spermiogenesis

    PubMed Central

    Mruk, Dolores D

    2011-01-01

    Drebrin E, an actin-binding protein lacking intrinsic activity in the regulation of actin dynamics (e.g., polymerization, capping, nucleation, branching, cross-linking, bundling and severing), is known to recruit actin regulatory proteins to a specific cellular site. Herein, we critically evaluate recent findings in the field which illustrate that drebrin E works together with two other actin-binding proteins, namely Arp3 (actin-related protein 3, a component of the Arp2/3 complex that simultaneously controls actin nucleation for polymerization and branching of actin filaments) and Eps8 (epidermal growth factor receptor pathway substrate 8 that controls capping of the barbed ends of actin filaments, as well as actin filament bundling) to regulate the homeostasis of F-actin filament bundles at the ectoplasmic specialization (ES), a testis-specific atypical adherens junction (AJ) in the seminiferous epithelium. This is mediated by the strict temporal and spatial expression of these three actin-binding proteins at the apical and basal ES at the Sertoli cell-spermatid (step 8–19) and Sertoli-Sertoli cell interface, respectively, during the seminiferous epithelial cycle of spermatogenesis. In this Commentary, we put forth a possible model by which drebrin E may be acting as a platform upon which proteins (e.g., Arp3) that are needed to alter the conformation of actin filament bundles at the ES can be recruited to the site, thus facilitating changes in cell shape and cell position in the epithelium during spermiogenesis and spermiation. In short, drebrin E may be acting as a “logistic” distribution center to manage different regulatory proteins at the apical ES, thereby regulating the dynamics of actin filament bundles and modulating the plasticity of the apical ES. This would allow adhesion to be altered continuously throughout the epithelial cycle to accommodate spermatid movement in the seminiferous epithelium during spermiogenesis and spermiation. We also

  14. Microscopy basics and the study of actin-actin-binding protein interactions.

    PubMed

    Thomasson, Maggie S; Macnaughtan, Megan A

    2013-12-15

    Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin-ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. 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.

  16. Mesoscopic Model of Actin-Based Propulsion

    PubMed Central

    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

  17. Instantaneous inactivation of cofilin reveals its function of F-actin disassembly in lamellipodia.

    PubMed

    Vitriol, Eric A; Wise, Ariel L; Berginski, Mathew E; Bamburg, James R; Zheng, James Q

    2013-07-01

    Cofilin is a key regulator of the actin cytoskeleton. It can sever actin filaments, accelerate filament disassembly, act as a nucleation factor, recruit or antagonize other actin regulators, and control the pool of polymerization-competent actin monomers. In cells these actions have complex functional outputs. The timing and localization of cofilin activity are carefully regulated, and thus global, long-term perturbations may not be sufficient to probe its precise function. To better understand cofilin's spatiotemporal action in cells, we implemented chromophore-assisted laser inactivation (CALI) to instantly and specifically inactivate it. In addition to globally inhibiting actin turnover, CALI of cofilin generated several profound effects on the lamellipodia, including an increase of F-actin, a rearward expansion of the actin network, and a reduction in retrograde flow speed. These results support the hypothesis that the principal role of cofilin in lamellipodia at steady state is to break down F-actin, control filament turnover, and regulate the rate of retrograde flow.

  18. 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

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

    PubMed Central

    Ti, Shih-Chieh; Pollard, Thomas D.

    2011-01-01

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

  20. A model of cross-bridge attachment to actin in the A*M*ATP state based on x-ray diffraction from permeabilized rabbit psoas muscle.

    PubMed Central

    Gu, Jin; Xu, Sengen; Yu, Leepo C

    2002-01-01

    A model of cross-bridges binding to actin in the weak binding A*M*ATP state is presented. The modeling was based on the x-ray diffraction patterns from the relaxed skinned rabbit psoas muscle fibers where ATP hydrolysis was inhibited by N-phenylmaleimide treatment (S. Xu, J. Gu, G. Melvin, L. C. Yu. 2002. Biophys. J. 82:2111-2122). Calculations included both the myosin filaments and the actin filaments of the muscle cells, and the binding to actin was assumed to be single headed. To achieve a good fit, considerable flexibility in the orientation of the myosin head and the position of the S1-S2 junction is necessary, such that the myosin head can bind to a nearby actin whereas the tail end was kept in the proximity of the helical track of the myosin filament. Hence, the best-fit model shows that the head binds to actin in a wide range of orientations, and the tail end deviates substantially from its lattice position in the radial direction (approximately 60 A). Surprisingly, the best fit model reveals that the detached head, whose location thus far has remained undetected, seems to be located close to the surface of the myosin filament. Another significant requirement of the best-fit model is that the binding site on actin is near the N terminus of the actin subunit, a position distinct from the putative rigor-binding site. The results support the idea that the essential role played by the weak binding states M*ATP <--> A*M*ATP for force generation lies in its flexibility, because the probability of attachment is greatly increased, compared with the weak binding M*ADP*P(i) <--> A*M*ADP*P(i) states. PMID:11916868

  1. Role of ATP-bound divalent metal ion in the conformation and function of actin. Comparison of Mg-ATP, Ca-ATP, and metal ion-free ATP-actin.

    PubMed

    Valentin-Ranc, C; Carlier, M F

    1991-04-25

    The fluorescence of N-acetyl-N'-(sulfo-1-naphthyl)ethylenediamine (AEDANS) covalently bound to Cys-374 of actin is used as a probe for different conformational states of G-actin according to whether Ca-ATP, Mg-ATP, or unchelated ATP is bound to the nucleotide site. Upon addition of large amounts (greater than 10(2)-fold molar excess) of EDTA to G-actin, metal ion-free ATP-G-actin is obtained with EDTA bound. Metal ion free ATP-G-actin is characterized by a higher AEDANS fluorescence than Mg-ATP-G-actin, which itself has a higher fluorescence than Ca-ATP-G-actin. Evidence for EDTA binding to G-actin is shown using difference spectrophotometry. Upon binding of EDTA, the rate of dissociation of the divalent metal ion from G-actin is increased (2-fold for Ca2+, 10-fold for Mg2+) in a range of pH from 7.0 to 8.0. A model is proposed that quantitatively accounts for the kinetic data. The affinity of ATP is weakened 10(6)-fold upon removal of the metal ion. Metal ion-free ATP-G-actin is in a partially open conformation, as indicated by the greater accessibility of -SH residues, yet it retains functional properties of polymerization and ATP hydrolysis that appear almost identical to those of Ca-ATP-actin, therefore different from those of Mg-ATP-actin. These results are discussed in terms of the role of the ATP-bound metal ion in actin structure and function.

  2. Mechanics model for actin-based motility.

    PubMed

    Lin, Yuan

    2009-02-01

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

  3. 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.

  4. 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.

  5. p38 mitogen-activated protein kinase mediates hyperosmolarity-induced vasoconstriction through myosin light chain phosphorylation and actin polymerization in rat aorta.

    PubMed

    Sasahara, Tomoya; Yayama, Katsutoshi; Okamoto, Hiroshi

    2013-01-01

    Hyperosmotic stress induces the contractile response of vascular smooth muscle cells (VSMCs). Previous studies have demonstrated that cytoskeleton reorganization and Rho/Rho-kinase-mediated inactivation of myosin light chain phosphatase (MLCP) play an important role in hyperosmotic vasoconstriction, but the precise mechanism is unknown. This study aimed to investigate the contractile response of endothelium-denuded rings of rat aortas to hyperosmolar sucrose (160 mM) in the presence or absence of inhibitors for various protein kinases. We found that the hyperosmotic constriction of aortic rings was attenuated not only by ML-7 or hydroxyfasudil, specific inhibitor for myosin light chain kinase (MLCK) or Rho-kinase, respectively, but also by SB203580, a specific inhibitor for p38 mitogen-activated kinase (p38 MAPK). Hyperosmolar sucrose evoked a transient increase in cytosolic free Ca(2+) in rat VSMCs, and this response was not affected by SB203580. Western blot analysis of proteins extracted from rings showed that the hyperosmolar sucrose stimulated phosphorylation of the Rho-kinase-mediated myosin phosphatase target subunit 1, myosin light chain (MLC), and p38 MAPK. The experiments performed using a combination of the kinase inhibitors showed that hyperosmolarity-induced MLC phosphorylation is partially mediated via the SB203580-sensitive pathway and is independent of both MLCK and Rho-kinase-mediated inactivation of MLCP. Furthermore, the hyperosmolarity-induced increase in the F-actin/G-actin ratio in rings was attenuated not only by hydroxyfasudil but also by SB203580. These results suggest that p38 MAPK is involved in hyperosmotic vasoconstriction via stimulation of MLC phosphorylation and cytoskeleton reorganization through pathways independent of activation of MLCK and/or Rho-kinase-mediated mechanisms.

  6. 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

  7. Polymer dynamics and fluid flow in actin-based cell motility

    NASA Astrophysics Data System (ADS)

    Theriot, Julie

    2005-03-01

    In living cells, nonequilibrium protein polymerization reactions are frequently used to convert chemical energy into mechanical energy and thereby generate useful force for cellular movements. We have examined the polymer and fluid dynamics in two biological cases where the assembly of branched actin filament networks generates force: the intracellular movement of the bacterial pathogen Listeria monocytogenes, and the extension of the leading edge of skin epithelial cells during wound-healing. In both cases, net actin filament assembly occurs at the front of the network structure and net disassembly occurs at the rear. Actin protein subunits and other network components must be recycled through the fluid phase to the front of the polymerizing network in order for forward movement to continue at steady state. For actin-based movement of Listeria monocytogenes, we have found that actin recycling is not rate-limiting; instead, the speed of movement is governed by the cooperative dissociation of groups of noncovalent protein-protein bonds attaching the filamentous network to the bacterial surface. In contrast, rapid actin-based extension at the leading edge of moving epithelial cells is associated with unusual perturbations in intracellular fluid flow.

  8. Actin-related proteins in Actinobacillus pleuropneumoniae and their interactions with actin-binding proteins.

    PubMed

    Guerrero-Barrera, A L; de la Garza, M; Mondragón, R; García-Cuéllar, C; Segura-Nieto, M

    1999-11-01

    A group of prokaryotic actin-related proteins (PARP) with an Mr of 43000 was detected in Actinobacillus pleuropneumoniae. These proteins were enriched by a depolymerization/polymerization cycle, under similar conditions to those used to polymerize muscle actin, and purified by affinity chromatography on a DNase I-Sepharose column. Three isoforms of A. pleuropneumoniae PARP (Ap-PARP) with pI values of 5.8, 6.15 and 6.2 were detected. Ap-PARP were recognized by four different anti-actin antibodies (one anti-muscle and three anti-cytoplasmic isoforms). Ap-PARP were also recognized by antibodies against Anabaena variabilis PARP (Av-PARP) and against actin-binding proteins such as alpha-actinin and spectrin, and also by a monoclonal antibody against heat-shock cognate protein 70 (Hsc70). Specific binding of phalloidin to Ap-PARP was detected both in permeabilized cells and in vitro. Purified Ap-PARP can polymerize under similar conditions to those required for skeletal muscle actin polymerization and the filaments formed appear to be decorated with myosin subfragment-1(S1) as observed by transmission electron microscopy. The amino acid composition of Ap-PARP revealed more similarities to muscle gamma-actin and the cytoplasmic beta-actin isoform than to eukaryotic actin-related proteins.

  9. 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.

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

    PubMed

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

    2012-06-15

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

  11. Novel Actin-like Filament Structure from Clostridium tetani*

    PubMed Central

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

    2012-01-01

    Eukaryotic F-actin is constructed from two protofilaments that gently wind around each other to form a helical polymer. Several bacterial actin-like proteins (Alps) are also known to form F-actin-like helical arrangements from two protofilaments, yet with varied helical geometries. Here, we report a unique filament architecture of Alp12 from Clostridium tetani that is constructed from four protofilaments. Through fitting of an Alp12 monomer homology model into the electron microscopy data, the filament was determined to be constructed from two antiparallel strands, each composed of two parallel protofilaments. These four protofilaments form an open helical cylinder separated by a wide cleft. The molecular interactions within single protofilaments are similar to F-actin, yet interactions between protofilaments differ from those in F-actin. The filament structure and assembly and disassembly kinetics suggest Alp12 to be a dynamically unstable force-generating motor involved in segregating the pE88 plasmid, which encodes the lethal tetanus toxin, and thus a potential target for drug design. Alp12 can be repeatedly cycled between states of polymerization and dissociation, making it a novel candidate for incorporation into fuel-propelled nanobiopolymer machines. PMID:22514279

  12. Autographa californica Multiple Nucleopolyhedrovirus Ac34 Protein Retains Cellular Actin-Related Protein 2/3 Complex in the Nucleus by Subversion of CRM1-Dependent Nuclear Export

    PubMed Central

    Mu, Jingfang; Zhang, Yongli; Hu, Yangyang; Hu, Xue; Zhou, Yuan; Pei, Rongjuan; Wu, Chunchen; Chen, Jizheng; van Oers, Monique M.; Chen, Xinwen; Wang, Yun

    2016-01-01

    Actin, nucleation-promoting factors (NPFs), and the actin-related protein 2/3 complex (Arp2/3) are key elements of the cellular actin polymerization machinery. With nuclear actin polymerization implicated in ever-expanding biological processes and the discovery of the nuclear import mechanisms of actin and NPFs, determining Arp2/3 nucleo-cytoplasmic shuttling mechanism is important for understanding the function of nuclear actin. A unique feature of alphabaculovirus infection of insect cells is the robust nuclear accumulation of Arp2/3, which induces actin polymerization in the nucleus to assist in virus replication. We found that Ac34, a viral late gene product encoded by the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is involved in Arp2/3 nuclear accumulation during virus infection. Further assays revealed that the subcellular distribution of Arp2/3 under steady-state conditions is controlled by chromosomal maintenance 1 (CRM1)-dependent nuclear export. Upon AcMNPV infection, Ac34 inhibits CRM1 pathway and leads to Arp2/3 retention in the nucleus. PMID:27802336

  13. Steady-state helices of the actin homolog MreB inside bacteria: Dynamics without motors

    NASA Astrophysics Data System (ADS)

    Allard, Jun F.; Rutenberg, Andrew D.

