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Sample records for actin polymerization controls

  1. Spatial control of actin polymerization during neutrophil chemotaxis

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2015-07-23

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

  3. ACD toxin-produced actin oligomers poison formin-controlled actin polymerization

    PubMed Central

    Heisler, David B.; Kudryashova, Elena; Grinevich, Dmitry O.; Suarez, Cristian; Winkelman, Jonathan D.; Birukov, Konstantin G.; Kotha, Sainath R.; Parinandi, Narasimham L.; Vavylonis, Dimitrios; Kovar, David R.; Kudryashov, Dmitri S.

    2015-01-01

    The actin crosslinking domain (ACD) is an actin-specific toxin produced by several pathogens, including life-threatening spp. of Vibrio cholerae, Vibrio vulnificus, and Aeromonas hydrophila. Actin crosslinking by ACD is thought to lead to slow cytoskeleton failure owing to a gradual sequestration of actin in the form of nonfunctional oligomers. Here we found that ACD converted cytoplasmic actin into highly toxic oligomers that potently “poisoned” the ability of major actin assembly proteins, formins, to sustain actin polymerization. Thus, ACD can target the most abundant cellular protein by employing actin oligomers as secondary toxins to efficiently subvert cellular functions of actin while functioning at very low doses. PMID:26228148

  4. Adhesion controls bacterial actin polymerization-based movement.

    PubMed

    Soo, Frederick S; Theriot, Julie A

    2005-11-01

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

  5. Colchicine activates actin polymerization by microtubule depolymerization.

    PubMed

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

    1997-06-30

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

  6. 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

  7. Impact of Carbon Nanomaterials on Actin Polymerization.

    PubMed

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

    2016-03-01

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

  8. Extracellular signaling cues for nuclear actin polymerization.

    PubMed

    Plessner, Matthias; Grosse, Robert

    2015-01-01

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

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

    PubMed

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

    2016-02-15

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the co-ordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. In the present study, we show that the actin-binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro cross-linking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of globular or monomeric actin (G-actin), akin to metal ions, either through charge neutralization or through conformational changes. PMID:26607837

  11. Calcium control of Saccharomyces cerevisiae actin assembly.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

    Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo. Images PMID:6757718

  12. 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

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

    PubMed

    Schroeter, Mechthild; Chalovich, Joseph M

    2004-11-01

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

  14. Feedback Interactions of Polymerized Actin with the Cell Membrane: Waves, Pulses, and Oscillations

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    Polymerized filaments of the protein actin have crucial functions in cell migration, and in bending the cell membrane to drive endocytosis or the formation of protrusions. The nucleation and polymerization of actin filaments are controlled by upstream agents in the cell membrane, including nucleation-promoting factors (NPFs) that activate the Arp2/3 complex to form new branches on pre-existing filaments. But polymerized actin (F-actin) also feeds back on the assembly of NPFs. We explore the effects of the resulting feedback loop of F-actin and NPFs on two phenomena: actin pulses that drive endocytosis in yeast, and actin waves traveling along the membrane of several cell types. In our model of endocytosis in yeast, the actin network is grown explicitly in three dimensions, exerts a negative feedback interaction on localized patch of NPFs in the membrane, and bends the membrane by exerting a distribution of forces. This model explains observed actin and NPF pulse dynamics, and the effects of several interventions including i) NPF mutations, ii) inhibition of actin polymerization, and iii) deletion of a protein that allows F-actin to bend the cell membrane. The model predicts that mutation of the active region of an NPF will enhance the accumulation of that NPF, and we confirm this prediction by quantitative fluorescence microscopy. For actin waves, we treat a similar model, with NPFs distributed over a larger region of the cell membrane. This model naturally generates actin waves, and predicts a transition from wave behavior to spatially localized oscillations when NPFs are confined to a small region. We also predict a transition from waves to static polarization as the negative-feedback coupling between F-actin and the NPFs is reduced. Supported by NIGMS Grant R01 GM107667.

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

    NASA Astrophysics Data System (ADS)

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

    2003-04-01

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

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

    PubMed

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

    2016-08-01

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

  17. 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

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2009-09-18

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

  1. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. 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

  6. 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

  7. 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

  8. Electrostatics control actin filament nucleation and elongation kinetics.

    PubMed

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

    2013-04-26

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

  9. Reactive oxygen species (ROS)-induced actin glutathionylation controls actin dynamics in neutrophils

    PubMed Central

    Sakai, Jiro; Li, Jingyu; Subramanian, Kulandayan K.; Mondal, Subhanjan; Bajrami, Besnik; Hattori, Hidenori; Jia, Yonghui; Dickinson, Bryan C.; Zhong, Jia; Ye, Keqiang; Chang, Christopher J; Ho, Ye-Shih; Zhou, Jun; Luo, Hongbo R.

    2012-01-01

    Summary The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis, and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, however the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils. PMID:23159440

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

    PubMed

    Joo, E Emily; Yamada, Kenneth M

    2016-05-01

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

  11. 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

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

    PubMed

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

    2015-01-12

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

  13. 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

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

    PubMed

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

    2016-03-21

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

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

    PubMed

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

    2014-12-01

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

  16. Induction of HoxB Transcription by Retinoic Acid Requires Actin Polymerization

    PubMed Central

    Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M.; Crippa, Massimo P.; Scita, Giorgio

    2009-01-01

    We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of “elongating” RNAPII, Prep1, β-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes. PMID:19477923

  17. Induction of HoxB transcription by retinoic acid requires actin polymerization.

    PubMed

    Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M; Crippa, Massimo P; Scita, Giorgio; Blasi, Francesco

    2009-08-01

    We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of "elongating" RNAPII, Prep1, beta-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes. PMID:19477923

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

    PubMed Central

    Burnett, Mark M.; Carlsson, Anders E.

    2012-01-01

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

  19. 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.

  20. 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.

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

    PubMed

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

    2009-02-25

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

  2. 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.

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2002-12-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  9. 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

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

    PubMed

    Holzinger, A; Lütz-Meindl, U

    2001-02-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-08-17

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

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

    PubMed

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

    2002-03-01

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

  14. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  17. 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

  18. 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

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

    PubMed Central

    Briz, Victor; Baudry, Michel

    2014-01-01

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

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

    PubMed

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

    2000-06-01

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

  1. 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.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

    Suchy, Sharon F.; Nussbaum, Robert L.

    2002-01-01

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

  4. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2016-01-01

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

  8. 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

  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. A Nucleator Arms Race: Cellular Control of Actin Assembly

    PubMed Central

    Campellone, Kenneth G.; Welch, Matthew D.

    2010-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    2011-01-01

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

  14. Geometrical and Mechanical Properties Control Actin Filament Organization

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2016-05-01

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

  17. 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

  18. 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).

  19. 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.

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

    PubMed Central

    Takano, Kazunari; Toyooka, Kiminori; Suetsugu, Shiro

    2008-01-01

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

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

    PubMed Central

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

    1973-01-01

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

  2. Cofilin-2 controls actin filament length in muscle sarcomeres

    PubMed Central

    Kremneva, Elena; Makkonen, Maarit H.; Skwarek-Maruszewska, Aneta; Gateva, Gergana; Michelot, Alphee; Dominguez, Roberto; Lappalainen, Pekka

    2014-01-01

    SUMMARY ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of ‘aged’ ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus. In contrast to other isoforms, cofilin-2 efficiently binds and disassembles both ADP- and ATP/ADP-Pi-actin filaments. We mapped surface-exposed cofilin-2-specific residues required for ATP-actin binding and propose that these residues function as an ‘actin nucleotide-state sensor’ among ADF/cofilins. The results suggest that cofilin-2 evolved specific biochemical and cellular properties allowing it to control actin dynamics in sarcomeres, where filament pointed ends may contain a mixture of ADP- and ATP/ADP-Pi-actin subunits. Our findings also offer a rationale for why cofilin-2 mutations in humans lead to myopathies. PMID:25373779

  3. Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse.

    PubMed

    Le Floc'h, Audrey; Tanaka, Yoshihiko; Bantilan, Niels S; Voisinne, Guillaume; Altan-Bonnet, Grégoire; Fukui, Yoshinori; Huse, Morgan

    2013-11-18

    The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP3) in the synaptic membrane. PIP3 functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP3 is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP3-dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses. PMID:24190432

  4. Electrically controlled polymeric gel actuators

    DOEpatents

    Adolf, Douglas B.; Shahinpoor, Mohsen; Segalman, Daniel J.; Witkowski, Walter R.

    1993-01-01

    Electrically controlled polymeric gel actuators or synthetic muscles capable of undergoing substantial expansion and contraction when subjected to changing pH environments, temperature, or solvent. The actuators employ compliant containers for the gels and their solvents. The gels employed may be cylindrical electromechanical gel fibers such as polyacrylamide fibers or a mixture of poly vinyl alcohol-polyacrylic acid arranged in a parallel aggregate and contained in an electrolytic solvent bath such as salt water. The invention includes smart, electrically activated devices exploiting this phenomenon. These devices are capable of being manipulated via active computer control as large displacement actuators for use in adaptive structure such as robots.

  5. Electrically controlled polymeric gel actuators

    DOEpatents

    Adolf, D.B.; Shahinpoor, M.; Segalman, D.J.; Witkowski, W.R.

    1993-10-05

    Electrically controlled polymeric gel actuators or synthetic muscles are described capable of undergoing substantial expansion and contraction when subjected to changing pH environments, temperature, or solvent. The actuators employ compliant containers for the gels and their solvents. The gels employed may be cylindrical electromechanical gel fibers such as polyacrylamide fibers or a mixture of poly vinyl alcohol-polyacrylic acid arranged in a parallel aggregate and contained in an electrolytic solvent bath such as salt water. The invention includes smart, electrically activated devices exploiting this phenomenon. These devices are capable of being manipulated via active computer control as large displacement actuators for use in adaptive structure such as robots. 11 figures.

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

    PubMed

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

    2015-12-15

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2016-07-18

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

  12. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2007-01-01

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

  15. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Shabtay, Ortal; Breitbart, Haim

    2016-07-01

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

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

    PubMed

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

    2013-09-01

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

  19. 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.

  20. Actin Filament Elongation in Arp2/3-derived Networks is Controlled by Three Distinct Mechanisms

    PubMed Central

    Michelot, Alphée; Grassart, Alexandre; Okreglak, Voytek; Costanzo, Michael; Boone, Charles; Drubin, David G.

    2012-01-01

    Summary Spatial and temporal control of actin filament barbed end elongation is crucial for force generation by actin networks. In this study, genetics, cell biology, and biochemistry were used to reveal three complementary mechanisms that regulate actin filament barbed end elongation in Arp2/3-derived networks. Aip1 inhibits elongation of aged ADP-actin filaments decorated with cofilin, and together with capping protein (CP), maintains a high level of assembly-competent actin species. We identified Abp1 and Aim3 as two additional proteins that work together to inhibit barbed end elongation. Abp1/Aim3 collaborates with CP to control elongation of newly assembled ATP-actin filaments to organize filament polarity within actin networks. Thus, three distinct mechanisms control filament elongation in different regions of Arp2/3 networks, maintaining pools of assembly-competent actin species while ensuring proper filament polarity and facilitating force production. PMID:23333351

  1. Actin filament elongation in Arp2/3-derived networks is controlled by three distinct mechanisms.

    PubMed

    Michelot, Alphée; Grassart, Alexandre; Okreglak, Voytek; Costanzo, Michael; Boone, Charles; Drubin, David G

    2013-01-28

    Spatial and temporal control of actin filament barbed end elongation is crucial for force generation by actin networks. In this study, genetics, cell biology, and biochemistry were used to reveal three complementary mechanisms that regulate actin filament barbed end elongation in Arp2/3-derived networks. Aip1 inhibits elongation of aged ADP-actin filaments decorated with cofilin and, together with capping protein (CP), maintains a high level of assembly-competent actin species. We identified Abp1 and Aim3 as two additional proteins that work together to inhibit barbed end elongation. Abp1/Aim3 collaborates with CP to control elongation of newly assembled ATP-actin filaments to organize filament polarity within actin networks. Thus, three distinct mechanisms control filament elongation in different regions of Arp2/3 networks, maintaining pools of assembly-competent actin species while ensuring proper filament polarity and facilitating force production. PMID:23333351

  2. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  6. 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.

  7. Phosphatidylinositol 5-phosphatase oculocerebrorenal syndrome of Lowe protein (OCRL) controls actin dynamics during early steps of Listeria monocytogenes infection.

    PubMed

    Kühbacher, Andreas; Dambournet, Daphné; Echard, Arnaud; Cossart, Pascale; Pizarro-Cerdá, Javier

    2012-04-13

    Listeria monocytogenes is a bacterial pathogen that induces its own entry into a broad range of mammalian cells through interaction of the bacterial surface protein InlB with the cellular receptor Met, promoting an actin polymerization/depolymerization process that leads to pathogen engulfment. Phosphatidylinositol bisphosphate (PI[4,5]P(2)) and trisphosphate (PI[3,4,5]P(3)) are two major phosphoinositide species that function as molecular scaffolds, recruiting cellular effectors that regulate actin dynamics during L. monocytogenes infection. Because the phosphatidylinositol 5'-phosphatase OCRL dephosphorylates PI(4,5)P(2) and to a lesser extent PI(3,4,5)P(3), we investigated whether this phosphatase modulates cell invasion by L. monocytogenes. Inactivation of OCRL by small interfering RNA (siRNA) leads to an increase in the internalization levels of L. monocytogenes in HeLa cells. Interestingly, OCRL depletion does not increase but rather decreases the surface expression of the receptor Met, suggesting that OCRL controls bacterial internalization by modulating signaling cascades downstream of Met. Immuno-fluorescence microscopy reveals that endogenous and overexpressed OCRL are present at L. monocytogenes invasion foci; live-cell imaging additionally shows that actin depolymerization coincides with EGFP-OCRL-a accumulation around invading bacteria. Together, these observations suggest that OCRL promotes actin depolymerization during L. monocytogenes infection; in agreement with this hypothesis, OCRL depletion leads to an increase in actin, PI(4,5)P(2), and PI(3,4,5)P(3) levels at bacterial internalization foci. Furthermore, in cells knocked down for OCRL, transfection of enzymatically active EGFP-OCRL-a (but not of a phosphatase-dead enzyme) decreases the levels of intracellular L. monocytogenes and of actin associated with invading bacteria. These results demonstrate that through its phosphatase activity, OCRL restricts L. monocytogenes invasion by modulating

  8. Phosphatidylinositol 5-Phosphatase Oculocerebrorenal Syndrome of Lowe Protein (OCRL) Controls Actin Dynamics during Early Steps of Listeria monocytogenes Infection*

    PubMed Central

    Kühbacher, Andreas; Dambournet, Daphné; Echard, Arnaud; Cossart, Pascale; Pizarro-Cerdá, Javier

    2012-01-01

    Listeria monocytogenes is a bacterial pathogen that induces its own entry into a broad range of mammalian cells through interaction of the bacterial surface protein InlB with the cellular receptor Met, promoting an actin polymerization/depolymerization process that leads to pathogen engulfment. Phosphatidylinositol bisphosphate (PI[4,5]P2) and trisphosphate (PI[3,4,5]P3) are two major phosphoinositide species that function as molecular scaffolds, recruiting cellular effectors that regulate actin dynamics during L. monocytogenes infection. Because the phosphatidylinositol 5′-phosphatase OCRL dephosphorylates PI(4,5)P2 and to a lesser extent PI(3,4,5)P3, we investigated whether this phosphatase modulates cell invasion by L. monocytogenes. Inactivation of OCRL by small interfering RNA (siRNA) leads to an increase in the internalization levels of L. monocytogenes in HeLa cells. Interestingly, OCRL depletion does not increase but rather decreases the surface expression of the receptor Met, suggesting that OCRL controls bacterial internalization by modulating signaling cascades downstream of Met. Immuno-fluorescence microscopy reveals that endogenous and overexpressed OCRL are present at L. monocytogenes invasion foci; live-cell imaging additionally shows that actin depolymerization coincides with EGFP-OCRL-a accumulation around invading bacteria. Together, these observations suggest that OCRL promotes actin depolymerization during L. monocytogenes infection; in agreement with this hypothesis, OCRL depletion leads to an increase in actin, PI(4,5)P2, and PI(3,4,5)P3 levels at bacterial internalization foci. Furthermore, in cells knocked down for OCRL, transfection of enzymatically active EGFP-OCRL-a (but not of a phosphatase-dead enzyme) decreases the levels of intracellular L. monocytogenes and of actin associated with invading bacteria. These results demonstrate that through its phosphatase activity, OCRL restricts L. monocytogenes invasion by modulating actin

  9. Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane

    PubMed Central

    Gokhin, David S.; Nowak, Roberta B.; Khoory, Joseph A.; de la Piedra, Alfonso; Ghiran, Ionita C.; Fowler, Velia M.

    2015-01-01

    Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (∼25–30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ∼60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane. PMID:25717184

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed

    Oplatka, Avraham

    2006-12-15

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

  12. 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

  13. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression.

    PubMed

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the "status" of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

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

    PubMed Central

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

    1988-01-01

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

  15. Kinetics of nucleation-controlled polymerization. A perturbation treatment for use with a secondary pathway.

    PubMed Central

    Bishop, M F; Ferrone, F A

    1984-01-01

    We present a perturbation method for analyzing nucleation-controlled polymerization augmented by a secondary pathway for polymer growth. With this method, the solution to the kinetic equations assumes a simple analytic closed form that can easily be used in fitting data. So long as the formation of polymers by the secondary pathway depends linearly on the concentration of monomers polymerized, the form of the solutions is the same. This permits the analysis of augmented growth models with a minimum number of modeling assumptions, and thus makes it readily possible to distinguish between a variety of secondary processes (heterogeneous nucleation, lateral growth, and fragmentation). In addition, the parameters of the homogeneous process, such as the homogeneous nucleus size, can be determined independent of the nature of the secondary mechanism. We describe applications of this method to the polymerization of actin, collagen, and sickle hemoglobin. We present an extensive analysis of data on actin polymerization (Wegner, A., and P. Savko, 1982, Biochemistry, 21:1909-1913) to illustrate the use of the method. Although our conclusions generally agree with theirs, we find that lateral growth describes the secondary pathway better than the fragmentation model originally proposed. We also show how this method can be used to study the degree of polymerization, the parentage of polymers, and the behavior of polymers in cycling experiments. PMID:6498276

  16. Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels

    PubMed Central

    Schreiber, Joerg; Végh, Marlene J.; Dawitz, Julia; Kroon, Tim; Loos, Maarten; Labonté, Dorthe; Li, Ka Wan; Van Nierop, Pim; Van Diepen, Michiel T.; De Zeeuw, Chris I.; Kneussel, Matthias; Meredith, Rhiannon M.; Smit, August B.

    2015-01-01

    Synaptic plasticity requires remodeling of the actin cytoskeleton. Although two actin isoforms, β- and γ-actin, are expressed in dendritic spines, the specific contribution of γ-actin in the expression of synaptic plasticity is unknown. We show that synaptic γ-actin levels are regulated by the E3 ubiquitin ligase TRIM3. TRIM3 protein and Actg1 transcript are colocalized in messenger ribonucleoprotein granules responsible for the dendritic targeting of messenger RNAs. TRIM3 polyubiquitylates γ-actin, most likely cotranslationally at synaptic sites. Trim3−/− mice consequently have increased levels of γ-actin at hippocampal synapses, resulting in higher spine densities, increased long-term potentiation, and enhanced short-term contextual fear memory consolidation. Interestingly, hippocampal deletion of Actg1 caused an increase in long-term fear memory. Collectively, our findings suggest that temporal control of γ-actin levels by TRIM3 is required to regulate the timing of hippocampal plasticity. We propose a model in which TRIM3 regulates synaptic γ-actin turnover and actin filament stability and thus forms a transient inhibitory constraint on the expression of hippocampal synaptic plasticity. PMID:26527743

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Albinsson, Sebastian; Bhattachariya, Anirban; Hellstrand, Per

    2014-04-01

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

  20. All-polymeric control of nanoferronics

    PubMed Central

    Xu, Beibei; Li, Huashan; Hall, Asha; Gao, Wenxiu; Gong, Maogang; Yuan, Guoliang; Grossman, Jeffrey; Ren, Shenqiang

    2015-01-01

    In the search for light and flexible nanoferronics, significant research effort is geared toward discovering the coexisting magnetic and electric orders in crystalline charge-transfer complexes. We report the first example of multiferroicity in centimeter-sized crystalline polymeric charge-transfer superstructures that grow at the liquid-air interface and are controlled by the regioregularity of the polymeric chain. The charge order–driven ferroic mechanism reveals spontaneous and hysteretic polarization and magnetization at the donor-acceptor interface. The charge transfer and ordering in the ferroic assemblies depend critically on the self-organizing and molecular packing of electron donors and acceptors. The invention described here not only represents a new coupling mechanism of magnetic and electric ordering but also creates a new class of emerging all-organic nanoferronics. PMID:26824068

  1. All-polymeric control of nanoferronics.

    PubMed

    Xu, Beibei; Li, Huashan; Hall, Asha; Gao, Wenxiu; Gong, Maogang; Yuan, Guoliang; Grossman, Jeffrey; Ren, Shenqiang

    2015-12-01

    In the search for light and flexible nanoferronics, significant research effort is geared toward discovering the coexisting magnetic and electric orders in crystalline charge-transfer complexes. We report the first example of multiferroicity in centimeter-sized crystalline polymeric charge-transfer superstructures that grow at the liquid-air interface and are controlled by the regioregularity of the polymeric chain. The charge order-driven ferroic mechanism reveals spontaneous and hysteretic polarization and magnetization at the donor-acceptor interface. The charge transfer and ordering in the ferroic assemblies depend critically on the self-organizing and molecular packing of electron donors and acceptors. The invention described here not only represents a new coupling mechanism of magnetic and electric ordering but also creates a new class of emerging all-organic nanoferronics. PMID:26824068

  2. Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2

    PubMed Central

    Cortesio, Christa L.; Lewellyn, Eric B.; Drubin, David G.

    2015-01-01

    Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2's role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function. PMID:25694450

  3. Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2.

    PubMed

    Cortesio, Christa L; Lewellyn, Eric B; Drubin, David G

    2015-04-15

    Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2's role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function. PMID:25694450

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

    PubMed

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

    2016-02-01

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

  5. F-actin waves, actin cortex disassembly and focal exocytosis driven by actin-phosphoinositide positive feedback.

    PubMed

    Masters, Thomas A; Sheetz, Michael P; Gauthier, Nils C

    2016-04-01

    Actin polymerization is controlled by the phosphoinositide composition of the plasma membrane. However, the molecular mechanisms underlying the spatiotemporal regulation of actin network organization over extended length scales are still unclear. To observe phosphoinositide-dependent cytoskeletal dynamics we combined the model system of frustrated phagocytosis, total internal reflection microscopy and manipulation of the buffer tonicity. We found that macrophages interacting with IgG-coated glass substrates formed circular F-actin waves on their ventral surface enclosing a region of plasma membrane devoid of cortical actin. Plasma membrane free of actin cortex was strongly depleted of PI(4,5)P2 , but enriched in PI(3,4)P2 and displayed a fivefold increase in exocytosis. Wave formation could be promoted by application of a hypotonic shock. The actin waves were characteristic of a bistable wavefront at the boundary between the regions of membrane containing and lacking cortical actin. Phosphoinositide modifiers and RhoGTPase activities dramatically redistributed with respect to the wavefronts, which often exhibited spatial oscillations. Perturbation of either lipid or actin cytoskeleton-related pathways led to rapid loss of both the polarized lipid distribution and the wavefront. As waves travelled over the plasma membrane, wavefront actin was seen to rapidly polymerize and depolymerize at pre-existing clusters of FcγRIIA, coincident with rapid changes in lipid composition. Thus the potential of receptors to support rapid F-actin polymerization appears to depend acutely on the local concentrations of multiple lipid species. We propose that interdependence through positive feedback from the cytoskeleton to lipid modifiers leads to coordinated local cortex remodeling, focal exocytosis, and organizes extended actin networks. PMID:26915738

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-06-01

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

  8. Actin nucleation at the centrosome controls lymphocyte polarity

    PubMed Central

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

    2016-01-01

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

  9. Actin nucleation at the centrosome controls lymphocyte polarity.

    PubMed

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

    2016-01-01

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

  10. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    DalleDonne, I; Milzani, A; Colombo, R

    1995-01-01

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

  13. 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

  14. 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

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

    PubMed Central

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

    1991-01-01

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

  16. A synaptic F-actin network controls otoferlin-dependent exocytosis in auditory inner hair cells

    PubMed Central

    Vincent, Philippe FY; Bouleau, Yohan; Petit, Christine; Dulon, Didier

    2015-01-01

    We show that a cage-shaped F-actin network is essential for maintaining a tight spatial organization of Cav1.3 Ca2+ channels at the synaptic ribbons of auditory inner hair cells. This F-actin network is also found to provide mechanosensitivity to the Cav1.3 channels when varying intracellular hydrostatic pressure. Furthermore, this F-actin mesh network attached to the synaptic ribbons directly influences the efficiency of otoferlin-dependent exocytosis and its sensitivity to intracellular hydrostatic pressure, independently of its action on the Cav1.3 channels. We propose a new mechanistic model for vesicle exocytosis in auditory hair cells where the rate of vesicle recruitment to the ribbons is directly controlled by a synaptic F-actin network and changes in intracellular hydrostatic pressure. DOI: http://dx.doi.org/10.7554/eLife.10988.001 PMID:26568308

  17. A synaptic F-actin network controls otoferlin-dependent exocytosis in auditory inner hair cells.

    PubMed

    Vincent, Philippe Fy; Bouleau, Yohan; Petit, Christine; Dulon, Didier

    2015-01-01

    We show that a cage-shaped F-actin network is essential for maintaining a tight spatial organization of Cav1.3 Ca(2+) channels at the synaptic ribbons of auditory inner hair cells. This F-actin network is also found to provide mechanosensitivity to the Cav1.3 channels when varying intracellular hydrostatic pressure. Furthermore, this F-actin mesh network attached to the synaptic ribbons directly influences the efficiency of otoferlin-dependent exocytosis and its sensitivity to intracellular hydrostatic pressure, independently of its action on the Cav1.3 channels. We propose a new mechanistic model for vesicle exocytosis in auditory hair cells where the rate of vesicle recruitment to the ribbons is directly controlled by a synaptic F-actin network and changes in intracellular hydrostatic pressure. PMID:26568308

  18. 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

  19. Controlled polymerization by incarceration of monomers in nanochannels.

    PubMed

    Uemura, Takashi; Kitagawa, Susumu

    2010-01-01

    Porous Coordination Polymers (PCPs) composed of transition metal ions and bridging organic ligands have been extensively studied. The characteristic features of PCPs are highly regular channel structures, controllable channel sizes approximating molecular dimensions, designable surface potentials and functionality, and flexible frameworks responsive to guest molecules. Owing to these advantages, successful applications of PCPs range from molecular storage and separation to heterogeneous catalysts. In particular, use of their regulated and tunable nanochannels in the field of polymerization has allowed multi-level control of polymerization via control of stereoregularlity, molecular weight, etc. In this chapter, we focus on recent progress in polymerization utilizing the nanochannels of PCPs, and demonstrate why this polymerization system is attractive and promising from the viewpoint of precision control of polymeric structures. PMID:21618745

  20. Direct dynamin–actin interactions regulate the actin cytoskeleton

    PubMed Central

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

    2010-01-01

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

  1. 2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils.

    PubMed

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

    2012-01-15

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

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

    PubMed

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

    2016-07-01

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

  3. Profilin connects actin assembly with microtubule dynamics.

    PubMed

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

    2016-08-01

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

  4. Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media

    SciTech Connect

    Sanders, Lori K.; Xian, Wujing; Guaqueta, Camilo; Strohman, Michael J.; Vrasich, Chuck R.; Luijten, Erik; Wong, Gerard C.L.

    2008-07-11

    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.

  5. Control of Electrostatic Interactions Between F-Actin And Genetically Modified Lysozyme in Aqueous Media

    SciTech Connect

    Sanders, L.K.; Xian, W.; Guaqueta, C.; Strohman, M.; Vrasich, C.R.; Luijten, E.; Wong, G.C.L.

    2009-06-04

    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.