    2007-09-01

    Within individual bacteria, we combine force-dependent polymerization dynamics of individual MreB protofilaments with an elastic model of protofilament bundles buckled into helical configurations. We use variational techniques and stochastic simulations to relate the pitch of the MreB helix, the total abundance of MreB, and the number of protofilaments. By comparing our simulations with mean-field calculations, we find that stochastic fluctuations are significant. We examine the quasistatic evolution of the helical pitch with cell growth, as well as time scales of helix turnover and de novo establishment. We find that while the body of a polarized MreB helix treadmills toward its slow-growing end, the fast-growing tips of laterally associated protofilaments move toward the opposite fast-growing end of the MreB helix. This offers a possible mechanism for targeted polar localization without cytoplasmic motor proteins.

  14. Effect of mesoscale ordering on the density of States of polymeric semiconductors.

    PubMed

    Gemünden, Patrick; Poelking, Carl; Kremer, Kurt; Daoulas, Kostas; Andrienko, Denis

    2015-06-01

    A multiscale simulation scheme, which incorporates both long-range conformational disorder and local molecular ordering, is proposed for predicting large-scale morphologies and charge transport properties of polymeric semiconductors. Using poly(3-hexylthiophene) as an example, it is illustrated how the energy landscape and its spatial correlations evolve with increasing degree of structural order in mesophases with amorphous, uniaxial, and biaxial nematic ordering. It is shown that the formation of low-lying energy states in more ordered systems is mostly due to larger (on average) conjugation lengths and not due to electrostatic interactions. The proposed scheme is general and can be applied to a wide range of polymeric organic materials. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed Central

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

    2003-01-01

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

  16. Actin-Regulator Feedback Interactions during Endocytosis

    PubMed Central

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

    2016-01-01

    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

  17. 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.

  18. Cytoskeletal Actin Gates a Cl− Channel in Neocortical Astrocytes

    PubMed Central

    Lascola, Christopher D.; Nelson, Deborah J.; Kraig, Richard P.

    2009-01-01

    Increases in astroglial Cl− conductance accompany changes in cell morphology and disassembly of cytoskeletal actin, but Cl− channels underlying these conductance increases have not been described. We characterize an outwardly rectifying Cl− channel in rodent neocortical cultured astrocytes and describe how cell shape and cytoskeletal actin modulate channel gating. In inside-out patch-clamp recordings from cultured astrocytes, outwardly rectifying Cl− channels either were spontaneously active or inducible in quiescent patches by depolarizing voltage steps. Average single-channel conductance was 36 pS between −60 and −80 mV and was 75 pS between 60 and 80 mV in symmetrical (150 mm NaCl) solutions. The permeability ratio (PNa/PCl) was 0.14 at lower ionic strength but increased at higher salt concentrations. Both ATP and 4,4-diisothiocyanostilbene-2,2′-disulfonic acid produced a flicker block, whereas Zn2+ produced complete inhibition of channel activity. The frequency of observing both spontaneous and inducible Cl− channel activity was markedly higher in stellate than in flat, polygonally shaped astrocytes. In addition, cytoskeletal actin modulated channel open-state probability (PO) and conductance at negative membrane potentials, controlling the degree of outward rectification. Direct application of phalloidin, which stabilizes actin, preserved low PO and promoted lower conductance levels at negative potentials. Lower PO also was induced by direct application of polymerized actin. The actions of phalloidin and actin were reversed by coapplication of gelsolin and cytochalasin D, respectively. These results provide the first report of an outwardly rectifying Cl− channel in neocortical astrocytes and demonstrate how changes in cell shape and cytoskeletal actin may control Cl− conductance in these cells. PMID:9464993

  19. Cytoskeletal actin gates a Cl- channel in neocortical astrocytes.

    PubMed

    Lascola, C D; Nelson, D J; Kraig, R P

    1998-03-01

    Increases in astroglial Cl- conductance accompany changes in cell morphology and disassembly of cytoskeletal actin, but Cl- channels underlying these conductance increases have not been described. We characterize an outwardly rectifying Cl- channel in rodent neocortical cultured astrocytes and describe how cell shape and cytoskeletal actin modulate channel gating. In inside-out patch-clamp recordings from cultured astrocytes, outwardly rectifying Cl- channels either were spontaneously active or inducible in quiescent patches by depolarizing voltage steps. Average single-channel conductance was 36 pS between -60 and -80 mV and was 75 pS between 60 and 80 mV in symmetrical (150 mM NaCl) solutions. The permeability ratio (PNa/PCl) was 0.14 at lower ionic strength but increased at higher salt concentrations. Both ATP and 4, 4-diisothiocyanostilbene-2,2'-disulfonic acid produced a flicker block, whereas Zn2+ produced complete inhibition of channel activity. The frequency of observing both spontaneous and inducible Cl- channel activity was markedly higher in stellate than in flat, polygonally shaped astrocytes. In addition, cytoskeletal actin modulated channel open-state probability (PO) and conductance at negative membrane potentials, controlling the degree of outward rectification. Direct application of phalloidin, which stabilizes actin, preserved low PO and promoted lower conductance levels at negative potentials. Lower PO also was induced by direct application of polymerized actin. The actions of phalloidin and actin were reversed by coapplication of gelsolin and cytochalasin D, respectively. These results provide the first report of an outwardly rectifying Cl- channel in neocortical astrocytes and demonstrate how changes in cell shape and cytoskeletal actin may control Cl- conductance in these cells.

  20. 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.

  1. Functional characterization of skeletal F-actin labeled on the NH2-terminal segment of residues 1-28.

    PubMed

    Bertrand, R; Chaussepied, P; Audemard, E; Kassab, R

    1989-05-15

    Rabbit skeletal alpha-actin was covalently labeled in the filamentous state by the fluorescent nucleophile, N-(5-sulfo-1-naphthyl)ethylenediamine (EDANS) in the presence of the carboxyl group activator 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide (EDC). The coupling reaction was continued until the incorporation of nearly 1 mol EDANS/mol actin. After limited proteolytic digestion of the labeled protein and chromatographic identification of the EDANS-peptides, about 80% of the attached fluorophore was found on the actin segment of residues 1-28, most probably within the N-terminal acidic region of residues 1-7. A minor labeling site was located on the segment that consists of residues 40-113. No label was incorporated into the COOH-terminal moiety consisting of residues 113-375. The isolated EDANS-G-actin undergoes polymerization in the presence of salts but at a rate significantly greater than unlabeled actin. The EDANS-F-actin could be complexed to skeletal chymotryptic myosin subfragment 1 (S-1) and to tropomyosin. The complex formed between EDANS-F-actin and S-1 could not be further crosslinked by EDC but the two proteins were readily joined by glutaraldehyde as observed for native actin-S-1, suggesting that the EDANS-substituted carboxyl site is also involved in the EDC crosslinking of native actin to S-1. Moreover, the EDANS labeling of F-actin resulted in a 20-fold increase in the Km of the actin-activated Mg2+.ATPase of S-1. Thus, this labeling, while it did not much affect the rigor actin-S-1 interaction, changes the actin binding to the S-1-nucleotide complexes significantly. The selective introduction of a variety of spectral probes, like EDANS, or other classes of fluorophores, on the N-terminal region of actin, through the reported carbodiimide coupling reaction, would provide several different derivatives valuable for assessing the functional role of the negatively charged N-terminus of actin during its interaction with myosin and other actin

  2. Synthesis, Biophysical Properties and Pharmacokinetics of Ultrahigh Molecular Weight Tense and Relaxed State Polymerized Bovine Hemoglobins

    PubMed Central

    Buehler, Paul W.; Zhou, Yipin; Cabrales, Pedro; Jia, Yiping; Sun, Guoyong; Harris, David R.; Tsai, Amy; Intaglietta, Marcos; Palmer, Andre F.

    2010-01-01

    Hemoglobin-based oxygen carriers (HBOC) are currently being developed as red blood cell (RBC) substitutes for use in transfusion medicine. Despite significant commercial development, late stage clinical results of polymerized hemoglobin (PolyHb) solutions hamper development. We synthesized two types of PolyHbs with ultrahigh molecular weights: tense (T) state PolyHb (MW = 16.59 MDa and P50 = 41 mm Hg) and relaxed (R) state PolyHb (MW = 26.33 MDa and P50 = 0.66 mm Hg). By maintaining Hb in either the T- or R-state during the polymerization reaction, we were able to synthesize ultrahigh molecular weight PolyHbs in distinct quaternary states with no tetrameric Hb, high viscosity, low colloid osmotic pressure and the ability to maintain O2 dissociation, CO association and NO dioxygenation reactions. The PolyHbs elicited some in vitro RBC aggregation that was less than 6% dextran (500 kDa) but more than 5% human serum albumin. In vitro, T-state PolybHb autoxidized faster than R-state PolybHb as expected from previously reported studies, conversely, when administered to guinea pigs as a 20% exchange transfusion, R-state PolybHb oxidized faster and to a greater extent than T-state PolybHb, suggesting a more complex oxidative processes in vivo. Our findings also demonstrate that T-state PolybHb exhibited a longer circulating half-life, slower clearance and longer systemic exposure time compared to R-state PolybHb. PMID:20149433

  3. Biomimetic systems for studying actin-based motility.

    PubMed

    Upadhyaya, Arpita; van Oudenaarden, Alexander

    2003-09-16

    Actin polymerization provides a major driving force for eukaryotic cell motility. Successive intercalation of monomeric actin subunits between the plasma membrane and the filamentous actin network results in protrusions of the membrane enabling the cell to move or to change shape. One of the challenges in understanding eukaryotic cell motility is to dissect the elementary biochemical and biophysical steps that link actin polymerization to mechanical force generation. Recently, significant progress was made using biomimetic, in vitro systems that are inspired by the actin-based motility of bacterial pathogens such as Listeria monocytogenes. Polystyrene microspheres and synthetic phospholipid vesicles coated with proteins that initiate actin polymerization display motile behavior similar to Listeria, mimicking the leading edge of lamellipodia and filopodia. A major advantage of these biomimetic systems is that both biochemical and physical parameters can be controlled precisely. These systems provide a test bed for validating theoretical models on force generation and polarity establishment resulting from actin polymerization. In this review, we discuss recent experimental progress using biomimetic systems propelled by actin polymerization and discuss these results in the light of recent theoretical models on actin-based motility.

  4. Actin from pig and rat uterus.

    PubMed Central

    Elce, J S; Elbrecht, A S; Middlestadt, M U; McIntyre, E J; Anderson, P J

    1981-01-01

    Smooth-muscle actin was isolated from pig uterus and from pregnant-rat uterus. Methods involving acetone-dried powders were unsuccessful, and a column-chromatographic procedure was developed, with proteinase inhibitors and avoiding polymerization as a purification step. The yield of pure actin was 0.8--1.5 mg/g wet wt. of uterus, which should be compared with an expected yield of actin from skeletal muscle of 2--4 mg/g wet wt. The actin was pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and exhibited alpha-, beta-, and gamma-forms on isoelectric focusing. It possessed a blocked N-terminal amino acid residue, and its amino acid analysis conformed to those of other actins. The rat uterine actin was available only in small amounts (5--10 mg) and did not polymerize. The pig uterine actin could be obtained in amounts up to 30 mg, polymerized reversibly, and activated a skeletal myosin Mg2+-dependent ATPase. Images Fig. 2. Fig. 4. PMID:6458278

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

    PubMed Central

    Jorques, María; Rada, Patricia; Ramirez, Lorena; Valverde, Ángela M.; Nebreda, Ángel R.; Sastre, Juan

    2017-01-01

    Background Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. Methods Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. Results We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation. Although the steady state levels of cyclin D1 in wild type and p38α knock out old livers remained unaffected, cyclin B1- a marker for G2/M transition- was significantly overexpressed in p38α knock out mice. Our findings suggest that hepatocytes do enter into S phase but they do not complete cell division upon p38α deficiency leading to cytokinesis failure and binucleation. Moreover, old liver-specific p38α MAPK knock out mice exhibited reduced F-actin polymerization and a dramatic loss of actin cytoskeleton. This was associated with abnormal hyperactivation of RhoA and Cdc42 GTPases. Long-term p38α deficiency drives to inactivation of HSP27, which seems to account for the impairment in actin cytoskeleton as Hsp27-silencing decreased the number and length of actin filaments in isolated hepatocytes. Conclusions p38α MAPK is essential for actin dynamics with age in hepatocytes. PMID:28166285

  6. Evaluation of Resource Utilization and Treatment Patterns in Patients with Actinic Keratosis in the United States.

    PubMed

    Asche, Carl V; Zografos, Panagiotis; Norlin, Jenny M; Urbanek, Bill; Mamay, Carl; Makin, Charles; Erntoft, Sandra; Chen, Chi-Chang; Hines, Dionne M; Mark Siegel, Daniel

    2016-01-01

    To compare health care resource utilization and treatment patterns between patients with actinic keratosis (AK) treated with ingenol mebutate gel (IngMeb) and those treated with other field-directed AK therapies. A retrospective, propensity-score-matched, cohort study compared refill/repeat and adding-on/switching patterns and outpatient visits and prescriptions (health care resource utilization) over 6 months in patients receiving IngMeb versus those receiving imiquimod, 5-fluorouracil, diclofenac sodium, and methyl aminolevulinate or aminolevulinic acid photodynamic therapy (MAL/ALA-PDT). The final sample analyzed included four matched treatment cohort pairs (IngMeb and comparator; n = 790-971 per treatment arm). Refill rates were similar except for imiquimod (15% vs. 9% for imiquimod and IngMeb, respectively; P < 0.05). MAL/ALA-PDT treatment repetition rates were higher than IngMeb refill rates (20% vs. 10%; P < 0.05). Topical agent add-on/switch rates were comparable. PDT had higher switch rates than did IngMeb (5% vs. 2%; P < 0.05). The IngMeb cohort had a significantly lower proportion of patients with at least one AK-related outpatient visit during the 6-month follow-up than did any other cohort: versus imiquimod (50% vs. 66%; P < 0.0001), versus 5-fluorouracil (50% vs. 69%; P < 0.0001), versus diclofenac sodium (51% vs. 56%; P = 0.034), and versus MAL/ALA-PDT (50% vs. 100%; P < 0.0001). There were significantly fewer AK-related prescriptions among patients receiving IngMeb than among patients in other cohorts. Results based on the first 6 months after treatment initiation suggested that most field-directed AK therapies had clinically comparable treatment patterns except imiquimod, which was associated with higher refill rates, and PDT, which was associated with significantly more frequent treatment sessions and higher switching rates. IngMeb was also associated with significantly fewer outpatient visits than were other field-directed therapies. Copyright

  7. 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

  8. Bacterial Actins.

    PubMed

    Izoré, Thierry; van den Ent, Fusinita

    2017-01-01

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

  9. Statistics of actin-propelled trajectories in noisy environments

    NASA Astrophysics Data System (ADS)

    Wen, Fu-Lai; Chen, Hsuan-Yi; Leung, Kwan-tai

    2016-06-01

    Actin polymerization is ubiquitously utilized to power the locomotion of eukaryotic cells and pathogenic bacteria in living systems. Inevitably, actin polymerization and depolymerization proceed in a fluctuating environment that renders the locomotion stochastic. Previously, we have introduced a deterministic model that manages to reproduce actin-propelled trajectories in experiments, but not to address fluctuations around them. To remedy this, here we supplement the deterministic model with noise terms. It enables us to compute the effects of fluctuating actin density and forces on the trajectories. Specifically, the mean-squared displacement (MSD) of the trajectories is computed and found to show a super-ballistic scaling with an exponent 3 in the early stage, followed by a crossover to a normal, diffusive scaling of exponent 1 in the late stage. For open-end trajectories such as straights and S-shaped curves, the time of crossover matches the decay time of orientational order of the velocities along trajectories, suggesting that it is the spreading of velocities that leads to the crossover. We show that the super-ballistic scaling of MSD arises from the initial, linearly increasing correlation of velocities, before time translational symmetry is established. When the spreading of velocities reaches a steady state in the long-time limit, short-range correlation then yields a diffusive scaling in MSD. In contrast, close-loop trajectories like circles exhibit localized periodic motion, which inhibits spreading. The initial super-ballistic scaling of MSD arises from velocity correlation that both linearly increases and oscillates in time. Finally, we find that the above statistical features of the trajectories transcend the nature of noises, be it additive or multiplicative, and generalize to other self-propelled systems that are not necessarily actin based.

  10. Serpin Inhibition Mechanism: A Delicate Balance between Native Metastable State and Polymerization.

    PubMed

    Khan, Mohammad Sazzad; Singh, Poonam; Azhar, Asim; Naseem, Asma; Rashid, Qudsia; Kabir, Mohammad Anaul; Jairajpuri, Mohamad Aman

    2011-01-01

    The serpins (serine proteinase inhibitors) are structurally similar but functionally diverse proteins that fold into a conserved structure and employ a unique suicide substrate-like inhibitory mechanism. Serpins play absolutely critical role in the control of proteases involved in the inflammatory, complement, coagulation and fibrinolytic pathways and are associated with many conformational diseases. Serpin's native state is a metastable state which transforms to a more stable state during its inhibitory mechanism. Serpin in the native form is in the stressed (S) conformation that undergoes a transition to a relaxed (R) conformation for the protease inhibition. During this transition the region called as reactive center loop which interacts with target proteases, inserts itself into the center of β-sheet A to form an extra strand. Serpin is delicately balanced to perform its function with many critical residues involved in maintaining metastability. However due to its typical mechanism of inhibition, naturally occurring serpin variants produces conformational instability that allows insertion of RCL of one molecule into the β-sheet A of another to form a loop-sheet linkage leading to its polymerization and aggregation. Thus understanding the molecular basis and amino acid involved in serpin polymerization mechanism is critical to devising strategies for its cure.

  11. Serpin Inhibition Mechanism: A Delicate Balance between Native Metastable State and Polymerization

    PubMed Central

    Khan, Mohammad Sazzad; Singh, Poonam; Azhar, Asim; Naseem, Asma; Rashid, Qudsia; Kabir, Mohammad Anaul; Jairajpuri, Mohamad Aman

    2011-01-01

    The serpins (serine proteinase inhibitors) are structurally similar but functionally diverse proteins that fold into a conserved structure and employ a unique suicide substrate-like inhibitory mechanism. Serpins play absolutely critical role in the control of proteases involved in the inflammatory, complement, coagulation and fibrinolytic pathways and are associated with many conformational diseases. Serpin's native state is a metastable state which transforms to a more stable state during its inhibitory mechanism. Serpin in the native form is in the stressed (S) conformation that undergoes a transition to a relaxed (R) conformation for the protease inhibition. During this transition the region called as reactive center loop which interacts with target proteases, inserts itself into the center of β-sheet A to form an extra strand. Serpin is delicately balanced to perform its function with many critical residues involved in maintaining metastability. However due to its typical mechanism of inhibition, naturally occurring serpin variants produces conformational instability that allows insertion of RCL of one molecule into the β-sheet A of another to form a loop-sheet linkage leading to its polymerization and aggregation. Thus understanding the molecular basis and amino acid involved in serpin polymerization mechanism is critical to devising strategies for its cure. PMID:22312466

  12. Model of Yeast Actin Cable Distribution and Dynamics

    NASA Astrophysics Data System (ADS)

    Tang, Haosu; Vavylonis, Dimitrios

    2013-03-01

    The growth of fission yeast relies on the polymerization of actin filaments at the cell tips. These filaments are nucleated by formin proteins that localize at tip cortical sites. These actin filaments bundle to form actin cables that span the cell and guide the movement of vesicles toward the cell tips. Since fluorescence microscopy shows the structure and dynamics of actin cables, we are able to compare the results of the theoretical models of actin cables to experiment, thus enabling quantitative tests of the mechanisms of actin polymerization in cells. We used computer simulations to study the spatial and dynamical properties of actin cables. We simulated individual actin filaments as three-dimensional semiflexible polymer, composed of beads connected with springs. Formin polymerization was simulated as filament growth out of cortical sites located at cell tips. Actin filament severing by cofilin was simulated as filament turnover. We added attractive interactions between beads to simulate filament bundling by actin cross-linkers such as fimbrin. Comparison of the results of the model to prior experiments suggests that filament severing, nucleation and crosslinking are sufficient to describe the many features of actin cables. We found bundled and unbundled phases as cross-linking strength was varied and propose experiments to test the model predictions.

  13. Dendritic Actin Filament Nucleation Causes Traveling Waves and Patches

    PubMed Central

    Carlsson, Anders E

    2010-01-01

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

  14. Coordinated integrin activation by actin-dependent force during T-cell migration

    PubMed Central

    Nordenfelt, Pontus; Elliott, Hunter L.; Springer, Timothy A.

    2016-01-01

    For a cell to move forward it must convert chemical energy into mechanical propulsion. Force produced by actin polymerization can generate traction across the plasma membrane by transmission through integrins to their ligands. However, the role this force plays in integrin activation is unknown. Here we show that integrin activity and cytoskeletal dynamics are reciprocally linked, where actin-dependent force itself appears to regulate integrin activity. We generated fluorescent tension-sensing constructs of integrin αLβ2 (LFA-1) to visualize intramolecular tension during cell migration. Using quantitative imaging of migrating T cells, we correlate tension in the αL or β2 subunit with cell and actin dynamics. We find that actin engagement produces tension within the β2 subunit to induce and stabilize an active integrin conformational state and that this requires intact talin and kindlin motifs. This supports a general mechanism where localized actin polymerization can coordinate activation of the complex machinery required for cell migration. PMID:27721490

  15. 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.

  16. A novel solid state photocatalyst for living radical polymerization under UV irradiation.

    PubMed

    Fu, Qiang; McKenzie, Thomas G; Ren, Jing M; Tan, Shereen; Nam, Eunhyung; Qiao, Greg G

    2016-02-11

    This study presents the development of a novel solid state photocatalyst for the photoinduced controlled radical polymerization of methacrylates under mild UV irradiation (λmax ≈ 365 nm) in the absence of conventional photoinitiators, metal-catalysts or dye sensitizers. The photocatalyst design was based on our previous finding that organic amines can act in a synergistic photochemical reaction with thiocarbonylthio compounds to afford well controlled polymethacrylates under UV irradiation. Therefore, in the current contribution an amine-rich polymer was covalently grafted onto a solid substrate, thus creating a heterogeneous catalyst that would allow for facile removal, recovery and recyclability when employed for such photopolymerization reactions. Importantly, the polymethacrylates synthesized using the solid state photocatalyst (ssPC) show similarly excellent chemical and structural integrity as those catalysed by free amines. Moreover, the ssPC could be readily recovered and re-used, with multiple cycles of polymerization showing minimal effect on the integrity of the catalyst. Finally, the ssPC was employed in various photo-"click" reactions, permitting high yielding conjugations under photochemical control.

  17. Thermally Stimulated Discharge Current Analysis of Polymeric Solid-State Ionic Conductors

    NASA Astrophysics Data System (ADS)

    Xu, Haisheng; Gu, Qingchao; Fan, Maosen; Yang, Changzheng

    1997-06-01

    Thermally Stimulated Discharge Current (TSDC) analysis was used to study the electrical properties of two systems of polymeric solid-state ionic conductors. One system consisted of LiClO4 doped polyethylene glycol polyurethaneureas (PEUU) characterized by bi-ionic conduction and the other system was based on a sulfonated polyethylene oxide polyurethane ionomer (SPUI) characterized by a single-ion transport mechanism. TSDC spectra of the PEUU as well as SPUI matrices showed four peaks (, , , and ). These peaks are found to be related to the local motion of polyether soft segments, glass transition, Maxwell-Wagner-Sillars interfacial polarization and transport of charged ionic species, respectively. The discharge currents without polarization varying with temperature for both, the PEUU-LiClO4 complex and SPUI, are also measured. These curves of discharge current show only one peak which is attributed to transport of charged ionic species. The temperature range in which the discharge current exhibits a steep increase is consistent with the temperature range in which a turning point appears on the plot of lg i versus 1000/T obtained by ac impedance analysis. The experimental results demonstrate that the TSDC analysis is a valuable tool for studying polymeric solid-state ionic conductors.

  18. A novel solid state photocatalyst for living radical polymerization under UV irradiation

    PubMed Central

    Fu, Qiang; McKenzie, Thomas G.; Ren, Jing M.; Tan, Shereen; Nam, Eunhyung; Qiao, Greg G.

    2016-01-01

    This study presents the development of a novel solid state photocatalyst for the photoinduced controlled radical polymerization of methacrylates under mild UV irradiation (λmax ≈ 365 nm) in the absence of conventional photoinitiators, metal-catalysts or dye sensitizers. The photocatalyst design was based on our previous finding that organic amines can act in a synergistic photochemical reaction with thiocarbonylthio compounds to afford well controlled polymethacrylates under UV irradiation. Therefore, in the current contribution an amine-rich polymer was covalently grafted onto a solid substrate, thus creating a heterogeneous catalyst that would allow for facile removal, recovery and recyclability when employed for such photopolymerization reactions. Importantly, the polymethacrylates synthesized using the solid state photocatalyst (ssPC) show similarly excellent chemical and structural integrity as those catalysed by free amines. Moreover, the ssPC could be readily recovered and re-used, with multiple cycles of polymerization showing minimal effect on the integrity of the catalyst. Finally, the ssPC was employed in various photo-“click” reactions, permitting high yielding conjugations under photochemical control. PMID:26863939

  19. A novel solid state photocatalyst for living radical polymerization under UV irradiation

    NASA Astrophysics Data System (ADS)

    Fu, Qiang; McKenzie, Thomas G.; Ren, Jing M.; Tan, Shereen; Nam, Eunhyung; Qiao, Greg G.

    2016-02-01

    This study presents the development of a novel solid state photocatalyst for the photoinduced controlled radical polymerization of methacrylates under mild UV irradiation (λmax ≈ 365 nm) in the absence of conventional photoinitiators, metal-catalysts or dye sensitizers. The photocatalyst design was based on our previous finding that organic amines can act in a synergistic photochemical reaction with thiocarbonylthio compounds to afford well controlled polymethacrylates under UV irradiation. Therefore, in the current contribution an amine-rich polymer was covalently grafted onto a solid substrate, thus creating a heterogeneous catalyst that would allow for facile removal, recovery and recyclability when employed for such photopolymerization reactions. Importantly, the polymethacrylates synthesized using the solid state photocatalyst (ssPC) show similarly excellent chemical and structural integrity as those catalysed by free amines. Moreover, the ssPC could be readily recovered and re-used, with multiple cycles of polymerization showing minimal effect on the integrity of the catalyst. Finally, the ssPC was employed in various photo-“click” reactions, permitting high yielding conjugations under photochemical control.

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

    PubMed Central

    Belmont, Lisa D.; Drubin, David G.