  6. Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions

    PubMed Central

    Baranwal, Somesh

    2015-01-01

    Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. PMID:25792565

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

    PubMed Central

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

    2014-01-01

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

  8. 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

  9. Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs

    PubMed Central

    Zheng, Maozhong; Beck, Martina; Müller, Jens; Chen, Tong; Wang, Xiaohua; Wang, Feng; Wang, Qinli; Wang, Yuqing; Baluška, František; Logan, David C.; Šamaj, Jozef; Lin, Jinxing

    2009-01-01

    Background Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements. Methodology/Principal Findings We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria. Conclusions/Significance Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events. PMID:19536333

  10. How a single residue in individual β-thymosin/WH2 domains controls their functions in actin assembly

    PubMed Central

    Didry, Dominique; Cantrelle, Francois-Xavier; Husson, Clotilde; Roblin, Pierre; Moorthy, Anna M Eswara; Perez, Javier; Le Clainche, Christophe; Hertzog, Maud; Guittet, Eric; Carlier, Marie-France; van Heijenoort, Carine; Renault, Louis

    2012-01-01

    β-Thymosin (βT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin-β4, or enhance motility by directing polarized filament assembly like Ciboulot βT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-β4, Ciboulot, TetraThymosinβ and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-β4. Functionally different βT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long βT and WH2 domains. The results open perspectives for elucidating the functions of βT/WH2 domains in other modular proteins. PMID:22193718

  11. Measuring F-actin properties in dendritic spines

    PubMed Central

    Koskinen, Mikko; Hotulainen, Pirta

    2014-01-01

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

  12. 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.

  13. Application of Controlled Radical Polymerization for Nucleic Acid Delivery

    PubMed Central

    CHU, DAVID S.H.; SCHELLINGER, JOAN G.; SHI, JULIE; CONVERTINE, ANTHONY J.; STAYTON, PATRICK S.; PUN, SUZIE H.

    2012-01-01

    CONSPECTUS Nucleic acid-based therapeutics can potentially address otherwise untreatable genetic disorders and have significant potential for a wide range of diseases. Therapeutic gene delivery can restore protein function by replacing defunct genes to restore cellular health while RNA interference (RNAi) can mask mutated and harmful genes. Cationic polymers have been extensively studied for nucleic acid delivery applications due to their self-assembly with nucleic acids into virus-sized nanoparticles and high transfection efficiency in vitro, but toxicity and particle stability have limited their clinical applications. The advent of controlled radical polymerization has improved the quality, control and reproducibility of synthesized materials. Controlled radical polymerization yields well-defined, narrowly disperse materials of designable architectures and molecular weight, allowing study of the effects of polymer architecture and molecular weight on transfection efficiency and cytotoxicity for improved design of next-generation vectors. Robust methods such as atom transfer radical polymerization (ATRP), reverse addition-fragmentation chain transfer polymerization (RAFT), and ring-opening metastasis polymerization (ROMP) have been used to engineer materials that specifically enhance extracellular stability, cellular specificity, and decrease toxicity. This Account reviews findings from structure-function studies that have elucidated key design motifs necessary for the development of effective nucleic acid vectors. In addition, polymers that are biodegradable, form supramolecular structures, target specific cells, or facilitate endosomal release are also discussed. Finally, promising materials with in vivo applications ranging from pulmonary gene delivery to DNA vaccines are described. PMID:22242774

  14. Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip

    PubMed Central

    Bornschlögl, Thomas; Romero, Stéphane; Vestergaard, Christian L.; Joanny, Jean-François; Van Nhieu, Guy Tran; Bassereau, Patricia

    2013-01-01

    Filopodia are dynamic, finger-like plasma membrane protrusions that sense the mechanical and chemical surroundings of the cell. Here, we show in epithelial cells that the dynamics of filopodial extension and retraction are determined by the difference between the actin polymerization rate at the tip and the retrograde flow at the base of the filopodium. Adhesion of a bead to the filopodial tip locally reduces actin polymerization and leads to retraction via retrograde flow, reminiscent of a process used by pathogens to invade cells. Using optical tweezers, we show that filopodial retraction occurs at a constant speed against counteracting forces up to 50 pN. Our measurements point toward retrograde flow in the cortex together with frictional coupling between the filopodial and cortical actin networks as the main retraction-force generator for filopodia. The force exerted by filopodial retraction, however, is limited by the connection between filopodial actin filaments and the membrane at the tip. Upon mechanical rupture of the tip connection, filopodia exert a passive retraction force of 15 pN via their plasma membrane. Transient reconnection at the tip allows filopodia to continuously probe their surroundings in a load-and-fail manner within a well-defined force range. PMID:24198333

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

    PubMed

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

    2016-02-15

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

  16. Functional polymeric microparticles engineered from controllable microfluidic emulsions.

    PubMed

    Wang, Wei; Zhang, Mao-Jie; Chu, Liang-Yin

    2014-02-18

    Functional polymeric microparticles with typical sizes of 1-1000 μm have received considerable attention for many applications. Especially in biomedical fields, polymeric microparticles with advanced functions such as targeted delivery, controlled encapsulation, or "capture and release" show great importance as delivery systems for active molecules and drugs, as imaging agents for analytics and diagnostics, as microreactors for confined bioreactions, and more. Generally, the functions of these microparticles rely on both their structures and the properties of their component materials. Thus, creating unique structures from functional materials provides an important strategy for developing advanced functional polymeric microparticles. Several methods, such as dispersion polymerization, precipitation polymerization, copolymer self-assembly, and phase-separated polymer precipitation can be used to make functional microparticles, but each has limitations, for example, their limited control over the particle size and structure. Using emulsions as templates, however, allows precise control over the size, shape, composition, and structure of the resulting microparticles by tuning those of the emulsions via specific emulsification techniques. Microfluidic methods offer excellent control of emulsion droplets, thereby providing a powerful platform for continuous, reproducible, scalable production of polymeric microparticles with unprecedented control over their monodispersity, structures, and compositions. This approach provides broad opportunities for producing polymeric microparticles with novel structure-property combinations and elaborately designed functions. In this Account, we highlight recent efforts in microfluidic fabrication of advanced polymeric microparticles with well-designed functions for potential biomedical applications, and we describe the development of microfluidic techniques for producing monodisperse and versatile emulsion templates. We begin by

  17. Actin from Saccharomyces cerevisiae.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

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

  18. A possible role of actin in the mechanical control of the cell cycle.

    PubMed

    Tripathi, S C

    1989-01-01

    Sail-sheet Cultures (SSC) are those in which the cells are i) grown within the meshes of inert grids ii) exposed to nutrients from most sides iii) attached to one another only at the edges like sail of a yacht (hence, the name 'sail-sheet') and iv) have the advantage of three-dimensional structure similar to an in vivo situation. We grew fibroblasts from chicken heart explants as SSC and studied the effect of mechanical stretching on the F-actin content of these cells. This study was designed to investigate the hypothesis that the effect of tension on the cell cycle may be channeled through the microfilaments. Data from this preliminary study suggested that short-term mechanical stretching of sail-sheets, using low frequency tension (1.0 Hz), diminishes F-actin. Thus, it may be possible to relate the decrease in the F-actin content of these cells to the slowing down of their locomotory activity, possible rounding up, and division. This study might contribute to the understanding of the mechanical control of the cell cycle and be of relevance in the phenomena such as healing of wounds and control of the cell division in tumors. PMID:2620166

  19. The yin-yang of dendrite morphology: unity of actin and microtubules.

    PubMed

    Georges, Penelope C; Hadzimichalis, Norell M; Sweet, Eric S; Firestein, Bonnie L

    2008-12-01

    Actin and microtubules (MT) are targets of numerous molecular pathways that control neurite outgrowth. To generate a neuronal protrusion, coordinated structural changes of the actin and MT cytoskeletons must occur. Neurite formation occurs when actin filaments (F-actin) are destabilized, filopodia are extended, and MTs invade filopodia. This process results in either axon or dendrite formation. Axonal branching involves interplay between F-actin and MTs, with F-actin and MTs influencing polymerization, stabilization, and maintenance of each other. Our knowledge of the mechanisms regulating development of the axon, however, far eclipses our understanding of dendritic development and branching. The two classes of neurites, while fundamentally similar in their ability to elongate and branch, dramatically differ in growth rate, orientation of polarized MT bundles, and mechanisms that initiate branching. In this review, we focus on how F-actin, MTs, and proteins that link the two cytoskeletons coordinate to specifically initiate dendritic events. PMID:18987787

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  2. Cdc42-dependent actin dynamics controls maturation and secretory activity of dendritic cells

    PubMed Central

    Schulz, Anna M.; Stutte, Susanne; Hogl, Sebastian; Luckashenak, Nancy; Dudziak, Diana; Leroy, Céline; Forné, Ignasi; Imhof, Axel; Müller, Stephan A.; Brakebusch, Cord H.; Lichtenthaler, Stefan F.

    2015-01-01

    Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration, and proliferation. To further study the molecular mechanisms of dendritic cell (DC) regulation by Cdc42, we used Cdc42-deficient DCs. Cdc42 deficiency renders DCs phenotypically mature as they up-regulate the co-stimulatory molecule CD86 from intracellular storages to the cell surface. Cdc42 knockout DCs also accumulate high amounts of invariant chain–major histocompatibility complex (MHC) class II complexes at the cell surface, which cannot efficiently present peptide antigens (Ag’s) for priming of Ag-specific CD4 T cells. Proteome analyses showed a significant reduction in lysosomal MHC class II–processing proteins, such as cathepsins, which are lost from DCs by enhanced secretion. As these effects on DCs can be mimicked by chemical actin disruption, our results propose that Cdc42 control of actin dynamics keeps DCs in an immature state, and cessation of Cdc42 activity during DC maturation facilitates secretion as well as rapid up-regulation of intracellular molecules to the cell surface. PMID:26553928

  3. 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.

  4. Control of electrostatic interactions between F-actin and genetically modified lysozyme in aqueous media

    PubMed Central

    Sanders, Lori K.; Xian, Wujing; Guáqueta, Camilo; Strohman, Michael J.; Vrasich, Chuck R.; Luijten, Erik; Wong, Gerard C. L.

    2007-01-01

    The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin–lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin–lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin–lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity. PMID:17911256

  5. Shank–cortactin interactions control actin dynamics to maintain flexibility of neuronal spines and synapses

    PubMed Central

    MacGillavry, Harold D.; Kerr, Justin M.; Kassner, Josh; Frost, Nicholas A.; Blanpied, Thomas A.

    2016-01-01

    The family of Shank scaffolding molecules (comprising Shank1, 2 and 3) are core components of the postsynaptic density (PSD) in neuronal synapses. Shanks link surface receptors to other scaffolding molecules within the PSD, as well as to the actin cytoskeleton. However, determining the function of Shank proteins in neurons has been complicated because the different Shank isoforms share a very high degree of sequence and domain homology. Therefore, to control Shank content while minimizing potential compensatory effects, a miRNA-based knockdown strategy was developed to reduce the expression of all synaptically targeted Shank isoforms simultaneously in rat hippocampal neurons. Using this approach, a strong (>75%) reduction in total Shank protein levels was achieved at individual dendritic spines, prompting an approximately 40% decrease in mushroom spine density. Furthermore, Shank knockdown reduced spine actin levels and increased sensitivity to the actin depolymerizing agent Latrunculin A. A SHANK2 mutant lacking the proline-rich cortactin-binding motif (SHANK2-ΔPRO) was unable to rescue these defects. Furthermore, Shank knockdown reduced cortactin levels in spines and increased the mobility of spine cortactin as measured by single-molecule tracking photoactivated localization microscopy, suggesting that Shank proteins recruit and stabilize cortactin at the synapse. Furthermore, it was found that Shank knockdown significantly reduced spontaneous remodelling of synapse morphology that could not be rescued by the SHANK2-ΔPRO mutant. It was concluded that Shank proteins are key intermediates between the synapse and the spine interior that, via cortactin, permit the actin cytoskeleton to dynamically regulate synapse morphology and function. PMID:26547831

  6. Actinic Keratosis

    MedlinePlus

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

  7. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators

    PubMed Central

    Dopie, Joseph; Rajakylä, Eeva K.; Joensuu, Merja S.; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K.

    2015-01-01

    ABSTRACT Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes. PMID:26021350

  8. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators.

    PubMed

    Dopie, Joseph; Rajakylä, Eeva K; Joensuu, Merja S; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K

    2015-07-01

    Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes. PMID:26021350

  9. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls.

    PubMed

    Butler, J H; Hu, S; Brady, S R; Dixon, M W; Muday, G K

    1998-02-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo. PMID:11536873

  10. In vitro and in vivo evidence for actin association of the naphthylphthalamic acid-binding protein from zucchini hypocotyls

    NASA Technical Reports Server (NTRS)

    Butler, J. H.; Hu, S.; Brady, S. R.; Dixon, M. W.; Muday, G. K.

    1998-01-01

    The N-1-naphthylphthalamic acid (NPA)-binding protein is part of the auxin efflux carrier, the protein complex that controls polar auxin transport in plant tissues. This study tested the hypothesis that the NPA-binding protein (NBP) is associated with the actin cytoskeleton in vitro and that an intact actin cytoskeleton is required for polar auxin transport in vivo. Cytoskeletal polymerization was altered in extracts of zucchini hypocotyls with reagents that stabilized either the polymeric or monomeric forms of actin or tubulin. Phalloidin treatment altered actin polymerization, as demonstrated by immunoblot analyses following native and denaturing electrophoresis. Phalloidin increased both filamentous actin (F-actin) and NPA-binding activity, while cytochalasin D and Tris decreased both F-actin and NPA-binding activity in cytoskeletal pellets. The microtubule stabilizing drug taxol increased pelletable tubulin, but did not alter either the amount of pelletable actin or NPA-binding activity. Treatment of etiolated zucchini hypocotyls with cytochalasin D decreased the amount of auxin transport and its regulation by NPA. These experimental results are consistent with an in vitro actin cytoskeletal association of the NPA-binding protein and with the requirement of an intact actin cytoskeleton for maximal polar auxin transport in vivo.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  13. Actin Dynamics in Growth Cone Motility and Navigation

    PubMed Central

    Gomez, Timothy M.; Letourneau, Paul C.

    2014-01-01

    Motile growth cones lead growing axons through developing tissues to synaptic targets. These behaviors depend on the organization and dynamics of actin filaments that fill the growth cone leading margin (peripheral (P-) domain). Actin filament organization in growth cones is regulated by actin-binding proteins that control all aspects of filament assembly, turnover, interactions with other filaments and cytoplasmic components, and participation in producing mechanical forces. Actin filament polymerization drives protrusion of sensory filopodia and lamellipodia, and actin filament connections to the plasma membrane link the filament network to adhesive contacts of filopodia and lamellipodia with other surfaces. These contacts stabilize protrusions and transduce mechanical forces generated by actomyosin activity into traction that pulls an elongating axon along the path towards its target. Adhesive ligands and extrinsic guidance cues bind growth cone receptors and trigger signaling activities involving Rho GTPases, kinases, phosphatases, cyclic nucleotides and [Ca++] fluxes. These signals regulate actin binding proteins to locally modulate actin polymerization, interactions and force transduction to steer the growth cone leading margin towards the sources of attractive cues and away from repellent guidance cues. PMID:24164353

  14. Skylab D024 thermal control coatings and polymeric films experiment

    NASA Technical Reports Server (NTRS)

    Lehn, William L.; Hurley, Charles J.

    1992-01-01

    The Skylab D024 Thermal Control Coatings and Polymeric Films Experiment was designed to determine the effects of the external Skylab space environment on the performance and properties of a wide variety of selected thermal control coatings and polymeric films. Three duplicate sets of thermal control coatings and polymeric films were exposed to the Skylab space environment for varying periods of time during the mission. The specimens were retrieved by the astronauts during extravehicular activities (EVA) and placed in hermetically sealed return containers, recovered, and returned to the Wright Laboratory/Materials Laboratory/WPAFB, Ohio for analysis and evaluation. Postflight analysis of the three sets of recovered thermal control coatings indicated that measured changes in specimen thermo-optical properties were due to a combination of excessive contamination and solar degradation of the contaminant layer. The degree of degradation experienced over-rode, obscured, and compromised the measurement of the degradation of the substrate coatings themselves. Results of the analysis of the effects of exposure on the polymeric films and the contamination observed are also presented. The D024 results were used in the design of the LDEF M0003-5 Thermal Control Materials Experiment. The results are presented here to call to the attention of the many other LDEF experimenters the wealth of directly related, low earth orbit, space environmental exposure data that is available from the ten or more separate experiments that were conducted during the Skylab mission. Results of these experiments offer data on the results of low altitude space exposure on materials recovered from space with exposure longer than typical STS experiments for comparison with the LDEF results.

  15. Shape Control in Engineering of Polymeric Nanoparticles for Therapeutic Delivery

    PubMed Central

    Williford, John-Michael; Santos, Jose Luis; Shyam, Rishab; Mao, Hai-Quan

    2015-01-01

    Nanoparticle-mediated delivery of therapeutics holds great potential for the diagnosis and treatment of a wide range of diseases. Significant advances have been made in the design of new polymeric nanoparticle carriers through modulation of their physical and chemical structures and biophysical properties. Nanoparticle shape has been increasingly proposed as an important attribute dictating their transport properties in biological milieu. In this review, we highlight three major methods for preparing polymeric nanoparticles that allow for exquisite control of particle shape. Special attention is given to various approaches to controlling nanoparticle shape by tuning copolymer structural parameters and assembly conditions. This review also provides comparisons of these methods in terms of their unique capabilities, materials choices, and specific delivery cargos, and summarizes the biological effects of nanoparticle shape on transport properties at the tissue and cellular levels. PMID:26146550

  16. Immunological Responses and Actin Dynamics in Macrophages Are Controlled by N-Cofilin but Are Independent from ADF

    PubMed Central

    Jönsson, Friederike; Gurniak, Christine B.; Fleischer, Bernhard; Kirfel, Gregor; Witke, Walter

    2012-01-01

    Dynamic changes in the actin cytoskeleton are essential for immune cell function and a number of immune deficiencies have been linked to mutations, which disturb the actin cytoskeleton. In macrophages and dendritic cells, actin remodelling is critical for motility, phagocytosis and antigen presentation, however the actin binding proteins, which control antigen presentation have been poorly characterized. Here we dissect the specific roles of the family of ADF/cofilin F-actin depolymerizing factors in macrophages and in local immune responses. Macrophage migration, cell polarization and antigen presentation to T-cells require n-cofilin mediated F-actin remodelling. Using a conditional mouse model, we show that n-cofilin also controls MHC class II-dependent antigen presentation. Other cellular processes such as phagocytosis and antigen processing were found to be independent of n-cofilin. Our data identify n-cofilin as a novel regulator of antigen presentation, while ADF on the other hand is dispensable for macrophage motility and antigen presentation. PMID:22558315

  17. Actin dynamics: from nanoscale to microscale.

    PubMed

    Carlsson, Anders E

    2010-01-01

    The dynamic nature of actin in cells manifests itself constantly. Polymerization near the cell edge is balanced by depolymerization in the interior, externally induced actin polymerization is followed by depolymerization, and spontaneous oscillations of actin at the cell periphery are frequently seen. I discuss how mathematical modeling relates quantitative measures of actin dynamics to the rates of underlying molecular level processes. The dynamic properties addressed include the rate of actin assembly at the leading edge of a moving cell, the disassembly rates of intracellular actin networks, the polymerization time course in externally stimulated cells, and spontaneous spatiotemporal patterns formed by actin. Although several aspects of actin assembly have been clarified by increasingly sophisticated models, our understanding of rapid actin disassembly is limited, and the origins of nonmonotonic features in externally stimulated actin polymerization remain unclear. Theory has generated several concrete, testable hypotheses for the origins of spontaneous actin waves and cell-edge oscillations. The development and use of more biomimetic systems applicable to the geometry of a cell will be key to obtaining a quantitative understanding of actin dynamics in cells. PMID:20462375

  18. Quantifying actin wave modulation on periodic topography

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  19. Nogo-A controls structural plasticity at dendritic spines by rapidly modulating actin dynamics.

    PubMed

    Kellner, Yves; Fricke, Steffen; Kramer, Stella; Iobbi, Cristina; Wierenga, Corette J; Schwab, Martin E; Korte, Martin; Zagrebelsky, Marta

    2016-06-01

    Nogo-A and its receptors have been shown to control synaptic plasticity, including negatively regulating long-term potentiation (LTP) in the cortex and hippocampus at a fast time scale and restraining experience-dependent turnover of dendritic spines over days. However, the molecular mechanisms and the precise time course mediating these actions of Nogo-A are largely unexplored. Here we show that Nogo-A signaling in the adult nervous system rapidly modulates the spine actin cytoskeleton within minutes to control structural plasticity at dendritic spines of CA3 pyramidal neurons. Indeed, acute Nogo-A loss-of-function transiently increases F-actin stability and results in an increase in dendritic spine density and length. In addition, Nogo-A acutely restricts AMPAR insertion and mEPSC amplitude at hippocampal synaptic sites. These data indicate a crucial function of Nogo-A in modulating the very tight balance between plasticity and stability of the neuronal circuitry underlying learning processes and the ability to store long-term information in the mature CNS. © 2016 Wiley Periodicals, Inc. PMID:26748478

  20. ROP Gtpase–Dependent Dynamics of Tip-Localized F-Actin Controls Tip Growth in Pollen Tubes

    PubMed Central

    Fu, Ying; Wu, Guang; Yang, Zhenbiao

    2001-01-01

    Tip-growing pollen tubes provide a useful model system to study polar growth. Although roles for tip-focused calcium gradient and tip-localized Rho-family GTPase in pollen tube growth is established, the existence and function of tip-localized F-actin have been controversial. Using the green fluorescent protein–tagged actin-binding domain of mouse talin, we found a dynamic form of tip-localized F-actin in tobacco pollen tubes, termed short actin bundles (SABs). The dynamics of SABs during polar growth in pollen tubes is regulated by Rop1At, a Rop GTPase belonging to the Rho family. When overexpressed, Rop1At transformed SAB into a network of fine filaments and induced a transverse actin band behind the tip, leading to depolarized growth. These changes were due to ectopic Rop1At localization to the apical region of the plasma membrane and were suppressed by guanine dissociation inhibitor overexpression, which removed ectopically localized Rop1At. Rop GTPase–activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes. Moreover, overexpression of RopGAP1 alone disrupted SABs and inhibited growth. Finally, SAB oscillates and appears at the tip before growth. Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants. PMID:11238457

  1. Photomediated Controlled Radical Polymerization and Block Copolymerization of Vinylidene Fluoride.

    PubMed

    Asandei, Alexandru D

    2016-02-24

    This review summarizes recent research on novel photochemical methods for the initiation and control of the polymerization of main chain fluorinated monomers as exemplified by vinylidene fluoride (VDF) and for the synthesis of their block copolymers. Such reactions can be carried out at ambient temperature in glass tubes using visible light. Novel, original protocols include the use of hypervalent iodide carboxylates alone or in conjunction with molecular iodine, as well as the use of photoactive transition metal carbonyls in the presence of alkyl, fluoroalkyl, and perfluoroalkyl halides. An in-depth study of the reaction parameters highlights the use of dimethyl carbonate as a preferred polymerization solvent and outlines the structure-property relationship for hypervalent iodide carboxylates and halide initiators in both the free radical and iodine degenerative transfer controlled radical polymerization (IDT-CRP) of VDF. Finally, the rational selection of metal carbonyls that are successful not only as IDT mediators but, more importantly, in the quantitative activation of both PVDF-CH2-CF2-I and PVDF-CF2-CH2-I chain ends toward the synthesis of well-defined PVDF block copolymers is presented. PMID:26760676

  2. Controlled release of ethylene via polymeric films for food packaging

    NASA Astrophysics Data System (ADS)

    Pisano, Roberto; Bazzano, Marco; Capozzi, Luigi Carlo; Ferri, Ada; Sangermano, Marco

    2015-12-01

    In modern fruit supply chain a common method to trigger ripening is to keep fruits inside special chambers and initiate the ripening process through administration of ethylene. Ethylene is usually administered through cylinders with inadequate control of its final concentration in the chamber. The aim of this study is the development of a new technology to accurately regulate ethylene concentration in the atmosphere where fruits are preserved: a polymeric film, containing an inclusion complex of α-cyclodextrin with ethylene, was developed. The complex was prepared by molecular encapsulation which allows the entrapment of ethylene into the cavity of α-cyclodextrin. After encapsulation, ethylene can be gradually released from the inclusion complex and its release rate can be regulated by temperature and humidity. The inclusion complex was dispersed into a thin polymeric film produced by UV-curing. This method was used because is solvent-free and involves low operating temperature; both conditions are necessary to prevent rapid release of ethylene from the film. The polymeric films were characterized with respect to thermal behaviour, crystalline structure and kinetics of ethylene release, showing that can effectively control the release of ethylene within confined volume.

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

    SciTech Connect

    Seo, Myungeun; Hillmyer, Marc A.

    2013-04-08

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

  4. Atomic Force Microscopy and Light Scattering of Small Unilamellar Actin-Containing Liposomes

    PubMed Central

    Palmer, Andre F.; Wingert, Philip; Nickels, Jonathan

    2003-01-01

    Three-dimensional networks of filamentous actin (F-actin) encapsulated inside phosphatidylcholine liposomes are currently being used in an effort to model the cytoskeleton and plasma membrane of eukaryotic cells. In this article, unilamellar lipid vesicles consisting of egg yolk-derived phosphatidylcholine encapsulating monomeric actin (G-actin) were made via extrusion in low ionic strength buffer (G-buffer). Vesicle shape and structure in these dispersions was studied using a combination of fluid-tapping atomic force microscopy, and multiangle static light scattering. After subjecting the liposome dispersion to high ionic strength polymerization buffer (F-buffer) containing K+ ions, atomic force microscopy imaging and light scattering of these liposomes indicated the formation of specialized structures, including an overall liposome structure transformation from spherical to torus, disk-shaped geometries and tubular assemblies. Several atomic force microscopy control measurements were made to ascertain that the specialized structures formed were not due to free G-actin and F-actin self-assembling on the sample surface, plain liposomes exposed to G- and F-buffer, or liposomes encapsulating G-actin. Liposomes encapsulating G-actin assumed mostly thin disk shapes and some large irregularly shaped aggregates. In contrast, liposomes encapsulating polymerized actin assumed mostly torus or disk shapes along with some high aspect ratio tubular structures. PMID:12885667

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

    PubMed Central

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

    2013-01-01

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

  6. Chemical control of rate and onset temperature of nadimide polymerization

    NASA Technical Reports Server (NTRS)

    Lauver, R. W.

    1985-01-01

    The chemistry of norbornenyl capped imide compounds (nadimides) is briefly reviewed with emphasis on the contribution of Diels-Alder reversion in controlling the rate and onset of the thermal polymerization reaction. Control of onset temperature of the cure exotherm by adjusting the concentration of maleimide is demonstrated using selected model compounds. The effects of nitrophenyl compounds as free radical retarders on nadimide reactivity are discussed. A simple copolymerization model is proposed for the overall nadimide cure reaction. An approximate numerical analysis is carried out to demonstrate the ability of the model to simulate the trends observed for both maleimide and nitrophenyl additions.

  7. The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity

    PubMed Central

    Strale, Pierre-Olivier; Duchesne, Laurence; Peyret, Grégoire; Montel, Lorraine; Nguyen, Thao; Png, Evelyn; Tampé, Robert; Troyanovsky, Sergey; Hénon, Sylvie; Ladoux, Benoit

    2015-01-01

    Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell–cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell–cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell–cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell–cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell–cell contact fluidity. PMID:26195669

  8. 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.

  9. Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation

    PubMed Central

    Adachi, Taiji

    2014-01-01

    The physiological role of the actin cytoskeleton is well known: it provides mechanical support and endogenous force generation for formation of a cell shape and for migration. Furthermore, a growing number of studies have demonstrated another significant role of the actin cytoskeleton: it offers dynamic epigenetic memory for guiding cell fate, in particular, proliferation and differentiation. Because instantaneous imbalance in the mechanical homeostasis is adjusted through actin remodeling, a synthetic extracellular matrix (ECM) niche as a source of topographical and mechanical cues is expected to be effective at modulation of the actin cytoskeleton. In this context, the synthetic ECM niche determines cell migration, proliferation, and differentiation, all of which have to be controlled in functional tissue engineering scaffolds to ensure proper regulation of tissue/organ formation, maintenance of tissue integrity and repair, and regeneration. Here, with an emphasis on the epigenetic role of the actin cytoskeletal system, we propose a design concept of micro/nanotopography of a tissue engineering scaffold for control of cell migration, proliferation, and differentiation in a stable and well-defined manner, both in vitro and in vivo. PMID:24720435

  10. Topography design concept of a tissue engineering scaffold for controlling cell function and fate through actin cytoskeletal modulation.

    PubMed

    Miyoshi, Hiromi; Adachi, Taiji

    2014-12-01

    The physiological role of the actin cytoskeleton is well known: it provides mechanical support and endogenous force generation for formation of a cell shape and for migration. Furthermore, a growing number of studies have demonstrated another significant role of the actin cytoskeleton: it offers dynamic epigenetic memory for guiding cell fate, in particular, proliferation and differentiation. Because instantaneous imbalance in the mechanical homeostasis is adjusted through actin remodeling, a synthetic extracellular matrix (ECM) niche as a source of topographical and mechanical cues is expected to be effective at modulation of the actin cytoskeleton. In this context, the synthetic ECM niche determines cell migration, proliferation, and differentiation, all of which have to be controlled in functional tissue engineering scaffolds to ensure proper regulation of tissue/organ formation, maintenance of tissue integrity and repair, and regeneration. Here, with an emphasis on the epigenetic role of the actin cytoskeletal system, we propose a design concept of micro/nanotopography of a tissue engineering scaffold for control of cell migration, proliferation, and differentiation in a stable and well-defined manner, both in vitro and in vivo. PMID:24720435

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

    PubMed Central

    2010-01-01

    Background Most in vivo studies that have addressed the role of actin dynamics in NADPH oxidase function in phagocytes have used toxins to modulate the polymerization state of actin and mostly effects on actin has been evaluated by end point measurements of filamentous actin, which says little about actin dynamics, and without consideration for the subcellular distribution of the perturbed actin cytoskeleton. Results Here, we in addition to toxins use conditional expression of the major actin regulatory protein LIM kinase-1 (LIMK1), and shRNA knock-down of cofilin to modulate the cellular F/G-actin ratio in the Ra2 microglia cell line, and we use Fluorescence Recovery after Photobleaching (FRAP) in β-actin-YFP-transduced cells to obtain a dynamic measure of actin recovery rates (actin turn-over rates) in different F/G-actin states of the actin cytoskeleton. Our data demonstrate that stimulated NADPH oxidase function was severely impaired only at extreme actin recovery rates and F/G-actin ratios, and surprisingly, that any moderate changes of these parameters of the actin cytoskeleton invariably resulted in an increased NADPH oxidase activity. Conclusion moderate actin polymerization and depolymerization both increase the FMLP and PMA-stimulated NADPH oxidase activity of microglia, which is directly correlated with neither actin recovery rate nor F/G- actin ratio. Our results indicate that NADPH oxidase functions in an enhanced state of activity in stimulated phagocytes despite widely different states of the actin cytoskeleton. PMID:20825680

  12. Actinic keratosis

    MedlinePlus

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

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

    PubMed Central

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

    2016-01-01

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

  14. Tissue-specific mechanical and geometrical control of cell viability and actin cytoskeleton alignment

    NASA Astrophysics Data System (ADS)

    Wang, Dong; Zheng, Wenfu; Xie, Yunyan; Gong, Peiyuan; Zhao, Fang; Yuan, Bo; Ma, Wanshun; Cui, Yan; Liu, Wenwen; Sun, Yi; Piel, Matthieu; Zhang, Wei; Jiang, Xingyu

    2014-08-01

    Different tissues have specific mechanical properties and cells of different geometries, such as elongated muscle cells and polygonal endothelial cells, which are precisely regulated during embryo development. However, the mechanisms that underlie these processes are not clear. Here, we built an in vitro model to mimic the cellular microenvironment of muscle by combining both mechanical stretch and geometrical control. We found that mechanical stretch was a key factor that determined the optimal geometry of myoblast C2C12 cells under stretch, whereas vascular endothelial cells and fibroblasts had no such dependency. We presented the first experimental evidence that can explain why myoblasts are destined to take the elongated geometry so as to survive and maintain parallel actin filaments along the stretching direction. The study is not only meaningful for the research on myogenesis but also has potential application in regenerative medicine.

  15. Fixed Point Transformations Based Iterative Control of a Polymerization Reaction

    NASA Astrophysics Data System (ADS)

    Tar, József K.; Rudas, Imre J.

    As a paradigm of strongly coupled non-linear multi-variable dynamic systems the mathematical model of the free-radical polymerization of methyl-metachrylate with azobis (isobutyro-nitrile) as an initiator and toluene as a solvent taking place in a jacketed Continuous Stirred Tank Reactor (CSTR) is considered. In the adaptive control of this system only a single input variable is used as the control signal (the process input, i.e. dimensionless volumetric flow rate of the initiator), and a single output variable is observed (the process output, i.e. the number-average molecular weight of the polymer). Simulation examples illustrate that on the basis of a very rough and primitive model consisting of two scalar variables various fixed-point transformations based convergent iterations result in a novel, sophisticated adaptive control.

  16. Controlling internal pore sizes in bicontinuous polymeric nanospheres.

    PubMed

    McKenzie, Beulah E; Friedrich, Heiner; Wirix, Maarten J M; de Visser, Joël F; Monaghan, Olivia R; Bomans, Paul H H; Nudelman, Fabio; Holder, Simon J; Sommerdijk, Nico A J M

    2015-02-16

    Complex polymeric nanospheres were formed in water from comb-like amphiphilic block copolymers. Their internal morphology was determined by three-dimensional cryo-electron tomographic analysis. Varying the polymer molecular weight (MW) and the hydrophilic block weight content allowed for fine control over the internal structure. Construction of a partial phase diagram allowed us to determine the criteria for the formation of bicontinuous polymer nanosphere (BPN), namely for copolymers with MW of up to 17 kDa and hydrophilic weight fractions of ≤0.25; and varying the organic solvent to water ratio used in their preparation allowed for control over nanosphere diameters from 70 to 460 nm. Significantly, altering the block copolymer hydrophilic-hydrophobic balance enabled control of the internal pore diameter of the BPNs from 10 to 19 nm. PMID:25640026

  17. Controlling Internal Pore Sizes in Bicontinuous Polymeric Nanospheres**

    PubMed Central

    McKenzie, Beulah E; Friedrich, Heiner; Wirix, Maarten J M; de Visser, Joël F; Monaghan, Olivia R; Bomans, Paul H H; Nudelman, Fabio; Holder, Simon J; Sommerdijk, Nico A J M

    2015-01-01

    Complex polymeric nanospheres were formed in water from comb-like amphiphilic block copolymers. Their internal morphology was determined by three-dimensional cryo-electron tomographic analysis. Varying the polymer molecular weight (MW) and the hydrophilic block weight content allowed for fine control over the internal structure. Construction of a partial phase diagram allowed us to determine the criteria for the formation of bicontinuous polymer nanosphere (BPN), namely for copolymers with MW of up to 17 kDa and hydrophilic weight fractions of ≤0.25; and varying the organic solvent to water ratio used in their preparation allowed for control over nanosphere diameters from 70 to 460 nm. Significantly, altering the block copolymer hydrophilic–hydrophobic balance enabled control of the internal pore diameter of the BPNs from 10 to 19 nm. PMID:25640026

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

    PubMed

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

    2016-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  20. TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse

    PubMed Central

    Na, Bo-Ra; Kim, Hye-Ran; Piragyte, Indre; Oh, Hyun-Mee; Kwon, Min-Sung; Akber, Uroos; Lee, Hyun-Su; Park, Do-Sim; Song, Woo Keun; Park, Zee-Yong; Im, Sin-Hyeog; Rho, Mun-Chual; Hyun, Young-Min; Kim, Minsoo

    2015-01-01

    The formation of an immunological synapse (IS) requires tight regulation of actin dynamics by many actin polymerizing/depolymerizing proteins. However, the significance of actin stabilization at the IS remains largely unknown. In this paper, we identify a novel function of TAGLN2—an actin-binding protein predominantly expressed in T cells—in stabilizing cortical F-actin, thereby maintaining F-actin contents at the IS and acquiring LFA-1 (leukocyte function-associated antigen-1) activation after T cell receptor stimulation. TAGLN2 blocks actin depolymerization and competes with cofilin both in vitro and in vivo. Knockout of TAGLN2 (TAGLN2−/−) reduced F-actin content and destabilized F-actin ring formation, resulting in decreased cell adhesion and spreading. TAGLN2−/− T cells displayed weakened cytokine production and cytotoxic effector function. These findings reveal a novel function of TAGLN2 in enhancing T cell responses by controlling actin stability at the IS. PMID:25869671

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

    PubMed

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

    2016-01-14

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-06-01

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

  4. Dendritic Actin Filament Nucleation Causes Traveling Waves and Patches

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders E.

    2010-06-01

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

  5. Control over Colloidal Particle Morphology by Dispersion Polymerization

    NASA Astrophysics Data System (ADS)

    Peng, B.

    2013-03-01

    The main subject of this thesis is the structure and morphology control of colloidal polymer particles, in particular, poly(methyl methacrylate) (PMMA) particles, by dispersion polymerization in polar solvents. The structure control, ranging from surface morphology and internal structure to shape manipulation of polymer particles, was attempted through copolymerization of monomers with various types of materials, such as cross-linkers, dyes, and colloidal silica spheres or rods. The obtained spherical or non-spherical particles are interesting as model systems as they mimic molecules at colloidal scales. Their phase behavior can be studied in the presence or absence of external fields (like electric and gravity fields). Additionally, they can be used for various applications, such as photonic crystals

  6. Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility.

    PubMed

    Bear, James E; Svitkina, Tatyana M; Krause, Matthias; Schafer, Dorothy A; Loureiro, Joseph J; Strasser, Geraldine A; Maly, Ivan V; Chaga, Oleg Y; Cooper, John A; Borisy, Gary G; Gertler, Frank B

    2002-05-17

    Cell motility requires lamellipodial protrusion, a process driven by actin polymerization. Ena/VASP proteins accumulate in protruding lamellipodia and promote the rapid actin-driven motility of the pathogen Listeria. In contrast, Ena/VASP negatively regulate cell translocation. To resolve this paradox, we analyzed the function of Ena/VASP during lamellipodial protrusion. Ena/VASP-deficient lamellipodia protruded slower but more persistently, consistent with their increased cell translocation rates. Actin networks in Ena/VASP-deficient lamellipodia contained shorter, more highly branched filaments compared to controls. Lamellipodia with excess Ena/VASP contained longer, less branched filaments. In vitro, Ena/VASP promoted actin filament elongation by interacting with barbed ends, shielding them from capping protein. We conclude that Ena/VASP regulates cell motility by controlling the geometry of actin filament networks within lamellipodia. PMID:12086607

  7. Role of actin and myosin in the control of paracellular permeability in pig, rat and human vascular endothelium.

    PubMed Central

    Schnittler, H J; Wilke, A; Gress, T; Suttorp, N; Drenckhahn, D

    1990-01-01

    1. We have investigated the endothelial actomyosin system with particular emphasis on its possible role in actively opening a paracellular route for permeability. 2. Actin and myosin comprised 16% of total endothelial protein with a molar actin/myosin ratio of 16.2 which is close to the actin/myosin ratio of muscle (studies on freshly isolated pig pulmonary arterial endothelial cells, PAEC). 3. By immunocytochemistry at the light and electron microscope levels the bulk of actin and myosin was colocalized in close vicinity to the intercellular clefts of both micro- and macrovascular endothelial cells in situ and in vitro. 4. Calcium-ionophore-induced rise in permeability of human umbilical venous endothelial cells (HUVEC) and PAEC monolayers grown on filters in a two-chamber permeability system was caused by opening of intercellular gaps. Gap formation depended on the rise in intracellular Ca2+ and could be blocked by the calmodulin-binding drugs trifluperazine (TFP) and W7. 5. In skinned monolayers of cultured PAEC and in isolated sheets of HUVEC gap formation was shown to require ATP and occurred only when free myosin binding sites were available on endothelial actin filaments (experiments with myosin subfragment 1 modified by N-ethylmaleimide, S1-NEM). 6. These experiments suggest that actin and myosin in endothelial cells play a central role in regulating the width of the intercellular clefts, thereby controlling the paracellular pathway of vascular permeability. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14 Fig. 15 PMID:2100310

  8. Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices.

    PubMed

    Sung, Kyung Eun; Su, Gui; Pehlke, Carolyn; Trier, Steven M; Eliceiri, Kevin W; Keely, Patricia J; Friedl, Andreas; Beebe, David J

    2009-09-01

    Interest in constructing a reliable 3-dimensional (3D) collagen culture platform in microfabricated systems is increasing as researchers strive to investigate reciprocal interaction between extracellular matrix (ECM) and cells under various conditions. However, in comparison to conventional 2-dimensional (2D) cell culture research, relatively little work has been reported about the polymerization of collagen type I matrix in microsystems. We, thus, present a study of 3D collagen polymerization to achieve reproducible 3D cell culture in microfluidic devices. Array-based microchannels are employed to efficiently examine various polymerization conditions, providing more replicates with less sample volume than conventional means. Collagen fibers assembled in microchannels were almost two-times thinner than those in conventional gels prepared under similar conditions, and the fiber thickness difference influenced viability and morphology of embedded human mammary fibroblast (HMF) cells. HMF cells contained more actin stress fibers and showed increased viability in 3D collagen matrix composed of thicker collagen fibers. Relatively low pH of the collagen solution within a physiological pH range (6.5-8.5) and pre-incubation at low temperature (approximately 4 degrees C) before polymerization at 37 degrees C allow sufficient time for molecular assembly, generating thicker collagen fibers and enhancing HMF cell viability. The results provide the basis for improved process control and reproducibility of 3D collagen matrix culture in microchannels, allowing predictable modifications to provide optimum conditions for specific cell types. In addition, the presented method lays the foundation for high throughput 3D cellular screening. PMID:19540580

  9. Morphology Control of Multicomponent Polymeric Surfactants Using Pressure

    NASA Astrophysics Data System (ADS)

    Cho, Junhan

    The development of nanoscale morphologies for a molten polymeric surfactant under pressure is investigated by using a recently formulated self-consistent field theory. A linear ABC block copolymer is taken as our model system that allows a disparity in the propensities for curved interfaces and pressure responses of ij-pairs. The interplay of those features lead the copolymer to new morphologies at a moderate segregation level and at ambient condition such as networks and pillars of 2-dimensional array. It is shown that pressure is an effective means of morphology control and identification for those new structures. The role of volume fluctuations in the development of those structures is discussed. J.C. acknowledges the support from Center for Photofunctional Energy Materials through Gyeonggi Regional Research Program.

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

    PubMed Central

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

    2010-01-01

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

  11. ADF and Cofilin1 Control Actin Stress Fibers, Nuclear Integrity, and Cell Survival

    PubMed Central

    Kanellos, Georgios; Zhou, Jing; Patel, Hitesh; Ridgway, Rachel A.; Huels, David; Gurniak, Christine B.; Sandilands, Emma; Carragher, Neil O.; Sansom, Owen J.; Witke, Walter; Brunton, Valerie G.; Frame, Margaret C.

    2015-01-01

    Summary Genetic co-depletion of the actin-severing proteins ADF and CFL1 triggers catastrophic loss of adult homeostasis in multiple tissues. There is impaired cell-cell adhesion in skin keratinocytes with dysregulation of E-cadherin, hyperproliferation of differentiated cells, and ultimately apoptosis. Mechanistically, the primary consequence of depleting both ADF and CFL1 is uncontrolled accumulation of contractile actin stress fibers associated with enlarged focal adhesions at the plasma membrane, as well as reduced rates of membrane protrusions. This generates increased intracellular acto-myosin tension that promotes nuclear deformation and physical disruption of the nuclear lamina via the LINC complex that normally connects regulated actin filaments to the nuclear envelope. We therefore describe a pathway involving the actin-severing proteins ADF and CFL1 in regulating the dynamic turnover of contractile actin stress fibers, and this is vital to prevent the nucleus from being damaged by actin contractility, in turn preserving cell survival and tissue homeostasis. PMID:26655907

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  15. Cdc42 and Actin Control Polarized Expression of TI-VAMP Vesicles to Neuronal Growth Cones and Their Fusion with the Plasma MembraneV⃞

    PubMed Central

    Alberts, Philipp; Rudge, Rachel; Irinopoulou, Theano; Danglot, Lydia; Gauthier-Rouvière, Cécile; Galli, Thierry

    2006-01-01

    Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth. PMID:16381811

  16. F-actin mechanics control spindle centring in the mouse zygote

    NASA Astrophysics Data System (ADS)

    Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S.; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition.

  17. F-actin mechanics control spindle centring in the mouse zygote.

    PubMed

    Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition. PMID:26727405

  18. F-actin mechanics control spindle centring in the mouse zygote

    PubMed Central

    Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S.; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition. PMID:26727405

  19. Crystal structure of a nuclear actin ternary complex.

    PubMed

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

    2016-08-01

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

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

    PubMed

    Grosse, Robert; Vartiainen, Maria K

    2013-11-01

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

  1. Signaling Network Triggers and Membrane Physical Properties Control the Actin Cytoskeleton-Driven Isotropic Phase of Cell Spreading

    PubMed Central

    Rangamani, Padmini; Fardin, Marc-Antoine; Xiong, Yuguang; Lipshtat, Azi; Rossier, Olivier; Sheetz, Michael P.; Iyengar, Ravi

    2011-01-01

    Cell spreading is regulated by signaling from the integrin receptors that activate intracellular signaling pathways to control actin filament regulatory proteins. We developed a hybrid model of whole-cell spreading in which we modeled the integrin signaling network as ordinary differential equations in multiple compartments, and cell spreading as a three-dimensional stochastic model. The computed activity of the signaling network, represented as time-dependent activity levels of the actin filament regulatory proteins, is used to drive the filament dynamics. We analyzed the hybrid model to understand the role of signaling during the isotropic phase of fibroblasts spreading on fibronectin-coated surfaces. Simulations showed that the isotropic phase of spreading depends on integrin signaling to initiate spreading but not to maintain the spreading dynamics. Simulations predicted that signal flow in the absence of Cdc42 or WASP would reduce the spreading rate but would not affect the shape evolution of the spreading cell. These predictions were verified experimentally. Computational analyses showed that the rate of spreading and the evolution of cell shape are largely controlled by the membrane surface load and membrane bending rigidity, and changing information flow through the integrin signaling network has little effect. Overall, the plasma membrane acts as a damper such that only ∼5% of the actin dynamics capability is needed for isotropic spreading. Thus, the biophysical properties of the plasma membrane can condense varying levels of signaling network activities into a single cohesive macroscopic cellular behavior. PMID:21320428

  2. Src64 controls a novel actin network required for proper ring canal formation in the Drosophila male germline.

    PubMed

    Eikenes, Åsmund Husabø; Malerød, Lene; Lie-Jensen, Anette; Sem Wegner, Catherine; Brech, Andreas; Liestøl, Knut; Stenmark, Harald; Haglund, Kaisa

    2015-12-01

    In many organisms, germ cells develop as cysts in which cells are interconnected via ring canals (RCs) as a result of incomplete cytokinesis. However, the molecular mechanisms of incomplete cytokinesis remain poorly understood. Here, we address the role of tyrosine phosphorylation of RCs in the Drosophila male germline. We uncover a hierarchy of tyrosine phosphorylation within germline cysts that positively correlates with RC age. The kinase Src64 is responsible for mediating RC tyrosine phosphorylation, and loss of Src64 causes a reduction in RC diameter within germline cysts. Mechanistically, we show that Src64 controls an actin network around the RCs that depends on Abl and the Rac/SCAR/Arp2/3 pathway. The actin network around RCs is required for correct RC diameter in cysts of developing germ cells. We also identify that Src64 is required for proper germ cell differentiation in the Drosophila male germline independent of its role in RC regulation. In summary, we report that Src64 controls actin dynamics to mediate proper RC formation during incomplete cytokinesis during germline cyst development in vivo. PMID:26628094

  3. Polymeric barrier membranes for device packaging, diffusive control and biocompatibility

    NASA Astrophysics Data System (ADS)

    Wasikiewicz, J. M.; Roohpour, N.; Paul, D.; Grahn, M.; Ateh, D.; Rehman, I.; Vadgama, P.

    2008-11-01

    Current state-of-the-art implantable micron feature electronic devices are capable of monitoring and stimulating functions in vivo. Within an EU Framework VI project a further step was taken in developing key microsystem technologies and communication methods that could bring intelligence directly to the human interface, in the form of reactive medical implants and ambulatory measurement systems. Information from these devices is planned to be transmitted out into the wider environment for remote processing. However, the packaging of such state-of-the-art devices to enhance tissue biocompatibility, and to protect conducting elements from in vivo corrosion during extended use, along with protecting the body from toxins leaching from implant components, remains a concern. Candidate polymeric barriers as hydration resistant and solute impermeable interfaces to mitigate such major problems of chronic implantation were investigated. Materials studied included silicone rubber, PVC, polyurethane, and diamond-like carbon (DLC). Polymer permeability to water solutes was marginally improved through incorporation of lipid into these structures. Surface biocompatibility was assessed on the basis of protein film deposition in vitro and by cell viability studies in tissue culture. Short-term toxicity was not observed for any of the tested materials, though there were substantial differences in hydration. Additionally, polypyrrole over active electrodes shows feasibility for controlled tissue interfacing whilst retaining electrical conductivity.

  4. Polymeric surfaces exhibiting photocatalytic activity and controlled anisotropic wettability

    NASA Astrophysics Data System (ADS)

    Anastasiadis, Spiros H.; Frysali, Melani A.; Papoutsakis, Lampros; Kenanakis, George; Stratakis, Emmanuel; Vamvakaki, Maria; Mountrichas, Grigoris; Pispas, Stergios

    2015-03-01

    In this work we focus on surfaces, which exhibit controlled, switchable wettability in response to one or more external stimuli as well as photocatalytic activity. For this we are inspired from nature to produce surfaces with a dual-scale hierarchical roughness and combine them with the appropriate inorganic and/or polymer coating. The combination of the hierarchical surface with a ZnO coating and a pH- or temperature-responsive polymer results in efficient photo-active properties as well as reversible superhydrophobic / superhydrophilic surfaces. Furthermore, we fabricate surfaces with unidirectional wettability variation. Overall, such complex surfaces require advanced design, combining hierarchically structured surfaces with suitable polymeric materials. Acknowledgment: This research was partially supported by the European Union (European Social Fund, ESF) and Greek national funds through the ``ARISTEIA II'' Action (SMART-SURF) of the Operational Programme ``Education and Lifelong Learning,'' NSRF 2007-2013, via the General Secretariat for Research & Technology, Ministry of Education and Religious Affairs, Greece.

  5. Yeast Formins Bni1 and Bnr1 Utilize Different Modes of Cortical Interaction during the Assembly of Actin Cables

    PubMed Central

    Buttery, Shawnna M.; Yoshida, Satoshi

    2007-01-01

    The budding yeast formins Bni1 and Bnr1 control the assembly of actin cables. These formins exhibit distinct patterns of localization and polymerize two different populations of cables: Bni1 in the bud and Bnr1 in the mother cell. We generated a functional Bni1-3GFP that improved the visualization of Bni1 in vivo at endogenous levels. Bni1 exists as speckles in the cytoplasm, some of which colocalize on actin cables. These Bni1 speckles display linear, retrograde-directed movements. Loss of polymerized actin or specifically actin cables abolished retrograde movement, and resulted in depletion of Bni1 speckles from the cytoplasm, with enhanced targeting of Bni1 to the bud tip. Mutations that impair the actin assembly activity of Bni1 abolished the movement of Bni1 speckles, even when actin cables were present. In contrast, Bnr1-GFP or 3GFP-Bnr1 did not detectably associate with actin cables and was not observed as cytoplasmic speckles. Finally, fluorescence recovery after photobleaching demonstrated that Bni1 was very dynamic, exchanging between polarized sites and the cytoplasm, whereas Bnr1 was confined to the bud neck and did not exchange with a cytoplasmic pool. In summary, our results indicate that formins can have distinct modes of cortical interaction during actin cable assembly. PMID:17344480

  6. Actinic keratosis

    MedlinePlus

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

  7. Actinic Cheilitis

    MedlinePlus

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

  8. Mesoscopic model of actin-based propulsion.

    PubMed

    Zhu, Jie; Mogilner, Alex

    2012-01-01

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

  9. CADM1 Controls Actin Cytoskeleton Assembly and Regulates Extracellular Matrix Adhesion in Human Mast Cells

    PubMed Central

    Moiseeva, Elena P.; Straatman, Kees R.; Leyland, Mark L.; Bradding, Peter

    2014-01-01

    CADM1 is a major receptor for the adhesion of mast cells (MCs) to fibroblasts, human airway smooth muscle cells (HASMCs) and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM). Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion. PMID:24465823

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

    PubMed Central

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

    2014-01-01

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

  11. 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

  12. Actin, Membrane Trafficking and the Control of Prion Induction, Propagation and Transmission in Yeast.

    PubMed

    Moosavi, Behrooz; Mousavi, Bibimaryam; Yang, Guang-Fu

    2016-01-01

    The model eukaryotic yeast Saccharomyces cerevisiae has proven a useful model system in which prion biogenesis and elimination are studied. Several yeast prions exist in budding yeast and a number of studies now suggest that these alternate protein conformations may play important roles in the cell. During the last few years cellular factors affecting prion induction, propagation and elimination have been identified. Amongst these, proteins involved in the regulation of the actin cytoskeleton and dynamic membrane processes such as endocytosis have been found to play a critical role not only in facilitating de novo prion formation but also in prion propagation. Here we briefly review prion formation and maintenance with special attention given to the cellular processes that require the functionality of the actin cytoskeleton. PMID:26503767

  13. 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.

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

    PubMed Central

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

    2016-01-01

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

  15. Dynamic Polymeric Microtubes for the Remote-Controlled Capture, Guidance, and Release of Sperm Cells.

    PubMed

    Magdanz, Veronika; Guix, Maria; Hebenstreit, Franziska; Schmidt, Oliver G

    2016-06-01

    Remote-controlled release of single sperm cells is demonstrated by the use of polymeric microtubes that unfold upon temperature increase to 38 °C. Thermoresponsive, ferromagnetic multilayers are tailored to catch sperm cells and remotely control them by external magnetic fields. These polymeric spermbots are propelled by the sperm flagella. When the temperature is increased, the tubes unfold and the cell is set free. PMID:27003908

  16. A General Approach to Sequence-Controlled Polymers Using Macrocyclic Ring Opening Metathesis Polymerization

    PubMed Central

    2015-01-01

    A new and general strategy for the synthesis of sequence-defined polymers is described that employs relay metathesis to promote the ring opening polymerization of unstrained macrocyclic structures. Central to this approach is the development of a small molecule “polymerization trigger” which when coupled with a diverse range of sequence-defined units allows for the controlled, directional synthesis of sequence controlled polymers. PMID:26053158

  17. Force Generation by Endocytic Actin Patches in Budding Yeast

    PubMed Central

    Carlsson, Anders E.; Bayly, Philip V.

    2014-01-01

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

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

    PubMed

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

    2008-08-15

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

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

    PubMed

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

    2006-08-01

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

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

    PubMed Central

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

    2008-01-01

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

  1. Model for adhesion clutch explains biphasic relationship between actin flow and traction at the cell leading edge

    NASA Astrophysics Data System (ADS)

    Craig, Erin M.; Stricker, Jonathan; Gardel, Margaret; Mogilner, Alex

    2015-05-01

    Cell motility relies on the continuous reorganization of a dynamic actin-myosin-adhesion network at the leading edge of the cell, in order to generate protrusion at the leading edge and traction between the cell and its external environment. We analyze experimentally measured spatial distributions of actin flow, traction force, myosin density, and adhesion density in control and pharmacologically perturbed epithelial cells in order to develop a mechanical model of the actin-adhesion-myosin self-organization at the leading edge. A model in which the F-actin network is treated as a viscous gel, and adhesion clutch engagement is strengthened by myosin but weakened by actin flow, can explain the measured molecular distributions and correctly predict the spatial distributions of the actin flow and traction stress. We test the model by comparing its predictions with measurements of the actin flow and traction stress in cells with fast and slow actin polymerization rates. The model predicts how the location of the lamellipodium-lamellum boundary depends on the actin viscosity and adhesion strength. The model further predicts that the location of the lamellipodium-lamellum boundary is not very sensitive to the level of myosin contraction.