    1998-01-01

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

  1. F- and G-actin homeostasis regulates mechanosensitive actin nucleation by formins.

    PubMed

    Higashida, Chiharu; Kiuchi, Tai; Akiba, Yushi; Mizuno, Hiroaki; Maruoka, Masahiro; Narumiya, Shuh; Mizuno, Kensaku; Watanabe, Naoki

    2013-04-01

    Physical force evokes rearrangement of the actin cytoskeleton. Signalling pathways such as tyrosine kinases, stretch-activated Ca(2+) channels and Rho GTPases are involved in force sensing. However, how signals are transduced to actin assembly remains obscure. Here we show mechanosensitive actin polymerization by formins (formin homology proteins). Cells overexpressing mDia1 increased the amount of F-actin on release of cell tension. Fluorescence single-molecule speckle microscopy revealed rapid induction of processive actin assembly by mDia1 on cell cortex deformation. mDia1 lacking the Rho-binding domain and other formins exhibited mechanosensitive actin nucleation, suggesting Rho-independent activation. Mechanosensitive actin nucleation by mDia1 required neither Ca(2+) nor kinase signalling. Overexpressing LIM kinase abrogated the induction of processive mDia1. Furthermore, s-FDAPplus (sequential fluorescence decay after photoactivation) analysis revealed a rapid actin monomer increase on cell cortex deformation. Our direct visualization of the molecular behaviour reveals a mechanosensitive actin filament regeneration mechanism in which G-actin released by actin remodelling plays a pivotal role.

  2. 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.

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

    PubMed

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

    2010-10-15

    Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

  4. Non-Straub type actin from molluscan catch muscle

    SciTech Connect

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

  5. Production of fungal antibiotics using polymeric solid supports in solid-state and liquid fermentation.

    PubMed

    Bigelis, Ramunas; He, Haiyin; Yang, Hui Y; Chang, Li-Ping; Greenstein, Michael

    2006-10-01

    The use of inert absorbent polymeric supports for cellular attachment in solid-state fungal fermentation influenced growth, morphology, and production of bioactive secondary metabolites. Two filamentous fungi exemplified the utility of this approach to facilitate the discovery of new antimicrobial compounds. Cylindrocarpon sp. LL-Cyan426 produced pyrrocidines A and B and Acremonium sp. LL-Cyan416 produced acremonidins A-E when grown on agar bearing moist polyester-cellulose paper and generated distinctly different metabolite profiles than the conventional shaken or stationary liquid fermentations. Differences were also apparent when tenfold concentrated methanol extracts from these fermentations were tested against antibiotic-susceptible and antibiotic-resistant Gram-positive bacteria, and zones of inhibition were compared. Shaken broth cultures of Acremonium sp. or Cylindrocarpon sp. showed complex HPLC patterns, lower levels of target compounds, and high levels of unwanted compounds and medium components, while agar/solid support cultures showed significantly increased yields of pyrrocidines A and B and acremonidins A-E, respectively. This method, mixed-phase fermentation (fermentation with an inert solid support bearing liquid medium), exploited the increase in surface area available for fungal growth on the supports and the tendency of some microorganisms to adhere to solid surfaces, possibly mimicking their natural growth habits. The production of dimeric anthraquinones by Penicillium sp. LL-WF159 was investigated in liquid fermentation using various inert polymeric immobilization supports composed of polypropylene, polypropylene cellulose, polyester-cellulose, or polyurethane. This culture produced rugulosin, skyrin, flavomannin, and a new bisanthracene, WF159-A, after fermentation in the presence and absence of polymeric supports for mycelial attachment. The physical nature of the different support systems influenced culture morphology and relative

  6. Reversible S-glutathionylation of Cys 374 regulates actin filament formation by inducing structural changes in the actin molecule.

    PubMed

    Dalle-Donne, I; Giustarini, D; Rossi, R; Colombo, R; Milzani, A

    2003-01-01

    S-glutathionylation, the reversible formation of mixed disulphides of cysteinyl residues in target proteins with glutathione, occurs under conditions of oxidative stress; this could be a posttranslational mechanism through which protein function is regulated by the cellular redox status. A novel physiological relevance of actin polymerization regulated by glutathionylation of Cys(374) has been recently suggested. In the present study we showed that glutathionylated actin (GS-actin) has a decreased capacity to polymerize compared to native actin, filament elongation being the polymerization step actually inhibited. Actin polymerizability recovers completely after dethiolation, indicating that S-glutathionylation does not induce any protein denaturation and is therefore a reversible oxidative modification. The increased exposure of hydrophobic regions of protein surface observed upon S-glutathionylation indicates changes in actin conformation. Structural alterations are confirmed by the increased rate of ATP exchange as well as by the decreased susceptibility to proteolysis of the subtilisin cleavage site between Met(47) and Gly(48), in the DNase-I-binding loop of the actin subdomain 2. Structural changes in the surface loop 39-51 induced by S-glutathionylation could influence actin polymerization in view of the involvement of the N-terminal portion of this loop in intermonomer interactions, as predicted by the atomic models of F-actin.

  7. Actin-based propulsion of a microswimmer.

    PubMed

    Leshansky, A M

    2006-07-01

    A simple hydrodynamic model of actin-based propulsion of microparticles in dilute cell-free cytoplasmic extracts is presented. Under the basic assumption that actin polymerization at the particle surface acts as a force dipole, pushing apart the load and the free (nonanchored) actin tail, the propulsive velocity of the microparticle is determined as a function of the tail length, porosity, and particle shape. The anticipated velocities of the cargo displacement and the rearward motion of the tail are in good agreement with recently reported results of biomimetic experiments. A more detailed analysis of the particle-tail hydrodynamic interaction is presented and compared to the prediction of the simplified model.

  8. F-actin aggregates in transformed cells

    PubMed Central

    1981-01-01

    Polymerized actin has been found aggregated into distinctive patches inside transformed cells in culture. The F-actin-specific fluorescent probe, nitrobenzoxadiazole-phallacidin, labels these F-actin aggregates near the ventral cell surface of cells transformed by RNA or DNA tumor viruses, or by chemical mutagens, or spontaneously. Their appearance in all eight transformed cell types studied suggests their ubiquity and involvement in transformation morphology. Actin patches developed in normal rat kidney (NRK) cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (LA23-NRK) within 30 min after a shift from the nonpermissive (39 degrees C) to the permissive temperature (32 degrees C). Patch appearance paralleling viral src gene expression tends to implicate pp60src kinase activity in destabilizing the cytoskeleton. However, appearance of the actin aggregates in cells not transformed by retrovirus calls for alternative mechanisms, perhaps involving an endogenous kinase, for this apparently common trait. PMID:6270163

  9. Actin in mung bean mitochondria and implications for its function.

    PubMed

    Lo, Yih-Shan; Cheng, Ning; Hsiao, Lin-June; Annamalai, Arunachalam; Jauh, Guang-Yuh; Wen, Tuan-Nan; Dai, Hwa; Chiang, Kwen-Sheng

    2011-10-01

    Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin-green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography-tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed.

  10. Incorporation of isosorbide into poly(butylene terephthalate) via solid-state polymerization.

    PubMed

    Sablong, Rafaël; Duchateau, Robbert; Koning, Cor E; de Wit, Gert; va Es, Daan; Koelewijn, Roelof; van Haveren, Jacco

    2008-11-01

    The biomass-based monomer isosorbide was incorporated into poly(butylene terephthalate) (PBT) by solid-state polymerization (SSP) using the macrodiol monomer BTITB-(OH) 2, which consists of isosorbide (I), terephthalic acid (T), and 1,4-butandiol (B) residues. This macromonomer can be synthesized by a simple one-pot, two-step reaction. Polymers with number-average molecular weights up to 100,000 g x mol (-1) were readily synthesized from various ratios of PBT/BTITB-(OH) 2. Their molecular weights, thermal properties, and colors were compared with corresponding copolyesters that were obtained by melt polycondensation. We found that T m, T c, and especially T g were superior for materials that were obtained by SSP. This is ascribed to differences in the microstructures of both types of copolyesters; the SSP products exhibit a more blocky structure than do the more random melt-polymerized counterparts. The SSP method resulted in much higher molecular weights and much less colored polymers, and it seems to be the preferred route for incorporating biobased monomers that exhibit limited thermal stability into engineering plastics.

  11. Dendritic spine actin dynamics in neuronal maturation and synaptic plasticity.

    PubMed

    Hlushchenko, Iryna; Koskinen, Mikko; Hotulainen, Pirta

    2016-09-01

    The majority of the postsynaptic terminals of excitatory synapses in the central nervous system exist on small bulbous structures on dendrites known as dendritic spines. The actin cytoskeleton is a structural element underlying the proper development and morphology of dendritic spines. Synaptic activity patterns rapidly change actin dynamics, leading to morphological changes in dendritic spines. In this mini-review, we will discuss recent findings on neuronal maturation and synaptic plasticity-induced changes in the dendritic spine actin cytoskeleton. We propose that actin dynamics in dendritic spines decrease through actin filament crosslinking during neuronal maturation. In long-term potentiation, we evaluate the model of fast breakdown of actin filaments through severing and rebuilding through polymerization and later stabilization through crosslinking. We will discuss the role of Ca(2+) in long-term depression, and suggest that actin filaments are dissolved through actin filament severing. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. Chlamydial TARP is a bacterial nucleator of actin.

    PubMed

    Jewett, Travis J; Fischer, Elizabeth R; Mead, David J; Hackstadt, Ted

    2006-10-17

    Chlamydia trachomatis entry into host cells results from a parasite-directed remodeling of the actin cytoskeleton. A type III secreted effector, TARP (translocated actin recruiting phosphoprotein), has been implicated in the recruitment of actin to the site of internalization. To elucidate the role of TARP in actin recruitment, we identified host cell proteins that associated with recombinant GST-TARP fusions. TARP directly associated with actin, and this interaction promoted actin nucleation as determined by in vitro polymerization assays. Domain analysis of TARP identified an actin-binding domain that bears structural and primary amino acid sequence similarity to WH2 domain family proteins. In addition, a proline-rich domain was found to promote TARP oligomerization and was required for TARP-dependent nucleation of new actin filaments. Our findings reveal a mechanism by which chlamydiae induce localized cytoskeletal changes by the translocated effector TARP during entry into host cells.

  13. New directions for high-performance materials via postextrusion solid state polymerization

    NASA Astrophysics Data System (ADS)

    Almonacil, Celine

    Solid state polymerization (SSP) usually consists of heating condensation polymers to temperatures below their melting point and holding there for a significant time to raise their molecular weight. The process is common in the polymer industry for the production of high molecular weight polyesters and polyamides for industrial fibers and molded products. Recent research has shown that post-extrusion SSP, where polymerization is performed on extruded products such as thin films or fibers, has the potential to lead to high performance materials. Although literature on SSP is abundant, the mechanisms and possible morphological consequences have remained largely unexplored. The purpose of this work is to explore the potential for generating high performance oriented polymer morphologies by performing a fundamental analysis of the mechanisms and morphological consequences of post-extrusion SSP in oriented polymers. It is based on recent research that has shown that interchange reactions can play a fundamental role during many solid state polymerizations by providing the primary mechanism for migration of functionality. It is also based on the recent recognition that these reactions can cause profound changes in the morphology of the polymer. A coarse-grained model which can be used to explore quantitatively the effect of interchange reactions on the topological distribution of chains in inter-crystalline regions is presented here. It includes a novel thermodynamic scheme, coupled with Monte Carlo Rotational Isomeric State simulations, to determine quantitatively the relative probabilities of morphologically different reaction pathways. The results show the role of intrinsic molecular rigidity on interconversions of bridges and loops during SSP of different polymers. The generalized scheme presented here can serve to identify, via gedanken experiments, appropriate semi-rigid systems to explore through real synthesis and processing of high mechanical performance polymers

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

    PubMed

    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

    2014-06-01

    Can human Sertoli cells cultured in vitro and that have formed an epithelium be used as a model to monitor toxicant-induced junction disruption and to better understand the mechanism(s) by which toxicants disrupt cell adhesion at the Sertoli cell blood-testis barrier (BTB)? Our findings illustrate that human Sertoli cells cultured in vitro serve as a reliable system to monitor the impact of environmental toxicants on the BTB function. Suspicions of a declining trend in semen quality and a concomitant increase in exposures to environmental toxicants over the past decades reveal the need of an in vitro system that efficiently and reliably monitors the impact of toxicants on male reproductive function. Furthermore, studies in rodents have confirmed that environmental toxicants impede Sertoli cell BTB function in vitro and in vivo. We examined the effects of two environmental toxicants: cadmium chloride (0.5-20 µM) and bisphenol A (0.4-200 µM) on human Sertoli cell function. Cultured Sertoli cells from three men were used in this study, which spanned an 18-month period. Human Sertoli cells from three subjects were cultured in F12/DMEM containing 5% fetal bovine serum. Changes in protein expression were monitored by immunoblotting using specific antibodies. Immunofluorescence analyses were used to assess changes in the distribution of adhesion proteins, F-actin and actin regulatory proteins following exposure to two toxicants: cadmium chloride and bisphenol A (BPA). Human Sertoli cells were sensitive to cadmium and BPA toxicity. Changes in the localization of cell adhesion proteins were mediated by an alteration of the actin-based cytoskeleton. This alteration of F-actin network in Sertoli cells as manifested by truncation and depolymerization of actin microfilaments at the Sertoli cell BTB was caused by mislocalization of actin filament barbed end capping and bundling protein Eps8, and branched actin polymerization protein Arp3. Besides impeding actin dynamics

  15. Novel solid state proton-conductors based on polymeric non-oxy acids. Final report

    SciTech Connect

    Appleby, A.J.; Srinivasan, S.; Parthasarathy, A.; Gonzalez, E.R.; DesMarteau, D.; Gillette, M.S.; Ghosh, J.K.; Jalan, V.; Desai, M.