  2. 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.

  3. Actin-Regulator Feedback Interactions during Endocytosis.

    PubMed

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

    2016-03-29

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

  4. Quality control of residual solvent content in polymeric microparticles.

    PubMed

    Dixit, Kalpana; Athawale, Rajani B; Singh, Sarabjit

    2015-01-01

    Organic solvents are the innate part of pharmaceutical industry, playing vital role in the bulk drug substance as well as finished product manufacturing. Even though they are used for various crucial purposes, they still lack therapeutic beneficial effect and can be toxic if present in unacceptable limits in final product. Hence, their concentration must be regulated in the final pharmaceutical formulation. With the major development in the market of polymeric microparticles in past few decades, drug product manufacturers are paying more attention towards the development of new techniques for reducing residual solvent content of microparticles. This article sheds light on the importance of removal of organic volatile impurities from the formulation and its regulatory aspects. It also highlights how residual solvent affects various physicochemical characteristics of polymeric microparticles and suggests certain solutions as per the current state of art for limiting organic solvent content in the final product. PMID:25560934

  5. 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.

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

    PubMed

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

    2015-05-01

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

  7. The IQGAP-related protein DGAP1 interacts with Rac and is involved in the modulation of the F-actin cytoskeleton and control of cell motility.

    PubMed

    Faix, J; Clougherty, C; Konzok, A; Mintert, U; Murphy, J; Albrecht, R; Mühlbauer, B; Kuhlmann, J

    1998-10-01

    DGAP1 of Dictyostelium discoideum is a cell cortex associated 95 kDa protein that shows homology to both RasGTPase-activating proteins (RasGAPs) and RasGAP-related proteins. When tested for RasGAP activity, recombinant DGAP1 protein did not promote the GTPase activity of human H-Ras or of Dictyostelium RasG in vitro. Instead, DGAP1 bound to Dictyostelium Rac1A and human Rac1, but not to human Cdc42. DGAP1 preferentially interacted with the activated GTP-bound forms of Rac1 and Rac1A, but did not affect the GTPase activities. Since Rho-type GTPases are implicated in the formation of specific F-actin structures and in the control of cell morphology, the microfilament system of mutants that either lack or overexpress DGAP1 has been analysed. DGAP1-null mutants showed elevated levels of F-actin that was organised in large leading edges, membrane ruffles or numerous large filopods. Expression of actin fused to green fluorescent protein (GFP) was used to monitor the actin dynamics in these cells, and revealed that the F-actin cytoskeleton of DGAP1-null cells was rapidly re-arranged to form ruffles and filopods. Conversely, in DGAP1-overexpressing cells, the formation of cellular projections containing F-actin was largely suppressed. Measurement of cell migration demonstrated that DGAP1 expression is inversely correlated with the speed of cell motility. PMID:9739079

  8. Cadherin controls nectin recruitment into adherens junctions by remodeling the actin cytoskeleton

    PubMed Central

    Troyanovsky, Regina B.; Indra, Indrajyoti; Chen, Chi-Shuo; Hong, Soonjin; Troyanovsky, Sergey M.

    2015-01-01

    ABSTRACT The mechanism that coordinates activities of different adhesion receptors is poorly understood. We investigated this mechanism by focusing on the nectin-2 and E-cadherin adherens junction receptors. We found that, cadherin was not required for the basic process of nectin junction formation because nectin-2 formed junctions in cadherin-deficient A431D cells. Formation of nectin-2 junctions in these cells, however, became regulated by cadherin as soon as E-cadherin was re-expressed. E-cadherin recruited nectin-2 into adherens junctions, where both proteins formed distinct but tightly associated clusters. Live-cell imaging showed that the appearance of E-cadherin clusters often preceded that of nectin-2 clusters at sites of junction assembly. Inactivation of E-cadherin clustering by different strategies concomitantly suppressed the formation of nectin clusters. Furthermore, cadherin significantly increased the stability of nectin clusters, thereby making them resistant to the BC-12 antibody, which targets the nectin-2 adhesion interface. By testing different E-cadherin–α-catenin chimeras, we showed that the recruitment of nectin into chimera junctions is mediated by the actin-binding domain of α-catenin. Our data suggests that E-cadherin regulates assembly of nectin junctions through α-catenin-induced remodeling of the actin cytoskeleton around the cadherin clusters. PMID:25395582

  9. Specific Myosins Control Actin Organization, Cell Morphology, and Migration in Prostate Cancer Cells.

    PubMed

    Makowska, Katarzyna A; Hughes, Ruth E; White, Kathryn J; Wells, Claire M; Peckham, Michelle

    2015-12-15

    We investigated the myosin expression profile in prostate cancer cell lines and found that Myo1b, Myo9b, Myo10, and Myo18a were expressed at higher levels in cells with high metastatic potential. Moreover, Myo1b and Myo10 were expressed at higher levels in metastatic tumors. Using an siRNA-based approach, we found that knockdown of each myosin resulted in distinct phenotypes. Myo10 knockdown ablated filopodia and decreased 2D migration speed. Myo18a knockdown increased circumferential non-muscle myosin 2A-associated actin filament arrays in the lamella and reduced directional persistence of 2D migration. Myo9b knockdown increased stress fiber formation, decreased 2D migration speed, and increased directional persistence. Conversely, Myo1b knockdown increased numbers of stress fibers but did not affect 2D migration. In all cases, the cell spread area was increased and 3D migration potential was decreased. Therefore, myosins not only act as molecular motors but also directly influence actin organization and cell morphology, which can contribute to the metastatic phenotype. PMID:26670045

  10. Tau co-organizes dynamic microtubule and actin networks

    PubMed Central

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

    2015-01-01

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

  11. Intranuclear Actin Regulates Osteogenesis

    PubMed Central

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Blanpied, Thomas A.

    2013-03-01

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

  13. Glucose-sensitive polymeric matrices for controlled drug delivery.

    PubMed

    Goldraich, M; Kost, J

    1993-01-01

    Hydrogel matrices were prepared by chemical polymerization of solutions containing 2-hydroxyethyl methacrylate, N,N-dimethyl-aminoethyl methacrylate, tetraethylene glycol dimethacrylate, ethylene glycol and water solutions containing glucose oxidase, bacitracin or insulin. The hydrogels displayed faster and higher swelling and release rates at lower pH or at higher glucose concentrations. Swelling and release kinetics were also responsive to step changes in glucose concentration in the physiological range. The kinetics of the soluble and immobilized enzyme followed Michaelis Menten's kinetics. In the soluble state the enzyme was more active than the immobilized one due to mass transfer limitations, which may be overcome by preparation of microbead configuration. PMID:10146247

  14. Effect of ATP on actin filament stiffness.

    PubMed

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

    1990-09-01

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

  15. Photo-Regeneration of Severed Gel Using Photo-Controlled Radical Polymerization

    NASA Astrophysics Data System (ADS)

    Singh, Awaneesh; Kuksenok, Olga; Johnson, Jeremiah A.; Balazs, Anna C.

    Using the framework of dissipative particle dynamics (DPD) simulation, we developed a novel computational model that enables photo-regeneration of the gel matrix when a significant portion of the material is severed. We considered photo-controlled radical polymerization (photo-CRP) within polymer networks with embedded iniferters (initiators for the photo-CRP reaction). These iniferters turn on the polymerization process in the presence of light with monomers and cross-linkers in the solution. This ''photo-growth'' allow us to effectively regenerate severed gels under the application of light. The growth process can be turned off once the polymerization is near completion, which forms a new cross-linked gel that resembles the uncut material. The polymerization rate can be modulated by altering the light intensity.

  16. Ssp1 CaMKK: A Sensor of Actin Polarization That Controls Mitotic Commitment through Srk1 in Schizosaccharomyces pombe

    PubMed Central

    Giménez-Zaragoza, David; López-Avilés, Sandra; Yance-Chávez, Tula; Montserrat, Marta; Pujol, M. Jesús; Bachs, Oriol; Aligue, Rosa

    2015-01-01

    Background Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is required for diverse cellular functions. Mammalian CaMKK activates CaMKs and also the evolutionarily-conserved AMP-activated protein kinase (AMPK). The fission yeast Schizosaccharomyces pombe CaMKK, Ssp1, is required for tolerance to limited glucose through the AMPK, Ssp2, and for the integration of cell growth and division through the SAD kinase Cdr2. Results Here we report that Ssp1 controls the G2/M transition by regulating the activity of the CaMK Srk1. We show that inhibition of Cdc25 by Srk1 is regulated by Ssp1; and also that restoring growth polarity and actin localization of ssp1-deleted cells by removing the actin-monomer-binding protein, twinfilin, is sufficient to suppress the ssp1 phenotype. Conclusions These findings demonstrate that entry into mitosis is mediated by a network of proteins, including the Ssp1 and Srk1 kinases. Ssp1 connects the network of components that ensures proper polarity and cell size with the network of proteins that regulates Cdk1-cyclin B activity, in which Srk1 plays an inhibitory role. PMID:26575035

  17. Light activation of transcription: photocaging of nucleotides for control over RNA polymerization

    PubMed Central

    Pinheiro, André Vidal; Baptista, Pedro; Lima, João Carlos

    2008-01-01

    We describe the use of ATP caged with [7-(diethylamino)coumarin-4-yl]methyl (DEACM) for light-controlled in vitro transcription reactions. Polymerization is blocked when DEACM is bonded to the gamma phosphate group of the ATP molecule. Controlled light irradiation releases ATP and transcription is initiated. In order to provide full control over the process, conditions involved in substrate release, nucleotide availability after release and the effect of the released coumarin in RNA polymerization were assessed in further detail. Together, our data provide the first direct evidence of control over enzymatic polymerization of nucleic acids through light. This approach may provide researchers with a unique tool for the study of biological processes at a molecular level. PMID:18586819

  18. Phase separation of lipid microdomains controlled by polymerized lipid bilayer matrices.

    PubMed

    Okazaki, Takashi; Tatsu, Yoshiro; Morigaki, Kenichi

    2010-03-16

    We developed a micropatterned model biological membrane on a solid substrate that can induce phase separation of lipid microdomains in a designed geometry. Micropatterned lipid bilayers were generated by the photolithographic polymerization of a diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC). By changing the UV dose for the photopolymerization, we could modulate the coverage of the surface by the polymeric bilayer domains. After removing nonpolymerized DiynePC, natural phospholipid membranes were incorporated into the micropatterned polymeric bilayer matrix by a self-assembly process (vesicle fusion). As we incorporated a ternary lipid mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM), and cholesterol (Chol) (1:1:1), liquid ordered domains (Lo: rich in SM and Chol) were accumulated in the polymer free regions, whereas liquid disordered domains (Ld: rich in DOPC) preferentially participated into the partially polymeric bilayer regions. It was postulated that Ld domains preferentially came in contact with the polymeric bilayer boundaries because of their lower elastic moduli and a smaller thickness mismatch at the boundary. The effect of polymeric bilayer matrix to hinder the size growth of Lo domains should also be playing an important role. The controlled phase separation should open new possibilities to locally concentrate membrane proteins and other nanometer-sized materials on the substrate by associating them with the lipid microdomains. PMID:20020734

  19. 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.

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

    PubMed Central

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

    2010-01-01

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

  1. 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.

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

    PubMed

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

    2016-05-27

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

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

    PubMed Central

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

    2012-01-01

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

  4. Pushing with actin: from cells to pathogens.

    PubMed

    Small, J Victor

    2015-02-01

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

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

    PubMed Central

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

    2012-01-01

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

  6. Actin-dependent mechanisms in AMPA receptor trafficking

    PubMed Central

    Hanley, Jonathan G.

    2014-01-01

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

  7. The flatness of Lamellipodia explained by the interaction between actin dynamics and membrane deformation.

    PubMed

    Schmeiser, Christian; Winkler, Christoph

    2015-09-01

    The crawling motility of many cell types relies on lamellipodia, flat protrusions spreading on flat substrates but (on cells in suspension) also growing into three-dimensional space. Lamellipodia consist of a plasma membrane wrapped around an oriented actin filament meshwork. It is well known that the actin density is controlled by coordinated polymerization, branching, and capping processes, but the mechanisms producing the small aspect ratios of lamellipodia (hundreds of nm thickness vs. several μm lateral and inward extension) remain unclear. The main hypothesis of this work is a strong influence of the local geometry of the plasma membrane on the actin dynamics. This is motivated by observations of co-localization of proteins with I-BAR domains (like IRSp53) with polymerization and branching agents along the membrane. The I-BAR domains are known to bind to the membrane and to prefer and promote membrane curvature. This hypothesis is translated into a stochastic mathematical model where branching and capping rates, and polymerization speeds depend on the local membrane geometry and branching directions are influenced by the principal curvature directions. This requires the knowledge of the deformation of the membrane, being described in a quasi-stationary approximation by minimization of a modified Helfrich energy, subject to the actin filaments acting as obstacles. Simulations with this model predict pieces of flat lamellipodia without any prescribed geometric restrictions. PMID:26002996

  8. Interaction between Calcium and Actin in Guard Cell and Pollen Signaling Networks

    PubMed Central

    Chen, Dong-Hua; Acharya, Biswa R.; Liu, Wei; Zhang, Wei

    2013-01-01

    Calcium (Ca2+) plays important roles in plant growth, development, and signal transduction. It is a vital nutrient for plant physical design, such as cell wall and membrane, and also serves as a counter-cation for biochemical, inorganic, and organic anions, and more particularly, its concentration change in cytosol is a ubiquitous second messenger in plant physiological signaling in responses to developmental and environmental stimuli. Actin cytoskeleton is well known for its importance in cellular architecture maintenance and its significance in cytoplasmic streaming and cell division. In plant cell system, the actin dynamics is a process of polymerization and de-polymerization of globular actin and filamentous actin and that acts as an active regulator for calcium signaling by controlling calcium evoked physiological responses. The elucidation of the interaction between calcium and actin dynamics will be helpful for further investigation of plant cell signaling networks at molecular level. This review mainly focuses on the recent advances in understanding the interaction between the two aforementioned signaling components in two well-established model systems of plant, guard cell, and pollen. PMID:27137395

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

    PubMed Central

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

    1998-01-01

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

  10. Excitable actin dynamics in lamellipodial protrusion and retraction.

    PubMed

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

    2012-04-01

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

  11. Accessing conjugated polymers with precisely controlled heterobisfunctional chain ends via post-polymerization modification of the OTf group and controlled Pd(0)/t-Bu3P-catalyzed Suzuki cross-coupling polymerization

    SciTech Connect

    Hu, Qiao -Sheng; Hong, Kunlun; Zhang, Hong -Hai

    2015-08-12

    In this study, a general strategy toward the synthesis of well-defined conjugated polymers with controlled heterobisfunctional chain ends via combination of controlled Pd(0)/t-Bu3P Suzuki cross-coupling polymerization with the post-polymerization modification of the triflate (OTf) group was disclosed.

  12. Accessing conjugated polymers with precisely controlled heterobisfunctional chain ends via post-polymerization modification of the OTf group and controlled Pd(0)/t-Bu3P-catalyzed Suzuki cross-coupling polymerization

    DOE PAGESBeta

    Hu, Qiao -Sheng; Hong, Kunlun; Zhang, Hong -Hai

    2015-08-12

    In this study, a general strategy toward the synthesis of well-defined conjugated polymers with controlled heterobisfunctional chain ends via combination of controlled Pd(0)/t-Bu3P Suzuki cross-coupling polymerization with the post-polymerization modification of the triflate (OTf) group was disclosed.

  13. Drug-Initiated, Controlled Ring-Opening Polymerization for the Synthesis of Polymer-Drug Conjugates

    PubMed Central

    Tong, Rong; Cheng, Jianjun

    2012-01-01

    Paclitaxel, a polyol chemotherapeutic agent, was covalently conjugated through its 2′-OH to polylactide with 100% regioselectivity via controlled polymerization of lactide mediated by paclitaxel/(BDI-II)ZnN(TMS)2 (BDI-II = 2-((2,6-diisopropylphenyl)amino)-4-((2,6-diisopropylphenyl)imino)-2-pentene). The steric bulk of the substituents on the N-aryl groups of the BDI ligand drastically affected the regiochemistry of coordination of the metal catalysts to paclitaxel and the subsequent ring-opening polymerization of lactide. The drug-initiated, controlled polymerization of lactide was extended, again with 100% regioselectivity, to docetaxel, a chemotherapeutic agent that is even more structurally complex than paclitaxel. Regioselective incorporation of paclitaxel (or docetaxel) to other biopolymers (i.e., poly(δ-valerolactone), poly(trimethylene carbonate), and poly(ε-caprolactone)), was also achieved through drug/(BDI-II)ZnN(TMS)2-mediated controlled polymerization. These drug-polylactide conjugates with precisely controlled structures are expected to be excellent building blocks for drug delivery, coating, and controlled-release applications. PMID:23357880

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  16. Sterically controlled azomethine ylide cycloaddition polymerization of phenyl-C61-butyric acid methyl ester.

    PubMed

    Stephen, Meera; Ramanitra, Hasina H; Santos Silva, Hugo; Dowland, Simon; Bégué, Didier; Genevičius, Kristijonas; Arlauskas, Kęstutis; Juška, Gytis; Morse, Graham E; Distler, Andreas; Hiorns, Roger C

    2016-05-01

    Phenyl-C61-butyric acid methyl ester (PCBM) is polymerized simply using a one-pot reaction to yield soluble, high molecular weight polymers. The sterically controlled azomethine ylide cycloaddition polymerization (SACAP) is demonstrated to be highly adaptable and yields polymers with probable Mn≈ 24 600 g mol(-1) and Mw≈ 73 800 g mol(-1). Products are metal-free and of possible benefit to organic and hybrid photovoltaics and electronics as they form thin films from solution and have raised LUMOs. The promising electronic properties of this new polymer are discussed. PMID:27066898

  17. Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo

    PubMed Central

    Eskin, Julian A.; Rankova, Aneliya; Johnston, Adam B.; Alioto, Salvatore L.; Goode, Bruce L.

    2016-01-01

    Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1’s functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles. PMID:26764093

  18. Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo.

    PubMed

    Eskin, Julian A; Rankova, Aneliya; Johnston, Adam B; Alioto, Salvatore L; Goode, Bruce L

    2016-03-01

    Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1's functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles. PMID:26764093

  19. Tailoring the Structure of Polymer Networks with Photo-Controlled Radical Polymerization

    NASA Astrophysics Data System (ADS)

    Singh, Awaneesh; Kuksenok, Olga; Johnson, Jeremiah A.; Balazs, Anna C.

    Using dissipative particle dynamics (DPD) approach, we developed a novel computational model to study the photo-controlled radical polymerization (photo-CRP) within polymer networks with embedded iniferters. The polymerization process can be turned ``on'' or ``off'' in response to light and the polymerization rate can be modulated by altering the light intensity. This ``photo-growth'' approach allows us to impart changes in the gel network pore size and composition to form photo-tunable smart materials. For example, our approach allows us to design gel composites that are comprised of two distinct layers made of two compatible components at low photo-iniferter concentrations or gel composites that are comprised of two incompatible components that are relatively well intermixed at high photo-iniferter concentration.

  20. Stereo- and Temporally Controlled Coordination Polymerization Triggered by Alternating Addition of a Lewis Acid and Base.

    PubMed

    Liu, Bo; Cui, Dongmei; Tang, Tao

    2016-09-19

    Significant progress has been made with regard to temporally controlled radical and ring-opening polymerizations, for example, by means of chemical reagents, light, and voltage, whereas quantitative switch coordination polymerization is still challenging. Herein, we report the temporally and stereocontrolled 3,4-polymerization of isoprene through allosterically regulating the active metal center by alternating addition of Lewis basic pyridine to "poison" the Lewis acidic active metal species through acid-base interactions and Lewis acidic Al(i) Bu3 to release the original active species through pyridine abstraction. This process is quick, quantitative, and can be repeated multiple times while maintaining high 3,4-selectivity. Moreover, this strategy is also effective for the switch copolymerization of isoprene and styrene with dual 3,4- and syndiotactic selectivity. Tuning the switch cycles and intervals enables the isolation of various copolymers with different distributions of 3,4-polyisoprene and syndiotactic polystyrene sequences. PMID:27539866

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

    PubMed

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

    2013-01-01

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

  2. The Three-Dimensional Dynamics of Actin Waves, a Model of Cytoskeletal Self-Organization

    PubMed Central

    Bretschneider, Till; Anderson, Kurt; Ecke, Mary; Müller-Taubenberger, Annette; Schroth-Diez, Britta; Ishikawa-Ankerhold, Hellen C.; Gerisch, Günther

    2009-01-01

    Actin polymerization is typically initiated at specific sites in a cell by membrane-bound protein complexes, and the resulting structures are involved in specialized cellular functions, such as migration, particle uptake, or mitotic division. Here we analyze the potential of the actin system to self-organize into waves that propagate on the planar, substrate-attached membrane of a cell. We show that self-assembly involves the ordered recruitment of proteins from the cytoplasmic pool and relate the organization of actin waves to their capacity for applying force. Three proteins are shown to form distinct three-dimensional patterns in the actin waves. Myosin-IB is enriched at the wave front and close to the plasma membrane, the Arp2/3 complex is distributed throughout the waves, and coronin forms a sloping layer on top of them. CARMIL, a protein that links myosin-IB to the Arp2/3 complex, is also recruited to the waves. Wave formation does not depend on signals transmitted by heterotrimeric G-proteins, nor does their propagation require SCAR, a regulator upstream of the Arp2/3 complex. Propagation of the waves is based on an actin treadmilling mechanism, indicating a program that couples actin assembly to disassembly in a three-dimensional pattern. When waves impinge on the cell perimeter, they push the edge forward; when they reverse direction, the cell border is paralyzed. These data show that force-generating, highly organized supramolecular networks are autonomously formed in live cells from molecular motors and proteins controlling actin polymerization and depolymerization. PMID:19348770

  3. Myosin IIB and F-actin control apical vacuolar morphology and histamine-induced trafficking of H-K-ATPase-containing tubulovesicles in gastric parietal cells

    PubMed Central

    Crothers, James M.; Rosen, Jared E.; Nakada, Stephanie L.; Rakholia, Milap; Okamoto, Curtis T.; Forte, John G.; Machen, Terry E.

    2014-01-01

    Selective inhibitors of myosin or actin function and confocal microscopy were used to test the role of an actomyosin complex in controlling morphology, trafficking, and fusion of tubulovesicles (TV) containing H-K-ATPase with the apical secretory canaliculus (ASC) of primary-cultured rabbit gastric parietal cells. In resting cells, myosin IIB and IIC, ezrin, and F-actin were associated with ASC, whereas H-K-ATPase localized to intracellular TV. Histamine caused fusion of TV with ASC and subsequent expansion resulting from HCl and water secretion; F-actin and ezrin remained associated with ASC whereas myosin IIB and IIC appeared to dissociate from ASC and relocalize to the cytoplasm. ML-7 (inhibits myosin light chain kinase) caused ASC of resting cells to collapse and most myosin IIB, F-actin, and ezrin to dissociate from ASC. TV were unaffected by ML-7. Jasplakinolide (stabilizes F-actin) caused ASC to develop large blebs to which actin, myosin II, and ezrin, as well as tubulin, were prominently localized. When added prior to stimulation, ML-7 and jasplakinolide prevented normal histamine-stimulated transformations of ASC/TV and the cytoskeleton, but they did not affect cells that had been previously stimulated with histamine. These results indicate that dynamic pools of actomyosin are required for maintenance of ASC structure in resting cells and for trafficking of TV to ASC during histamine stimulation. However, the dynamic pools of actomyosin are not required once the histamine-stimulated transformation of TV/ASC and cytoskeleton has occurred. These results also show that vesicle trafficking in parietal cells shares mechanisms with similar processes in renal collecting duct cells, neuronal synapses, and skeletal muscle. PMID:24578340

  4. Actinic Prurigo.

    PubMed

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

    2015-01-01

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

  5. [Actinic Keratosis].

    PubMed

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

    2015-07-01

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

  6. Computer Simulations of Mechano-Chemical Networks Choreographing Actin Dynamics in Cell Motility

    NASA Astrophysics Data System (ADS)

    Zhuravlev, Pavel I.; Hu, Longhua; Papoian, Garegin A.

    In eukaryotic cells, cell motility is largely driven by self-assembly and growth of filamentous networks comprised of actin. Numerous proteins regulate actin network dynamics either biochemically, or through mechanical interactions. This regulation is rather complex, intricately coordinated both spatially and temporally. Although experiments in vivo and in vitro have provided a trove of structural and biochemical information about actin-based cell motility processes, experimental data is not always easy to interpret unambiguously, sometimes various interpretations being in contradiction with each other. Hence, mathematical modeling approaches are necessary for providing a physical foundation for interpreting and guiding experiments. In particular, computer simulations based on physicochemical interactions provide a systems-level description of protrusion dynamics. In this contribution, we review recent progress in modeling actin-based cell motility using detailed computer simulations. We elaborate on the way actin network dynamics is determined by the interplay between chemical reactions, mechanical feedbacks, and transport bottlenecks. We also discuss the role of inherent randomness of elementary chemical reactions in determining the dynamical behavior of the mechano-chemical network controlling actin polymerization and growth.

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2013-01-01

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

  9. Multi input single output model predictive control of non-linear bio-polymerization process

    NASA Astrophysics Data System (ADS)

    Arumugasamy, Senthil Kumar; Ahmad, Z.

    2015-05-01

    This paper focuses on Multi Input Single Output (MISO) Model Predictive Control of bio-polymerization process in which mechanistic model is developed and linked with the feedforward neural network model to obtain a hybrid model (Mechanistic-FANN) of lipase-catalyzed ring-opening polymerization of ɛ-caprolactone (ɛ-CL) for Poly (ɛ-caprolactone) production. In this research, state space model was used, in which the input to the model were the reactor temperatures and reactor impeller speeds and the output were the molecular weight of polymer (Mn) and polymer polydispersity index. State space model for MISO created using System identification tool box of Matlab™. This state space model is used in MISO MPC. Model predictive control (MPC) has been applied to predict the molecular weight of the biopolymer and consequently control the molecular weight of biopolymer. The result shows that MPC is able to track reference trajectory and give optimum movement of manipulated variable.

  10. Multi input single output model predictive control of non-linear bio-polymerization process

    SciTech Connect

    Arumugasamy, Senthil Kumar; Ahmad, Z.

    2015-05-15

    This paper focuses on Multi Input Single Output (MISO) Model Predictive Control of bio-polymerization process in which mechanistic model is developed and linked with the feedforward neural network model to obtain a hybrid model (Mechanistic-FANN) of lipase-catalyzed ring-opening polymerization of ε-caprolactone (ε-CL) for Poly (ε-caprolactone) production. In this research, state space model was used, in which the input to the model were the reactor temperatures and reactor impeller speeds and the output were the molecular weight of polymer (M{sub n}) and polymer polydispersity index. State space model for MISO created using System identification tool box of Matlab™. This state space model is used in MISO MPC. Model predictive control (MPC) has been applied to predict the molecular weight of the biopolymer and consequently control the molecular weight of biopolymer. The result shows that MPC is able to track reference trajectory and give optimum movement of manipulated variable.

  11. 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

  12. Tropomyosin diffusion over actin subunits facilitates thin filament assembly

    PubMed Central

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

    2016-01-01

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

  13. Fabrication of Polymeric Coatings with Controlled Microtopographies Using an Electrospraying Technique.

    PubMed

    Guo, Qiongyu; Mather, Jason P; Yang, Pine; Boden, Mark; Mather, Patrick T

    2015-01-01

    Surface topography of medical implants provides an important biophysical cue on guiding cellular functions at the cell-implant interface. However, few techniques are available to produce polymeric coatings with controlled microtopographies onto surgical implants, especially onto implant devices of small dimension and with complex structures such as drug-eluting stents. Therefore, the main objective of this study was to develop a new strategy to fabricate polymeric coatings using an electrospraying technique based on the uniqueness of this technique in that it can be used to produce a mist of charged droplets with a precise control of their shape and dimension. We hypothesized that this technique would allow facile manipulation of coating morphology by controlling the shape and dimension of electrosprayed droplets. More specifically, we employed the electrospraying technique to coat a layer of biodegradable polyurethane with tailored microtopographies onto commercial coronary stents. The topography of such stent coatings was modulated by controlling the ratio of round to stretched droplets or the ratio of round to crumped droplets under high electric field before deposition. The shape of electrosprayed droplets was governed by the stability of these charged droplets right after ejection or during their flight in the air. Using the electrospraying technique, we achieved conformal polymeric coatings with tailored microtopographies onto conductive surgical implants. The approach offers potential for controlling the surface topography of surgical implant devices to modulate their integration with surrounding tissues. PMID:26090663

  14. Fabrication of Polymeric Coatings with Controlled Microtopographies Using an Electrospraying Technique

    PubMed Central

    Guo, Qiongyu; Mather, Jason P.; Yang, Pine; Boden, Mark; Mather, Patrick T.

    2015-01-01

    Surface topography of medical implants provides an important biophysical cue on guiding cellular functions at the cell-implant interface. However, few techniques are available to produce polymeric coatings with controlled microtopographies onto surgical implants, especially onto implant devices of small dimension and with complex structures such as drug-eluting stents. Therefore, the main objective of this study was to develop a new strategy to fabricate polymeric coatings using an electrospraying technique based on the uniqueness of this technique in that it can be used to produce a mist of charged droplets with a precise control of their shape and dimension. We hypothesized that this technique would allow facile manipulation of coating morphology by controlling the shape and dimension of electrosprayed droplets. More specifically, we employed the electrospraying technique to coat a layer of biodegradable polyurethane with tailored microtopographies onto commercial coronary stents. The topography of such stent coatings was modulated by controlling the ratio of round to stretched droplets or the ratio of round to crumped droplets under high electric field before deposition. The shape of electrosprayed droplets was governed by the stability of these charged droplets right after ejection or during their flight in the air. Using the electrospraying technique, we achieved conformal polymeric coatings with tailored microtopographies onto conductive surgical implants. The approach offers potential for controlling the surface topography of surgical implant devices to modulate their integration with surrounding tissues. PMID:26090663

  15. 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

  16. Impact of Alkyl Spacer Length on Aggregation Pathways in Kinetically Controlled Supramolecular Polymerization.

    PubMed

    Ogi, Soichiro; Stepanenko, Vladimir; Thein, Johannes; Würthner, Frank

    2016-01-20

    We have investigated the kinetic and thermodynamic supramolecular polymerizations of a series of amide-functionalized perylene bisimide (PBI) organogelator molecules bearing alkyl spacers of varied lengths (ethylene to pentylene chains, PBI-1-C2 to PBI-1-C5) between the amide and PBI imide groups. These amide-functionalized PBIs form one-dimensional fibrous nanostructures as the thermodynamically favored states in solvents of low polarity. Our in-depth studies revealed, however, that the kinetic behavior of their supramolecular polymerization is dependent on the spacer length. Propylene- and pentylene-tethered PBIs follow a similar polymerization process as previously observed for the ethylene-tethered PBI. Thus, the monomers of these PBIs are kinetically trapped in conformationally restricted states through intramolecular hydrogen bonding between the amide and imide groups. In contrast, the intramolecularly hydrogen-bonded monomers of butylene-tethered PBI spontaneously self-assemble into nanoparticles, which constitute an off-pathway aggregate state with regard to the thermodynamically stable fibrous supramolecular polymers obtained. Thus, for this class of π-conjugated system, an unprecedented off-pathway aggregate with high kinetic stability could be realized for the first time by introducing an alkyl linker of optimum length (C4 chain) between the amide and imide groups. Our current system with an energy landscape of two competing nucleated aggregation pathways is applicable to the kinetic control over the supramolecular polymerization by the seeding approach. PMID:26699283

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

    PubMed Central

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

    2011-01-01

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

  18. Biodegradable polymeric microcarriers with controllable porous structure for tissue engineering.

    PubMed

    Shi, Xudong; Sun, Lei; Jiang, Jian; Zhang, Xiaolin; Ding, Wenjun; Gan, Zhihua

    2009-12-01

    Porous microspheres fabricated by biodegradable polymers show great potential as microcarriers for cell cultivation in tissue engineering. Herein biodegradable poly(DL-lactide) (PLA) was used to fabricate porous microspheres through a modified double emulsion solvent evaporation method. The influence of fabrication parameters, such as the stirring speed of the primary and secondary emulsion, the polymer concentration of the oil phase, and solvent type, as well as the post-hydrolysis treatment of the porous structure of the PLA microspheres are discussed. Good attachment and an active spread of MG-63 cells on the microspheres is observed, which indicates that the PLA microspheres with controllable porous structure are of great potential as cell delivery carriers for tissue engineering. PMID:19821453

  19. Fabrication and control of CT number through polymeric composites based on coronary plaque CT phantom applications.

    PubMed

    Hoy, Carlton F O; Naguib, Hani E; Paul, Narinder

    2016-01-01

    Biomedical phantoms are commonly used for various medical imaging modalities to improve imaging quality and procedures. Current biomedical phantoms fabricated commercially are high in cost and limited in the specificity of human environments and structures that can be mimicked. This study aimed to control the measurable computed tomography (CT) number in Hounsfield units through polymeric biomedical phantom materials using controlled amounts of hydroxyapatite (hA). The purpose was to fabricate CT phantoms capable of mimicking various coronary plaque types while introducing a fabrication technique and basis for a numerical model to which the technique may be applied. The CT number is tunable based on the controlled material properties of electron density and atomic numbers. Three different polymeric matrices of polyethylene (PE), thermoplastic polyurethane (TPU), and polyvinylidene fluoride (PVDF) were selected due to their varied specific densities and ease of fabrication acting as integral properties for CT phantom fabrication. These polymers were processed together with additions of hA in mass percentages of 2.5, 5, 10, and 20% hA as well as a 0% hA as a control for each polymeric material. By adding hA to PE, TPU, and PVDF an increasing trend was exhibited between CT number and weight percent of hA. PMID:26958580

  20. Structural dynamics of an actin spring.

    PubMed

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

    2011-02-16

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

  1. YIH1 is an actin-binding protein that inhibits protein kinase GCN2 and impairs general amino acid control when overexpressed.

    PubMed

    Sattlegger, Evelyn; Swanson, Mark J; Ashcraft, Emily A; Jennings, Jennifer L; Fekete, Richard A; Link, Andrew J; Hinnebusch, Alan G

    2004-07-16

    The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2 (eIF2alpha) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn- phenotype) by suppressing eIF2alpha phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn- phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2alpha phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn- phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin. PMID:15126500

  2. Wettability control by laser texturing process generating localized gold nanoparticles on polymeric thin films.

    PubMed

    Spano, F; Castellano, A; Massaro, A; Fragouli, D; Cingolani, R; Athanassiou, A

    2012-06-01

    In this work a new approach is introduced for surface properties control by laser texturing process. By UV laser irradiation, we are able to control the surface wettability of a chitosan polymeric film in which is introduced a chloroauric acid salt by immersion. Specifically the UV irradiation is responsible for the creation of gold nanoparticles at the irradiated surface of the polymeric film. This photolytic process allows us to localize and design accurately surface patterns and moreover to tune metallic particle size in the range of nanoscale. After the characterization of our gold textured surfaces by atomic force and scanning electron microscopies, we demonstrate the link between wettability surface properties and gold nanoparticles size. The experimental results indicate the influence of the laser intensity, the irradiation time and the polymer film thickness (by increasing the gold concentration) on the gold nanoparticle density and size. PMID:22905535

  3. The Design of MACs (Minimal Actin Cortices)

    PubMed Central

    Vogel, Sven K; Heinemann, Fabian; Chwastek, Grzegorz; Schwille, Petra

    2013-01-01

    The actin cell cortex in eukaryotic cells is a key player in controlling and maintaining the shape of cells, and in driving major shape changes such as in cytokinesis. It is thereby constantly being remodeled. Cell shape changes require forces acting on membranes that are generated by the interplay of membrane coupled actin filaments and assemblies of myosin motors. Little is known about how their interaction regulates actin cell cortex remodeling and cell shape changes. Because of the vital importance of actin, myosin motors and the cell membrane, selective in vivo experiments and manipulations are often difficult to perform or not feasible. Thus, the intelligent design of minimal in vitro systems for actin-myosin-membrane interactions could pave a way for investigating actin cell cortex mechanics in a detailed and quantitative manner. Here, we present and discuss the design of several bottom-up in vitro systems accomplishing the coupling of actin filaments to artificial membranes, where key parameters such as actin densities and membrane properties can be varied in a controlled manner. Insights gained from these in vitro systems may help to uncover fundamental principles of how exactly actin-myosin-membrane interactions govern actin cortex remodeling and membrane properties for cell shape changes. © 2013 Wiley Periodicals, Inc. PMID:24039068

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe; Carlsson, Anders E.

    2015-12-01

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

  7. Polymeric nanostructures with pH-labile core for controlled drug release.

    PubMed

    Banerjee, Rakesh; Maiti, Saikat; Dey, Debabrata; Dhara, Dibakar

    2016-01-15

    Efficient and stimuli-triggered controlled delivery of therapeutics is one of the important issues in modern advanced therapy. In the present work, a versatile route for the synthesis of core cross-linked polymeric nanostructures (CLPN) through thiol-acrylate Michael addition reaction via the formation of β-thiopropionate has been described. The acid groups of the poly(acrylic acid) block of poly(ethylene glycol)-b-poly(N-isopropylacrylamide)-b-poly(acrylic acid) triblock copolymer were reacted with 2-hydroxyethyl acrylate (HEA) to yield the corresponding acrylate-functionalized copolymer (P1). Following this, P1 was reacted with a thiol functionalized cross-linker (CL) resulting in the formation of core cross-linked polymeric nanoparticles through acrylate-thiol Michael reaction. The ability of these nanoparticles to encapsulate drug molecules inside their core and their effective release following a pH-triggered controlled degradation of the core were demonstrated. The temperature sensitive release behaviour of the system was also studied. The non-toxic nature of the precursor polymers and the core cross-linked polymeric nanoparticles was also established, that further substantiated their potential as carriers for controlled release of drugs. PMID:26454376

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

    PubMed Central

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

    1987-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2015-12-01

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

  12. Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks.

    PubMed

    Lappalainen, Pekka

    2016-08-15

    The actin cytoskeleton supports a vast number of cellular processes in nonmuscle cells. It is well established that the organization and dynamics of the actin cytoskeleton are controlled by a large array of actin-binding proteins. However, it was only 40 years ago that the first nonmuscle actin-binding protein, filamin, was identified and characterized. Filamin was shown to bind and cross-link actin filaments into higher-order structures and contribute to phagocytosis in macrophages. Subsequently many other nonmuscle actin-binding proteins were identified and characterized. These proteins regulate almost all steps of the actin filament assembly and disassembly cycles, as well as the arrangement of actin filaments into diverse three-dimensional structures. Although the individual biochemical activities of most actin-regulatory proteins are relatively well understood, knowledge of how these proteins function together in a common cytoplasm to control actin dynamics and architecture is only beginning to emerge. Furthermore, understanding how signaling pathways and mechanical cues control the activities of various actin-binding proteins in different cellular, developmental, and pathological processes will keep researchers busy for decades. PMID:27528696

  13. Structure and Mechanics of Actin Cortex Contained in Vesicles

    NASA Astrophysics Data System (ADS)

    Limozin, Laurent; Roth, Alexander; Sackmann, Erich

    2003-03-01

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

  14. Controlled polymerization of acrylonitrile proceeded along with the Belousov-Zhabotinsky oscillator by changing its stirring conditions

    NASA Astrophysics Data System (ADS)

    Furue, Yuuka; Okano, Kunihiko; Banno, Taisuke; Asakura, Kouichi

    2016-02-01

    Chemical oscillations of the manganese-ion catalyzed Belousov-Zhabotinsky (BZ) reaction system were found to be controlled by changing its stirring conditions. The oscillation stopped at a high stirring rate, while it reappeared immediately by reducing the stirring rate. It is known in the BZ reaction system, that the radical polymerization takes place along with the oscillation when acrylic monomers are added. By the addition of acrylonitrile to the system stirred at a high stirring rate, the oscillation as well as the polymerization of acrylonitrile stopped. The radical polymerization of acrylonitrile by the BZ oscillator is thus found to be made controllable by changing the mixing conditions.

  15. Graphene oxide as a radical initiator: Free radical and controlled radical polymerization of sodium 4-vinylbenzenesulfonate with graphene oxide

    DOE PAGESBeta

    Voylov, Dmitry N.; Saito, Tomonori; Lokitz, Bradley S.; Uhrig, David; Wang, Yangyang; Agapov, Alexander L.; Holt, Adam P.; Bocharova, Vera; Kisliuk, Alexander; Sokolov, Alexei P.

    2016-01-19

    The free radical and controlled radical polymerization of sodium 4-vinylbenzenesulfonate using graphene oxide as a radical initiator was studied. This work demonstrates that graphene oxide can initiate radical polymerization in an aqueous solution without any additional initiator. Poly(sodium 4-vinylbenzenesulfonate) obtained via reversible addition fragmentation chain transfer polymerization had a controlled molecular weight with a very narrow polydispersity ranging between 1.01 and 1.03. Furthermore, the reduction process of graphene oxide as well as the resulting composite material properties were analyzed in detail.

  16. Controlled synthesis of photochromic polymer brushes by atom transfer radical polymerization.

    SciTech Connect

    Piech, Marcin; Bell, Nelson Simmons; Long, Timothy Michael

    2005-06-01

    This work reports on the grafting of methyl methacrylate polymer brushes containing spirobenzopyran pendant groups from flat silica surfaces and colloidal particles utilizing atom transfer radical polymerization (ATRP). The reaction conditions were optimized with respect to the kind of surface bound initiator, the type of halide and ligand used in the catalytic complex, the presence/absence of untethered initiator, and solvent type. This enabled synthesis of coatings up to 80 {+-} 3 nm thick with controlled spirobenzopyran content. While polymerization kinetics indicate the presence of chain termination reactions, the 'living' character of the process is confirmed by controlled formation of block copolymer brushes. UV/vis spectroscopy was used to characterize the UV-induced isomerization of spirobenzopyran to zwitterionic merocyanine and the thermal back-reaction. Spectral and kinetic analyses of this latter bleaching process points to the existence of free and associated merocyanines in the polymeric brush in both tetrahydrofuran and toluene. However, stabilization of merocyanine species by the polymer matrix is considerably greater in toluene with thermal back-reaction rates approaching those determined for solid dry films.

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

    PubMed

    Suh, B I

    1999-01-01

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

  18. Anchoring energy enhancement and pretilt angle control of liquid crystal alignment on polymerized surfaces

    NASA Astrophysics Data System (ADS)

    Weng, Libo; Liao, Pei-Chun; Lin, Chen-Chun; Ting, Tien-Lun; Hsu, Wen-Hao; Su, Jenn-Jia; Chien, Liang-Chy

    2015-09-01

    We demonstrate enhanced surface anchoring energy and control of pretilt angle in a nematic liquid crystal cell with vertical alignment and polymerized surfaces (PS-VA). The polymerized surfaces are formed by ultraviolet (UV) irradiation-induced phase separation of a minute amount of a reactive monomer in the vertical-aligned nematic liquid crystal. By introducing a bias voltage during UV curing, surface-localized polymer protrusions with a dimension of 100nm and a field-induced pretilt angle are observed. Experimental evidences and theoretical analyses validate that PS-VA has increased surface anchoring strength by two folds and pretilt angle has been changed from 89° to 86° compared to those of a VA cell. The enabling PS-VA cell technique with excel electro-optical properties such as very good dark state, high optical contrast, and fast rise and decay times may lead to development of a wide range of applications.

  19. Anchoring energy enhancement and pretilt angle control of liquid crystal alignment on polymerized surfaces

    SciTech Connect

    Weng, Libo; Chien, Liang-Chy; Liao, Pei-Chun; Lin, Chen-Chun; Ting, Tien-Lun; Hsu, Wen-Hao; Su, Jenn-Jia

    2015-09-15

    We demonstrate enhanced surface anchoring energy and control of pretilt angle in a nematic liquid crystal cell with vertical alignment and polymerized surfaces (PS-VA). The polymerized surfaces are formed by ultraviolet (UV) irradiation-induced phase separation of a minute amount of a reactive monomer in the vertical-aligned nematic liquid crystal. By introducing a bias voltage during UV curing, surface-localized polymer protrusions with a dimension of 100nm and a field-induced pretilt angle are observed. Experimental evidences and theoretical analyses validate that PS-VA has increased surface anchoring strength by two folds and pretilt angle has been changed from 89° to 86° compared to those of a VA cell. The enabling PS-VA cell technique with excel electro-optical properties such as very good dark state, high optical contrast, and fast rise and decay times may lead to development of a wide range of applications.

  20. Bundling actin filaments from membranes: some novel players

    PubMed Central

    Thomas, Clément

    2012-01-01

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

  1. Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu

    PubMed Central

    Ayling, Laura J.; Briddon, Stephen J.; Halls, Michelle L.; Hammond, Gerald R. V.; Vaca, Luis; Pacheco, Jonathan; Hill, Stephen J.; Cooper, Dermot M. F.

    2012-01-01

    The central and pervasive influence of cAMP on cellular functions underscores the value of stringent control of the organization of adenylyl cyclases (ACs) in the plasma membrane. Biochemical data suggest that ACs reside in membrane rafts and could compartmentalize intermediary scaffolding proteins and associated regulatory elements. However, little is known about the organization or regulation of the dynamic behaviour of ACs in a cellular context. The present study examines these issues, using confocal image analysis of various AC8 constructs, combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. These studies reveal that AC8, through its N-terminus, enhances the cortical actin signal at the plasma membrane; an interaction that was confirmed by GST pull-down and immunoprecipitation experiments. AC8 also associates dynamically with lipid rafts; the direct association of AC8 with sterols was confirmed in Förster resonance energy transfer experiments. Disruption of the actin cytoskeleton and lipid rafts indicates that AC8 tracks along the cytoskeleton in a cholesterol-enriched domain, and the cAMP that it produces contributes to sculpting the actin cytoskeleton. Thus, an adenylyl cyclase is shown not just to act as a scaffold, but also to actively orchestrate its own micro-environment, by associating with the cytoskeleton and controlling the association by producing cAMP, to yield a highly organized signalling hub. PMID:22399809

  2. 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.

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

    PubMed

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

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

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

    PubMed Central

    Holzinger, Andreas; Blaas, Kathrin

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Grikscheit, Katharina; Frank, Tanja; Wang, Ying

    2015-01-01

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

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

    PubMed

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

    2015-05-11

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

    Root, D. D.; Reisler, E.

    1992-01-01

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

  11. Surface morphology control of cross-linked polymer particles via dispersion polymerization.

    PubMed

    Peng, Bo; Imhof, Arnout

    2015-05-14

    Cross-linked polymer colloids (poly(methyl methacrylate) and polystyrene) with diverse shapes were prepared in polar solvents (ethanol, methanol and water) via dispersion polymerization, in which a linear addition of the cross-linker was used during reaction. Apart from spherical particles we found dented spheres or particles covered with nodules, or a combination of both. A comprehensive investigation was carried out, mainly concentrating on the effect of the experimental conditions (e.g., the addition start time and total addition time, cross-linker density and the solvency of the solvents) on particle morphologies. Consequently, we suggest a number of effective ways for the synthesis of regular (spherical) colloidal particles through maintaining a relatively low concentration of the cross-linker during the entire reaction, or forcing the co-polymerization (of monomer and cross-linker) locus to the continuous medium, or using a high quality or quantity of the stabilizer. Moreover, the size of the particles was also precisely manipulated by varying the polarity of the solvents, the concentration of the cross-linker, and the amount and average molecular weight of the stabilizer. In addition, the formation of the heavily dented particles with a very rough surface prepared under a pure or oxygen-'contaminated' nitrogen environment was monitored over time. The results accumulated in this article are of use for a better understanding of the mechanism of the polymerization and control over the structure and property of polymer particles. PMID:25793973

  12. 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.

  13. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    DOE PAGESBeta

    Wang, Jun; Bonnesen, Peter V; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; Cleaves, II, H. James; Baddorf, Arthur P; Sumpter, Bobby G; Pan, Minghu; Maksymovych, Petro; et al

    2016-01-04

    The self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two ormore » more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. The resulting characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Moreover, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers.« less

  14. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    NASA Astrophysics Data System (ADS)

    Wang, Jun; Bonnesen, Peter V.; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; James Cleaves, H., II; Baddorf, Arthur P.; Sumpter, Bobby G.; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel

    2016-01-01

    Self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. These characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Further, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers.

  15. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers

    PubMed Central

    Wang, Jun; Bonnesen, Peter V.; Rangel, E.; Vallejo, E.; Sanchez-Castillo, Ariadna; James Cleaves II, H.; Baddorf, Arthur P.; Sumpter, Bobby G.; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel

    2016-01-01

    Self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N9-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. These characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Further, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers. PMID:26725380

  16. Supramolecular polymerization of a prebiotic nucleoside provides insights into the creation of sequence-controlled polymers.

    PubMed

    Wang, Jun; Bonnesen, Peter V; Rangel, E; Vallejo, E; Sanchez-Castillo, Ariadna; James Cleaves Ii, H; Baddorf, Arthur P; Sumpter, Bobby G; Pan, Minghu; Maksymovych, Petro; Fuentes-Cabrera, Miguel

    2016-01-01

    Self-assembly of a nucleoside on Au(111) was studied to ascertain whether polymerization on well-defined substrates constitutes a promising approach for making sequence-controlled polymers. Scanning tunneling microscopy and density functional theory were used to investigate the self-assembly on Au(111) of (RS)-N(9)-(2,3-dihydroxypropyl)adenine (DHPA), a plausibly prebiotic nucleoside analog of adenosine. It is found that DHPA molecules self-assemble into a hydrogen-bonded polymer that grows almost exclusively along the herringbone reconstruction pattern, has a two component sequence that is repeated over hundreds of nanometers, and is erasable with electron-induced excitation. Although the sequence is simple, more complicated ones are envisioned if two or more nucleoside types are combined. Because polymerization occurs on a substrate in a dry environment, the success of each combination can be gauged with high-resolution imaging and accurate modeling techniques. These characteristics make nucleoside self-assembly on a substrate an attractive approach for designing sequence-controlled polymers. Further, by choosing plausibly prebiotic nucleosides, insights may be provided into how nature created the first sequence-controlled polymers capable of storing information. Such insights, in turn, can inspire new ways of synthesizing sequence-controlled polymers. PMID:26725380

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

    Takaine, Masak; Numata, Osamu; Nakano, Kentaro

    2009-10-21

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

  19. Architecture and Connectivity Govern Actin Network Contractility.

    PubMed

    Ennomani, Hajer; Letort, Gaëlle; Guérin, Christophe; Martiel, Jean-Louis; Cao, Wenxiang; Nédélec, François; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2016-03-01

    Actomyosin contractility plays a central role in a wide range of cellular processes, including the establishment of cell polarity, cell migration, tissue integrity, and morphogenesis during development. The contractile response is variable and depends on actomyosin network architecture and biochemical composition. To determine how this coupling regulates actomyosin-driven contraction, we used a micropatterning method that enables the spatial control of actin assembly. We generated a variety of actin templates and measured how defined actin structures respond to myosin-induced forces. We found that the same actin filament crosslinkers either enhance or inhibit the contractility of a network, depending on the organization of actin within the network. Numerical simulations unified the roles of actin filament branching and crosslinking during actomyosin contraction. Specifically, we introduce the concept of "network connectivity" and show that the contractions of distinct actin architectures are described by the same master curve when considering their degree of connectivity. This makes it possible to predict the dynamic response of defined actin structures to transient changes in connectivity. We propose that, depending on the connectivity and the architecture, network contraction is dominated by either sarcomeric-like or buckling mechanisms. More generally, this study reveals how actin network contractility depends on its architecture under a defined set of biochemical conditions. PMID:26898468

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2006-01-01

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

  4. Polymeric Nanoparticles with Precise Ratiometric Control over Drug Loading for Combination Therapy

    PubMed Central

    Aryal, Santosh; Hu, Che-Ming Jack; Zhang, Liangfang

    2011-01-01

    We report a novel approach for nanoparticle-based combination chemotherapy by concurrently incorporating two different types of drugs into a single polymeric nanoparticle with ratiometric control over the loading of the two drugs. By adapting metal alkoxide chemistry, we synthesize highly hydrophobic drug-poly-l-lactide (drug-PLA) conjugates, of which the polymer has the same chain length while the drug may differ. These drug-polymer conjugates are then encapsulated into lipid-coated polymeric nanoparticles through a single-step nanoprecipication method. Using doxorubicin (DOX) and camptothecin (CPT) as two model chemotherapy drugs, various ratios of DOX-PLA and CPT-PLA conjugates are loaded into the nanoparticles with over 90% loading efficiency. The resulting nanoparticles are uniform in size, size distribution and surface charge. The loading yield of DOX and CPT in the particles can be precisely controlled by simply adjusting the DOX-PLA:CPT-PLA molar ratio. Cellular cytotoxicity results show that the dual-drug loaded nanoparticles are superior to the corresponding cocktail mixtures of single-drug loaded nanoparticles. This dual-drug delivery approach offers a solution to the long-standing challenge in ratiometric control over the loading of different types of drugs onto the same drug delivery vehicle. We expect that this approach can be exploited for many types of chemotherapeutic agents containing hydroxyl groups and thus enable co-delivery of various drug combinations for combinatorial treatments of diseases. PMID:21696189

  5. 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

  6. miR-34/449 control apical actin network formation during multiciliogenesis through small GTPase pathways

    PubMed Central

    Chevalier, Benoît; Adamiok, Anna; Mercey, Olivier; Revinski, Diego R.; Zaragosi, Laure-Emmanuelle; Pasini, Andrea; Kodjabachian, Laurent; Barbry, Pascal; Marcet, Brice

    2015-01-01

    Vertebrate multiciliated cells (MCCs) contribute to fluid propulsion in several biological processes. We previously showed that microRNAs of the miR-34/449 family trigger MCC differentiation by repressing cell cycle genes and the Notch pathway. Here, using human and Xenopus MCCs, we show that beyond this initial step, miR-34/449 later promote the assembly of an apical actin network, required for proper basal bodies anchoring. Identification of miR-34/449 targets related to small GTPase pathways led us to characterize R-Ras as a key regulator of this process. Protection of RRAS messenger RNA against miR-34/449 binding impairs actin cap formation and multiciliogenesis, despite a still active RhoA. We propose that miR-34/449 also promote relocalization of the actin binding protein Filamin-A, a known RRAS interactor, near basal bodies in MCCs. Our study illustrates the intricate role played by miR-34/449 in coordinating several steps of a complex differentiation programme by regulating distinct signalling pathways. PMID:26381333

  7. miR-34/449 control apical actin network formation during multiciliogenesis through small GTPase pathways.

    PubMed

    Chevalier, Benoît; Adamiok, Anna; Mercey, Olivier; Revinski, Diego R; Zaragosi, Laure-Emmanuelle; Pasini, Andrea; Kodjabachian, Laurent; Barbry, Pascal; Marcet, Brice

    2015-01-01

    Vertebrate multiciliated cells (MCCs) contribute to fluid propulsion in several biological processes. We previously showed that microRNAs of the miR-34/449 family trigger MCC differentiation by repressing cell cycle genes and the Notch pathway. Here, using human and Xenopus MCCs, we show that beyond this initial step, miR-34/449 later promote the assembly of an apical actin network, required for proper basal bodies anchoring. Identification of miR-34/449 targets related to small GTPase pathways led us to characterize R-Ras as a key regulator of this process. Protection of RRAS messenger RNA against miR-34/449 binding impairs actin cap formation and multiciliogenesis, despite a still active RhoA. We propose that miR-34/449 also promote relocalization of the actin binding protein Filamin-A, a known RRAS interactor, near basal bodies in MCCs. Our study illustrates the intricate role played by miR-34/449 in coordinating several steps of a complex differentiation programme by regulating distinct signalling pathways. PMID:26381333

  8. Synaptotagmin 1 causes phosphatidyl inositol lipid-dependent actin remodeling in cultured non-neuronal and neuronal cells

    SciTech Connect

    Johnsson, Anna-Karin; Karlsson, Roger

    2012-01-15

    Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a range of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.

  9. Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility.