    1992-01-01

    Objectives of this project were to prepare and characterize novel solid state proton-conductors and to evaluate these compounds as fuel cell electrolytes. The thrust was on the synthesis of new proton-conducting ``model`` and ``polymeric`` compounds, based on acid functions of the type (R{sub f}SO{sub 2}){sub 2}NH and (R{sub f}SO{sub 2}){sub 2}CH{sub 2} in appropriate fluorinated carbon structures, their physics-chemical characterization (Infra-red, Nuclear Magnetic Resonance, Differential Scanning Calorimetry, and X-ray Diffraction), and is pro. evaluation as candidate fuel cell electrolytes for use at elevated temperatures. This project consisted of four tasks (i) Synthesis of Proton-Conducting Polymer Electrolytes; (ii) Physical and Chemical Characterization of Proton-Conducting Polymer Electrolytes; (iii) Electrochemical Characterization of Proton-Conducting Polymer Electrolytes; and (iv) Evaluation of Proton-Conducting Polymer Electrolytes for Fuel Cells.

  16. 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

  17. Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast

    PubMed Central

    Boldogh, Istvan R.; Yang, Hyeong-Cheol; Nowakowski, W. Dan; Karmon, Sharon L.; Hays, Lara G.; Yates, John R.; Pon, Liza A.

    2001-01-01

    The Arp2/3 complex is implicated in actin polymerization-driven movement of Listeria monocytogenes. Here, we find that Arp2p and Arc15p, two subunits of this complex, show tight, actin-independent association with isolated yeast mitochondria. Arp2p colocalizes with mitochondria. Consistent with this result, we detect Arp2p-dependent formation of actin clouds around mitochondria in intact yeast. Cells bearing mutations in ARP2 or ARC15 genes show decreased velocities of mitochondrial movement, loss of all directed movement and defects in mitochondrial morphology. Finally, we observe a decrease in the velocity and extent of mitochondrial movement in yeast in which actin dynamics are reduced but actin cytoskeletal structure is intact. These results support the idea that the movement of mitochondria in yeast is actin polymerization driven and that this movement requires Arp2/3 complex. PMID:11248049

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

    PubMed

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

    2012-05-01

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

  19. 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.

  20. Profilin connects actin assembly with microtubule dynamics

    PubMed Central

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

    2016-01-01

    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 micro­tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element. PMID:27307590

  1. An Investigation of the Solid-State Condensation Polymerization Reaction in Vapor-Deposited Poly(amic acid)

    NASA Astrophysics Data System (ADS)

    Anthamatten, Mitchell; Letts, Stephan A.; Day, Katherine; Cook, Robert C.; Gies, Anthony P.; Nonidez, William K.

    2004-03-01

    The condensation polymerization reaction of 4,4'-oxydianiline (ODA) with pyromellitic dianhydride (PMDA) to form poly(amic acid) and the subsequent imidization reaction to form polyimide were investigated for films prepared using vapor deposition polymerization techniques. Fourier-transform infrared spectroscopy (FTIR), thermal analysis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of films prepared at different temperatures indicate that additional solid-state polymerization occurs prior to imidization reactions. Experiments suggest that poly(amic acid) oligomers form upon vapor-deposition and have a number-average molecular weights of about 1500 Daltons. Between 100-130 °C these chains undergo additional condensation reactions to form slightly higher molecular weight oligomers. This work was performed under the auspices of the U.S. Department of Energy by the University of California Lawrence Livermore National Laboratory under contract No. W-7405-Eng-48.

  2. Time-resolved fluorescence measurements of actin-phalloidin interactions

    NASA Astrophysics Data System (ADS)

    Helms, Michael K.; French, Todd E.

    2000-03-01

    Compounds that interact with the cytoskeleton affect mobility and division, making them useful for treatment of certain types of cancer. Actin binding drugs such as the phallotoxins (small, bicyclic peptides) bind to and stabilize actin polymers (F-actin) without binding to actin monomers (G-actin). It has been shown that the intensity of fluorescently labeled phallotoxins such as fluorescein- phalloidin and rhodamine-phalloidin increases upon bind F- actin. We used LJL BioSystems' new FLAReTM technology to measure excited state lifetime changes of fluorescein- phalloidin and rhodamine-phalloidin upon binding to F- actin.

  3. Crystal Structures of Monomeric Actin Bound to Cytochalasin D

    PubMed Central

    Nair, Usha B.; Joel, Peteranne B.; Wan, Qun; Lowey, Susan; Rould, Mark A.; Trybus, Kathleen M.

    2008-01-01

    The fungal toxin cytochalasin D (CD) interferes with the normal dynamics of the actin cytoskeleton by binding to the barbed end of actin filaments. Despite its widespread use as a tool for studying actin-mediated processes, the exact location and nature of its binding to actin has not been previously determined. Here we describe two crystal structures of an expressed monomeric actin in complex with CD, one obtained by soaking preformed actin crystals with CD, and the other by co-crystallization. The binding site for CD, in the hydrophobic cleft between actin subdomains 1 and 3, is the same in the two structures. Polar and hydrophobic contacts play an equally important role in CD binding, and six hydrogen bonds stabilize the actin-CD complex. Many unrelated actin-binding proteins and marine toxins target this cleft, and the hydrophobic pocket at the front end of the cleft (viewing actin with subdomain 2 in the upper right corner). CD differs in that it binds to the back half of the cleft. The ability of CD to induce actin dimer formation and actin-catalyzed ATP hydrolysis may be related to its unique binding site, and the necessity to fit its bulky macrocycle into this cleft. Contacts with residues lining this cleft appear to be crucial to capping and/or severing. The co-crystallized actin-CD structure also revealed changes in actin conformation. A rotation of ~6° of the smaller actin domain (subdomains 1 and 2) with respect to the larger domain (subdomains 3 and 4) results in small changes in crystal packing that allow the D-loop to adopt an extended loop structure, instead of being disordered as it is in most crystal structures of actin. We speculate that these changes represent a potential conformation that the actin monomer can adopt on the pathway to polymerization or in the filament. PMID:18938176

  4. Actin Age Orchestrates Myosin-5 and Myosin-6 Runlengths

    PubMed Central

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

    2015-01-01

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

  5. New Insights into Mechanism and Regulation of Actin Capping Protein

    PubMed Central

    Cooper, John A.; Sept, David

    2008-01-01

    The heterodimeric actin capping protein, referred to here as “CP,” is an essential element of the actin cytoskeleton, binding to the barbed ends of actin filaments and regulating their polymerization. In vitro, CP has a critical role in the dendritic nucleation process of actin assembly mediated by Arp2/3 complex, and in vivo, CP is important for actin assembly and actin-based process of morphogenesis and differentiation. Recent studies have provided new insight into the mechanism of CP binding the barbed end, which raises new possibilities for the dynamics of CP and actin in cells. In addition, a number of molecules that bind and regulate CP have been discovered, suggesting new ideas for how CP may integrate into diverse processes of cell physiology. PMID:18544499

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

    PubMed

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

    2012-02-01

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

  7. Confinement induces actin flow in a meiotic cytoplasm

    PubMed Central

    Pinot, Mathieu; Steiner, Villier; Dehapiot, Benoit; Yoo, Byung-Kuk; Chesnel, Franck; Blanchoin, Laurent; Kervrann, Charles; Gueroui, Zoher

    2012-01-01

    In vivo, F-actin flows are observed at different cell life stages and participate in various developmental processes during asymmetric divisions in vertebrate oocytes, cell migration, or wound healing. Here, we show that confinement has a dramatic effect on F-actin spatiotemporal organization. We reconstitute in vitro the spontaneous generation of F-actin flow using Xenopus meiotic extracts artificially confined within a geometry mimicking the cell boundary. Perturbations of actin polymerization kinetics or F-actin nucleation sites strongly modify the network flow dynamics. A combination of quantitative image analysis and biochemical perturbations shows that both spatial localization of F-actin nucleators and actin turnover play a decisive role in generating flow. Interestingly, our in vitro assay recapitulates several symmetry-breaking processes observed in oocytes and early embryonic cells. PMID:22753521

  8. Fabrication of superhydrophobic films with robust adhesion and dual pinning state via in situ polymerization.

    PubMed

    Raza, Aikifa; Si, Yang; Ding, Bin; Yu, Jianyong; Sun, Gang

    2013-04-01

    Superhydrophobic films on glass substrate with robust adhesion and dual pinning to the water droplets were fabricated utilizing a novel in situ polymerized fluorinated polybenzoxazine (F-PBZ) having drooping aliphatic chains and incorporated SiO2 nanoparticles (SiO2 NPs). By employing the F-PBZ/SiO2 NPs modification, the as-prepared composite films possess the robust adhesion to the glass substrate and superhydrophobic pinned state with water contact angle (WCA) of 150° and the non-pinned state with WCA approaching to 165°. Surface morphological studies have indicated that the wettability of the resultant films could be controlled by tuning the surface composition as well as the hierarchical structures. The key role of micro and sub-micro-sized structures and the nanometer sized voids is discussed by the investigation into static contact angle, contact angle hysteresis, droplet evaporation, and propensity for air pocket formation. The as-prepared films exhibited high adhesion toward the glass substrate with considerable durability in corrosive water and proved their simultaneous use in the transportation of micro-droplets, which could be helpful to design large-area and highly scalable superhydrophobic films.

  9. Force of an Actin Spring

    PubMed Central

    Shin, Jennifer H.; Tam, Barney K.; Brau, Ricardo R.; Lang, Matthew J.; Mahadevan, L.; Matsudaira, Paul

    2007-01-01

    Cellular movements are produced by forces. Typically, cytoskeletal proteins such as microtubules and actin filaments generate forces via polymerization or in conjunction with molecular motors. However, the fertilization of a Limulus polyphemus egg involves a third type of actin-based cellular engine—a biological spring. During the acrosome reaction, a 60-μm long coiled and twisted bundle of actin filaments straightens and extends from a sperm cell, penetrating the vitelline layer surrounding the egg. A subtle overtwist of 0.2°/subunit underlies the mechanochemical basis for the extension of this actin spring. Upon calcium activation, this conformational strain energy is converted to mechanical work, generating the force required to extend the bundle through the vitelline layer. In this article, we stall the extension of the acrosome bundle in agarose gels of different concentrations. From the stall forces, we estimate a maximum force of 2 nN and a puncturing pressure of 1.6 MPa. We show the maximum force of extension is three times larger than the force required to puncture the vitelline layer. Thus, the elastic strain energy stored in the acrosome bundle is more than sufficient to power the acrosome reaction through the egg envelope. PMID:17351007

  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. Actin dynamics counteract membrane tension during clathrin-mediated endocytosis

    PubMed Central

    Boulant, Steeve; Kural, Comert; Zeeh, Jean-Christophe; Ubelmann, Florent; Kirchhausen, Tom

    2011-01-01

    Clathrin-mediated endocytosis is independent of actin dynamics in many circumstances but requires actin polymerization in others. We show that membrane tension determines the actin dependence of clathrin-coat assembly. As found previously, clathrin assembly supports formation of mature coated pits in the absence of actin polymerization on both dorsal and ventral surfaces of non-polarized mammalian cells, and also on basolateral surfaces of polarized cells. Actin engagement is necessary, however, to complete membrane deformation into a coated pit on apical surfaces of polarized cells and, more generally, on the surface of any cell in which the plasma membrane is under tension from osmotic swelling or mechanical stretching. We use these observations to alter actin dependence experimentally and show that resistance of the membrane to propagation of the clathrin lattice determines the distinction between "actin-dependent" and "actin-independent". We also find that light-chain bound Hip1R mediates actin engagement. These data thus provide a unifying explanation for the role of actin dynamics in coated-pit budding. PMID:21841790

  12. Hexosamine biosynthesis pathway flux contributes to insulin resistance via altering membrane phosphatidylinositol 4,5-bisphosphate and cortical filamentous actin.

    PubMed

    Bhonagiri, Padma; Pattar, Guruprasad R; Horvath, Emily M; Habegger, Kirk M; McCarthy, Alicia M; Elmendorf, Jeffrey S

    2009-04-01

    We recently found that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2))-regulated filamentous actin (F-actin) polymerization was diminished in hyperinsulinemic cell culture models of insulin resistance. Here we delineated whether increased glucose flux through the hexosamine biosynthesis pathway (HBP) causes the PIP(2)/F-actin dysregulation and insulin resistance induced by hyperinsulinemia. Increased HBP activity was detected in 3T3-L1 adipocytes cultured under conditions closely resembling physiological hyperinsulinemia (5 nm insulin for 12 h) and in cells where HBP activity was amplified by 2 mm glucosamine (GlcN). Both the physiological hyperinsulinemia and experimental GlcN challenge induced comparable losses of PIP(2) and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP(2) corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP(2)/F-actin structure, inhibition of the rate-limiting HBP enzyme (glutamine:fructose-6-phosphate amidotransferase) restored PIP(2)-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of glutamine:fructose-6-phosphate amidotransferase was associated with a loss of detectable plasma membrane PIP(2) and insulin sensitivity. A slight decrease in intracellular ATP resulted from amplifying HBP by hyperinsulinemia and GlcN. However, experimental maintenance of the intracellular ATP pool under both conditions with inosine did not reverse the PIP(2)/F-actin-based insulin-resistant state. Furthermore, less invasive challenges with glucose, in the absence of insulin, also led to PIP(2)/F-actin dysregulation. Accordingly, we suggest that the functionality of cell systems dependent on PIP(2) and/or F-actin status, such as the glucose transport system, can be critically compromised by inappropriate HBP activity.

  13. Hexosamine Biosynthesis Pathway Flux Contributes to Insulin Resistance via Altering Membrane Phosphatidylinositol 4,5-Bisphosphate and Cortical Filamentous Actin

    PubMed Central

    Bhonagiri, Padma; Pattar, Guruprasad R.; Horvath, Emily M.; Habegger, Kirk M.; McCarthy, Alicia M.; Elmendorf, Jeffrey S.

    2009-01-01

    We recently found that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated filamentous actin (F-actin) polymerization was diminished in hyperinsulinemic cell culture models of insulin resistance. Here we delineated whether increased glucose flux through the hexosamine biosynthesis pathway (HBP) causes the PIP2/F-actin dysregulation and insulin resistance induced by hyperinsulinemia. Increased HBP activity was detected in 3T3-L1 adipocytes cultured under conditions closely resembling physiological hyperinsulinemia (5 nm insulin for 12 h) and in cells where HBP activity was amplified by 2 mm glucosamine (GlcN). Both the physiological hyperinsulinemia and experimental GlcN challenge induced comparable losses of PIP2 and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP2 corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP2/F-actin structure, inhibition of the rate-limiting HBP enzyme (glutamine:fructose-6-phosphate amidotransferase) restored PIP2-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of glutamine:fructose-6-phosphate amidotransferase was associated with a loss of detectable plasma membrane PIP2 and insulin sensitivity. A slight decrease in intracellular ATP resulted from amplifying HBP by hyperinsulinemia and GlcN. However, experimental maintenance of the intracellular ATP pool under both conditions with inosine did not reverse the PIP2/F-actin-based insulin-resistant state. Furthermore, less invasive challenges with glucose, in the absence of insulin, also led to PIP2/F-actin dysregulation. Accordingly, we suggest that the functionality of cell systems dependent on PIP2 and/or F-actin status, such as the glucose transport system, can be critically compromised by inappropriate HBP activity. PMID:19036880

  14. Ca2+-dependent actin coating of lamellar bodies after exocytotic fusion: a prerequisite for content release or kiss-and-run.

    PubMed

    Miklavc, Pika; Wittekindt, Oliver H; Felder, Edward; Dietl, Paul

    2009-01-01

    Type II pneumocytes secrete surfactant, a lipoprotein-like substance reducing the surface tension in the lung, by regulated exocytosis of secretory vesicles termed lamellar bodies (LBs). This secretory process is characterized by a protracted postfusion phase in which fusion pores open slowly and may act as mechanical barriers for release. Combining dark-field with fluorescence microscopy, we show in ss-actin green fluorescent protein-transfected pneumocytes that LB fusion with the plasma membrane is followed by actin coating of the fused LB. This is inhibited by cytoplasmic Ca(2+) chelation or the phospholipase D inhibitor C2 ceramide. Actin coating occurs by polymerization of actin monomers, as evidenced by staining with Alexa 568 phalloidin. After actin coating of the fused LB, it either shrinks while releasing surfactant ("kiss-coat-and-release"), remains in this fused state without further action ("kiss-coat-and-wait"), or is retrieved and pushed forward in the cell on top of an actin tail ("kiss-coat-and-run"). In the absence of actin coating, no release or run was observed. These data suggest that actin coating creates a force needed for either extrusion of vesicle contents or retrieval and intracellular propulsion.

  15. Resonance Femtosecond-Stimulated Raman Spectroscopy without Actinic Excitation Showing Low-Frequency Vibrational Activity in the S2 State of All-Trans β-Carotene.

    PubMed

    Quick, Martin; Dobryakov, Alexander L; Kovalenko, Sergey A; Ernsting, Nikolaus P

    2015-04-02

    Raman scattering with stimulating femtosecond probe pulses (FSR) was used to observe vibrational activity of all-trans β-carotene in n-hexane. The short-lived excited electronic state S2 was accessed in two ways: (i) by transient FSR after an actinic pulse to populate the S2 state, exploiting resonance from an Sx ← S2 transition, and (ii) by FSR without actinic excitation, using S2 ↔ S0 resonance exclusively and narrow-band Raman/broad-band femtosecond probe pulses only. The two approaches have nonlinear optical susceptibilities χ((5)) and χ((3)), respectively. Both methods show low-frequency bands of the S2 state at 200, 400, and ∼600 cm(-1), which are reported for the first time. With (ii) the intensities of low-frequency vibrational resonances in S2 are larger compared to those in S0, implying strong anharmonicities/mode mixing in the excited state. In principle, for short-lived electronic states, the χ((3)) method should allow the best characterization of low-frequency modes.

  16. Logical gates in actin monomer.

    PubMed

    Adamatzky, Andrew

    2017-09-18

    We evaluate information processing capacity of a single actin molecule by calculating distributions of logical gates implemented by the molecule via propagating patterns of excitation. We represent a filamentous actin molecule as an excitable automaton network (F-actin automaton). where every atom updates its state depending on states of atoms its connected to with chemical bonds (hard neighbours) and atoms being in physical proximity to the atom (soft neighbours). A resting atom excites if a sum of its excited hard neighbours and a weighted sum of its soft neighbours belong to some specified interval. We demonstrate that F-actin automata implement OR, AND, XOR and AND-NOT gates via interacting patterns of excitation. Gate AND is the most common gate and gate XOR is the rarest. Using the architectures of gates discovered we implement one bit half-adder and controlled-not circuits in the F-actin automata. Speed and space values of the F-actin molecular computers are discussed.

  17. 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

  18. Cysteine-rich protein 2 accelerates actin filament cluster formation

    PubMed Central

    Shinohara, Satoko; Takaoka, Shunpei; Miyake, Jun

    2017-01-01

    Filamentous actin (F-actin) forms many types of structures and dynamically regulates cell morphology and movement, and plays a mechanosensory role for extracellular stimuli. In this study, we determined that the smooth muscle-related transcription factor, cysteine-rich protein 2 (CRP2), regulates the supramolecular networks of F-actin. The structures of CRP2 and F-actin in solution were analyzed by small-angle X-ray solution scattering (SAXS). The general shape of CRP2 was partially unfolded and relatively ellipsoidal in structure, and the apparent cross sectional radius of gyration (Rc) was about 15.8 Å. The predicted shape, derived by ab initio modeling, consisted of roughly four tandem clusters: LIM domains were likely at both ends with the middle clusters being an unfolded linker region. From the SAXS analysis, the Rc of F-actin was about 26.7 Å, and it was independent of CRP2 addition. On the other hand, in the low angle region of the CRP2-bound F-actin scattering, the intensities showed upward curvature with the addition of CRP2, which indicates increasing branching of F-actin following CRP2 binding. From biochemical analysis, the actin filaments were augmented and clustered by the addition of CRP2. This F-actin clustering activity of CRP2 was cooperative with α-actinin. Thus, binding of CRP2 to F-actin accelerates actin polymerization and F-actin cluster formation. PMID:28813482

  19. Actin Automata: Phenomenology and Localizations

    NASA Astrophysics Data System (ADS)

    Adamatzky, Andrew; Mayne, Richard

    Actin is a globular protein which forms long filaments in the eukaryotic cytoskeleton, whose roles in cell function include structural support, contractile activity to intracellular signaling. We model actin filaments as two chains of one-dimensional binary-state semi-totalistic automaton arrays to describe hypothetical signaling events therein. Each node of the actin automaton takes state "0" (resting) or "1" (excited) and updates its state in discrete time depending on its neighbor's states. We analyze the complete rule space of actin automata using integral characteristics of space-time configurations generated by these rules and compute state transition rules that support traveling and mobile localizations. Approaches towards selection of the localization supporting rules using the global characteristics are outlined. We find that some properties of actin automata rules may be predicted using Shannon entropy, activity and incoherence of excitation between the polymer chains. We also show that it is possible to infer whether a given rule supports traveling or stationary localizations by looking at ratios of excited neighbors that are essential for generations of the localizations. We conclude by applying biomolecular hypotheses to this model and discuss the significance of our findings in context with cell signaling and emergent behavior in cellular computation.

  20. Structural Dynamics of an Actin Spring

    PubMed Central

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

    2011-01-01

    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 Ca2+. 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

  1. The C-terminus of cardiac troponin I stabilizes the Ca2+-activated state of tropomyosin on actin filaments

    PubMed Central

    Galińska, Agnieszka; Hatch, Victoria; Craig, Roger; Murphy, Anne M.; Van Eyk, Jennifer E.; Wang, C.-L. Albert; Lehman, William; Foster, D. Brian

    2010-01-01

    Rationale Ca2+ control of troponin-tropomyosin position on actin regulates cardiac muscle contraction. The inhibitory subunit of troponin, cardiac troponin I (cTnI) is primarily responsible for maintaining a tropomyosin conformation that prevents crossbridge cycling. Despite extensive characterization of cTnI, the precise role of its C-terminal domain (residues 193–210) is unclear. Mutations within this region are associated with restrictive cardiomyopathy, and C-terminal deletion of cTnI, in some species, has been associated with myocardial stunning. Objective We sought to investigate the effect of a cTnI deletion -removal of 17 amino acids from the C-terminus- on the structure of troponin-regulated tropomyosin bound to actin. Methods and Results A truncated form of human cTnI (cTnI1–192) was expressed and reconstituted with Troponin C and Troponin T to form a mutant troponin. Using electron microscopy and 3D-image reconstruction, we show that the mutant troponin perturbs the positional equilibrium dynamics of tropomyosin in the presence of Ca2+. Specifically, it biases tropomyosin position toward an “enhanced Cstate” that exposes more of the myosin-binding site on actin than found with wild-type troponin. Conclusions In addition to its well-established role of promoting the so-called “blocked-state” or “B-state”, cTnI participates in proper stabilization of tropomyosin in the “Ca2+-activated state” or “C-state”. The last 17 amino acids fulfill this stabilizing role. The data are consistent with a “fly-casting” model in which the mobile C-terminus of cTnI ensures proper conformational switching of troponin-tropomyosin. Loss of actin-sensing function within this domain, by pathological proteolysis or cardiomyopathic mutation, may be sufficient to perturb tropomyosin conformation. PMID:20035081

  2. Aligned carbon nanotube film enables thermally induced state transformations in layered polymeric materials.

    PubMed

    Lee, Jeonyoon; Stein, Itai Y; Kessler, Seth S; Wardle, Brian L

    2015-04-29

    The energy losses and geometric constraints associated with conventional curing techniques of polymeric systems motivate the study of a highly scalable out-of-oven curing method using a nanostructured resistive heater comprised of aligned carbon nanotubes (A-CNT). The experimental results indicate that, when compared to conventional oven based techniques, the use of an "out-of-oven" A-CNT integrated heater leads to orders of magnitude reductions in the energy required to process polymeric layered structures such as composites. Integration of this technology into structural systems enables the in situ curing of large-scale polymeric systems at high efficiencies, while adding sensing and control capabilities.

  3. Oscillatory Increases in Alkalinity Anticipate Growth and May Regulate Actin Dynamics in Pollen Tubes of Lily[W][OA

    PubMed Central

    Lovy-Wheeler, Alenka; Kunkel, Joseph G.; Allwood, Ellen G.; Hussey, Patrick J.; Hepler, Peter K.

    2006-01-01

    Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events. PMID:16920777

  4. Thermal Properties and Morphological Analyses of poly(ethylene terephthalate)[PET] Solid State Polymerized

    NASA Astrophysics Data System (ADS)

    Medellin-Rodriguez, F. J.; Lopez-Guillen, R.

    1997-03-01

    High molecular weight PET was obtained through solid state polymerization of commercial PET. After optimizing synthesis variables such as time and reaction temperature, catalyst and gas flow rate, bulk crystallization studies were made with the products in terms of crystallization time and temperature. Molecular crystallization analyses were then performed for the highest molecular weight PET and also for the commercial reference. Isothermal crystallization and melting experiments were systematically practiced in order to characterize the high molecular weight PET. Bulk crystallization studies indicated changes in nucleation patterns from instantaneous to sporadic as the molecular weight was increased. When the molecular crystallization results were correlated with secondary nucleation theory and Regime III, a proportional relationship between the elementary jump activation energy and molecular weight was found. Melting experiments indicated that the main effect of increasing molecular weight is an increase in the second melting endotherm when this feature is present. The support by CONACyT through grant No. 0667P-A is greatly appreciated.

  5. Solid-State Thermolytic Epimerization and Polymerization of (+)-Catechin and (-)-Epicatechin Proceed through Quinone Methide Intermediates.

    PubMed

    Oomah, B Dave; Quigley, Nicole; Latorre, Marie-Laure; Rayne, Sierra; Mazza, Giuseppe

    2008-06-04

    A mechanism involving a quinone methide intermediate is proposed for the thermally driven epimerization and polymerization reactions of solid-state catechin and epicatechin. Epimerization of catechin to epicatechin maximized after heating at 180 degrees C for 30 min (0.76:1, epicatechin/catechin ratio), whereas maximum epicatechin to catechin epimerization occurred after heating at 250 degrees C for 2 min at a ratio of 1.34:1 (catechin/epicatechin). These results imply that the conversion of epicatechin to catechin is the thermodynamically favored process. Conversion to nonepimerized products was optimal (99%) after 8 min at 220 degrees C for catechin and after 2 min at 265 degrees C for epicatechin (99.8%). Pyrocatechol was identified by HPLC as a degradation product in both catechin and epicatechin. Heating catechin at 220 degrees C for 8 min produced a 5-fold increase in chromatographic peak area at 450 nm, indicative of quinone methide formation that was thermally stable over time and degraded in the presence of sodium borohydride.

  6. Osmotically induced electrical signals from actin filaments.

    PubMed Central

    Cantiello, H F; Patenaude, C; Zaner, K

    1991-01-01

    Actin filaments, F-actin, a major component of the cortical cytoskeleton, play an important role in a variety of cell functions. In this report we have assessed the role of osmotic stress on the electrochemical properties of F-actin. The spontaneous Donnan potential of a polymerized actin solution (5 mg/ml) was -3.93 +/- 1.84 mV, which was linearly reduced by osmotic stress on the order of 1-20 mOsm (0.28 +/- 0.06 mV/mM). Calculated surface charge density was reduced and eventually reversed by increasing the osmotic stress as expected for a phase transition behavior. The electro-osmotic behavior of F-actin disappeared at pH 5.5 and was dependent on its filamentous nature. Furthermore, osmotically stressed F-actin displayed a nonlinear electric response upon application of electric fields on the order of 500-2,000 V/cm. These data indicate that F-actin in solution may display nonideal electro-osmotic properties consistent with ionic "cable" behavior which may be of biological significance in the processing and conduction of electrical signals within the cellular compartment. PMID:1873465

  7. The History of Current State of the Art of Propylene Polymerization Catalysts.

    ERIC Educational Resources Information Center

    Goodall, Brian L.