    PubMed

    Lauer, P; Theriot, J A; Skoble, J; Welch, M D; Portnoy, D A

    2001-12-01

    The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged-to-alanine mutations in the NH2-terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin-binding sequence of ActA spans 40 amino acids. We also identified a 'motility rate and cloud-to-tail transition' region in which nine contiguous mutations spanning amino acids 165-260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to 'skid' or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin-based motility of L. monocytogenes. PMID:11886549

  10. Metal-centered polymers: Using controlled polymerization methodologies for the generation of responsive materials

    NASA Astrophysics Data System (ADS)

    Johnson, Robert Matthew

    Controlled polymerization methods were used to prepare highly modular polymeric metal complexes via convergent and divergent strategies. In these materials, the metal center provides a versatile hub for preparing diverse architectures through coordinative bonds. Moreover, the metal complex introduces various properties to the polymer such as luminescence, magnetism, or electroactivity. Suitably functionalized metal complexes have been used for the atom transfer radical polymerization of acrylate and methacrylate monomers by metalloinitiation to generate luminescent biocompatible materials through a divergent synthesis. By cleaving the tert-butyl groups from poly(tert -butyl acrylate), water soluble [Ru(bpyPAA2)3] 2+ has been prepared as well as the amphiphilic star block copolymer [Ru{bpy(PLA-PAA)2}3]2+ (PLA = poly(lactic acid), PAA = poly(acrylic acid) Bipyridine-centered polymeric macroligands may be chelated to a variety of metal salts. The polymer size greatly influences the formation of [Fe(bpy) 3]2+ centered polymers. As the molecular weight increases (> ˜25 kDa) tris complex formation decreases. Tris(bpy) synthesis is also impacted by chemical composition. BpyPtBA2 (PtBA = poly(tert-butyl acrylate) generates an iron mono(bpy) complex before giving rise to the bis(bpy) iron complex; no tris complex is observed. In contrast, the combination of bpyPEG2 (3 equiv) (PEG = (poly(ethylene glycol)) results in the formation of some iron tris(bpy) compound; however, complete tris(bpy) product formation is suppressed, presumably because of the chelating ability of the PEG chains. These examples contrast with other polymeric macroligands such as bpyPS2, bpyPMMA2, bpyPCL2 and bpyPLA 2 (PS = polystyrene; PMMA = poly(methyl methacrylate); PCL = poly(epsilon-caprolactone); PLA = poly(DL-lactic acid)) for which chelation reactions are facile for low molecular weight macroligands (<15 kDa), with chelation efficiencies (defined as (epsilonPMC/epsilonbpy) x 100%) only declining

  11. Supramolecular Architectures Based no Dehydro[24]annulenes: Toward the Controlled Synthesis of pi-Conjugated Nanotubular Materials via Topochemical Polymerization

    NASA Astrophysics Data System (ADS)

    Suzuki, Mitsuharu

    Chapter 1 overviews currently available synthetic methodologies of carbon nanomaterials. Conventional syntheses, stepwise chemical syntheses, and seeding/cloning approaches are described. Problems associated with each methodology are pointed out. Chapter 2 begins with introductions to the dehydroannulene-based synthesis of carbon nanomaterials and topochemical polymerization of butadiynes. This chapter then describes a new approach to achieve the controlled synthesis of tubular nanocarbon materials, namely multifold topochemical polymerization of dehydroannulenes. An extensive crystal-engineering study leads to successful formation of supramolecular nanotubes based on dehydro[24]annulenes. The obtained structures possess preferable packing parameters for the intended multifold topochemical polymerization. Chapter 3 explores on-surface self-assemblies of dehydro[24]annulenes. The relationship between the molecular structure and self-assembling behavior of is examined with the aid of scanning tunnel microscopy. This study paves the way for the topochemical polymerization of these compounds within surface-confined self-assemblies.

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

    PubMed

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

    2016-10-01

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

  13. Designing materials for advanced microelectronic patterning applications using controlled polymerization RAFT technology

    NASA Astrophysics Data System (ADS)

    Sheehan, Michael T.; Farnham, William B.; Chambers, Charles R.; Tran, Hoang V.; Okazaki, Hiroshi; Brun, Yefim; Romberger, Matthew L.; Sounik, James R.

    2011-04-01

    Reversible Addition Fragmentation Chain Transfer (RAFT) polymerization technology enables the production of polymers possessing low polydispersity (PD) in high yield for many applications. RAFT technology also enables control over polymer architecture. With synthetic control over these polymer characteristics, a variety of polymers can be designed and manufactured for use in advanced electronic applications. By matching the specific RAFT reagent and monomer combinations, we can accommodate monomer reactivity and optimize acrylate or methacrylate polymerizations (193 and 193i photoresist polymers) or optimize styrenic monomer systems (248 nm photoresist polymers) to yield polymers with PD as low as 1.05. For 193i lithography, we have used RAFT technology to produce block copolymers comprising of a random "resist" block with composition and size based on conventional dry photoresist materials and a "low surface energy" block The relative block lengths and compositions may be varied to tune solution migration behavior, surface energy, contact angles, and solubility in developer. Directed self assembly is proving to be an interesting and innovative method to make 2- and even 3-dimensional periodic, uniform patterns. Two keys to acceptable performance of directed self assembly from block copolymers are the uniformity and the purity of the materials will be discussed.

  14. Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

    PubMed Central

    Wang, Wei; Lester, John M.; Amorosa, Anthony E.; Chance, Deborah L.; Mossine, Valeri V.; Mawhinney, Thomas P.

    2015-01-01

    Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and efficient synthesis of well-controlled fluorescent statistical glycopolymers using reversible addition-fragmentation chain-transfer (RAFT)-based polymerization is demonstrated. The synthesis starts with the preparation of β-galactose-containing glycomonomer 2-lactobionamidoethyl methacrylamide obtained by reaction of lactobionolactone and N-(2-aminoethyl) methacrylamide (AEMA). 2-Gluconamidoethyl methacrylamide (GAEMA) is used as a structural analog lacking a terminal β-galactoside. The following RAFT-mediated copolymerization reaction involves three different monomers: N-(2-hydroxyethyl) acrylamide as spacer, AEMA as target for further fluorescence labeling, and the glycomonomers. Tolerant of aqueous systems, the RAFT agent used in the reaction is (4-cyanopentanoic acid)-4-dithiobenzoate. Low dispersities (≤1.32), predictable copolymer compositions, and high reproducibility of the polymerizations were observed among the products. Fluorescent polymers are obtained by modifying the glycopolymers with carboxyfluorescein succinimidyl ester targeting the primary amine functional groups on AEMA. Lectin-binding specificities of the resulting glycopolymers are verified by testing with corresponding agarose beads coated with specific glycoepitope recognizing lectins. Because of the ease of the synthesis, the tight control of the product compositions and the good reproducibility of the reaction, this protocol can be translated towards preparation of other RAFT-based glycopolymers with specific structures and compositions, as desired. PMID:26132587

  15. Control of polymerization shrinkage and stress in nanogel-modified monomer and composite materials

    PubMed Central

    Moraes, Rafael R.; Garcia, Jeffrey W.; Barros, Matthew D.; Lewis, Steven H.; Pfeifer, Carmem S.; Liu, JianCheng; Stansbury, Jeffrey W.

    2011-01-01

    Objectives This study demonstrates the effects of nano-scale prepolymer particles as additives to model dental monomer and composite formulations. Methods Discrete nanogel particles were prepared by solution photopolymerization of isobornyl methacrylate and urethane dimethacrylate in the presence of a chain transfer agent, which also provided a means to attach reactive groups to the prepolymer. Nanogel was added to triethylene glycol dimethacrylate (TEGDMA) in increments between 5 and 40 wt% with resin viscosity, reaction kinetics, shrinkage, mechanical properties, stress and optical properties evaluated. Maximum loading of barium glass filler was determined as a function of nanogel content and composites with varied nanogel content but uniform filler loading were compared in terms of consistency, conversion, shrinkage and mechanical properties. Results High conversion, high molecular weight internally crosslinked and cyclized nanogel prepolymer was efficiently prepared and redispersed into TEGDMA with an exponential rise in viscosity accompanying nanogel content. Nanogel addition at any level produced no deleterious effects on reaction kinetics, conversion or mechanical properties, as long as reactive nanogels were used. A reduction in polymerization shrinkage and stress was achieved in proportion to nanogel content. Even at high nanogel concentrations, the maximum loading of glass filler was only marginally reduced relative to the control and high strength composite materials with low shrinkage were obtained. Significance The use of reactive nanogels offers a versatile platform from which resin and composite handling properties can be adjusted while the polymerization shrinkage and stress development that challenge the adhesive bonding of dental restoratives are controllably reduced. PMID:21388669

  16. Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization.

    PubMed

    Wang, Wei; Lester, John M; Amorosa, Anthony E; Chance, Deborah L; Mossine, Valeri V; Mawhinney, Thomas P

    2015-01-01

    Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and efficient synthesis of well-controlled fluorescent statistical glycopolymers using reversible addition-fragmentation chain-transfer (RAFT)-based polymerization is demonstrated. The synthesis starts with the preparation of β-galactose-containing glycomonomer 2-lactobionamidoethyl methacrylamide obtained by reaction of lactobionolactone and N-(2-aminoethyl) methacrylamide (AEMA). 2-Gluconamidoethyl methacrylamide (GAEMA) is used as a structural analog lacking a terminal β-galactoside. The following RAFT-mediated copolymerization reaction involves three different monomers: N-(2-hydroxyethyl) acrylamide as spacer, AEMA as target for further fluorescence labeling, and the glycomonomers. Tolerant of aqueous systems, the RAFT agent used in the reaction is (4-cyanopentanoic acid)-4-dithiobenzoate. Low dispersities (≤1.32), predictable copolymer compositions, and high reproducibility of the polymerizations were observed among the products. Fluorescent polymers are obtained by modifying the glycopolymers with carboxyfluorescein succinimidyl ester targeting the primary amine functional groups on AEMA. Lectin-binding specificities of the resulting glycopolymers are verified by testing with corresponding agarose beads coated with specific glycoepitope recognizing lectins. Because of the ease of the synthesis, the tight control of the product compositions and the good reproducibility of the reaction, this protocol can be translated towards preparation of other RAFT-based glycopolymers with specific structures and compositions, as desired. PMID:26132587

  17. Study of mass loss of spacecraft polymeric thermal control coatings under electron and proton radiations

    NASA Astrophysics Data System (ADS)

    Khasanshin, Rashid; Novikov, Lev; Galygin, Alexander

    Polymeric composites have a number of properties that give a possibility to apply them as spacecraft external coatings. In space environment, however, such materials become one of the main sources of volatile products that form the outer spacecraft atmosphere and are able to con-dense on contamination-sensitive surfaces of onboard equipment. Thermal control coatings oc-cupy a considerable part of a satellite surface and are mostly subjected to ionizing radiations ac-companying by outgassing. The main stages of the process are the following: formation of vola-tile radiolysis products, diffusion of the products to free material surface, and desorption. Radia-tion-induced destruction and outgassing of material increase its permeability and accelerate mi-gration processes in it. Experimental data of effect of radiation on mass loss of polymeric composites used as thermal control coatings was analyzed and interpreted in the work. As a particular case, it was shown that mass loss of a polymeric composite irradiated by protons is greater than by electrons if energies and flux densities of the particles are the same. It can be explained that volatile products, in the first case, generate within a thin near-surface layer of material which permeability increases together with the absorbed dose, and quickly escape in vacuum. In the second case, a bulk of volatile products emerges far enough from the free surface of material which permeability increases slower as compared with proton radiation. Therefore, migration time of volatile products to the free surface grows and quantity of chemical reactions which they are involved in increases. To analyze and interpret experimental data, a mathematical model describing mass loss of polymeric composites subject to its growth of permeability under radiation is proposed. Based upon analysis of experiments and numerical simulation results, thresholds of fluen-cies and flux densities of electron and proton were determined. Exceeding these

  18. 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

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

    PubMed

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

    1992-11-01

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

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

    PubMed Central

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

    2009-01-01

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

  1. Modular and Versatile Spatial Functionalization of Tissue Engineering Scaffolds through Fiber‐Initiated Controlled Radical Polymerization

    PubMed Central

    Harrison, Rachael H.; Steele, Joseph A. M.; Chapman, Robert; Gormley, Adam J.; Chow, Lesley W.; Mahat, Muzamir M.; Podhorska, Lucia; Palgrave, Robert G.; Payne, David J.; Hettiaratchy, Shehan P.; Dunlop, Iain E.

    2015-01-01

    Native tissues are typically heterogeneous and hierarchically organized, and generating scaffolds that can mimic these properties is critical for tissue engineering applications. By uniquely combining controlled radical polymerization (CRP), end‐functionalization of polymers, and advanced electrospinning techniques, a modular and versatile approach is introduced to generate scaffolds with spatially organized functionality. Poly‐ε‐caprolactone is end functionalized with either a polymerization‐initiating group or a cell‐binding peptide motif cyclic Arg‐Gly‐Asp‐Ser (cRGDS), and are each sequentially electrospun to produce zonally discrete bilayers within a continuous fiber scaffold. The polymerization‐initiating group is then used to graft an antifouling polymer bottlebrush based on poly(ethylene glycol) from the fiber surface using CRP exclusively within one bilayer of the scaffold. The ability to include additional multifunctionality during CRP is showcased by integrating a biotinylated monomer unit into the polymerization step allowing postmodification of the scaffold with streptavidin‐coupled moieties. These combined processing techniques result in an effective bilayered and dual‐functionality scaffold with a cell‐adhesive surface and an opposing antifouling non‐cell‐adhesive surface in zonally specific regions across the thickness of the scaffold, demonstrated through fluorescent labelling and cell adhesion studies. This modular and versatile approach combines strategies to produce scaffolds with tailorable properties for many applications in tissue engineering and regenerative medicine. PMID:27134621

  2. Polymer-based monolithic columns in capillary format tailored by using controlled in situ polymerization.

    PubMed

    Aoki, Hiroshi; Tanaka, Nobuo; Kubo, Takuya; Hosoya, Ken

    2009-02-01

    This review introduces to the readers our new perspectives of polymer-based monolithic column with a high performance for small solutes such as drug candidates, illustrating the fabrication of LC columns in capillary. First, we briefly reviewed the status quo of polymer-based monolithic columns, comparing with silica monoliths. The miniaturization of LC system with higher throughput (shorter analytical time) was stressed conceptually, along with a fine permeable bicontinuous monolithic structure with submicron domain size (skeletal thickness + pore size) for higher performance. Second, from these perspectives, our column preparation was described, while our specially designed porogenic solvents were introduced as a controller of the monolithic morphology via reaction-induced phase separation. Specifically, monolithic columns were exemplified in two polymer formats, that is, one monolith prepared by free radical polymerization of glycerin 1,3-dimethacrylate, GDMA, and the other prepared by stepwise polymerization of newly introduced multifunctional epoxy and diamino monomers. Both monolithic columns in capillary format demonstrated a fine bicontinuous structure, affording a good compatibility of the efficiency (H) and permeability (D). Especially, the epoxy-based column showed an excellent separation impedance, E (=H(2)/D). Our micro-HPLC data were discussed along with a prototyped wired chip device. PMID:19142909

  3. Temporal Control of Gelation and Polymerization Fronts Driven by an Autocatalytic Enzyme Reaction

    PubMed Central

    Jee, Elizabeth; Bánsági, Tamás

    2016-01-01

    Abstract Chemical systems that remain kinetically dormant until activated have numerous applications in materials science. Herein we present a method for the control of gelation that exploits an inbuilt switch: the increase in pH after an induction period in the urease‐catalyzed hydrolysis of urea was used to trigger the base‐catalyzed Michael addition of a water‐soluble trithiol to a polyethylene glycol diacrylate. The time to gelation (minutes to hours) was either preset through the initial concentrations or the reaction was initiated locally by a base, thus resulting in polymerization fronts that converted the mixture from a liquid into a gel (ca. 0.1 mm min−1). The rate of hydrolytic degradation of the hydrogel depended on the initial concentrations, thus resulting in a gel lifetime of hours to months. In this way, temporal programming of gelation was possible under mild conditions by using the output of an autocatalytic enzyme reaction to drive both the polymerization and subsequent degradation of a hydrogel. PMID:26732469

  4. The physicodynamic properties of mucoadhesive polymeric films developed as female controlled drug delivery system.

    PubMed

    Yoo, Jin-Wook; Dharmala, Kiran; Lee, Chi H

    2006-02-17

    To develop an efficient female controlled drug delivery system (FcDDS) against sexually transmitted diseases (STDs), the polymeric films containing sodium dodecyl sulfate (SDS) were prepared with various compositions of Carbopol 934P, hydroxypropyl methylcellulose (HPMC) and polyethylene glycol (PEG). The physicochemical properties of mucoadhesive polymeric films, such as tensile strength, contact angle, swelling ratio and erosion rate in a vaginal fluid stimulant (VFS), were characterized. In addition, the drug release profile of SDS from the films and mucosal residence time were evaluated using a simulated dynamic vaginal system. It was demonstrated that the films made of Carbopol, HPMC and PEG were colorless, thin and soft and had proper physicodynamic properties for FcDDS. An increase in Carbopol content elevated tensile strength and swelling ratio but decreased the contact angle, erosion rate and the SDS release rate from the films. The films containing 0.25% (w/v) PEG as well as 0.75% (w/v) of combining Carbopol and HPMC remained on the vaginal tissue for up to 6h. The films containing the ratio of Carbopol:HPMC:PEG=1.5:1.5:1 and 1:2:1 seem to be optimal compositions for FcDDS, as they showed good peelability, relatively high swelling index and moderate tensile strength, and achieved the target release rate of SDS for 6h. PMID:16376036

  5. Temporal Control of Gelation and Polymerization Fronts Driven by an Autocatalytic Enzyme Reaction

    PubMed Central

    Jee, Elizabeth; Bánsági, Tamás

    2016-01-01

    Abstract Chemical systems that remain kinetically dormant until activated have numerous applications in materials science. Herein we present a method for the control of gelation that exploits an inbuilt switch: the increase in pH after an induction period in the urease‐catalyzed hydrolysis of urea was used to trigger the base‐catalyzed Michael addition of a water‐soluble trithiol to a polyethylene glycol diacrylate. The time to gelation (minutes to hours) was either preset through the initial concentrations or the reaction was initiated locally by a base, thus resulting in polymerization fronts that converted the mixture from a liquid into a gel (ca. 0.1 mm min−1). The rate of hydrolytic degradation of the hydrogel depended on the initial concentrations, thus resulting in a gel lifetime of hours to months. In this way, temporal programming of gelation was possible under mild conditions by using the output of an autocatalytic enzyme reaction to drive both the polymerization and subsequent degradation of a hydrogel.

  6. Temporal Control of Gelation and Polymerization Fronts Driven by an Autocatalytic Enzyme Reaction.

    PubMed

    Jee, Elizabeth; Bánsági, Tamás; Taylor, Annette F; Pojman, John A

    2016-02-01

    Chemical systems that remain kinetically dormant until activated have numerous applications in materials science. Herein we present a method for the control of gelation that exploits an inbuilt switch: the increase in pH after an induction period in the urease-catalyzed hydrolysis of urea was used to trigger the base-catalyzed Michael addition of a water-soluble trithiol to a polyethylene glycol diacrylate. The time to gelation (minutes to hours) was either preset through the initial concentrations or the reaction was initiated locally by a base, thus resulting in polymerization fronts that converted the mixture from a liquid into a gel (ca. 0.1 mm min(-1)). The rate of hydrolytic degradation of the hydrogel depended on the initial concentrations, thus resulting in a gel lifetime of hours to months. In this way, temporal programming of gelation was possible under mild conditions by using the output of an autocatalytic enzyme reaction to drive both the polymerization and subsequent degradation of a hydrogel. PMID:26732469

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. Jak3 Enables Chemokine-Dependent Actin Cytoskeleton Reorganization by Regulating Cofilin and Rac/Rhoa GTPases Activation

    PubMed Central

    Ambriz-Peña, Xochitl; García-Zepeda, Eduardo Alberto; Meza, Isaura; Soldevila, Gloria

    2014-01-01

    We have previously shown that Jak3 is involved in the signaling pathways of CCR7, CCR9 and CXCR4 in murine T lymphocytes and that Jak3−/− lymphocytes display an intrinsic defect in homing to peripheral lymph nodes. However, the molecular mechanism underlying the defective migration observed in Jak3−/− lymphocytes remains elusive. Here, it is demonstrated for the first time, that Jak3 is required for the actin cytoskeleton reorganization in T lymphocytes responding to chemokines. It was found that Jak3 regulates actin polymerization by controlling cofilin inactivation in response to CCL21 and CXCL12. Interestingly, cofilin inactivation was not precluded in PTX- treated cells despite their impaired actin polymerization. Additionally, Jak3 was required for small GTPases Rac1 and RhoA activation, which are indispensable for acquisition of the migratory cell phenotype and the generation of a functional leading edge and uropod, respectively. This defect correlates with data obtained by time-lapse video-microscopy showing an incompetent uropod formation and impaired motility in Jak3-pharmacologically inhibited T lymphocytes. Our data support a new model in which Jak3 and heterotrimeric G proteins can use independent, but complementary, signaling pathways to regulate actin cytoskeleton dynamics during cell migration in response to chemokines. PMID:24498424

  9. Actin in Herpesvirus Infection

    PubMed Central

    Roberts, Kari L.; Baines, Joel D.

    2011-01-01

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

  10. VASP Governs Actin Dynamics by Modulating Filament Anchoring

    PubMed Central

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

    2007-01-01

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

  11. Actin Rings of Power.

    PubMed

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

    2016-06-20

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

  12. Non-polymeric coatings to control drug release from metallic coronary stents

    NASA Astrophysics Data System (ADS)

    Gupta, Celia Edith Macias

    Percutaneous transluminal coronary angiography (PTCA) is a procedure used to re-open narrowed coronary arteries. During PTCA, a coronary stent is expanded inside a diseased vessel and serves as a scaffold to keep the artery open. The major drawback of stenting is restenosis---a re-narrowing of the vessel resulting from the hyperproliferation of smooth muscle cells. Drug eluting stents (DES) reduce the rate of restenosis compared to bare metal stents. Paclitaxel (PAT) is commonly used in DES for its ability to prevent restenosis. However, DES have been associated with thrombosis due to the polymer carrier that controls drug delivery. Therefore, there is a need to change the drug delivery mechanisms to eliminate the need of polymers. The goal of this dissertation is to develop a novel polymer-free drug eluting stent that controls drug release using nanoscale metal coatings. The coating was designed to release PAT as the metal slowly degrades in biological conditions. Once all the Paclitaxel has eluted from the surface, the coating will continue to degrade until the final result is a bare metal stent. The results of this study include a novel non-polymeric drug delivery system using nanoscale coatings that release Paclitaxel at a rate similar to commercial stents, as well as the biocompatibility and efficacy of these coatings. The non-polymeric drug delivery system described here achieved a Paclitaxel release profile equivalent to clinically available Paclitaxel-eluting stents and effectively inhibits smooth muscle cell proliferation, thereby completely eliminating the need for polymers to control drug release from coronary stents.

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

    SciTech Connect

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

    2008-08-27

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

  14. Actin purification from a gel of rat brain extracts.

    PubMed

    Levilliers, N; Peron-Renner, M; Coffe, G; Pudles, J

    1984-01-01

    Actin, 99% pure, has been recovered from rat brain with a high yield (greater than 15 mg/100 g brain). We have shown that: 1. a low ionic strength extract from rat brain tissue is capable of giving rise to a gel; 2. actin is the main gel component and its proportion is one order of magnitude higher than in the original extract; 3. actin can be isolated from this extract by a three-step procedure involving gelation, dissociation of the gel in 0.6 M KCl, followed by one or two depolymerization-polymerization cycles. PMID:6529588

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

    PubMed

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

    2006-12-18

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

  16. Simulation of the effect of confinement in actin ring formation

    NASA Astrophysics Data System (ADS)

    Adeli Koudehi, Maral; Vavylonis, Dimitrios; Haosu Tang Team; Dimitrios Vavylonis Team

    Actin filaments are vital for different network structures in living cells. During cytokinesis, they form a contractile ring containing myosin motor proteins and actin filament cross-linkers to separate one cell into two cells. Recent experimental studies have quantified the bundle, ring, and network structures that form when actin filaments polymerize in confined environments in vitro, in the presence of varying concentrations of cross-linkers. In this study, we performed numerical simulations to investigate the effect of actin spherical confinement and cross-linking in ring formation. We used a spring-bead model and Brownian dynamics to simulate semiflexible actin filaments that polymerize in a confining sphere with a rate proportional to the monomer concentration. Applying the model for different size of the confining spheres shows that the probability of ring formation decreases by increasing the radius (at fixed initial monomer concentration), in agreement with prior experimental data. We describe the effect of persistence length, orientation-dependent cross-linking, and initial actin monomer concentration. Simulations show that equilibrium configurations can be reached through zipping and unzipping of actin filaments in bundles and transient ring formation.

  17. Nuclear Actin Extends, with No Contraction in Sight

    PubMed Central

    Pederson, Thoru; Aebi, Ueli

    2005-01-01

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

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

    PubMed

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

    2016-05-10

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

  19. Nuclear and cytoplasmic actin in dinoflagellates.

    PubMed

    Soyer-Gobillard, M O; Ausseil, J; Géraud, M L

    1996-01-01

    Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the

  20. Structure of Actin-related protein 8 and its contribution to nucleosome binding

    PubMed Central

    Gerhold, Christian B.; Winkler, Duane D.; Lakomek, Kristina; Seifert, Florian U.; Fenn, Sebastian; Kessler, Brigitte; Witte, Gregor; Luger, Karolin; Hopfner, Karl-Peter

    2012-01-01

    Nuclear actin-related proteins (Arps) are subunits of several chromatin remodelers, but their molecular functions within these complexes are unclear. We report the crystal structure of the INO80 complex subunit Arp8 in its ATP-bound form. Human Arp8 has several insertions in the conserved actin fold that explain its inability to polymerize. Most remarkably, one insertion wraps over the active site cleft and appears to rigidify the domain architecture, while active site features shared with actin suggest an allosterically controlled ATPase activity. Quantitative binding studies with nucleosomes and histone complexes reveal that Arp8 and the Arp8–Arp4–actin-HSA sub-complex of INO80 strongly prefer nucleosomes and H3–H4 tetramers over H2A–H2B dimers, suggesting that Arp8 functions as a nucleosome recognition module. In contrast, Arp4 prefers free (H3–H4)2 over nucleosomes and may serve remodelers through binding to (dis)assembly intermediates in the remodeling reaction. PMID:22977180

  1. Temperature control of the motility of actin filaments interacting with myosin molecules using an electrically conductive glass in the presence of direct current.

    PubMed

    Wada, Reito; Sato, Daisuke; Nakamura, Takao; Hatori, Kuniyuki

    2015-11-15

    The motility of actin filaments interacting with heavy meromyosin molecules was directly observed on indium tin oxide-coated glass (ITO-glass), over which a surface current flowed. Because the increase in surface current applied to ITO-glass increases the temperature, we focused on the temperature-dependence of the sliding velocity and the effect of the current flow on the orientation of filament motion. Using high precision fluorescence measurements, the displacement vectors of filaments were collected at intervals of 1/30 s. The direction of filament motion was independent to that of current flow up to 0.17 A (7.7 A/m of surface current density); however, the velocity increased by approximately 2-fold when the surface temperature increased from 25 °C to 37 °C. The moving actin filaments exhibited a broader velocity distribution at high temperature than at low temperature. Collectively, these data suggest that using ITO-glass with a surface current to generate a well-controlled temperature change may serve to evaluate temperature-dependent transient responses in protein activity under a microscope, without interference from electrical effects. PMID:26456400

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

    PubMed

    Kakumoto, Toshiyuki; Nakata, Takao

    2013-01-01

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

  3. Recyclable Crosslinked Polymer Networks via One-Step Controlled Radical Polymerization.

    PubMed

    Jin, Kailong; Li, Lingqiao; Torkelson, John M

    2016-08-01

    A nitroxide-mediated polymerization strategy allows one-step synthesis of recyclable crosslinked polymeric materials from any monomers or polymers that contain carbon-carbon double bonds amenable to radical polymerization. The resulting materials with dynamic covalent bonds can show full property recovery after multiple melt-reprocessing recycles. This one-step strategy provides for both robust, relatively sustainable recyclability of crosslinked polymers and design of networks for advanced technologies. PMID:27206061

  4. Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo {sup 1}H NMR and fluorescence spectroscopy

    SciTech Connect

    Wazawa, Tetsuichi; Sagawa, Takashi; Ogawa, Tsubasa; Morimoto, Nobuyuki; Kodama, Takao; Suzuki, Makoto

    2011-01-28

    Research highlights: {yields} Translationally hyper-mobile water has been detected around actin filaments. {yields} Translationally hyper-mobile water is formed upon polymerization of actin. {yields} Low water viscosity was found around F-actin using fluorescence anisotropy. {yields} Formation of hyper-mobile water may explain endothermic actin polymerization. -- Abstract: This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo {sup 1}H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by {approx}5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1 ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.

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

    PubMed

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

    1993-10-01

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

  6. Modulation of the interaction between G-actin and thymosin beta 4 by the ATP/ADP ratio: possible implication in the regulation of actin dynamics.

    PubMed Central

    Carlier, M F; Jean, C; Rieger, K J; Lenfant, M; Pantaloni, D

    1993-01-01

    The interaction of G-actin with thymosin beta 4 (T beta 4), the major G-actin-sequestering protein in motile and proliferating cells, has been analyzed in vitro. T beta 4 is found to have a 50-fold higher affinity for MgATP-actin than for MgADP-actin. These results imply that in resting platelets and neutrophils, actin is sequestered by T beta 4 as MgATP-G-actin. Kinetic experiments and theoretical calculations demonstrate that this ATP/ADP dependence of T beta 4 affinity for G-actin can generate a mechanism of desequestration of G-actin by ADP, in the presence of physiological concentrations of T beta 4 (approximately 0.1 mM). The desequestration of G-actin by ADP is kinetically enhanced by profilin, which accelerates the dissociation of ATP from G-actin. Whether a local drop in the ATP/ADP ratio can allow local, transient desequestration and polymerization of actin either close to the plasma membrane, following platelet or neutrophil stimulation, or behind the Listeria bacterium in the host cell, while the surrounding cytoplasm contains sequestered ATP-G-actin, is an open issue raised by the present work. PMID:8506348

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Siton, Orit; Bernheim-Groswasser, Anne

    2014-01-01

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

  9. Control of molecular weight of polystyrene using the reverse iodine transfer polymerization (RITP)-emulsion technique.

    PubMed

    Oh, Hyeong Geun; Shin, Hongcheol; Jung, Hyejun; Lee, Byung Hyung; Choe, Soonja

    2011-01-15

    The RITP-emulsion polymerization of styrene in the presence of molecular iodine has been successfully performed using potassium persulfate (KPS) as an initiator and 1-hexadecanesulfonate as an emulsifier under argon atmosphere at 80°C for 7 hrs in the absence of light. The effects of the iodine concentration, molar ratio between KPS and iodine, and solid contents on the molecular weight of polystyrene (PS) were studied. As the iodine concentration increased from 0.05 to 0.504 mmol under the fixed [KPS]/[I(2)] ratio at 4.5, the weight-average molecular weight of PS substantially decreased from 126,120 to 35,690 g/mol, the conversion increased from 85.0% to 95.2%, and the weight-average particle diameter decreased from 159 to 103 nm. In addition, as the ratio of [KPS]/[I(2)] increased from 0.5 to 6.0 at the fixed [I(2)] of 0.504 mmol, the weight-average molecular weight of PS decreased from 72,170 to 30,640 g/mol with high conversion between 81.7% and 96.5%. Moreover, when the styrene solid content increased from 10 to 40 wt.% at the fixed [KPS]/[I(2)] ratio of 4.5, the weight-average molecular weight of PS varied between 33,500 and 37,200 g/mol, the conversion varied between 94.9% and 89.7% and the weight-average diameter varied from 122 to 205 nm. Thus, the control of molecular weight of PS less than 100,000g/mol with high conversion (95%) and particle stability of up to 40 wt.% solid content were easily achieved through the usage of iodine with suitable ratio of [KPS]/[I(2)] in the RITP-emulsion polymerization technique, which is of great industrial importance. PMID:20950818

  10. Course 6: Physics of Composite Cell Membrane and Actin Based Cytoskeleton

    NASA Astrophysics Data System (ADS)

    Sackmann, E.; Bausch, A. R.; Vonna, L.

    1 Architecture of composite cell membranes 1.1 The lipid/protein bilayer is a multicomponent smectic phase with mosaic like architecture 1.2 The spectrin/actin cytoskeleton as hyperelastic cell stabilizer 1.3 The actin cortex: Architecture and function 2 Physics of the actin based cytoskeleton 2.1 Actin is a living semiflexible polymer 2.2 Actin network as viscoelastic body 2.3 Correlation between macroscopic viscoelasticity and molecular 3 Heterogeneous actin gels in cells and biological function 3.1 Manipulation of actin gels 3.2 Control of organization and function of actin cortex by cell signalling 4 Micromechanics and microrheometry of cells 5 Activation of endothelial cells: On the possibility of formation of stress fibers as phase transition of actin-network triggered by cell signalling pathways 6 On cells as adaptive viscoplastic bodies 7 Controll of cellular protrusions controlled by actin/myosin cortex

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

    PubMed Central

    Vitriol, Eric A.; McMillen, Laura M.; Kapustina, Maryna; Gomez, Shawn M.; Vavylonis, Dimitrios; Zheng, James Q.

    2015-01-01

    Summary Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer binding protein thymosin β4 (Tβ4) for optimal leading edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tβ4 and appears to regulate lamellipodia homeostasis. Tβ4-bound G-actin specifically localizes to the leading edge because it doesn’t interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions. PMID:25865895

  12. Formation of size-controlled, denaturation-resistant lipid nanodiscs by an amphiphilic self-polymerizing peptide.

    PubMed

    Kondo, Hiroaki; Ikeda, Keisuke; Nakano, Minoru

    2016-10-01

    Nanodiscs are discoidal particles with a planar phospholipid bilayer enwrapped by proteins such as apolipoprotein A-I. Nanodiscs have been widely used for analyzing structures and functions of membrane proteins by dispersing them in solution. They are expected to be used as drug carriers and therapeutic agents. Amphiphilic peptides are known to form nanodiscs. However, the lipid-peptide nanodiscs are relatively unstable in solution, making them unsuitable for many applications. Here, we report the synthesis of an amphiphilic self-polymerizing peptide termed ASPP1, which polymerizes by intermolecular native chemical ligation reactions. ASPP1 spontaneously formed nanodiscs when added to phospholipid vesicles without using detergents. The diameter of the planar lipid bilayer in the nanodiscs was controlled by the lipid:peptide molar ratio. ASPP1-nanodiscs exhibited greater stability at high temperatures or in the presence of urea than nanodiscs formed by the non-polymerizing amphiphilic peptide or apolipoprotein A-I. Average and maximal degrees of ASPP1 polymerization were 2.4 and 12, respectively. Self-polymerization of the peptide appears to be responsible for stabilization of the nanodiscs. Our results open a new avenue for the development of nanodisc technology. PMID:27393815

  13. Controlled atom transfer radical polymerization of MMA onto the surface of high-density functionalized graphene oxide

    PubMed Central

    2014-01-01

    We report on the grafting of poly(methyl methacrylate) (PMMA) onto the surface of high-density functionalized graphene oxides (GO) through controlled radical polymerization (CRP). To increase the density of surface grafting, GO was first diazotized (DGO), followed by esterification with 2-bromoisobutyryl bromide, which resulted in an atom transfer radical polymerization (ATRP) initiator-functionalized DGO-Br. The functionalized DGO-Br was characterized by X-ray photoelectron spectroscopy (XPS), Raman, and XRD patterns. PMMA chains were then grafted onto the DGO-Br surface through a ‘grafting from’ technique using ATRP. Gel permeation chromatography (GPC) results revealed that polymerization of methyl methacrylate (MMA) follows CRP. Thermal studies show that the resulting graphene-PMMA nanocomposites have higher thermal stability and glass transition temperatures (Tg) than those of pristine PMMA. PMID:25114639

  14. Controlled atom transfer radical polymerization of MMA onto the surface of high-density functionalized graphene oxide

    NASA Astrophysics Data System (ADS)

    Kumar, Mukesh; Chung, Jin Suk; Hur, Seung Hyun

    2014-07-01

    We report on the grafting of poly(methyl methacrylate) (PMMA) onto the surface of high-density functionalized graphene oxides (GO) through controlled radical polymerization (CRP). To increase the density of surface grafting, GO was first diazotized (DGO), followed by esterification with 2-bromoisobutyryl bromide, which resulted in an atom transfer radical polymerization (ATRP) initiator-functionalized DGO-Br. The functionalized DGO-Br was characterized by X-ray photoelectron spectroscopy (XPS), Raman, and XRD patterns. PMMA chains were then grafted onto the DGO-Br surface through a `grafting from' technique using ATRP. Gel permeation chromatography (GPC) results revealed that polymerization of methyl methacrylate (MMA) follows CRP. Thermal studies show that the resulting graphene-PMMA nanocomposites have higher thermal stability and glass transition temperatures ( T g) than those of pristine PMMA.

  15. Evaluation of microwave assisted grafted sago starch as controlled release polymeric carrier.

    PubMed

    Singh, Akhilesh Vikram; Nath, Lila Kanta

    2013-09-01

    In the present investigation an attempt has been made to develop a new co-polymeric material for controlled release tablet formulations. The acrylamide grafting was successfully performed on the backbone of sago starch. The modified starch was tested for acute toxicity and drug-excipient compatibility study. The grafted material was used in making of controlled release tablets of lamivudine. The formulations were evaluated for physical characteristics such as hardness, friability, %drug content and weight variations. The in vitro release study showed that the optimized formulation exhibited highest correlation (R) value in case of Higuchi model and the release mechanism of the optimized formulation predominantly exhibited combination of diffusion and erosion process. There was a significant difference in the pharmacokinetic parameters (T(max), C(max), AUC, V(d), T(1/2) and MDT) of the optimized formulation as compared to the marketed conventional tablet Lamivir(®) was observed. The pharmacokinetics parameters were showed controlled pattern and better bioavailability. The optimized formulation exhibited good stability and release profile at the accelerated stability conditions. PMID:23707750

  16. Actin dynamics and the evolution of the memory trace.

    PubMed

    Rudy, Jerry W

    2015-09-24

    The goal of this essay is to link the regulation of actin dynamics to the idea that the synaptic changes that support long-term potentiation and memory evolve in temporally overlapping stages-generation, stabilization, and consolidation. Different cellular/molecular processes operate at each stage to change the spine cytoarchitecture and, in doing so, alter its function. Calcium-dependent processes that degrade the actin cytoskeleton network promote a rapid insertion of AMPA receptors into the post synaptic density, which increases a spine's capacity to express a potentiated response to glutamate. Other post-translation events then begin to stabilize and expand the actin cytoskeleton by increasing the filament actin content of the spine and reorganizing it to be resistant to depolymerizing events. Disrupting actin polymerization during this stabilization period is a terminal event-the actin cytoskeleton shrinks and potentiated synapses de-potentiate and memories are lost. Late-arriving, new proteins may consolidate changes in the actin cytoskeleton. However, to do so requires a stabilized actin cytoskeleton. The now enlarged spine has properties that enable it to capture other newly transcribed mRNAs or their protein products and thus enable the synaptic changes that support LTP and memory to be consolidated and maintained. This article is part of a Special Issue entitled SI: Brain and Memory. PMID:25498985

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

    PubMed Central

    Mizuno, Hiroaki; Watanabe, Naoki

    2012-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

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

    PubMed Central

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

    2015-01-01

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

  20. Controlling the thickness of hollow polymeric microspheres prepared by electrohydrodynamic atomization.

    PubMed

    Chang, Ming-Wei; Stride, Eleanor; Edirisinghe, Mohan

    2010-08-01

    In this study, the ability to control the shell thickness of hollow polymeric microspheres prepared using electrohydrodynamic processing at ambient temperature was investigated. Polymethylsilsesquioxane (PMSQ) was used as a model material for the microsphere shell encapsulating a core of liquid perfluorohexane (PFH). The microspheres were characterized by Fourier transform infrared spectroscopy and optical and electron microscopy, and the effects of the processing parameters (flow-rate ratio, polymer concentration and applied voltage) on the mean microsphere diameter (D) and shell thickness (t) were determined. It was found that the mean diameters of the hollow microspheres could be controlled in the range from 310 to 1000 nm while the corresponding mean shell thickness varied from 40 to 95 nm. The results indicate that the ratio D : t varied with polymer concentration, with the largest value of approximately 10 achieved with a solution containing 18 wt% of the polymer, while the smallest value (6.6) was obtained at 36 wt%. For polymer concentrations above 63 wt%, hollow microspheres could not be generated, but instead PMSQ fibres encapsulating PFH liquid were obtained. PMID:20519216

  1. Controlled nanopatterning of a polymerized ionic liquid in a strong electric field

    DOE PAGESBeta

    Bocharova, Vera; Agapov, Alexander L.; Tselev, Alexander; Kumar, Rajeev; Berdzinski, Stefan; Strehmel, Veronika; Kisliuk, Alexander; Kravchenko, Ivan I.; Sumpter, Bobby G.; Sokolov, Alexei P.; et al

    2014-12-17

    Nanolithography has become a driving force in advancements of the modern day's electronics, allowing for miniaturization of devices and a steady increase of the calculation, power, and storage densities. Among various nanofabrication approaches, scanning probe techniques, including atomic force microscopy (AFM), are versatile tools for creating nanoscale patterns utilizing a range of physical stimuli such as force, heat, or electric field confined to the nanoscale. In this study, the potential of using the electric field localized at the apex of an AFM tip to induce and control changes in the mechanical properties of an ion containing polymer—a polymerized ionic liquidmore » (PolyIL)—on a very localized scale is explored. In particular, it is demonstrated that by means of AFM, one can form topographical features on the surface of PolyIL-based thin films with a significantly lower electric potential and power consumption as compared to nonconductive polymer materials. Lastly,, by tuning the applied voltage and ambient air humidity, control over dimensions of the formed structures is reproducibly achieved.« less

  2. Controlled nanopatterning of a polymerized ionic liquid in a strong electric field

    SciTech Connect

    Bocharova, Vera; Agapov, Alexander L.; Tselev, Alexander; Kumar, Rajeev; Berdzinski, Stefan; Strehmel, Veronika; Kisliuk, Alexander; Kravchenko, Ivan I.; Sumpter, Bobby G.; Sokolov, Alexei P.; Kalinin, Sergei V.; Strelcov, Evgheni; Collins, Liam

    2014-12-17

    Nanolithography has become a driving force in advancements of the modern day's electronics, allowing for miniaturization of devices and a steady increase of the calculation, power, and storage densities. Among various nanofabrication approaches, scanning probe techniques, including atomic force microscopy (AFM), are versatile tools for creating nanoscale patterns utilizing a range of physical stimuli such as force, heat, or electric field confined to the nanoscale. In this study, the potential of using the electric field localized at the apex of an AFM tip to induce and control changes in the mechanical properties of an ion containing polymer—a polymerized ionic liquid (PolyIL)—on a very localized scale is explored. In particular, it is demonstrated that by means of AFM, one can form topographical features on the surface of PolyIL-based thin films with a significantly lower electric potential and power consumption as compared to nonconductive polymer materials. Lastly,, by tuning the applied voltage and ambient air humidity, control over dimensions of the formed structures is reproducibly achieved.

  3. Controlling the thickness of hollow polymeric microspheres prepared by electrohydrodynamic atomization

    PubMed Central

    Chang, Ming-Wei; Stride, Eleanor; Edirisinghe, Mohan

    2010-01-01

    In this study, the ability to control the shell thickness of hollow polymeric microspheres prepared using electrohydrodynamic processing at ambient temperature was investigated. Polymethylsilsesquioxane (PMSQ) was used as a model material for the microsphere shell encapsulating a core of liquid perfluorohexane (PFH). The microspheres were characterized by Fourier transform infrared spectroscopy and optical and electron microscopy, and the effects of the processing parameters (flow-rate ratio, polymer concentration and applied voltage) on the mean microsphere diameter (D) and shell thickness (t) were determined. It was found that the mean diameters of the hollow microspheres could be controlled in the range from 310 to 1000 nm while the corresponding mean shell thickness varied from 40 to 95 nm. The results indicate that the ratio D : t varied with polymer concentration, with the largest value of approximately 10 achieved with a solution containing 18 wt% of the polymer, while the smallest value (6.6) was obtained at 36 wt%. For polymer concentrations above 63 wt%, hollow microspheres could not be generated, but instead PMSQ fibres encapsulating PFH liquid were obtained. PMID:20519216

  4. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Collins, Caitlin

    2014-01-01

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

  7. A supracellular system of actin-lined canals controls biogenesis and release of virulence factors in parasitoid venom glands.

    PubMed

    Ferrarese, Roberto; Morales, Jorge; Fimiarz, Daniel; Webb, Bruce A; Govind, Shubha

    2009-07-01

    Parasitoid wasps produce virulence factors that bear significant resemblance to viruses and have the ability to block host defense responses. The function of these virulence factors, produced predominantly in wasp venom glands, and the ways in which they interfere with host development and physiology remain mysterious. Here, we report the discovery of a specialized system of canals in venom glands of five parasitoid wasps that differ in their infection strategies. This supracellular canal system is made up of individual secretory units, one per secretory cell. Individual units merge into the canal lumen. The membrane surface of the proximal end of each canal within the secretory cell assumes brush border morphology, lined with bundles of F-actin. Systemic administration of cytochalasin D compromises the integrity of the secretory unit. We show a dynamic and continuous association of p40, a protein of virus-like particles from a Drosophila parasitoid, L. heterotoma, with the canal and venom gland lumen. Similar structures in three Leptopilina species and Ganaspis xanthopoda, parasitoids of Drosophila spp., and Campoletis sonorenesis, a parasitoid of Heliothis virescens, suggest that this novel supracellular canal system is likely to be a common trait of parasitoid venom glands that is essential for efficient biogenesis and delivery of virulence factors. PMID:19561216

  8. A supracellular system of actin-lined canals controls biogenesis and release of virulence factors in parasitoid venom glands

    PubMed Central

    Ferrarese, Roberto; Morales, Jorge; Fimiarz, Daniel; Webb, Bruce A.; Govind, Shubha

    2009-01-01

    Summary Parasitoid wasps produce virulence factors that bear significant resemblance to viruses and have the ability to block host defense responses. The function of these virulence factors, produced predominantly in wasp venom glands, and the ways in which they interfere with host development and physiology remain mysterious. Here, we report the discovery of a specialized system of canals in venom glands of five parasitoid wasps that differ in their infection strategies. This supracellular canal system is made up of individual secretory units, one per secretory cell. Individual units merge into the canal lumen. The membrane surface of the proximal end of each canal within the secretory cell assumes brush border morphology, lined with bundles of F-actin. Systemic administration of cytochalasin D compromises the integrity of the secretory unit. We show a dynamic and continuous association of p40, a protein of virus-like particles from a Drosophila parasitoid, L. heterotoma, with the canal and venom gland lumen. Similar structures in three Leptopilina species and Ganaspis xanthopoda, parasitoids of Drosophila spp., and Campoletis sonorenesis, a parasitoid of Heliothis virescens, suggest that this novel supracellular canal system is likely to be a common trait of parasitoid venom glands that is essential for efficient biogenesis and delivery of virulence factors. PMID:19561216

  9. Microtransfer printing of metal ink patterns onto plastic substrates utilizing an adhesion-controlled polymeric donor layer

    NASA Astrophysics Data System (ADS)

    Park, Ji-Sub; Choi, Jun-Chan; Park, Min-Kyu; Bae, Jeong Min; Bae, Jin-Hyuk; Kim, Hak-Rin

    2016-06-01

    We propose a method for transfer-printed electrode patterns onto flexible/plastic substrates, specifically intended for metal ink that requires a high sintering temperature. Typically, metal-ink-based electrodes cannot be picked up for microtransfer printing because the adhesion between the electrodes and the donor substrate greatly increases after the sintering process due to the binding materials. We introduced a polymeric donor layer between the printed electrodes and the donor substrate and effectively reduced the adhesion between the Ag pattern and the polymeric donor layer by controlling the interfacial contact area. After completing a wet-etching process for the polymeric donor layer, we obtained Ag patterns supported on the fine polymeric anchor structures; the Ag patterns could be picked up onto the stamp surface even after the sintering process by utilizing the viscoelastic properties of the elastomeric stamp with a pick-up velocity control. The proposed method enables highly conductive metal-ink-based electrode patterns to be applied on thermally weak plastic substrates via an all-solution process. Metal electrodes transferred onto a film showed superior electrical and mechanical stability under the bending stress test required for use in printed flexible electronics.

  10. Controlled RAFT Polymerization of 2-Vinyl-4,4-Dimethylazlactone (VDMA): A Facile Route to Bio-Inspired Polymer Surfaces

    SciTech Connect

    Lokitz, Bradley S; Messman, Jamie M; Hinestrosa Salazar, Juan Pablo; Alonzo Calderon, Jose E; Verduzco, Rafael; Brown, Rebecca H; Osa, Masashi; Ankner, John Francis; Kilbey, II, S Michael

    2009-01-01

    We report the controlled radical polymerization of 2-vinyl-4,4-dimethyl azlactone (VDMA), a 2-alkenyl-2-oxazolin-5-one monomer that contains a polymerizable vinyl moiety as well as a highly reactive, pendant azlactone as well as solution characterizations and surface attachment and functionaliztion. Reversible addition fragmentation chain transfer (RAFT) was used to polymerize of VDMA in benzene at 65 C using either 2-(2-cyanopropyl) dithiobenzoate (CPDB) or 2-dodecylsulfanylthiocarbonyl-sulfanyl-2-methylpropionic acid (DMP) as RAFT chain transfer agents (CTAs). The pseudo first order kinetics and resultant well-defined polymers of low polydispersity indicate that both CTAs afford control over the RAFT polymerization of VDMA. Dynamic and static light scattering and small angle neutron scattering were performed to determine the dn/dc, weight-average molecular weight, radius of gyration, and second virial coefficient of VDMA homopolymers in THF. Additionally, well-defined polymers of VDMA containing carboxyl end groups were covalently attached to epoxy modified silicon wafers via esterification to produce polymeric scaffolds that could be subsequently functionalized for various bio-inspired applications.

  11. Simulation and Experiments To Identify Factors Allowing Synthetic Control of Structural Features of Polymeric Nanoparticles.

    PubMed

    Swope, William C; Rice, Julia E; Piunova, Victoria A; Carr, Amber C; Miller, Robert D; Sly, Joseph

    2016-08-01

    To develop a detailed picture of the microscopic structure of gelcore star polymers and to elucidate parameters of the synthetic process that might be exploited to control this structure, simulations of their synthesis were performed that were based on a particular synthetic approach. A range of results was observed from gelation at high reactant concentrations to the formation of various sizes and compositions of star polymers. Contrary to the prevailing experimental viewpoint, the simulations always suggest the production of a broad distribution of star polymer sizes. However, the GPC traces computed from simulation results are in good qualitative agreement with experiment. Topologically, the gelcore star polymers produced by simulation are not compact but, rather, sparse blobs loosely connected by filaments of linker when modeled in a good solvent. This is reflected in scaling relationships that relate polymer size (e.g., radius of gyration) and degree of polymerization. The arm-core composition is observed to be stoichiometric, strongly reflecting relative reactant concentrations during the synthesis. Reactions within star polymers that result in greater intramolecular cross-linking compete with those between star polymers that result in the production of larger star polymers from the joining of smaller ones. The balance in this competition can be controlled through the overall reactant concentration to limit and control resulting star polymer size. Therefore, the mean size, as well as the mean number of arms, can be controlled during synthesis by careful tuning of the overall ratio of the arm and linker reactant concentrations and the total reactant concentration. PMID:27385087

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

    PubMed

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

    2006-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Vicker, Michael G.

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

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

    PubMed

    Veksler, Alex; Gov, Nir S

    2009-09-16

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

  15. 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.

  16. Dictyostelium Dock180-related RacGEFs regulate the actin cytoskeleton during cell motility.

    PubMed

    Para, Alessia; Krischke, Miriam; Merlot, Sylvain; Shen, Zhouxin; Oberholzer, Michael; Lee, Susan; Briggs, Steven; Firtel, Richard A

    2009-01-01

    Cell motility of amoeboid cells is mediated by localized F-actin polymerization that drives the extension of membrane protrusions to promote forward movements. We show that deletion of either of two members of the Dictyostelium Dock180 family of RacGEFs, DockA and DockD, causes decreased speed of chemotaxing cells. The phenotype is enhanced in the double mutant and expression of DockA or DockD complements the reduced speed of randomly moving DockD null cells' phenotype, suggesting that DockA and DockD are likely to act redundantly and to have similar functions in regulating cell movement. In this regard, we find that overexpressing DockD causes increased cell speed by enhancing F-actin polymerization at the sites of pseudopod extension. DockD localizes to the cell cortex upon chemoattractant stimulation and at the leading edge of migrating cells and this localization is dependent on PI3K activity, suggesting that DockD might be part of the pathway that links PtdIns(3,4,5)P(3) production to F-actin polymerization. Using a proteomic approach, we found that DdELMO1 is associated with DockD and that Rac1A and RacC are possible in vivo DockD substrates. In conclusion, our work provides a further understanding of how cell motility is controlled and provides evidence that the molecular mechanism underlying Dock180-related protein function is evolutionarily conserved. PMID:19037099

  17. Whole Cell Model of Actin Diffusion and Reaction based on Single Molecule Speckle Microscopy Measurements

    NASA Astrophysics Data System (ADS)

    McMillen, Laura; Vavylonis, Dimitrios; Vavylonis Group Team

    It is debated whether transport of actin across the cell by diffusion alone is sufficiently fast to account for the rapid reorganization of actin filaments at the leading edge of motile cells. In order to investigate this question, we created a 3D model of the whole cell that includes reaction and diffusion of actin using a particle Monte Carlo method. For the lamellipodium of the simulated cell we use the model by Smith et al. Biophys. J 104:247 (2013), which includes two diffuse pools of actin, one which is slowly diffusing and the other which diffuses more quickly, as well as a pool of filamentous actin undergoing retrograde flow towards the cell center. We adjusted this model to fit a circular geometry around the whole cell. We also consider actin in the cell center which is either diffusing or in stationary filamentous form, representing cortical actin or actin in stress fibers. The local rates of polymerization and the lifetime distributions of polymerized actin were estimated from single molecule speckle microscopy experiments by the group of N. Watanabe. With this model we are able to simulate prior experiments that monitored the redistribution of actin after photoactivation or fluorescence recovery after photobleaching in various parts of the cell. We find that transport by diffusion is sufficient to fit these data, without the need for an active transport mechanism, however significant concentration gradients may develop at steady state.

  18. Isolation of a 5-Kilodalton Actin-Sequestering Peptide from Human Blood Platelets

    NASA Astrophysics Data System (ADS)

    Safer, Daniel; Golla, Rajasree; Nachmias, Vivianne T.

    1990-04-01

    Resting human platelets contain ≈0.3 mM unpolymerized actin. When freshly drawn and washed platelets are treated with saponin, 85-90% of the unpolymerized actin diffuses out. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions shows that the bulk of this unpolymerized actin migrates with a higher mobility than does pure G-actin, profilactin, or actin-gelsolin complex. When muscle G-actin is added to fresh or boiled saponin extract, the added muscle actin is shifted to the high-mobility form. The saponin extract contains an acidic peptide having a molecular mass in the range of 5 kDa, which has been purified to homogeneity by reverse-phase HPLC. This peptide also shifts muscle actin to the high-mobility form. Addition of either boiled saponin extract or the purified peptide to muscle G-actin also strongly and stoichiometrically inhibits salt-induced polymerization, as assayed by falling-ball viscometry and by sedimentation. We conclude that this peptide binds to the bulk of the unpolymerized actin in platelets and prevents it from polymerizing.

  19. Actin Automata with Memory

    NASA Astrophysics Data System (ADS)

    Alonso-Sanz, Ramón; Adamatzky, Andy

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

  20. Supercritical fluid extraction-liquid chromatography method development for a polymeric controlled-release drug formulation.

    PubMed

    Roston, D A; Sun, J J; Collins, P W; Perkins, W E; Tremont, S J

    1995-11-01

    We have recently been involved in the development of a method for assaying the active component in a controlled-release drug formulation, which is composed of a drug substance covalently bonded to polymer matrix. The drug substance in the formulation is the active enantiomer of misoprostol, a synthetic analog of natural prostaglandins and the active ingredient in Cytotec. Our method development consisted of a systematic evaluation of dynamic, off-line supercritical fluid extraction (SFE) as sample preparation for the formulation assay. Extracts were analyzed with normal phase and reversed-phase HPLC methods. The reversed-phase system utilized postcolumn reaction to provide selective detection of the extracted prostaglandin sample components. Several SFE parameters were investigated to optimize the recovery of the drug substance from the formulation, including sample quantity, extraction cell volume, extraction duration, supercritical carbon dioxide modifier, temperature, pressure, and collection solvent. The SFE experiments were completed with a commercially available multicell extractor. Preliminary validation studies utilized a formulation made with radiolabeled drug to determine the recovery achieved under the optimized SFE conditions and assessed the precision of replicate determinations. Analysis was completed under the optimized conditions to quantitate levels of the active component and related compounds in lots of the experimental polymeric formulation and to determine the total weight per cent extracted. PMID:8788137

  1. Polymer nanostructures synthesized by controlled living polymerization for tumor-targeted drug delivery.

    PubMed

    Wang, Christine E; Stayton, Patrick S; Pun, Suzie H; Convertine, Anthony J

    2015-12-10

    The development of drug delivery systems based on well-defined polymer nanostructures could lead to significant improvements in the treatment of cancer. The design of these therapeutic nanosystems must account for numerous systemic and circulation obstacles as well as the specific pathophysiology of the tumor. Nanoparticle size and surface charge must also be carefully selected in order to maintain long circulation times, allow tumor penetration, and avoid clearance by the reticuloendothelial system (RES). Targeting ligands such as vitamins, peptides, and antibodies can improve the accumulation of nanoparticle-based therapies in tumor tissue but must be optimized to allow for intratumoral penetration. In this review, we will highlight factors influencing the design of nanoparticle therapies as well as the development of modern controlled "living" polymerization techniques (e.g. ATRP, RAFT, ROMP) that are leading to the creation of sophisticated new polymer architectures with discrete spatially-defined functional modules. These innovative materials (e.g. star polymers, polymer brushes, macrocyclic polymers, and hyperbranched polymers) combine many of the desirable properties of traditional nanoparticle therapies while substantially reducing or eliminating the need for complex formulations. PMID:26342661

  2. Anti-inflammatory drug incorporation into polymeric nano-hybrids for local controlled release.

    PubMed

    Sammartino, G; Marenzi, G; Tammaro, L; Bolognese, A; Calignano, A; Costantino, U; Califano, L; Mastrangelo, F; Tetè, S; Vittoria, V

    2005-01-01

    In this paper we present the formulation, preparation and characterization of new polymeric composite materials containing a nano-hybrid to be used for the controlled molecular delivery of an anti-inflammatory molecule, Diclofenac. The nano-hybrid consists of a layer of double hydroxide of an Mg-Al hydrotalcite type, in which we replaced the chloride anions present in the host galleries with Diclofenac anions by a simple ion-exchange reaction. Different amounts of the hybrid material were incorporated in polycaprolactone and processed as films of 0.15 mm thickness. The composite materials were analyzed by X-ray diffractometry, thermogravimetry and for their mechanical properties, and showed properties even better than those for the pristine polymer. The release process of the anti-inflammatory molecules was very interesting and promising for tuneable drug delivery. It consists of two stages: a first stage, very rapid as a burst in which a small fraction of the drug is released, and of a second stage that is much slower, extending for longer and longer periods. The parameters influencing the drug release were individuated and discussed. PMID:16848988

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

    PubMed

    Arnette, Christopher; Frye, Keyada; Kaverina, Irina

    2016-01-01

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

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

    PubMed Central

    Arnette, Christopher; Frye, Keyada; Kaverina, Irina

    2016-01-01

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

  5. Bio-mimetic surface engineering of plasmid-loaded nanoparticles for active intracellular trafficking by actin comet-tail motility.

    PubMed

    Ng, Chee Ping; Goodman, Thomas T; Park, In-Kyu; Pun, Suzie H

    2009-02-01

    Intracellular transport after endosomal escape presents one of the major barriers for efficient non-viral gene delivery because plasmid DNA and synthetic nanoparticulate carriers suffer from significantly restricted diffusion in the cytoplasm. We postulate that forces generated by actin polymerization, a mechanism used by several bacterial pathogens such as Listeria monocytogenes, can be harnessed to propel nanoparticles within the cytoplasm and thereby overcome diffusional limitations associated with gene transport in the cell cytoplasm. In this work, we synthesized and characterized plasmid DNA-containing nanoparticles modified with ActA protein, the single protein in L. monocytogenes responsible for activating actin polymerization and initiating actin comet-tail propulsion. The motility of the ActA-modified nanoparticles was assessed in Xenopus laevis cytoplasmic extract supplemented with fluorescently labeled actin. Nanoparticle motility was monitored using multi-color, time-lapse fluorescence microscopy for the formation of actin comet tails attached to the fluorescently labeled vehicle. We observed particle motility with velocities approximately 0.06 microm/s with anionic-charged plasmid carriers formed from either poly(lactic-co-glycolic acid) (PLGA) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, but interestingly not with cationic particles assembled by encapsulation of plasmid with either polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE) lipids. Control particles coated with albumin instead of ActA also showed no motility. Taken together, we have demonstrated the feasibility of translating the comet-tail propulsion mechanism to synthetic drug carriers as a potential approach to overcome intracellular transport barriers, and also have identified appropriate gene delivery systems that can be employed for this mechanism. PMID:19046764

  6. Length regulation of mechanosensitive stereocilia depends on very slow actin dynamics and filament-severing proteins.

    PubMed

    Narayanan, Praveena; Chatterton, Paul; Ikeda, Akihiro; Ikeda, Sakae; Corey, David P; Ervasti, James M; Perrin, Benjamin J

    2015-01-01

    Auditory sensory hair cells depend on stereocilia with precisely regulated lengths to detect sound. Since stereocilia are primarily composed of crosslinked, parallel actin filaments, regulated actin dynamics are essential for controlling stereocilia length. Here we assessed stereocilia actin turnover by monitoring incorporation of inducibly expressed β-actin-GFP in adult mouse hair cells in vivo and by directly measuring β-actin-GFP turnover in explants. Stereocilia actin incorporation is remarkably slow and restricted to filament barbed ends in a small tip compartment, with minimal accumulation in the rest of the actin core. Shorter rows of stereocilia, which have mechanically gated ion channels, show more variable actin turnover than the tallest stereocilia, which lack channels. Finally, the proteins ADF and AIP1, which both mediate actin filament severing, contribute to stereocilia length maintenance. Altogether, the data support a model whereby stereocilia actin cores are largely static, with dynamic regulation at the tips to maintain a critical length. PMID:25897778

  7. The Molecular Evolution of Actin

    PubMed Central

    Hightower, Robin C.; Meagher, Richard B.

    1986-01-01

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

  8. p-Hydroxyphenyl (H) Units Lower the Degree of Polymerization in Lignin: Chemical Control in Lignin Biosynthesis

    SciTech Connect

    Sangha, A. K.; Parks, J. M.; Davis, M. F.; Smith, J. C.

    2013-01-01

    Lignin, composed predominantly of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) subunits, is a major component of plant cell walls that imparts resistance toward chemical and microbial deconstruction of plant biomass, rendering its conversion inefficient and costly. Previous studies have shown that alterating lignin composition, i.e., the relative abundance of H, G and S subunits, promises more efficient extraction of sugars from plant biomass. Smaller and less branched lignin chains are more easily extracted during pretreatment, making cellulose more readily degradable. Here, using density functional theory calculations, we show that the incorporation of H subunits into lignin via b-b and b-5 interunit linkages reduces the degree of polymerization in lignin. Frontier molecular orbital analyses of lignin dimers and trimers show that H as a terminal subunit on a growing lignin polymer linked via b-b and b-5 linkage cannot undergo radical formation, preventing further chain growth by endwise polymerization resulting in lignin polymers with lower degree of polymerization. These results indicate that, for endwise polymerization in lignin synthesis, there exists a chemical control that may lay a significant role in determining the structure of lignin.

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

    PubMed Central

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

    2007-01-01

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

  10. 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

  11. Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts.

    PubMed

    Kacena, Melissa A; Eleniste, Pierre P; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E; Mayo, Lindsey D; Bruzzaniti, Angela

    2012-05-18

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

  12. Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts*

    PubMed Central

    Kacena, Melissa A.; Eleniste, Pierre P.; Cheng, Ying-Hua; Huang, Su; Shivanna, Mahesh; Meijome, Tomas E.; Mayo, Lindsey D.; Bruzzaniti, Angela

    2012-01-01

    The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton. PMID:22447931

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

    SciTech Connect

    Hirano, Hidemi; Matsuura, Yoshiyuki

    2011-10-22

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

  14. 2E4 (Kaptin): A novel actin-associated protein from human blood platelets found in lamellipodia and the tips of the stereocilia of the inner ear

    PubMed Central

    Bearer, Elaine L.; Abraham, Manoj T.

    2010-01-01

    Actin – 2E4/kaptin – platelet activation – stereocilia – sensory epithelium Platelet activation, crucial for hemostasis, requires actin polymerization, yet the molecular mechanisms by which localized actin polymerization is mediated are not clear. Here we report the characterization of a novel actin-binding protein. 2E4, originally isolated from human blood platelets and likely to be involved in the actin rearrangements occurring during activation. 2E4 binds to filamentous (F)-actin by F-actin affinity chromatography and is eluted from F-actin affinity columns and extracted from cells with ATP. Its presence at the leading edge of platelets spread on glass and in the lamellipodia of motile fibroblasts suggests a role in actin dynamics. Using localization to obtain clues about function, we stained the sensory epithelium of the embryonic inner car to determine whether 2E4 is at the barbed end of actin filaments during their elongation. Indeed, 2E4 was present at the tips of the elongating stereocilium. 2E4 is novel by DNA sequence and has no identifiable structural motifs. Its unusual amino acid sequence, its ATP-sensitive actin association and its location at sites of actin polymerization in cells suggest 2E4 plays a unique role in the actin rearrangements that accompany platelet activation and stereocilia formation. PMID:10099934

  15. Molecular mechanisms underlying the force-dependent regulation of actin-to-ECM linkage at the focal adhesions.

    PubMed

    Hirata, Hiroaki; Sokabe, Masahiro; Lim, Chwee Teck

    2014-01-01

    The linkage of the actin cytoskeleton to extracellular matrices (ECMs) at focal adhesions provides a physical path for cells to exert traction forces on substrates during cellular processes such as migration and morphogenesis. Mechanical strength of the actin-to-ECM linkage increases in response to forces loaded at this linkage. This is achieved by local accumulations of actin filaments, as well as linker proteins connecting actins to integrins, at force-bearing adhesion sites, which leads to an increase in the number of molecular bonds between the actin cytoskeleton- and ECM-bound integrins. Zyxin-dependent actin polymerization and filamin-mediated actin bundling are seemingly involved in the force-dependent actin accumulation. Each actin-integrin link is primarily mediated by the linker protein talin, which is strengthened by another linker protein vinculin connecting the actin filaments to talin in a force-dependent manner. This eliminates slippage between the actin cytoskeleton and talin (clutch mechanism), thus playing a crucial role in creating cell membrane protrusions mediated by actin polymerization. Finally, each integrin-ECM bond is also strengthened when a force is loaded on it, which ensures force transmission at focal adhesions, contributing to stable cell-substrate adhesion in cell migration. PMID:25081617

  16. Competition for actin between two distinct F-actin networks defines a bistable switch for cell polarization.

    PubMed

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

    2015-11-01

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

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

    NASA Astrophysics Data System (ADS)

    John, Karin; Peyla, Philippe; Misbah, Chaouqi

    2007-03-01

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

  18. Concentration profiles of actin-binding molecules in lamellipodia

    NASA Astrophysics Data System (ADS)

    Falcke, Martin

    2016-04-01

    Motile cells form lamellipodia in the direction of motion, which are flat membrane protrusions containing an actin filament network. The network flows rearward relative to the leading edge of the lamellipodium due to actin polymerization at the front. Thus, actin binding molecules are subject to transport towards the rear of the cell in the bound state and diffuse freely in the unbound state. We analyze this reaction-diffusion-advection process with respect to the concentration profiles of these species and provide an analytic approximation for them. Network flow may cause a depletion zone of actin binding molecules close to the leading edge. The existence of such zone depends on the free molecule concentration in the cell body, on the ratio of the diffusion length to the distance bound molecules travel rearward with the flow before dissociating, and the ratio of the diffusion length to the width of the region with network flow and actin binding. Our calculations suggest the existence of depletion zones for the F-actin cross-linkers filamin and α-actinin in fish keratocytes (and other cell types), which is in line with the small elastic moduli of the F-actin network close to the leading edge found in measurements of the force motile cells are able to exert.

  19. Cell Motility Resulting form Spontaneous Polymerization Waves

    NASA Astrophysics Data System (ADS)

    Kruse, Karsten

    2014-03-01

    The crawling of living cells on solid substrates is often driven by the actin cytoskeleton, a network of structurally polar filamentous proteins that is intrinsically driven by the hydrolysis of ATP. How cells organize their actin network during crawling is still poorly understood. A possible general mechanism underlying actin organization has been offered by the observation of spontaneous actin polymerization waves in various different cell types. We use a theoretical approach to investigate the possible role of spontaneous actin waves on cell crawling. To this end, we develop a meanfield framework for studying spatiotemporal aspects of actin assembly dynamics, which helped to identify possible origins of self-organized actin waves. The impact of these waves on cell crawling is then investigated by using a phase-field approach to confine the actin network to a cellular domain. We find that spontaneous actin waves can lead to directional or amoeboidal crawling. In the latter case, the cell performs a random walk. Within our deterministic framework, this behavior is due to complex spiral waves inside the cell. Finally, we compare the seemingly random motion of our model cells to the dynamics of cells of the human immune system. These cells patrol the body in search for infected cells and we discuss possible implications of our theory for the search process' efficiency. Work was funded by the DFG through KR3430/1, GK1276, and SFB 1027.

  20. Control of polyaniline deposition on microporous cellulose ester membranes by in situ chemical polymerization.

    PubMed

    Qaiser, Asif A; Hyland, Margaret M; Patterson, Darrell A

    2009-11-12

    Polyaniline (PANI) can be deposited either on the surface or in the bulk of a microporous membrane by various chemical oxidative polymerization techniques. Each technique has distinctive effects on the PANI site and extent of deposition on the base membrane. In the present study, mixed cellulose ester (ME) membranes with tortuous pore morphology were used as base membranes. The chemical oxidative polymerization techniques employed, included polymerization using an in-house-built two-compartment permeation cell. The resultant composite membranes have been characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR-ATR), and electrical conductivity measurements. The results showed that PANI was layered on the pore walls of the membrane using two-compartment permeation cell. Vapor-phase polymerization yielded a surface layer of PANI with little deposition in the bulk. A distorted PANI surface layer was achieved by solution-phase (dip) polymerization. Moreover, asymmetric PANI deposition within the membrane bulk was evidenced using two-compartment permeation cell. Composite membranes synthesized using two-compartment cell showed highest levels of conductivity (approximately 10(-2) S/cm) as compared to the membranes modified by single-step solution-phase polymerization. FTIR-ATR results indicated the extent of PANI coating and its oxidation state which was identified as doped emeraldine PANI, from all the employed techniques. Asymmetric deposition and extent have been explained in terms of the physical and chemical reaction steps involved in the heterogeneous aniline polymerization reactions in the two-compartment cell technique. PMID:19888765

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

    PubMed Central

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

    2009-01-01

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

  2. The Bacterial Actin-Like Cytoskeleton

    PubMed Central

    Carballido-López, Rut

    2006-01-01

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

  3. 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

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

    PubMed Central

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

    2014-01-01

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

  5. Cortical actin regulation modulates vascular contractility and compliance in veins

    PubMed Central

    Saphirstein, Robert J; Gao, Yuan Z; Lin, Qian Qian; Morgan, Kathleen G

    2015-01-01

    Abstract The literature on arterial mechanics is extensive, but far less is known about mechanisms controlling mechanical properties of veins. We use here a multi-scale approach to identify subcellular sources of venous stiffness. Portal vein tissue displays a severalfold decrease in passive stiffness compared to aortic tissues. The α-adrenergic agonist phenylephrine (PE) increased tissue stress and stiffness, both attenuated by cytochalasin D (CytoD) and PP2, inhibitors of actin polymerization and Src activity, respectively. We quantify, for the first time, cortical cellular stiffness in freshly isolated contractile vascular smooth muscle cells using magnetic microneedle technology. Cortical stiffness is significantly increased by PE and CytoD inhibits this increase but, surprisingly, PP2 does not. No detectable change in focal adhesion size, measured by immunofluorescence of FAK and zyxin, accompanies the PE-induced changes in cortical stiffness. Probing with phospho-specific antibodies confirmed activation of FAK/Src and ERK pathways and caldesmon phosphorylation. Thus, venous tissue stiffness is regulated both at the level of the smooth muscle cell cortex, via cortical actin polymerization, and by downstream smooth muscle effectors of Src/ERK signalling pathways. These findings identify novel potential molecular targets for the modulation of venous capacitance and venous return in health and disease. Key points Most cardiovascular research focuses on arterial mechanisms of disease, largely ignoring venous mechanisms. Here we examine ex vivo venous stiffness, spanning tissue to molecular levels, using biomechanics and magnetic microneedle technology, and show for the first time that venous stiffness is regulated by a molecular actin switch within the vascular smooth muscle cell in the wall of the vein. This switch connects the contractile apparatus within the cell to adhesion structures and facilitates stiffening of the vessel wall, regulating blood flow return

  6. The preparation of size-controlled functionalized polymeric nanoparticles in micelles

    NASA Astrophysics Data System (ADS)

    Vakurov, Alexander; Pchelintsev, Nikolay A.; Forde, Jessica; Ó'Fágáin, Ciaran; Gibson, Tim; Millner, Paul

    2009-07-01

    The reverse micellar system of dioctyl-sulfosuccinate (AOT)/octane and toluene have been used as a template for polymerization of acrylamide (AA)/bisacrylamide (BAA)-based functionalized polymeric nanoparticles. Such nanoparticles are typically sized between 20 and 90 nm. They can be synthesized with different functional groups according to the monomers added to the polymerization mixture. In our experiments the nanoparticles carried amino and carboxyl groups following incorporation of allylamine (AAm) or methacrylic acid (MAA) monomers, respectively. The available amine or carboxyl groups can then be used for immobilization of enzymes or other biomolecules. These enzymes, subtilisin, laccase and lipase, were immobilized onto polyAA/BAA/MAA nanoparticles covalently after activating the MAA carboxylic groups with Woodward's K reagent. Non-covalent immobilization via electrostatic interaction was also performed.

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

    PubMed

    DiNubile, M J; Huang, S

    1997-01-01

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

  8. Focal Adhesion Kinase Controls pH-Dependent Epidermal Barrier Homeostasis by Regulating Actin-Directed Na+/H+ Exchanger 1 Plasma Membrane Localization

    PubMed Central

    Ilic, Dusko; Mao-Qiang, Man; Crumrine, Debra; Dolganov, Gregory; Larocque, Nicholas; Xu, Pu; Demerjian, Marianne; Brown, Barbara E.; Lim, Ssang-Taek; Ossovskaya, Valeria; Schlaepfer, David D.; Fisher, Susan J.; Feingold, Kenneth R.; Elias, Peter M.; Mauro, Theodora M.

    2007-01-01

    Ubiquitously expressed focal adhesion kinase (FAK), linked to multiple intracellular signaling pathways, has previously been shown to control cell motility, invasion, proliferation, and survival. Using mice with a keratinocyte-restricted deletion of fak (FAKK5 KO), we report here a novel role for FAK: maintenance of adult epidermal permeability barrier homeostasis. Abundant lacunae of unprocessed lipids in stratum corneum (SC) of FAKK5 KO mice and delayed barrier recovery pointed to malfunction of pH-dependent enzymes active in extracellular space of SC. Measuring the SC pH gradient showed significantly more neutral pH values in FAKK5 KO mice, suggesting the importance of FAK for acidification. Moreover, normal functions were restored when FAKK5 KO mice were exposed to a surface pH typical of mouse SC (pH = 5.5). Baseline levels and response to barrier disruption of secretory phospholipase A2 isoforms, enzymes that mediate generation of free fatty acids in epidermis, appeared similar in both FAKK5 KO and control littermates. We found that the critical SC acidification regulator Na+/H+ exchanger 1 failed to localize to the plasma membrane in FAK-deficient keratinocytes both in vivo and in vitro. Thus, for plasma membrane localization in terminally differentiated keratinocytes, Na+/H+ exchanger 1 requires an intact actin cytoskeleton, which is impaired in FAK-deficient cells. PMID:17525272

  9. Dynamics of Actin Cables in Polarized Growth of the Filamentous Fungus Aspergillus nidulans

    PubMed Central

    Bergs, Anna; Ishitsuka, Yuji; Evangelinos, Minoas; Nienhaus, G. U.; Takeshita, Norio

    2016-01-01

    Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although, specific marker proteins have been developed to visualize actin cables in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here, we observed actin cables using tropomyosin (TpmA) and Lifeact fused to fluorescent proteins in living Aspergillus nidulans hyphae and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules. PMID:27242709

  10. Dynamics of Actin Cables in Polarized Growth of the Filamentous Fungus Aspergillus nidulans.

    PubMed

    Bergs, Anna; Ishitsuka, Yuji; Evangelinos, Minoas; Nienhaus, G U; Takeshita, Norio

    2016-01-01

    Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although, specific marker proteins have been developed to visualize actin cables in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here, we observed actin cables using tropomyosin (TpmA) and Lifeact fused to fluorescent proteins in living Aspergillus nidulans hyphae and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules. PMID:27242709

  11. Myosins, Actin and Autophagy.

    PubMed

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

    2016-08-01

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

  12. Molecular weight control in organochromium olefin polymerization catalysis by hemilabile ligand–metal interactions

    PubMed Central

    Mark, Stefan; Wadepohl, Hubert

    2016-01-01

    Summary A series of Cr(III) complexes based on quinoline-cyclopentadienyl ligands with additional hemilabile side arms were prepared and used as single-site catalyst precursors for ethylene polymerization. The additional donor functions interact with the metal centers only after activation with the co-catalyst. Evidence for this comes from DFT-calculations and from the differing behavior of the complexes in ethylene polymerization. All complexes investigated show very high catalytic activity and the additional side arm minimizes chain-transfer reactions, leading to increase of molecular weights of the resulting polymers. PMID:27559387

  13. Molecular weight control in organochromium olefin polymerization catalysis by hemilabile ligand-metal interactions.

    PubMed

    Mark, Stefan; Wadepohl, Hubert; Enders, Markus

    2016-01-01

    A series of Cr(III) complexes based on quinoline-cyclopentadienyl ligands with additional hemilabile side arms were prepared and used as single-site catalyst precursors for ethylene polymerization. The additional donor functions interact with the metal centers only after activation with the co-catalyst. Evidence for this comes from DFT-calculations and from the differing behavior of the complexes in ethylene polymerization. All complexes investigated show very high catalytic activity and the additional side arm minimizes chain-transfer reactions, leading to increase of molecular weights of the resulting polymers. PMID:27559387

  14. Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models

    PubMed Central

    Schiffer, Mario; Teng, Beina; Gu, Changkyu; Shchedrina, Valentina A.; Kasaikina, Marina; Pham, Vincent A.; Hanke, Nils; Rong, Song; Gueler, Faikah; Schroder, Patricia; Tossidou, Irini; Park, Joon-Keun; Staggs, Lynne; Haller, Hermann; Erschow, Sergej; Hilfiker-Kleiner, Denise; Wei, Changli; Chen, Chuang; Tardi, Nicholas; Hakroush, Samy; Selig, Martin K.; Vasilyev, Aleksandr; Merscher, Sandra; Reiser, Jochen; Sever, Sanja

    2015-01-01

    Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to crosslink actin microfilaments into higher order structures have been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and of CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the significant regenerative potential of injured glomeruli and that targeting the oligomerization cycle of dynamin represents an attractive potential therapeutic target to treat CKD. PMID:25962121

  15. Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models.

    PubMed

    Schiffer, Mario; Teng, Beina; Gu, Changkyu; Shchedrina, Valentina A; Kasaikina, Marina; Pham, Vincent A; Hanke, Nils; Rong, Song; Gueler, Faikah; Schroder, Patricia; Tossidou, Irini; Park, Joon-Keun; Staggs, Lynne; Haller, Hermann; Erschow, Sergej; Hilfiker-Kleiner, Denise; Wei, Changli; Chen, Chuang; Tardi, Nicholas; Hakroush, Samy; Selig, Martin K; Vasilyev, Aleksandr; Merscher, Sandra; Reiser, Jochen; Sever, Sanja

    2015-06-01

    Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to cross-link actin microfilaments into higher-order structures has been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the substantial regenerative potential of injured glomeruli and identifying the oligomerization cycle of dynamin as an attractive potential therapeutic target to treat CKD. PMID:25962121

  16. Evolutionary conservation of physical and functional interactions between phospholipase D and actin.

    PubMed

    Kusner, David J; Barton, James A; Qin, Chunbo; Wang, Xuemin; Iyer, Shankar S

    2003-04-15

    Phospholipase D (PLD) enzymes from bacteria to mammals exhibit a highly conserved core structure and catalytic mechanism, but whether protein-protein interactions exhibit similar commonality is unknown. Our objective was to determine whether the physical and functional interactions of mammalian PLDs with actin are evolutionarily conserved among bacterial and plant PLDs. Highly purified bacterial and plant PLDs cosedimented with mammalian skeletal muscle alpha-actin, indicating direct interaction with F-actin. The binding of bacterial PLD to G-actin exhibited two affinity states, with dissociation constants of 1.13 pM and 0.58 microM. The effects of actin on the activities of bacterial and plant PLDs were polymerization dependent; monomeric G-actin inhibited PLD activity, whereas polymerized F-actin augmented PLD activity. Actin modulation of bacterial and plant PLDs demonstrated kinetic characteristics, efficacies, and potencies similar to those of human PLD1. Thus, physical and functional interactions between PLD and actin in PLD family members from bacteria to mammals are highly conserved throughout evolution. PMID:12667487

  17. Arabidopsis AtADF1 is functionally affected by mutations on actin binding sites.

    PubMed

    Dong, Chun-Hai; Tang, Wei-Ping; Liu, Jia-Yao

    2013-03-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G- and F-actin binding. The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A, R137/A) form another actin binding site that is important for F-actin binding. Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G-actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization. PMID:23190411

  18. Dynamic Localization of G-actin During Membrane Protrusion in Neuronal Motility

    PubMed Central

    Lee, Chi Wai; Vitriol, Eric A.; Shim, Sangwoo; Wise, Ariel L.; Velayutham, Radhi P.; Zheng, James Q.

    2013-01-01

    Summary Background Actin-based cell motility is fundamental for the development, function, and malignant events of eukaryotic organisms. During neural development, axonal growth cones depend on rapid assembly and disassembly of actin filaments (F-actin) for their guided extension to specific targets for wiring. Monomeric globular actin (G-actin) is the building block for F-actin but is not considered to play a direct role in spatiotemporal control of actin dynamics in cell motility. Results Here we report that a pool of G-actin dynamically localizes to the leading edge of growth cones and neuroblastoma cells to spatially elevate the G-/F-actin ratio that drives membrane protrusion and cell movement. Loss of G-actin localization leads to the cessation and retraction of membrane protrusions. Moreover, G-actin localization occurs asymmetrically in growth cones during attractive turning. Finally, we identify the actin monomer binding proteins profilin and thymosin β4 as key molecules that localize actin monomers to the leading edge of lamellipodia for their motility. Conclusions Our results suggest that dynamic localization of G-actin provides a novel mechanism to regulate the spatiotemporal actin dynamics underlying membrane protrusion in cell locomotion and growth cone chemotaxis. PMID:23746641

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

    PubMed

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

    2003-07-01

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

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

    PubMed

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

    2016-07-12

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

  1. Polymerization topochemistry of cellulose nanocrystals: a function of surface dehydration control.

    PubMed

    Tian, Chen; Fu, Shiyu; Habibi, Youssef; Lucia, Lucian A

    2014-12-01

    The activation (dehydration) of cellulose nanocrystals (CNCs) toward surface "brush" polymerization is accomplished either by freeze drying or solvent exchange. However, the question of which one of these protocols to choose over the other is generally open-ended. The current study attempts to shed light o