    1986-01-01

    Outlines the development of the modern catalysts for propylene polymerization, considering the historical background; structure of titanium chloride catalysts; first-generation catalysts; cocatalysts; second-generation catalysts; catalysts morphology; and third-generation (supported catalysts). (JN)

  8. The History of Current State of the Art of Propylene Polymerization Catalysts.

    ERIC Educational Resources Information Center

    Goodall, Brian L.

    1986-01-01

    Outlines the development of the modern catalysts for propylene polymerization, considering the historical background; structure of titanium chloride catalysts; first-generation catalysts; cocatalysts; second-generation catalysts; catalysts morphology; and third-generation (supported catalysts). (JN)

  9. Inside view of cell locomotion through single-molecule: fast F-/G-actin cycle and G-actin regulation of polymer restoration

    PubMed Central

    Watanabe, Naoki

    2010-01-01

    The actin cytoskeleton drives cell locomotion and tissue remodeling. The invention of live-cell fluorescence single-molecule imaging opened a window for direct viewing of the actin remodeling processes in the cell. Since then, a number of unanticipated molecular functions have been revealed. One is the mechanism of F-actin network breakdown. In lamellipodia, one third of newly polymerized F-actin disassembles within 10 seconds. This fast F-actin turnover is facilitated by the filament severing/disrupting activity involving cofilin and AIP1. Astoundingly fast dissociation kinetics of the barbed end interactors including capping protein suggests that F-actin turnover might proceed through repetitive disruption/reassembly of the filament near the barbed end. The picture of actin polymerization is also being revealed. At the leading edge of the cell, Arp2/3 complex is highly activated in a narrow edge region. In contrast, mDia1 and its related Formin homology proteins display a long-distance directional molecular movement using their processive actin capping ability. Recently, these two independently-developed projects converged into a discovery of the spatiotemporal coupling between mDia1-mediated filament nucleation and actin disassembly. Presumably, the local concentration fluctuation of G-actin regulates the actin nucleation efficiency of specific actin nucleators including mDia1. Pharmacological perturbation and quantitative molecular behavior analysis synergize to reveal hidden molecular linkages in the actin turnover cycle and cell signaling. PMID:20075609

  10. Effect of 457 nm Diode-Pumped Solid State Laser on the Polymerization Composite Resins: Microhardness, Cross-Link Density, and Polymerization Shrinkage

    PubMed Central

    Son, Sung-Ae; Park, Jeong-Kil; Jung, Kyoung-Hwa; Ko, Ching-Chang; Jeong, Chang-Mo

    2015-01-01

    Abstract Objective: The purpose of the present study was to test the usefulness of 457 nm diode-pumped solid state (DPSS) laser as a light source to cure composite resins. Materials and methods: Five different composite resins were light cured using three different light-curing units (LCUs): a DPSS 457 nm laser (LAS), a light-emitting diode (LED), and quartz-tungsten-halogen (QTH) units. The light intensity of LAS was 560 mW/cm2, whereas LED and QTH LCUs was ∼900 mW/cm2. The degree of polymerization was tested by evaluating microhardness, cross-link density, and polymerization shrinkage. Results: Before water immersion, the microhardness of laser-treated specimens ranged from 40.8 to 84.7 HV and from 31.7 to 79.0 HV on the top and bottom surfaces, respectively, and these values were 3.3–23.2% and 2.9–31.1% lower than the highest microhardness obtained using LED or QTH LCUs. Also, laser-treated specimens had lower top and bottom microhardnesses than the other LCUs treated specimens by 2.4–19.4% and 1.4–27.8%, respectively. After ethanol immersion for 24 h, the microhardness of laser-treated specimens ranged from 20.3 to 63.2 HV on top and bottom surfaces, but from 24.9 to 71.5 HV when specimens were cured using the other LCUs. Polymerization shrinkage was 9.8–14.7 μm for laser-treated specimens, and these were significantly similar or lower (10.2–16.0 μm) than those obtained using the other LCUs. Conclusions: The results may suggest that the 457 nm DPSS laser can be used as a light source for light-curing dental resin composites. PMID:25549163

  11. State of the art, challenges and perspectives in the design of nitric oxide-releasing polymeric nanomaterials for biomedical applications.

    PubMed

    Seabra, Amedea B; Justo, Giselle Z; Haddad, Paula S

    2015-11-01

    Recently, an increasing number of publications have demonstrated the importance of the small molecule nitric oxide (NO) in several physiological and pathophysiological processes. NO acts as a key modulator in cardiovascular, immunological, neurological, and respiratory systems, and deficiencies in the production of NO or its inactivation has been associated with several pathologic conditions, ranging from hypertension to sexual dysfunction. Although the clinical administration of NO is still a challenge owing to its transient chemical nature, the combination of NO and nanocarriers based on biocompatible polymeric scaffolds has emerged as an efficient approach to overcome the difficulties associated with the biomedical administration of NO. Indeed, significant progress has been achieved by designing NO-releasing polymeric nanomaterials able to promote the spatiotemporal generation of physiologically relevant amounts of NO in diverse pharmacological applications. In this review, we summarize the recent advances in the preparation of versatile NO-releasing nanocarriers based on polymeric nanoparticles, dendrimers and micelles. Despite the significant innovative progress achieved using nanomaterials to tailor NO release, certain drawbacks still need to be overcome to successfully translate these research innovations into clinical applications. In this regard, this review discusses the state of the art regarding the preparation of innovative NO-releasing polymeric nanomaterials, their impact in the biological field and the challenges that need to be overcome. We hope to inspire new research in this exciting area based on NO and nanotechnology.

  12. 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.

  13. Curvature and torsion in growing actin networks

    PubMed Central

    Shaevitz, Joshua W; Fletcher, Daniel A

    2011-01-01

    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. PMID:18560043

  14. Actin machinery and mechanosensitivity in invadopodia, podosomes and focal adhesions

    PubMed Central

    Albiges-Rizo, Corinne; Destaing, Olivier; Fourcade, Bertrand; Planus, Emmanuelle; Block, Marc R.

    2009-01-01

    Summary The invasiveness of cells is correlated with the presence of dynamic actin-rich membrane structures called invadopodia, which are membrane protrusions that are associated with localized polymerization of sub-membrane actin filaments. Similar to focal adhesions and podosomes, invadopodia are cell matrix adhesion sites. Indeed, invadopodia share several features with podosomes, but whether they are distinct structures is still a matter of debate. Invadopodia are built upon an N-WASP-dependent branched actin network, and the Rho GTPase Cdc42 is involved in inducing invadopodial-membrane protrusion, which is mediated by actin filaments that are organized in bundles to form an actin core. Actin-core formation is thought to be an early step in invadopodium assembly, and the actin core is perpendicular to the extracellular matrix and the plasma membrane; this contrasts with the tangential orientation of actin stress fibers anchored to focal adhesions. In this Commentary, we attempt to summarize recent insights into the actin dynamics of invadopodia and podosomes, and the forces that are transmitted through these invasive structures. Although the mechanisms underlying force-dependent regulation of invadopodia and podosomes are largely unknown compared with those of focal adhesions, these structures do exhibit mechanosensitivity. Actin dynamics and associated forces might be key elements in discriminating between invadopodia, podosomes and focal adhesions. Targeting actin regulatory molecules that specifically promote invadopodium formation is an attractive strategy against cancer-cell invasion. PMID:19692590

  15. Actin filaments as dynamic reservoirs for Drp1 recruitment.

    PubMed

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

    2016-10-15

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

  16. Direct Observation of Tropomyosin Binding to Actin Filaments

    PubMed Central

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

    2015-01-01

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

  17. Association of actin filaments with axonal microtubule tracts.

    PubMed

    Bearer, E L; Reese, T S

    1999-02-01

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

  18. Actin filaments as dynamic reservoirs for Drp1 recruitment

    PubMed Central

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

    2016-01-01

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

  19. Structure/Function Analysis of the Interaction of Phosphatidylinositol 4,5-Bisphosphate with Actin-capping Protein

    PubMed Central

    Kim, Kyoungtae; McCully, Michelle E.; Bhattacharya, Nandini; Butler, Boyd; Sept, David; Cooper, John A.

    2008-01-01

    The heterodimeric actin-capping protein (CP) can be inhibited by polyphosphoinositides, which may be important for actin polymerization at membranes in cells. Here, we have identified a conserved set of basic residues on the surface of CP that are important for the interaction with phosphatidylinositol 4,5-bisphosphate (PIP2). Computational docking studies predicted the identity of residues involved in this interaction, and functional and physical assays with site-directed mutants of CP confirmed the prediction. The PIP2 binding site overlaps with the more important of the two known actin-binding sites of CP. Correspondingly, we observed that loss of PIP2 binding correlated with loss of actin binding among the mutants. Using TIRF (total internal reflection fluorescence) microscopy, we observed that PIP2 rapidly converted capped actin filaments to a growing state, consistent with uncapping. Together, these results extend our understanding of how CP binds to the barbed end of the actin filament, and they support the idea that CP can “wobble” when bound to the barbed end solely by the C-terminal “tentacle” of its β-subunit. PMID:17182619

  20. Damage effects of protoporphyrin IX - sonodynamic therapy on the cytoskeletal F-actin of Ehrlich ascites carcinoma cells.

    PubMed

    Zhao, Xia; Liu, Quanhong; Tang, Wei; Wang, Xiaobing; Wang, Pan; Gong, Liyan; Wang, Yuan

    2009-01-01

    In this study, we report evidence of the damage effects of sonodynamic therapy (SDT) on a novel intracellular target, cytoskeletal F-actin, that has great importance for cancer treatment. Ehrlich ascites carcinoma (EAC) cells suspended in PBS were exposed to ultrasound at 1.34 MHz for up to 60s in the presence and absence of protoporphyrin IX (PPIX). To evaluate the polymeric state and distribution of actin filaments (AF) we employed FITC-Phalloidin staining. The percentage of cells with intact AF was decreased with 10-80 microM PPIX after ultrasonic exposure, while only few cells with disturbed F-actin were observed with 80 microM PPIX alone. The fluorescence intensity of FITC-Phalloidin labeled cells was detected by flow cytometry. The morphological changes of EAC cells were observed by scanning electron microscope (SEM). The nuclei were stained with Hoechst 33258 to determine apoptosis. Cytoskeletal F-actin and cell morphological changes were dependent on the time after SDT. Some cells suffered deformations of plasma membrane as blebs that reacted positively to FITC-Phalloidin at 2h after SDT treatment. Many of the cells showed the typically apoptotic chromatin fragmentation. The alterations were more significant 4h later. Our results showed that cytoskeletal F-actin might represent an important target for the SDT treatment and the observed effect on F-actin and the subsequent bleb formation mainly due to apoptosis formation due to the treatment.

  1. Plasma Gelsolin Levels Decrease in Diabetic State and Increase upon Treatment with F-Actin Depolymerizing Versions of Gelsolin

    PubMed Central

    Khatri, Neeraj; Sagar, Amin; Peddada, Nagesh; Choudhary, Vikas; Chopra, Bhupinder Singh; Garg, Veena; Ashish

    2014-01-01

    The study aims to map plasma gelsolin (pGSN) levels in diabetic humans and mice models of type II diabetes and to evaluate the efficacy of gelsolin therapy in improvement of diabetes in mice. We report that pGSN values decrease by a factor of 0.45 to 0.5 in the blood of type II diabetic humans and mice models. Oral glucose tolerance test in mice models showed that subcutaneous administration of recombinant pGSN and its F-actin depolymerizing competent versions brought down blood sugar levels comparable to Sitagliptin, a drug used to manage hyperglycemic condition. Further, daily dose of pGSN or its truncated versions to diabetic mice for a week kept sugar levels close to normal values. Also, diabetic mice treated with Sitagliptin for 7 days, showed increase in their pGSN values with the decrease in blood glucose as compared to their levels at the start of treatment. Gelsolin helped in improving glycemic control in diabetic mice. We propose that gelsolin level monitoring and replacement of F-actin severing capable gelsolin(s) should be considered in diabetic care. PMID:25478578

  2. 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.

  3. 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.

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

    PubMed Central

    Takaine, Masak; Numata, Osamu; Nakano, Kentaro

    2009-01-01

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

  5. SWAP70 Organizes the Actin Cytoskeleton and Is Essential for Phagocytosis.

    PubMed

    Baranov, Maksim V; Revelo, Natalia H; Dingjan, Ilse; Maraspini, Riccardo; Ter Beest, Martin; Honigmann, Alf; van den Bogaart, Geert

    2016-11-01

    Actin plays a critical role during the early stages of pathogenic microbe internalization by immune cells. In this study, we identified a key mechanism of actin filament tethering and stabilization to the surface of phagosomes in human dendritic cells. We found that the actin-binding protein SWAP70 is specifically recruited to nascent phagosomes by binding to the lipid phosphatidylinositol (3,4)-bisphosphate. Multi-color super-resolution stimulated emission depletion (STED) microscopy revealed that the actin cage surrounding early phagosomes is formed by multiple concentric rings containing SWAP70. SWAP70 colocalized with and stimulated activation of RAC1, a known activator of actin polymerization, on phagosomes. Genetic ablation of SWAP70 impaired actin polymerization around phagosomes and resulted in a phagocytic defect. These data show a key role for SWAP70 as a scaffold for tethering the peripheral actin cage to phagosomes.

  6. A Gly65Val substitution in an actin, GhACT_LI1, disrupts cell polarity and membrane anchoring of F-actin resulting in dwarf, lintless Li1 cotton plants

    USDA-ARS?s Scientific Manuscript database

    Actin polymerizes to form the cytoskeleton and organize polar growth in all eukaryotic cells. Species with numerous actin genes are especially useful for the dissection of actin molecular function due to redundancy and neofunctionalization. Here, we investigated the role of a cotton (Gossypium hi...

  7. Relationship of F-actin distribution to development of polar shape in human polymorphonuclear neutrophils

    PubMed Central

    1992-01-01

    Polymerization of actin has been associated with development of polar shape in human neutrophils (PMN). To examine the relation of filamentous actin (F-actin) distribution to shape change in PMN, we developed a method using computerized video image analysis and fluorescence microscopy to quantify distribution of F-actin in single cells. PMN were labeled with fluorescent probe NBD-phallicidin to measure filamentous actin and Texas red to assess cell thickness. We show that Texas red fluorescence is a reasonable measure of cell thickness and that correction of the NBD-phallicidin image for cell thickness using the Texas red image permits assessment of focal F-actin content. Parameters were derived that quantify total F-actin content, movement of F-actin away from the center of the cell, asymmetry of F- actin distribution, and change from round to polar shape. The sequence of change in F-actin distribution and its relation to development of polar shape in PMN was determined using these parameters. After stimulation with chemotactic peptide at 25 degrees C, F-actin polymerized first at the rim of the PMN. This was followed by development of asymmetry of F-actin distribution and change to polar shape. The dominant pseudopod developed first in the region of lower F- actin concentration followed later by polymerization of actin in the end of the developed pseudopod. Asymmetric F-actin distribution was detected in round PMN before development of polar shape. Based upon these data, asymmetric distribution of F-actin is coincident with and probably precedes development of polar shape in PMN stimulated in suspension by chemotactic peptide. PMID:1577856

  8. Actin growth profile in clathrin-mediated endocytosis

    NASA Astrophysics Data System (ADS)

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

    2017-05-01

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

  9. The Platelet Actin Cytoskeleton Associates with SNAREs and Participates in α-Granule Secretion†

    PubMed Central

    Woronowicz, Kamil; Dilks, James R.; Rozenvayn, Nataliya; Dowal, Louisa; Blair, Price S.; Peters, Christian G.; Woronowicz, Lucyna; Flaumenhaft, Robert

    2010-01-01

    Following platelet activation, platelets undergo a dramatic shape change mediated by the actin cytoskeleton and accompanied by secretion of granule contents. While the actin cytoskeleton is thought to influence platelet granule secretion, the mechanism for this putative regulation is not known. We found that disruption of the actin cytoskeleton by latrunculin A inhibited α-granule secretion induced by several different platelet agonists without significantly affecting activation-induced platelet aggregation. In a cell-free secretory system, platelet cytosol was required for α-granule secretion. Inhibition of actin polymerization prevented α-granule secretion in this system and purified platelet actin could substitute for platelet cytosol to support α–granule secretion. To determine whether SNAREs physically associate with the actin cytoskeleton, we isolated the Triton X-100 insoluble actin cytoskeleton from platelets. VAMP-8 and syntaxin-2 associated only with actin cytoskeletons of activated platelets. Syntaxin-4 and SNAP-23 associated with cytoskeletons isolated from either resting or activated platelets. When syntaxin-4 and SNAP-23 were tested for actin binding in a purified protein system, only syntaxin-4 associated directly with polymerized platelet actin. These data show that the platelet cytoskeleton interacts with select SNAREs and that actin polymerization facilitates α-granule release. PMID:20429610

  10. Extremely low polymerizability of a highly-divergent Chlamydomonas actin (NAP).

    PubMed

    Kato-Minoura, Takako

    2011-09-09

    Novel actin-like protein (NAP) is a highly divergent actin expressed in Chlamydomonas. With its low sequence similarity, it is uncertain whether NAP can polymerize into filaments. Here I assessed it by ectopically expressing enhanced green fluorescent protein-tagged NAP (EGFP-NAP) in cultured cells. EGFP-NAP was excluded from stress fibres but partially co-localized with endogenous actin in the cell periphery. In fluorescence recovery after photobleaching experiment, turnover rate of EGFP-NAP was similar to the estimated diffusion rate of monomeric actin. Therefore, EGFP-NAP likely accumulates by diffusion. These findings suggest that NAP has extremely poor ability to polymerize.

  11. Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile, long-lived plaques.

    PubMed

    Meijer, Harold J G; Hua, Chenlei; Kots, Kiki; Ketelaar, Tijs; Govers, Francine

    2014-06-01

    The actin cytoskeleton is a dynamic but well-organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact-eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub-apical region whereas in the extreme apex in growing hyphae, waves of fine F-actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ∼30 s while disassembly was accomplished in ∼10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.

  12. Improved blood compatibility of segmented polyurethane by polymeric additives having phospholipid polar group. II. Dispersion state of the polymeric additive and protein adsorption on the surface.

    PubMed

    Ishihara, K; Shibata, N; Tanaka, S; Iwasaki, Y; Kurosaki, T; Nakabayashi, N

    1996-11-01

    To improve the blood compatibility of a segmented polyurethane (SPU), phospholipid polymer, i.e., 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with cyclohexyl methacrylate or 2-ethylhexyl methacrylate, was blended into SPU as a polymeric additive. The blending was achieved by a solvent-evaporation technique from a homogeneous solution containing both the SPU and the MPC polymer. Surface analysis of the SPU membrane blended with the MPC polymer (SPU/MPC polymer membrane) revealed that the MPC polymer was concentrated at the surface of the SPU membrane which contacted the substrate, Teflon, compared with that which contacted air during the membrane-formation period. The dispersion state of the MPC polymer in the SPU membrane was evaluated in detail by staining the MPC unit with osmium tetraoxide. When sonication was applied during preparation of the mixed solution containing SPU and the MPC polymer, the dispersion of the MPC polymer in the SPU membrane was different from that without sonication. That is, the size of the domains of the MPC polymer became smaller but the number of the domains increased. The amount of the MPC polymer mixed with SPU affected the dispersion state. Plasma proteins adsorbed on the SPU/MPC polymer membrane surface after contact with human plasma were detected by gold-colloid-labeled immunoassay. Both albumin and fibrinogen were observed on the SPU membrane; however, the amount of these proteins was reduced on the SPU/MPC polymer membrane. Thus it was concluded that the blood compatibility of the SPU was effectively improved by the blending of the MPC polymer.

  13. Long-range conformational effects of proteolytic removal of the last three residues of actin.

    PubMed Central

    Strzelecka-Gołaszewska, H; Mossakowska, M; Woźniak, A; Moraczewska, J; Nakayama, H

    1995-01-01

    Truncated derivatives of actin devoid of either the last two (actin-2C) or three residues (actin-3C) were used to study the role of the C-terminal segment in the polymerization of actin. The monomer critical concentration and polymerization rate increased in the order: intact actin < actin-2C < actin-3C. Conversely, the rate of hydrolysis of actin-bound ATP during spontaneous polymerization of Mg-actin decreased in the same order, so that, for actin-3C, the ATP hydrolysis significantly lagged behind the polymer growth. Probing the conformation of the nucleotide site in the monomer form by measuring the rates of the bound nucleotide exchange revealed a similar change upon removal of either the two or three residues from the C-terminus. The C-terminal truncation also resulted in a slight decrease in the rate of subtilisin cleavage of monomeric actin within the DNAse-I binding loop, whereas in F-actin subunits the susceptibility of this and of another site within this loop, specifically cleaved by a proteinase from Escherichia coli A2 strain, gradually increased upon sequential removal of the two and of the third residue from the C-terminus. From these and other observations made in this work it has been concluded that perturbation of the C-terminal structure in monomeric actin is transmitted to the cleft, where nucleotide and bivalent cation are bound, and to the DNAse-I binding loop on the top of subdomain 2. Further changes at these sites, observed on the polymer level, seem to result from elimination of the intersubunit contact between the C-terminal residues and the DNAse-I binding loop. It is suggested that formation of this contact plays an essential role in regulating the hydrolysis of actin-bound ATP associated with the polymerization process. Images Figure 5 Figure 6 Figure 8 PMID:7733893

  14. Solid state polymerization and crystallography of polyimide precursors. Ph.D. Thesis - Va. Univ.

    NASA Technical Reports Server (NTRS)

    Wakelyn, N. T.

    1974-01-01

    Although the production of crystallinity in a polymeric system has historically led to commerically useful properties, the polyimides, prized for their high temperature characteristics, as customarily synthesized by melt or solution casting, are amorphous. It is shown that polymide containing residual crystallinity can be synthesized by isothermal annealing of crystals of the salt of the diisopropyl ester of pyromellitic acid and phenylene diamine. The reaction is topochemical in that the geometry of the polymer product is dependent upon that of the crystalline precursor. Infrared spectroscopy reveals the presence of imide absorption in the polymer, while powder diffractometry suggests residual crystallinity. Single crystal X-ray analysis of the monomer yields a structure of chains of alternating acid and base suggesting that the monomer is amenable to polymerization with a minimum of geometrical disruption.

  15. 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

  16. Ground state structure of high-energy-density polymeric carbon monoxide

    NASA Astrophysics Data System (ADS)

    Xia, Kang; Sun, Jian; Pickard, Chris J.; Klug, Dennis D.; Needs, Richard J.

    2017-04-01

    Crystal structure prediction methods and first-principles calculations have been used to explore low-energy structures of carbon monoxide (CO). Contrary to the standard wisdom, the most stable structure of CO at ambient pressure was found to be a polymeric structure of P n a 21 symmetry rather than a molecular solid. This phase is formed from six-membered (four carbon + two oxygen) rings connected by C=C double bonds with two double-bonded oxygen atoms attached to each ring. Interestingly, the polymeric P n a 21 phase of CO has a much higher energy density than trinitrotoluene (TNT). On compression to about 7 GPa, P n a 21 is found to transform into another chainlike phase of C c symmetry which has similar ring units to P n a 21 . On compression to 12 GPa, it is energetically favorable for CO to polymerize into a purely single bonded C m c a phase, which is stable over a wide pressure range and transforms into the previously known C m c m phase at around 100 GPa. Thermodynamic stability of these structures was verified using calculations with different density functionals, including hybrid and van der Waals corrected functionals.

  17. ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast.

    PubMed

    Gieselmann, R; Mann, K

    1992-02-24

    A new 56 kDa actin-binding protein (ASP-56) was isolated from pig platelet lysate. In falling ball viscosimetry it caused a reduction in viscosity that could be attributed to a decrease in the concentration of polymeric actin. Fluorescence measurements with NBD-labelled actin showed reduction of polymeric actin, too. These results could be explained by sequestering of actin in a non-polymerizable 1:1 ASP-56/actin complex. Sequencing of about 20 tryptic peptides of ASP-56 and comparison with known sequences revealed about 60% homology to the adenylate cyclase-associated protein (CAP) from yeast.

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed Central

    Kim, Mijung; Robich, Rebecca M.; Rinehart, Joseph P.; Denlinger, David L.

    2007-01-01

    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 actin that was most pronounced in the midguts of diapausing mosquitoes that were exposed to low temperature. In nondiapausing mosquitoes reared at 25°C and in diapausing mosquitoes reared at 18°C, polymerized actin was clustered at high concentrations at the intersections of the muscle fibers that form the midgut musculature. When adults 7–10 days post-eclosion were exposed to low temperature (-5°C for 12h), the polymerized actin was evenly distributed along the muscle fibers in both nondiapausing and diapausing mosquitoes. Exposure of older adults (1month post-eclosion) to low temperature (−5°C for 12h) elicited an even greater distribution of polymerized actin, an effect that was especially pronounced in diapausing mosquitoes. These changes in gene expression and actin distribution suggest a role for actins in enhancing survival of diapausing adults during the low temperatures of winter by fortification of the cytoskeleton. PMID:17078965

  20. Solid-state dye-sensitized solar cells using polymerized ionic liquid electrolyte with platinum-free counter electrode.

    PubMed

    Kawano, Ryuji; Katakabe, Toru; Shimosawa, Hironobu; Nazeeruddin, Md Khaja; Grätzel, Michael; Matsui, Hiroshi; Kitamura, Takayuki; Tanabe, Nobuo; Watanabe, Masayoshi

    2010-02-28

    A polymerized ionic liquid electrolyte and platinum-free counter electrode are employed for solid-state DSSCs. We are able to prepare a thin polymer electrolyte layer on nanocrystalline TiO(2) in order to reduce the cell resistance. In addition, an electron conductive polymer (PEDOT/PSS) or a single-wall carbon nanotube gel is used with the cell as an inexpensive counter electrode instead of platinum. The overall photon-to-current conversion efficiency was 3.7% in this study.

  1. Stereoengineering of poly(1,3-methylenecyclohexane) via two-state living coordination polymerization of 1,6-heptadiene.

    PubMed

    Crawford, Kaitlyn E; Sita, Lawrence R

    2013-06-19

    External control over the rate of dynamic methyl group exchange between configurationally stable active species and configurationally unstable dormant species with respect to chain-growth propagation within a highly stereoselective and regiospecific living coordination polymerization of 1,6-heptadiene has been used to generate a spectrum of different physical forms of poly(1,3-methylenecyclohexane) (PMCH) in which the stereochemical microstructure has been systematically varied between two limiting forms. The application of this strategy to manipulate the bulk properties of PMCH and the solid-state microphase behavior of well-defined PMCH-b-poly(1-hexene) block copolymers is further demonstrated.

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

    PubMed

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

    2011-10-01

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

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

    PubMed Central

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

    2011-01-01

    Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility. PMID:21998582

  4. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  5. Actin network architecture can determine myosin motor activity.

    PubMed

    Reymann, Anne-Cécile; Boujemaa-Paterski, Rajaa; Martiel, Jean-Louis; Guérin, Christophe; Cao, Wenxiang; Chin, Harvey F; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2012-06-08

    The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin