Sample records for intracellular actin pedestals

  1. Enteropathogenic Escherichia coli Use Redundant Tyrosine Kinases to Form Actin PedestalsD⃞

    PubMed Central

    Swimm, Alyson; Bommarius, Bettina; Li, Yue; Cheng, David; Reeves, Patrick; Sherman, Melanie; Veach, Darren; Bornmann, William; Kalman, Daniel

    2004-01-01

    Enteropathogenic Escherichia coli (EPEC) are deadly contaminants in water and food and induce protrusion of actin-rich membrane pedestals beneath themselves upon attachment to intestinal epithelia. EPEC then causes intestinal inflammation, diarrhea, and, among children, death. Here, we show that EPEC uses multiple tyrosine kinases for formation of pedestals, each of which is sufficient but not necessary. In particular, we show that Abl and Arg, members of the Abl family of tyrosine kinases, localize and are activated in pedestals. We also show that pyrido[2,3-d]pyrimidine (PD) compounds, which inhibit Abl, Arg, and related kinases, block pedestal formation. Finally, we show that Abl and Arg are sufficient for pedestal formation in the absence of other tyrosine kinase activity, but they are not necessary. Our results suggest that additional kinases that are sensitive to inhibition by PD also can suffice. Together, these results suggest that EPEC has evolved a mechanism to use any of several functionally redundant tyrosine kinases during pathogenesis, perhaps facilitating its capacity to infect different cell types. Moreover, PD compounds are being developed to treat cancers caused by dysregulated Abl. Our results raise the possibility that PD may be useful in treating EPEC infections, and because PD affects host and not bacterium, selecting resistant strains may be far less likely than with conventional antibiotics. PMID:15155808

  2. Analysis of Actin-Based Intracellular Trafficking in Pollen Tubes.

    PubMed

    Jiang, Yuxiang; Zhang, Meng; Huang, Shanjin

    2017-01-01

    Underlying rapid and directional pollen tube growth is the active intracellular trafficking system that carries materials necessary for cell wall synthesis and membrane expansion to the expanding point of the pollen tube. The actin cytoskeleton has been shown to control various intracellular trafficking events in the pollen tube, but the underlying cellular and molecular mechanisms remain poorly understood. To better understand how the actin cytoskeleton is involved in the regulation of intracellular trafficking events, we need to establish assays to visualize and quantify the distribution and dynamics of organelles, vesicles, or secreted proteins. In this chapter, we introduce methods regarding the visualization and quantification of the distribution and dynamics of organelles or vesicles in pollen tubes.

  3. Tir Is Essential for the Recruitment of Tks5 to Enteropathogenic Escherichia coli Pedestals.

    PubMed

    Jensen, Helene H; Pedersen, Hans N; Stenkjær, Eva; Pedersen, Gitte A; Login, Frédéric H; Nejsum, Lene N

    2015-01-01

    Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that infects the epithelial lining of the small intestine and causes diarrhea. Upon attachment to the intestinal epithelium, EPEC uses a Type III Secretion System to inject its own high affinity receptor Translocated intimin receptor (Tir) into the host cell. Tir facilitates tight adhesion and recruitment of actin-regulating proteins leading to formation of an actin pedestal beneath the infecting bacterium. The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells. Formation of podosomes is dependent upon the early podosome-specific scavenger protein Tks5, which is involved in actin recruitment. Although Tks5 is expressed in epithelial cells, and podosomes and EPEC pedestals share many components in their structure and mechanism of formation, the potential role of Tks5 in EPEC infections has not been studied. The aim of this study was to determine the subcellular localization of Tks5 in epithelial cells and to investigate if Tks5 is recruited to the EPEC pedestal. In an epithelial MDCK cell line stably expressing Tks5-EGFP, Tks5 localized to actin bundles. Upon infection, EPEC recruited Tks5-EGFP. Tir, but not Tir phosphorylation was essential for the recruitment. Time-lapse microscopy revealed that Tks5-EGFP was recruited instantly upon EPEC attachment to host cells, simultaneously with actin and N-WASp. EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5. Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation.

  4. Tir Is Essential for the Recruitment of Tks5 to Enteropathogenic Escherichia coli Pedestals

    PubMed Central

    Jensen, Helene H.; Pedersen, Hans N.; Stenkjær, Eva; Pedersen, Gitte A.; Login, Frédéric H.; Nejsum, Lene N.

    2015-01-01

    Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that infects the epithelial lining of the small intestine and causes diarrhea. Upon attachment to the intestinal epithelium, EPEC uses a Type III Secretion System to inject its own high affinity receptor Translocated intimin receptor (Tir) into the host cell. Tir facilitates tight adhesion and recruitment of actin-regulating proteins leading to formation of an actin pedestal beneath the infecting bacterium. The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells. Formation of podosomes is dependent upon the early podosome-specific scavenger protein Tks5, which is involved in actin recruitment. Although Tks5 is expressed in epithelial cells, and podosomes and EPEC pedestals share many components in their structure and mechanism of formation, the potential role of Tks5 in EPEC infections has not been studied. The aim of this study was to determine the subcellular localization of Tks5 in epithelial cells and to investigate if Tks5 is recruited to the EPEC pedestal. In an epithelial MDCK cell line stably expressing Tks5-EGFP, Tks5 localized to actin bundles. Upon infection, EPEC recruited Tks5-EGFP. Tir, but not Tir phosphorylation was essential for the recruitment. Time-lapse microscopy revealed that Tks5-EGFP was recruited instantly upon EPEC attachment to host cells, simultaneously with actin and N-WASp. EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5. Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation. PMID:26536015

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 themore » 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.« less

  6. Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells

    PubMed Central

    Swimm, Alyson I; Kalman, Daniel

    2008-01-01

    Enteropathogenic Escherichia coli (EPEC) are deadly contaminants in water and food, and induce protrusion of actin-filled membranous pedestals beneath themselves upon attachment to intestinal epithelia. Pedestal formation requires clustering of Tir and subsequent recruitment of cellular tyrosine kinases including Abl, Arg, and Etk as well as signaling molecules Nck, N-WASP, and Arp2/3 complex. We have developed a cytosolic extract-based cellular system that recapitulates actin pedestal formation in permeabilized red blood cells (RBC) infected with EPEC. RBC support attachment of EPEC and translocation of virulence factors, but not pedestal formation. We show here that extract induces a rapid Ca++-dependent release of Tir from the EPEC Type III secretion system, and that cytoplasmic factor(s) present in the extract facilitate translocation of Tir into the RBC plasma membrane. We show that Abl and related kinases in the extract phosphorylate Tir and that actin polymerization can be reconstituted in infected RBC following addition of cytosolic extract. Reconstitution requires the bacterial virulence factors Tir and intimin, and phosphorylation of Tir on tyrosine residue 474 results in the recruitment of Nck, N-WASP, and Arp2/3 complex beneath attached bacteria at sites of actin polymerization. Together these data describe a biochemical system for dissection of host components that mediate Type III secretion and the mechanisms by which complexes of proteins are recruited to discrete sites within the plasma membrane to initiate localized actin polymerization and morphological changes. PMID:18208322

  7. Active mechanical stabilization of the viscoplastic intracellular space of Dictyostelia cells by microtubule-actin crosstalk.

    PubMed

    Heinrich, Doris; Sackmann, Erich

    2006-11-01

    The micro-viscoelasticity of the intracellular space of Dictyostelium discoideum cells is studied by evaluating the intracellular transport of magnetic force probes and their viscoelastic responses to force pulses of 20-700 pN. The role of the actin cortex, the microtubule (MT) aster and their crosstalk is explored by comparing the behaviour of wild-type cells, myosin II null mutants, latrunculin A and benomyl treated cells. The MT coupled beads perform irregular local and long range directed motions which are characterized by measuring their velocity distributions (P(v)). The correlated motion of the MT and the centrosome are evaluated by microfluorescence of GFP-labelled MTs. P(v) can be represented by log-normal distributions with long tails and it is determined by random sweeping motions (v approximately 0.5 microm/s) of the MTs (caused by tangential forces on the filament ends coupled to the actin cortex) and by intermittent bead transports parallel to the MTs (v(max) approximately 1.5 microm/s). The tails are due to spontaneous filament deflections (with speeds up to 10 microm/s) attributed to pre-stressing of the MT by local cortical tensions, generated by dynactin motors generating plus-end directed forces in the MTs. The viscoelastic responses are strongly non-linear and are mostly directed opposite or perpendicular to the force, showing that the cytoplasm behaves as an active viscoplastic body with time and force dependent drag coefficients. Nano-Newton loads exerted on the soft MT are balanced by traction forces arising at the MT ends coupled to the actin cortex and the centrosome, respectively. The mechanical coupling between the soft microtubules and the viscoelastic actin cortex provides cells with high mechanical stability despite the softness of the cytoplasm.

  8. Pedestal Grinder.

    ERIC Educational Resources Information Center

    Engelbrecht, Nancy; And Others

    These instructional materials provide an orientation to the pedestal grinder for use at the postsecondary level. The first of eight sections defines 14 important terms. The second section outlines 16 rules for safe use of the pedestal grinder. The third section covers grinding wheels for five different types of materials. The fourth section…

  9. Dependence of pedestal properties on plasma parameters

    NASA Astrophysics Data System (ADS)

    Kim, S. K.; Na, Y.-S.; Saarelma, S.; Kwon, O.

    2018-01-01

    We have numerically investigated the dependence of pedestal properties such as the pedestal height and the pedestal width on various global parameters using the EURO-DEMO as the reference equilibrium. We have used EPED, a predictive model of the edge pedestal. Among global parameters, we have chosen to vary the triangularity, δ , the elongation, κ , and the poloidal beta, {{β }p} , which have larger effects on the pedestal properties. Improvement of pedestal properties can be achieved for more shaped plasma boundary. However, the increase in the pedestal height and the width with δ saturates around δ ∼ 0.6. Also, the pedestal width saturates and the pedestal temperature starts to decrease for κ >1.9 . Improvement of the pedestal properties due to δ is larger at higher poloidal beta. The pedestal width slightly increases with the electron density at the pedestal top and the effective charge number.

  10. Microinstabilities in the pedestal region

    NASA Astrophysics Data System (ADS)

    Dickinson, David; Dudson, Benjamin; Wilson, Howard; Roach, Colin

    2014-10-01

    The regulation of transport at the pedestal top is important for the inter-ELM pedestal dynamics. Linear gyrokinetic analysis of the pedestal region during an ELM cycle on MAST has shown kinetic ballooning modes to be unstable at the knee of the pressure profile and in the steep pedestal region whilst microtearing modes (MTMs) dominate in the shallow gradient region inboard of the pedestal top. The transition between these instabilities at the pedestal knee has been observed in low and high collisionality MAST pedestals, and is likely to play an important role in the broadening of the pedestal. Nonlinear simulations are needed in this region to understand the microturbulence, the corresponding transport fluxes, and to gain further insight into the processes underlying the pedestal evolution. Such gyrokinetic simulations are numerically challenging and recent upgrades to the GS2 gyrokinetic code help improve their feasibility. We are also exploring reduced models that capture the relevant physics using the plasma simulation framework BOUT + + . An electromagnetic gyrofluid model has recently been implemented with BOUT + + that has significantly reduced computational cost compared to the gyrokinetic simulations against which it will be benchmarked. This work was funded by the RCUK Energy programme, EURATOM and a EUROFusion fellowship WP14-FRF-CCFE/Dickinson and was carried out using: HELIOS at IFERC, Japan; ARCHER (EPSRC Grant No. EP/L000237/1); HECToR (EPSRC Grant No. EP/H002081/1).

  11. Multiscale modelling for tokamak pedestals

    NASA Astrophysics Data System (ADS)

    Abel, I. G.

    2018-04-01

    Pedestal modelling is crucial to predict the performance of future fusion devices. Current modelling efforts suffer either from a lack of kinetic physics, or an excess of computational complexity. To ameliorate these problems, we take a first-principles multiscale approach to the pedestal. We will present three separate sets of equations, covering the dynamics of edge localised modes (ELMs), the inter-ELM pedestal and pedestal turbulence, respectively. Precisely how these equations should be coupled to each other is covered in detail. This framework is completely self-consistent; it is derived from first principles by means of an asymptotic expansion of the fundamental Vlasov-Landau-Maxwell system in appropriate small parameters. The derivation exploits the narrowness of the pedestal region, the smallness of the thermal gyroradius and the low plasma (the ratio of thermal to magnetic pressures) typical of current pedestal operation to achieve its simplifications. The relationship between this framework and gyrokinetics is analysed, and possibilities to directly match our systems of equations onto multiscale gyrokinetics are explored. A detailed comparison between our model and other models in the literature is performed. Finally, the potential for matching this framework onto an open-field-line region is briefly discussed.

  12. A new F-actin structure in fungi: actin ring formation around the cell nucleus of Cryptococcus neoformans.

    PubMed

    Kopecká, Marie; Kawamoto, Susumu; Yamaguchi, Masashi

    2013-04-01

    The F-actin cytoskeleton of Cryptococcus neoformans is known to comprise actin cables, cortical patches and cytokinetic ring. Here, we describe a new F-actin structure in fungi, a perinuclear F-actin collar ring around the cell nucleus, by fluorescent microscopic imaging of rhodamine phalloidin-stained F-actin. Perinuclear F-actin rings form in Cryptococcus neoformans treated with the microtubule inhibitor Nocodazole or with the drug solvent dimethyl sulfoxide (DMSO) or grown in yeast extract peptone dextrose (YEPD) medium, but they are absent in cells treated with Latrunculin A. Perinuclear F-actin rings may function as 'funicular cabin' for the cell nucleus, and actin cables as intracellular 'funicular' suspending nucleus in the central position in the cell and moving nucleus along the polarity axis along actin cables.

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

    NASA Astrophysics Data System (ADS)

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

    1990-05-01

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

  14. Concrete pedestals for high-performance semiconductor production equipment

    NASA Astrophysics Data System (ADS)

    Vogen, Wayne; Franklin, Craig L.; Morneault, Joseph

    1999-09-01

    Concrete pedestals have many vibration and stiffness characteristics that make them a superior choice for sensitive semiconductor production equipment including scanners, scanning electron microscopes, focused ion beam millers and optical inspection equipment. Among the advantages of concrete pedestals are high inherent damping, monolithic construction that eliminates low stiffness joints common in steep pedestals, ability to reuse and ease of installation. Steel pedestals that have plates attached to the top of the frame are easily excited by acoustic excitation, especially in the range from 50 Hertz to 400 Hertz. Concrete pedestals do not suffer from this phenomenon because of the high mass and damping of the top surface.

  15. Pedestal substrate for coated optics

    DOEpatents

    Hale, Layton C.; Malsbury, Terry N.; Patterson, Steven R.

    2001-01-01

    A pedestal optical substrate that simultaneously provides high substrate dynamic stiffness, provides low surface figure sensitivity to mechanical mounting hardware inputs, and constrains surface figure changes caused by optical coatings to be primarily spherical in nature. The pedestal optical substrate includes a disk-like optic or substrate section having a top surface that is coated, a disk-like base section that provides location at which the substrate can be mounted, and a connecting cylindrical section between the base and optics or substrate sections. The connecting cylindrical section may be attached via three spaced legs or members. However, the pedestal optical substrate can be manufactured from a solid piece of material to form a monolith, thus avoiding joints between the sections, or the disk-like base can be formed separately and connected to the connecting section. By way of example, the pedestal optical substrate may be utilized in the fabrication of optics for an extreme ultraviolet (EUV) lithography imaging system, or in any optical system requiring coated optics and substrates with reduced sensitivity to mechanical mounts.

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

    PubMed Central

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

    2009-01-01

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

  17. ELM Suppression and Pedestal Structure in I-Mode Plasmas

    NASA Astrophysics Data System (ADS)

    Walk, John

    2013-10-01

    The I-mode regime is characterized by the formation of a temperature pedestal and enhanced energy confinement (H98 up to 1.2), without an accompanying density pedestal or drop in particle transport. Unlike ELMy H-modes, I-mode operation appears to have naturally-occurring suppression of large ELMs in addition to its highly favorable scalings of pedestal structure (and therefore overall performance). Instead, continuous Weakly Coherent Modes help to regulate density. Extensive study of the ELMy H-mode has led to the development of the EPED model, which utilizes calculations of coupled peeling-ballooning MHD modes and kinetic-ballooning mode (KBM) stability limits to predict the pedestal structure preceding an ELM crash. We apply similar tools to the structure and ELM stability of I-mode pedestals. Peeling-ballooning MHD calculations are completed using the ELITE code, showing I-mode pedestals to be generally MHD-stable. Under certain conditions, intermittent ELMs are observed in I-mode at reduced field, typically triggered by sawtooth crashes; modification of the temperature pedestal (and therefore the pressure profile stability) by sawtooth heat pulses is being examined in ELITE. Modeled stability to KBM turbulence in I-mode and ELMy H-mode suggests that typical I-modes are stable against KBM turbulence. Measured I-mode pedestals are significantly wider (more stable) than the width scaling with the square root of poloidal beta characteristic of the KBM-limited pedestals in ELMy H-mode. Finally, we explore scalings of pedestal structure with engineering parameters compared to ELMy H-modes on C-Mod. In particular, we focus on scalings of the pressure pedestal with heating power (and its relation to the favorable scaling of confinement with power in I-mode) and on relationships between heat flux and pedestal temperature gradients. This work is supported by DOE agreement DE-FC02-99ER54512. Theory work at General Atomics is supported by DOE agreement DE-FG02-99ER54309.

  18. The cytoskeletal scaffold Shank3 is recruited to pathogen-induced actin rearrangements

    PubMed Central

    Huett, Alan; Leong, John M; Podolsky, Daniel K.; Xavier, Ramnik J.

    2009-01-01

    Summary The common gastrointestinal pathogens enteropathogenic Escherichia coli (EPEC) and Salmonella Typhimurium both reorganize the gut epithelial cell actin cytoskeleton to mediate pathogenesis, utilizing mimicry of the host signaling apparatus. The PDZ domain-containing protein Shank3, is a large cytoskeletal scaffold protein with known functions in neuronal morphology and synaptic signaling, and is also capable of acting as a scaffolding adaptor during Ret tyrosine kinase signaling in epithelial cells. Using immunofluorescent and functional RNA-interference approaches we show that Shank3 is present in both EPEC- and S. Typhimurium-induced actin rearrangements and is required for optimal EPEC pedestal formation. We propose that Shank3 is one of a number of host synaptic proteins likely to play key roles in bacteria-host interactions. PMID:19371741

  19. Studies of the pedestal structure and inter-ELM pedestal evolution in JET with the ITER-like wall

    NASA Astrophysics Data System (ADS)

    Maggi, C. F.; Frassinetti, L.; Horvath, L.; Lunniss, A.; Saarelma, S.; Wilson, H.; Flanagan, J.; Leyland, M.; Lupelli, I.; Pamela, S.; Urano, H.; Garzotti, L.; Lerche, E.; Nunes, I.; Rimini, F.; Contributors, JET

    2017-11-01

    The pedestal structure of type I ELMy H-modes has been analysed for JET with the ITER-like Wall (JET-ILW). The electron pressure pedestal width is independent of ρ * and increases proportionally to  √β pol,PED. Additional broadening of the width is observed, at constant β pol, PED, with increasing ν * and/or neutral gas injection and the contribution of atomic physics effects in setting the pedestal width cannot as yet be ruled out. Neutral penetration alone does not determine the shape of the edge density profile in JET-ILW. The ratio of electron density to electron temperature scale lengths in the edge transport barrier region, η e, is of order 2-3 within experimental uncertainties. Existing understanding, represented in the stationary linear peeling-ballooning mode stability and the EPED pedestal structure models, is extended to the dynamic evolution between ELM crashes in JET-ILW, in order to test the assumptions underlying these two models. The inter-ELM temporal evolution of the pedestal structure in JET-ILW is not unique, but depends on discharge conditions, such as heating power and gas injection levels. The strong reduction in p e,PED with increasing D 2 gas injection at high power is primarily due to clamping of \

  20. SOL effects on the pedestal structure in DIII-D discharges

    DOE PAGES

    Sontag, Aaron C.; Chen, Xi; Canik, John; ...

    2017-05-24

    SOLPS analysis explains the differences in pedestal structure associated with different ion ∇B drift directions in DIII-D. Core transport models predict that fusion power scales roughly as the square of the pressure at the top of the pedestal, so understanding the effects that determine pedestal structure in steady-state operational scenarios is important to projecting scenarios developed in DIII-D to ITER and other devices. Both experiments and modeling indicate that scrape off layer (SOL) conditions are important in optimizing the pedestal structure for high-beta steady-state scenarios. The SOLPS code is used to provide interpretive analysis of the pedestal and SOL tomore » examine the nature of flows and fueling on the pedestal structure including the effects of drifts in the fluid model. This analysis shows that flows driven by the ion ∇B drift are outward when this drift is toward the x-point in a single-null divertor configuration (favorable ∇B direction for reduced H-mode power threshold), and inward when the drift is away from the x-point (unfavorable ∇B direction). It is hypothesized that these flows decrease the density gradient in the pedestal in the favorable direction, thereby stabilizing the kinetic ballooning mode (KBM) and increasing the pedestal width. Comparisons of pedestal structures in similarly shaped DIII-D steady-state plasmas confirm this change, showing increased density pedestal width and lower peak density and lower separatrix density with the favorable drift direction. The pedestal temperature is higher in the lower density case, resulting in an increased pedestal pressure, which indicates that the increased particle flux does not significantly degrade energy confinement. Modeling of cases with constant ∇B drift direction but changing between the more open lower divertor and more closed upper divertor show that there is increased fueling inside the pedestal with the more open geometry. As a result, the pedestal fueling rate

  1. SOL effects on the pedestal structure in DIII-D discharges

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sontag, Aaron C.; Chen, Xi; Canik, John

    SOLPS analysis explains the differences in pedestal structure associated with different ion ∇B drift directions in DIII-D. Core transport models predict that fusion power scales roughly as the square of the pressure at the top of the pedestal, so understanding the effects that determine pedestal structure in steady-state operational scenarios is important to projecting scenarios developed in DIII-D to ITER and other devices. Both experiments and modeling indicate that scrape off layer (SOL) conditions are important in optimizing the pedestal structure for high-beta steady-state scenarios. The SOLPS code is used to provide interpretive analysis of the pedestal and SOL tomore » examine the nature of flows and fueling on the pedestal structure including the effects of drifts in the fluid model. This analysis shows that flows driven by the ion ∇B drift are outward when this drift is toward the x-point in a single-null divertor configuration (favorable ∇B direction for reduced H-mode power threshold), and inward when the drift is away from the x-point (unfavorable ∇B direction). It is hypothesized that these flows decrease the density gradient in the pedestal in the favorable direction, thereby stabilizing the kinetic ballooning mode (KBM) and increasing the pedestal width. Comparisons of pedestal structures in similarly shaped DIII-D steady-state plasmas confirm this change, showing increased density pedestal width and lower peak density and lower separatrix density with the favorable drift direction. The pedestal temperature is higher in the lower density case, resulting in an increased pedestal pressure, which indicates that the increased particle flux does not significantly degrade energy confinement. Modeling of cases with constant ∇B drift direction but changing between the more open lower divertor and more closed upper divertor show that there is increased fueling inside the pedestal with the more open geometry. As a result, the pedestal fueling rate

  2. Pedestal Crater and Yardangs

    NASA Technical Reports Server (NTRS)

    2003-01-01

    MGS MOC Release No. MOC2-444, 6 August 2003

    This April 2003 Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a small meteor impact crater that has been modified by wind erosion. Two things happened after the crater formed. First, the upper few meters of surface material into which the meteor impacted was later eroded away by wind. The crater ejecta formed a protective armor that kept the material under the ejecta from been blown away. This caused the crater and ejecta to appear as if standing upon a raised platform--a feature that Mars geologists call a pedestal crater. Next, the pedestal crater was buried beneath several meters of new sediment, and then this material was eroded away by wind to form the array of sharp ridges that run across the pedestal crater's surface. These small ridges are known as yardangs. This picture is illuminated by sunlight from the upper left; it is located in west Daedalia Planum near 14.6oS, 131.9oW.

  3. Effect of the relative shift between the electron density and temperature pedestal position on the pedestal stability in JET-ILW and comparison with JET-C

    NASA Astrophysics Data System (ADS)

    Stefanikova, E.; Frassinetti, L.; Saarelma, S.; Loarte, A.; Nunes, I.; Garzotti, L.; Lomas, P.; Rimini, F.; Drewelow, P.; Kruezi, U.; Lomanowski, B.; de la Luna, E.; Meneses, L.; Peterka, M.; Viola, B.; Giroud, C.; Maggi, C.; contributors, JET

    2018-05-01

    The electron temperature and density pedestals tend to vary in their relative radial positions, as observed in DIII-D (Beurskens et al 2011 Phys. Plasmas 18 056120) and ASDEX Upgrade (Dunne et al 2017 Plasma Phys. Control. Fusion 59 14017). This so-called relative shift has an impact on the pedestal magnetohydrodynamic (MHD) stability and hence on the pedestal height (Osborne et al 2015 Nucl. Fusion 55 063018). The present work studies the effect of the relative shift on pedestal stability of JET ITER-like wall (JET-ILW) baseline low triangularity (δ) unseeded plasmas, and similar JET-C discharges. As shown in this paper, the increase of the pedestal relative shift is correlated with the reduction of the normalized pressure gradient, therefore playing a strong role in pedestal stability. Furthermore, JET-ILW tends to have a larger relative shift compared to JET carbon wall (JET-C), suggesting a possible role of the plasma facing materials in affecting the density profile location. Experimental results are then compared with stability analysis performed in terms of the peeling-ballooning model and with pedestal predictive model EUROPED (Saarelma et al 2017 Plasma Phys. Control. Fusion). Stability analysis is consistent with the experimental findings, showing an improvement of the pedestal stability, when the relative shift is reduced. This has been ascribed mainly to the increase of the edge bootstrap current, and to minor effects related to the increase of the pedestal pressure gradient and narrowing of the pedestal pressure width. Pedestal predictive model EUROPED shows a qualitative agreement with experiment, especially for low values of the relative shift.

  4. Edge-localized mode avoidance and pedestal structure in I-mode plasmasa)

    NASA Astrophysics Data System (ADS)

    Walk, J. R.; Hughes, J. W.; Hubbard, A. E.; Terry, J. L.; Whyte, D. G.; White, A. E.; Baek, S. G.; Reinke, M. L.; Theiler, C.; Churchill, R. M.; Rice, J. E.; Snyder, P. B.; Osborne, T.; Dominguez, A.; Cziegler, I.

    2014-05-01

    I-mode is a high-performance tokamak regime characterized by the formation of a temperature pedestal and enhanced energy confinement, without an accompanying density pedestal or drop in particle and impurity transport. I-mode operation appears to have naturally occurring suppression of large Edge-Localized Modes (ELMs) in addition to its highly favorable scalings of pedestal structure and overall performance. Extensive study of the ELMy H-mode has led to the development of the EPED model, which utilizes calculations of coupled peeling-ballooning MHD modes and kinetic-ballooning mode (KBM) stability limits to predict the pedestal structure preceding an ELM crash. We apply similar tools to the structure and ELM stability of I-mode pedestals. Analysis of I-mode discharges prepared with high-resolution pedestal data from the most recent C-Mod campaign reveals favorable pedestal scalings for extrapolation to large machines—pedestal temperature scales strongly with power per particle Pnet/n ¯e, and likewise pedestal pressure scales as the net heating power (consistent with weak degradation of confinement with heating power). Matched discharges in current, field, and shaping demonstrate the decoupling of energy and particle transport in I-mode, increasing fueling to span nearly a factor of two in density while maintaining matched temperature pedestals with consistent levels of Pnet/n ¯e. This is consistent with targets for increased performance in I-mode, elevating pedestal βp and global performance with matched increases in density and heating power. MHD calculations using the ELITE code indicate that I-mode pedestals are strongly stable to edge peeling-ballooning instabilities. Likewise, numerical modeling of the KBM turbulence onset, as well as scalings of the pedestal width with poloidal beta, indicates that I-mode pedestals are not limited by KBM turbulence—both features identified with the trigger for large ELMs, consistent with the observed suppression of

  5. Spatial Cytoskeleton Organization Supports Targeted Intracellular Transport

    NASA Astrophysics Data System (ADS)

    Hafner, Anne E.; Rieger, Heiko

    2018-03-01

    The efficiency of intracellular cargo transport from specific source to target locations is strongly dependent upon molecular motor-assisted motion along the cytoskeleton. Radial transport along microtubules and lateral transport along the filaments of the actin cortex underneath the cell membrane are characteristic for cells with a centrosome. The interplay between the specific cytoskeleton organization and the motor performance realizes a spatially inhomogeneous intermittent search strategy. In order to analyze the efficiency of such intracellular search strategies we formulate a random velocity model with intermittent arrest states. We evaluate efficiency in terms of mean first passage times for three different, frequently encountered intracellular transport tasks: i) the narrow escape problem, which emerges during cargo transport to a synapse or other specific region of the cell membrane, ii) the reaction problem, which considers the binding time of two particles within the cell, and iii) the reaction-escape problem, which arises when cargo must be released at a synapse only after pairing with another particle. Our results indicate that cells are able to realize efficient search strategies for various intracellular transport tasks economically through a spatial cytoskeleton organization that involves only a narrow actin cortex rather than a cell body filled with randomly oriented actin filaments.

  6. Integrity of the actin cytoskeleton of host macrophages is essential for Leishmania donovani infection.

    PubMed

    Roy, Saptarshi; Kumar, G Aditya; Jafurulla, Md; Mandal, Chitra; Chattopadhyay, Amitabha

    2014-08-01

    Visceral leishmaniasis is a vector-borne disease caused by an obligate intracellular protozoan parasite Leishmania donovani. The molecular mechanism involved in internalization of Leishmania is poorly understood. The entry of Leishmania involves interaction with the plasma membrane of host cells. We have previously demonstrated the requirement of host membrane cholesterol in the binding and internalization of L. donovani into macrophages. In the present work, we explored the role of the host actin cytoskeleton in leishmanial infection. We observed a dose-dependent reduction in the attachment of Leishmania promastigotes to host macrophages upon destabilization of the actin cytoskeleton by cytochalasin D. This is accompanied by a concomitant reduction in the intracellular amastigote load. We utilized a recently developed high resolution microscopy-based method to quantitate cellular F-actin content upon treatment with cytochalasin D. A striking feature of our results is that binding of Leishmania promastigotes and intracellular amastigote load show close correlation with cellular F-actin level. Importantly, the binding of Escherichia coli remained invariant upon actin destabilization of host cells, thereby implying specific involvement of the actin cytoskeleton in Leishmania infection. To the best of our knowledge, these novel results constitute the first comprehensive demonstration on the specific role of the host actin cytoskeleton in Leishmania infection. Our results could be significant in developing future therapeutic strategies to tackle leishmaniasis. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Edge-localized mode avoidance and pedestal structure in I-mode plasmas

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Walk, J. R., E-mail: jrwalk@psfc.mit.edu; Hughes, J. W.; Hubbard, A. E.

    I-mode is a high-performance tokamak regime characterized by the formation of a temperature pedestal and enhanced energy confinement, without an accompanying density pedestal or drop in particle and impurity transport. I-mode operation appears to have naturally occurring suppression of large Edge-Localized Modes (ELMs) in addition to its highly favorable scalings of pedestal structure and overall performance. Extensive study of the ELMy H-mode has led to the development of the EPED model, which utilizes calculations of coupled peeling-ballooning MHD modes and kinetic-ballooning mode (KBM) stability limits to predict the pedestal structure preceding an ELM crash. We apply similar tools to themore » structure and ELM stability of I-mode pedestals. Analysis of I-mode discharges prepared with high-resolution pedestal data from the most recent C-Mod campaign reveals favorable pedestal scalings for extrapolation to large machines—pedestal temperature scales strongly with power per particle P{sub net}/n{sup ¯}{sub e}, and likewise pedestal pressure scales as the net heating power (consistent with weak degradation of confinement with heating power). Matched discharges in current, field, and shaping demonstrate the decoupling of energy and particle transport in I-mode, increasing fueling to span nearly a factor of two in density while maintaining matched temperature pedestals with consistent levels of P{sub net}/n{sup ¯}{sub e}. This is consistent with targets for increased performance in I-mode, elevating pedestal β{sub p} and global performance with matched increases in density and heating power. MHD calculations using the ELITE code indicate that I-mode pedestals are strongly stable to edge peeling-ballooning instabilities. Likewise, numerical modeling of the KBM turbulence onset, as well as scalings of the pedestal width with poloidal beta, indicates that I-mode pedestals are not limited by KBM turbulence—both features identified with the trigger for large ELMs

  8. Comparative genome analysis reveals a conserved family of actin-like proteins in apicomplexan parasites

    PubMed Central

    Gordon, Jennifer L; Sibley, L David

    2005-01-01

    Background The phylum Apicomplexa is an early-branching eukaryotic lineage that contains a number of important human and animal pathogens. Their complex life cycles and unique cytoskeletal features distinguish them from other model eukaryotes. Apicomplexans rely on actin-based motility for cell invasion, yet the regulation of this system remains largely unknown. Consequently, we focused our efforts on identifying actin-related proteins in the recently completed genomes of Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., and Theileria spp. Results Comparative genomic and phylogenetic studies of apicomplexan genomes reveals that most contain only a single conventional actin and yet they each have 8–10 additional actin-related proteins. Among these are a highly conserved Arp1 protein (likely part of a conserved dynactin complex), and Arp4 and Arp6 homologues (subunits of the chromatin-remodeling machinery). In contrast, apicomplexans lack canonical Arp2 or Arp3 proteins, suggesting they lost the Arp2/3 actin polymerization complex on their evolutionary path towards intracellular parasitism. Seven of these actin-like proteins (ALPs) are novel to apicomplexans. They show no phylogenetic associations to the known Arp groups and likely serve functions specific to this important group of intracellular parasites. Conclusion The large diversity of actin-like proteins in apicomplexans suggests that the actin protein family has diverged to fulfill various roles in the unique biology of intracellular parasites. Conserved Arps likely participate in vesicular transport and gene expression, while apicomplexan-specific ALPs may control unique biological traits such as actin-based gliding motility. PMID:16343347

  9. A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

    DOE PAGES

    Liu, Yao; Zhu, Wenhan; Tan, Yunhao; ...

    2017-01-27

    Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H 95E XXH 99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cellmore » rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Furthermore, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.« less

  10. Predictive modeling of pedestal structure in KSTAR using EPED model

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Han, Hyunsun; Kim, J. Y.; Kwon, Ohjin

    2013-10-15

    A predictive calculation is given for the structure of edge pedestal in the H-mode plasma of the KSTAR (Korea Superconducting Tokamak Advanced Research) device using the EPED model. Particularly, the dependence of pedestal width and height on various plasma parameters is studied in detail. The two codes, ELITE and HELENA, are utilized for the stability analysis of the peeling-ballooning and kinetic ballooning modes, respectively. Summarizing the main results, the pedestal slope and height have a strong dependence on plasma current, rapidly increasing with it, while the pedestal width is almost independent of it. The plasma density or collisionality gives initiallymore » a mild stabilization, increasing the pedestal slope and height, but above some threshold value its effect turns to a destabilization, reducing the pedestal width and height. Among several plasma shape parameters, the triangularity gives the most dominant effect, rapidly increasing the pedestal width and height, while the effect of elongation and squareness appears to be relatively weak. Implication of these edge results, particularly in relation to the global plasma performance, is discussed.« less

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

    PubMed

    Appaduray, Mark A; Masedunskas, Andrius; Bryce, Nicole S; Lucas, Christine A; Warren, Sean C; Timpson, Paul; Stear, Jeffrey H; Gunning, Peter W; Hardeman, Edna C

    2016-01-01

    The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.

  12. Global performance enhancements via pedestal optimisation on ASDEX Upgrade

    NASA Astrophysics Data System (ADS)

    Dunne, M. G.; Frassinetti, L.; Beurskens, M. N. A.; Cavedon, M.; Fietz, S.; Fischer, R.; Giannone, L.; Huijsmans, G. T. A.; Kurzan, B.; Laggner, F.; McCarthy, P. J.; McDermott, R. M.; Tardini, G.; Viezzer, E.; Willensdorfer, M.; Wolfrum, E.; The EUROfusion MST1 Team; The ASDEX Upgrade Team

    2017-02-01

    Results of experimental scans of heating power, plasma shape, and nitrogen content are presented, with a focus on global performance and pedestal alteration. In detailed scans at low triangularity, it is shown that the increase in stored energy due to nitrogen seeding stems from the pedestal. It is also shown that the confinement increase is driven through the temperature pedestal at the three heating power levels studied. In a triangularity scan, an orthogonal effect of shaping and seeding is observed, where increased plasma triangularity increases the pedestal density, while impurity seeding (carbon and nitrogen) increases the pedestal temperature in addition to this effect. Modelling of these effects was also undertaken, with interpretive and predictive models being employed. The interpretive analysis shows a general agreement of the experimental pedestals in separate power, shaping, and seeding scans with peeling-ballooning theory. Predictive analysis was used to isolate the individual effects, showing that the trends of additional heating power and increased triangularity can be recoverd. However, a simple change of the effective charge in the plasma cannot explain the observed levels of confinement improvement in the present models.

  13. The Research of EAST Pedestal Structure and Preliminary Application

    NASA Astrophysics Data System (ADS)

    Wang, Tengfei; Zang, Qing; Han, Xiaofeng; Xiao, Shumei; Hu, Ailan; Zhao, Junyu

    2016-10-01

    The pedestal characteristic is an important basis for high confinement mode (H-mode) research. Because of the finite spatial resolution of Thomson scattering (TS) diagnostic on Experimental Advanced Superconducting Tokamak (EAST), it is necessary to characterize the pedestal with a suitable functional form. Based on simulated and experimental data of EAST, it is shown that the two-line method with a bilinear fitting has better reproducibility of pedestal parameters than hyperbolic tangent (tanh) and modified hyperbolic tangent (mtanh) methods. This method has been applied to EAST type I edge localized mode (ELM) discharges, and the electron pedestal density is found to be proportional to the line-averaged density and the edge pressure gradient is found to be proportional to the pedestal pressure. Furthermore, the ion poloidal gyro-radius has been identified as the suitable parameter to describe the pedestal pressure width. supported by National Natural Science Foundation of China (Nos. 11275233 and 11405206), and the National Magnetic Confinement Fusion Science Program of China (No. 2013GB112003), and Science Foundation of Institute of Plasma Physics, Chinese Academy of Sciences (No. DSJJ-15-JC01)

  14. Drosophila Spire is an actin nucleation factor.

    PubMed

    Quinlan, Margot E; Heuser, John E; Kerkhoff, Eugen; Mullins, R Dyche

    2005-01-27

    The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factors--the Arp2/3 complex and the formin family of proteins--that nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.

  15. 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

  16. Spatial control of actin polymerization during neutrophil chemotaxis.

    PubMed

    Weiner, O D; Servant, G; Welch, M D; Mitchison, T J; Sedat, J W; Bourne, H R

    1999-06-01

    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.

  17. Alteration of the Cortical Actin Cytoskeleton Deregulates Ca2+ Signaling, Monospermic Fertilization, and Sperm Entry

    PubMed Central

    Puppo, A.; Chun, Jong T.; Gragnaniello, Giovanni; Garante, Ezio; Santella, Luigia

    2008-01-01

    Background When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca2+) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear. Methodology/Principal Findings We have measured changes in intracellular Ca2+ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP3 receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton. Conclusions/Significance Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca2+ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy. PMID:18974786

  18. Physics of the Tokamak Pedestal, and Implications for Magnetic Fusion Energy

    NASA Astrophysics Data System (ADS)

    Snyder, Philip

    2017-10-01

    High performance in tokamaks is achieved via the spontaneous formation of a transport barrier in the outer few percent of the confined plasma. This narrow insulating layer, referred to as a ``pedestal,'' typically results in a >30x increase in pressure across a 0.4-5cm layer. Predicted fusion power scales with the square of the pedestal top pressure (or ``pedestal height''), hence a fusion reactor strongly benefits from a high pedestal, provided this can be attained without large Edge Localized Modes (ELMs), which may erode plasma facing materials. The overlap of drift orbit, turbulence, and equilibrium scales across this narrow layer leads to rich and complex physics, and challenges traditional analytic and computational approaches. We review studies employing gyrokinetic, neoclassical, MHD, and other methods, which have explored how a range of instabilities, influenced by complex geometry, and strong ExB flows and bootstrap current, drive transport across the pedestal and guide its structure and dynamics. Development of high resolution diagnostics, and coordinated experiments on several tokamaks, have validated understanding of important aspects of the physics, while highlighting open issues. A predictive model (EPED) has proven capable of predicting the pedestal height and width to 20-25% accuracy in large statistical studies. This model was used to predict a new, high pedestal ``Super H-Mode'' regime, which was subsequently discovered on DIII-D, and motivated experiments on Alcator C-Mod which achieved world record, reactor relevant pedestal pressure. We review open issues including improved formalism, particle and momentum transport, the role of neutrals and impurities, ELM control, and pedestal formation. Finally we discuss coupling pedestal and core predictive models to enable more comprehensive optimization of the tokamak fusion concept. Supported by the US DOE under DE-FG02-95ER54309, FC02-06ER54873, DE-FC02-04ER54698, DE-FC02-99ER54512.

  19. Bacterial Actins.

    PubMed

    Izoré, Thierry; van den Ent, Fusinita

    2017-01-01

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

  20. Dynamics of actin-based movement by Rickettsia rickettsii in vero cells.

    PubMed

    Heinzen, R A; Grieshaber, S S; Van Kirk, L S; Devin, C J

    1999-08-01

    Actin-based motility (ABM) is a virulence mechanism exploited by invasive bacterial pathogens in the genera Listeria, Shigella, and Rickettsia. Due to experimental constraints imposed by the lack of genetic tools and their obligate intracellular nature, little is known about rickettsial ABM relative to Listeria and Shigella ABM systems. In this study, we directly compared the dynamics and behavior of ABM of Rickettsia rickettsii and Listeria monocytogenes. A time-lapse video of moving intracellular bacteria was obtained by laser-scanning confocal microscopy of infected Vero cells synthesizing beta-actin coupled to green fluorescent protein (GFP). Analysis of time-lapse images demonstrated that R. rickettsii organisms move through the cell cytoplasm at an average rate of 4.8 +/- 0.6 micrometer/min (mean +/- standard deviation). This speed was 2.5 times slower than that of L. monocytogenes, which moved at an average rate of 12.0 +/- 3.1 micrometers/min. Although rickettsiae moved more slowly, the actin filaments comprising the actin comet tail were significantly more stable, with an average half-life approximately three times that of L. monocytogenes (100.6 +/- 19.2 s versus 33.0 +/- 7.6 s, respectively). The actin tail associated with intracytoplasmic rickettsiae remained stationary in the cytoplasm as the organism moved forward. In contrast, actin tails of rickettsiae trapped within the nucleus displayed dramatic movements. The observed phenotypic differences between the ABM of Listeria and Rickettsia may indicate fundamental differences in the mechanisms of actin recruitment and polymerization.

  1. Electrostatics Control Actin Filament Nucleation and Elongation Kinetics*

    PubMed Central

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

    2013-01-01

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

  2. Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

    PubMed Central

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R.; Bryce, Nicole S.; Whan, Renee M.; Hardeman, Edna C.

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments. PMID:25978408

  3. A gyrokinetic perspective on the JET-ILW pedestal

    NASA Astrophysics Data System (ADS)

    Hatch, D. R.; Kotschenreuther, M.; Mahajan, S.; Valanju, P.; Liu, X.

    2017-03-01

    JET has been unable to recover historical confinement levels when operating with an ITER-like wall (ILW) due largely to the inaccessibility of high pedestal temperatures. Finding a path to overcome this challenge is of utmost importance for both a prospective JET DT campaign and for future ITER operation. Gyrokinetic simulations (using the Gene code) quantitatively capture experimental transport levels for a representative experimental discharge and qualitatively recover the major experimental trends. Microtearing turbulence is a major transport mechanisms for the low-temperature pedestals characteristic of unseeded JET-ILW discharges. At higher temperatures and/or lower {ρ\\ast} , we identify electrostatic ITG transport of a type that is strongly shear-suppressed on smaller machines. Consistent with observations, this transport mechanism is strongly reduced by the presence of a low-Z impurity (e.g. carbon or nitrogen at the level of {{Z}\\text{eff}}∼ 2 ), recovering the accessibility of high pedestal temperatures. Notably, simulations based on dimensionless {ρ\\ast} scans recover historical scaling behavior except in the unique JET-ILW parameter regime where ITG turbulence becomes important. Our simulations also elucidate the observed degradation of confinement caused by gas puffing, emphasizing the important role of the density pedestal structure. This study maps out important regions of parameter space, providing insights that may point to optimal physical regimes that can enable the recovery of high pedestal temperatures on JET.

  4. Testing an H-mode Pedestal Model Using DIII-D Data

    NASA Astrophysics Data System (ADS)

    Kritz, A. H.; Onjun, T.; Bateman, G.; Guzdar, P. N.; Mahajan, S. M.; Osborne, T.

    2004-11-01

    Tests against experimental data are carried out for a model of the pedestal at the edge of H-mode plasmas based on double-Beltrami solutions of the two-fluid Hall-MHD equations for the interaction of the magnetic and velocity fields.(S.M. Mahajan and Z. Yoshida, PRL 81 (1998) 4863, Phys. Plasmas 7 (2000) 635.) The width and height of the pedestal predicted by the model are tested against experimental data from the DIII-D tokamak. The model for the pedestal width, which has a particularly simple form, namely, inversely proportional to the square root of the density, does not appear to capture the parameter dependence of the experimental data. When the model for the pedestal temperature is rescaled to optimize agreement with data, the RMS error is found to be comparable with the RMS error found using other pedestal models.(T. Onjun, G. Bateman, A.H. Kritz, G. Hammett, Phys. Plasmas 9 (2002) 5018.)

  5. Magnetic-flutter-induced pedestal plasma transport

    NASA Astrophysics Data System (ADS)

    Callen, J. D.; Hegna, C. C.; Cole, A. J.

    2013-11-01

    Plasma toroidal rotation can limit reconnection of externally applied resonant magnetic perturbation (RMP) fields δB on rational magnetic flux surfaces. Hence it causes the induced radial perturbations δBρ to be small there, thereby inhibiting magnetic island formation and stochasticity at the top of pedestals in high (H-mode) confinement tokamak plasmas. However, the δBρs induced by RMPs increase away from rational surfaces and are shown to induce significant sinusoidal radial motion (flutter) of magnetic field lines with a radial extent that varies linearly with δBρ and inversely with distance from the rational surface because of the magnetic shear. This produces a radial electron thermal diffusivity that is (1/2)(δBρ/B0)2 times a kinetically derived, electron-collision-induced, magnetic-shear-reduced, effective parallel electron thermal diffusivity in the absence of magnetic stochasticity. These low collisionality flutter-induced transport processes and thin magnetic island effects are shown to be highly peaked in the vicinity of rational surfaces at the top of low collisionality pedestals. However, the smaller but finite level of magnetic-flutter-induced electron heat transport midway between rational surfaces is the primary factor that determines the electron temperature difference between rational surfaces at the pedestal top. The magnetic-flutter-induced non-ambipolar electron density transport can be large enough to push the plasma toward an electron density transport root. Requiring ambipolar density transport is shown to determine the radial electric field, the plasma toroidal rotation (via radial force balance), a reduced electron thermal diffusivity and increased ambipolar density transport in the pedestal. At high collisionality the various flutter effects are less strongly peaked at rational surfaces and generally less significant. They are thus less likely to exhibit flutter-induced resonant behaviour and transition toward an electron

  6. Calcium-mediated actin reset (CaAR) mediates acute cell adaptations.

    PubMed

    Wales, Pauline; Schuberth, Christian E; Aufschnaiter, Roland; Fels, Johannes; García-Aguilar, Ireth; Janning, Annette; Dlugos, Christopher P; Schäfer-Herte, Marco; Klingner, Christoph; Wälte, Mike; Kuhlmann, Julian; Menis, Ekaterina; Hockaday Kang, Laura; Maier, Kerstin C; Hou, Wenya; Russo, Antonella; Higgs, Henry N; Pavenstädt, Hermann; Vogl, Thomas; Roth, Johannes; Qualmann, Britta; Kessels, Michael M; Martin, Dietmar E; Mulder, Bela; Wedlich-Söldner, Roland

    2016-12-06

    Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress.

  7. Preservation of layered paleodeposits in high-latitude pedestal craters on Mars

    NASA Astrophysics Data System (ADS)

    Kadish, Seth J.; Head, James W.

    2011-06-01

    An outstanding question in Mars' climate history is whether or not pedestal craters represent the armored remnants of ice-rich paleodeposits. We address this question using new high-resolution images; in a survey of several hundred high-latitude pedestal craters, we have identified 12 examples in which visible and/or topographically expressed layers are exposed on the marginal scarp of the pedestal. One example, located on the south polar layered deposits, preserves ice-rich layers that have otherwise been completely removed from the polar cap. These observations provide empirical evidence that the pedestal crater formation mechanism is capable of armoring and preserving ice-rich layered paleodeposits. Although layered exposures have not yet been observed in mid-latitude pedestal craters, high-latitude instances of discontinuous, partially covered layers suggest that layers can be readily concealed, likely through mantling and/or mass wasting processes along the marginal scarp. This interpretation is supported by the observation that high-latitude pedestals with exposed layers along their margins are, on average, taller than mid-latitude examples, and have larger, steeper marginal scarps, which may help to maintain layer exposures. These observations favor the interpretation that mid- to high-latitude pedestal craters represent the armored remnants of ice- and dust-rich paleodeposits, which occurred transiently due to changes in the climate regime. Preservation of fine-scale layering of ice and dust at these latitudes implies that the climate change did not involve regional melting conditions.

  8. Antenna Mechanism of Length Control of Actin Cables

    PubMed Central

    Mohapatra, Lishibanya; Goode, Bruce L.; Kondev, Jane

    2015-01-01

    Actin cables are linear cytoskeletal structures that serve as tracks for myosin-based intracellular transport of vesicles and organelles in both yeast and mammalian cells. In a yeast cell undergoing budding, cables are in constant dynamic turnover yet some cables grow from the bud neck toward the back of the mother cell until their length roughly equals the diameter of the mother cell. This raises the question: how is the length of these cables controlled? Here we describe a novel molecular mechanism for cable length control inspired by recent experimental observations in cells. This “antenna mechanism” involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. We compute the probability distribution of cable lengths as a function of several experimentally tuneable parameters such as the formin-binding affinity of Smy1 and the concentration of myosin motors delivering Smy1. These results provide testable predictions of the antenna mechanism of actin-cable length control. PMID:26107518

  9. Antenna Mechanism of Length Control of Actin Cables.

    PubMed

    Mohapatra, Lishibanya; Goode, Bruce L; Kondev, Jane

    2015-06-01

    Actin cables are linear cytoskeletal structures that serve as tracks for myosin-based intracellular transport of vesicles and organelles in both yeast and mammalian cells. In a yeast cell undergoing budding, cables are in constant dynamic turnover yet some cables grow from the bud neck toward the back of the mother cell until their length roughly equals the diameter of the mother cell. This raises the question: how is the length of these cables controlled? Here we describe a novel molecular mechanism for cable length control inspired by recent experimental observations in cells. This "antenna mechanism" involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. We compute the probability distribution of cable lengths as a function of several experimentally tuneable parameters such as the formin-binding affinity of Smy1 and the concentration of myosin motors delivering Smy1. These results provide testable predictions of the antenna mechanism of actin-cable length control.

  10. The actin cytoskeleton in whole mount preparations and sections.

    PubMed

    Resch, Guenter P; Urban, Edit; Jacob, Sonja

    2010-01-01

    In non-muscle cells, the actin cytoskeleton plays a key role by providing a scaffold contributing to the definition of cell shape, force for driving cell motility, cytokinesis, endocytosis, and propulsion of pathogens, as well as tracks for intracellular transport. A thorough understanding of these processes requires insight into the spatial and temporal organisation of actin filaments into diverse higher-order structures, such as networks, parallel bundles, and contractile arrays. Transmission and scanning electron microscopy can be used to visualise the actin cytoskeleton, but due to the delicate nature of actin filaments, they are easily affected by standard preparation protocols, yielding variable degrees of ultrastructural preservation. In this chapter, we describe different conventional and cryo-approaches to visualise the actin cytoskeleton using transmission electron microscopy and discuss their specific advantages and drawbacks. In the first part, we present three different whole mount techniques, which allow visualisation of actin in the peripheral, thinly spread parts of cells grown in monolayers. In the second part, we describe specific issues concerning the visualisation of actin in thin sections. Techniques for three-dimensional visualisation of actin, protein localisation, and correlative light and electron microscopy are also included. Copyright © 2010 Elsevier Inc. All rights reserved.

  11. Latitude dependence of Martian pedestal craters: Evidence for a sublimation-driven formation mechanism

    NASA Astrophysics Data System (ADS)

    Kadish, Seth J.; Barlow, Nadine G.; Head, James W.

    2009-10-01

    We report on the results of a survey to document and characterize pedestal craters on Mars equatorward of ˜60°N and 65°S latitude. The identification of 2696 pedestal craters reveals a strong latitude dependence, with the vast majority found poleward of 33°N and 40°S. This latitudinal extent is correlated with many climate indicators consistent with the presence of an ice-rich substrate and with climate model predictions of where ice is deposited during periods of higher obliquity in the Amazonian. We have measured key physical attributes of pedestal craters, including the farthest radial extents of the pedestals, pedestal heights, and the circularity of the pedestal margins. In conjunction with the geographic distribution, our measurements strongly support a sublimation-related formation mechanism. This is in contrast to previous hypotheses, which have relied on eolian deflation to produce the elevated plateaus. The identification of marginal pits on the scarps of some pedestal craters, interpreted to be sublimation pits, provide direct evidence for the presence of ice-rich material underlying the armored surface of pedestal craters. On the basis of our findings, we propose a formation mechanism whereby projectiles impact into a volatile-rich dust/snow/ice substrate tens to hundreds of meters thick overlying a dominantly fragmental silicate regolith. The area surrounding the resulting crater becomes armored. Pedestals extend to a distance of multiple crater radii, farther than typical ejecta deposits, necessitating an armoring mechanism that is capable of indurating the surface to a distance greater than the reach of the ejecta. Return to low obliquity causes sublimation of the volatile-rich layer from the intercrater plains, lowering the elevation of the regional terrain. This yields generally circular pedestal craters elevated above the surrounding plains. As a result, the armored surfaces of pedestal craters have preserved a significant record of Amazonian

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

    PubMed Central

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

    2016-01-01

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

  13. Rice actin-binding protein RMD is a key link in the auxin-actin regulatory loop that controls cell growth.

    PubMed

    Li, Gang; Liang, Wanqi; Zhang, Xiaoqing; Ren, Haiyun; Hu, Jianping; Bennett, Malcolm J; Zhang, Dabing

    2014-07-15

    The plant hormone auxin plays a central role in plant growth and development. Auxin transport and signaling depend on actin organization. Despite its functional importance, the mechanistic link between actin filaments (F-actin) and auxin intracellular signaling remains unclear. Here, we report that the actin-organizing protein Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), provides a key link. Mutants lacking RMD display abnormal cell growth and altered configuration of F-actin array direction. The rmd mutants also exhibit an inhibition of auxin-mediated cell elongation, decreased polar auxin transport, altered auxin distribution gradients in root tips, and suppression of plasma membrane localization of auxin transporters O. sativa PIN-FORMED 1b (OsPIN1b) and OsPIN2 in root cells. We demonstrate that RMD is required for endocytosis, exocytosis, and auxin-mediated OsPIN2 recycling to the plasma membrane. Moreover, RMD expression is directly regulated by heterodimerized O. sativa auxin response factor 23 (OsARF23) and OsARF24, providing evidence that auxin modulates the orientation of F-actin arrays through RMD. In support of this regulatory loop, osarf23 and lines with reduced expression of both OsARF23 and OsARF24 display reduced RMD expression, disrupted F-actin organization and cell growth, less sensitivity to auxin response, and altered auxin distribution and OsPIN localization. Our findings establish RMD as a crucial component of the auxin-actin self-organizing regulatory loop from the nucleus to cytoplasm that controls rice cell growth and morphogenesis.

  14. Study of neoclassical effects on the pedestal structure in ELMy H-mode plasmas

    NASA Astrophysics Data System (ADS)

    Pankin, A. Y.; Bateman, G.; Kritz, A. H.; Rafiq, T.; Park, G. Y.; Ku, S.; Chang, C. S.; Snyder, P. B.

    2009-11-01

    The neoclassical effects on the H-mode pedestal structure are investigated in this study. First principles' kinetic simulations of the neoclassical pedestal dynamics are combined with the MHD stability conditions for triggering ELM crashes that limit the pedestal width and height in H-mode plasmas. The neoclassical kinetic XGC0 code [1] is used to produce systematic scans over plasma parameters including plasma current, elongation, and triangularity. As plasma profiles evolve, the MHD stability limits of these profiles are analyzed with the ideal MHD stability ELITE code [2]. The scalings of the pedestal width and height are presented as a function of the scanned plasma parameters. Simulations with the XGC0 code, which include coupled ion-electron dynamics, yield predictions for both ion and electron pedestal profiles. Differences in the electron and ion pedestal scalings are investigated. [1] C.S. Chang et al, Phys. Plasmas 11 (2004) 2649. [2] P.B. Snyder et al, Phys. Plasmas, 9 (2002) 2037.

  15. ITER Simulations Using the PEDESTAL Module in the PTRANSP Code

    NASA Astrophysics Data System (ADS)

    Halpern, F. D.; Bateman, G.; Kritz, A. H.; Pankin, A. Y.; Budny, R. V.; Kessel, C.; McCune, D.; Onjun, T.

    2006-10-01

    PTRANSP simulations with a computed pedestal height are carried out for ITER scenarios including a standard ELMy H-mode (15 MA discharge) and a hybrid scenario (12MA discharge). It has been found that fusion power production predicted in simulations of ITER discharges depends sensitively on the height of the H-mode temperature pedestal [1]. In order to study this effect, the NTCC PEDESTAL module [2] has been implemented in PTRANSP code to provide boundary conditions used for the computation of the projected performance of ITER. The PEDESTAL module computes both the temperature and width of the pedestal at the edge of type I ELMy H-mode discharges once the threshold conditions for the H-mode are satisfied. The anomalous transport in the plasma core is predicted using the GLF23 or MMM95 transport models. To facilitate the steering of lengthy PTRANSP computations, the PTRANSP code has been modified to allow changes in the transport model when simulations are restarted. The PTRANSP simulation results are compared with corresponding results obtained using other integrated modeling codes.[1] G. Bateman, T. Onjun and A.H. Kritz, Plasma Physics and Controlled Fusion, 45, 1939 (2003).[2] T. Onjun, G. Bateman, A.H. Kritz, and G. Hammett, Phys. Plasmas 9, 5018 (2002).

  16. Integrated modelling of H-mode pedestal and confinement in JET-ILW

    NASA Astrophysics Data System (ADS)

    Saarelma, S.; Challis, C. D.; Garzotti, L.; Frassinetti, L.; Maggi, C. F.; Romanelli, M.; Stokes, C.; Contributors, JET

    2018-01-01

    A pedestal prediction model Europed is built on the existing EPED1 model by coupling it with core transport simulation using a Bohm-gyroBohm transport model to self-consistently predict JET-ILW power scan for hybrid plasmas that display weaker power degradation than the IPB98(y, 2) scaling of the energy confinement time. The weak power degradation is reproduced in the coupled core-pedestal simulation. The coupled core-pedestal model is further tested for a 3.0 MA plasma with the highest stored energy achieved in JET-ILW so far, giving a prediction of the stored plasma energy within the error margins of the measured experimental value. A pedestal density prediction model based on the neutral penetration is tested on a JET-ILW database giving a prediction with an average error of 17% from the experimental data when a parameter taking into account the fuelling rate is added into the model. However the model fails to reproduce the power dependence of the pedestal density implying missing transport physics in the model. The future JET-ILW deuterium campaign with increased heating power is predicted to reach plasma energy of 11 MJ, which would correspond to 11-13 MW of fusion power in equivalent deuterium-tritium plasma but with isotope effects on pedestal stability and core transport ignored.

  17. Calcium-mediated actin reset (CaAR) mediates acute cell adaptations

    PubMed Central

    Wales, Pauline; Schuberth, Christian E; Aufschnaiter, Roland; Fels, Johannes; García-Aguilar, Ireth; Janning, Annette; Dlugos, Christopher P; Schäfer-Herte, Marco; Klingner, Christoph; Wälte, Mike; Kuhlmann, Julian; Menis, Ekaterina; Hockaday Kang, Laura; Maier, Kerstin C; Hou, Wenya; Russo, Antonella; Higgs, Henry N; Pavenstädt, Hermann; Vogl, Thomas; Roth, Johannes; Qualmann, Britta; Kessels, Michael M; Martin, Dietmar E; Mulder, Bela; Wedlich-Söldner, Roland

    2016-01-01

    Actin has well established functions in cellular morphogenesis. However, it is not well understood how the various actin assemblies in a cell are kept in a dynamic equilibrium, in particular when cells have to respond to acute signals. Here, we characterize a rapid and transient actin reset in response to increased intracellular calcium levels. Within seconds of calcium influx, the formin INF2 stimulates filament polymerization at the endoplasmic reticulum (ER), while cortical actin is disassembled. The reaction is then reversed within a few minutes. This Calcium-mediated actin reset (CaAR) occurs in a wide range of mammalian cell types and in response to many physiological cues. CaAR leads to transient immobilization of organelles, drives reorganization of actin during cell cortex repair, cell spreading and wound healing, and induces long-lasting changes in gene expression. Our findings suggest that CaAR acts as fundamental facilitator of cellular adaptations in response to acute signals and stress. DOI: http://dx.doi.org/10.7554/eLife.19850.001 PMID:27919320

  18. Cofilin 1-Mediated Biphasic F-Actin Dynamics of Neuronal Cells Affect Herpes Simplex Virus 1 Infection and Replication

    PubMed Central

    Xiang, Yangfei; Zheng, Kai; Ju, Huaiqiang; Wang, Shaoxiang; Pei, Ying; Ding, Weichao; Chen, Zhenping; Wang, Qiaoli; Qiu, Xianxiu; Zhong, Meigong; Zeng, Fanli; Ren, Zhe; Qian, Chuiwen; Liu, Ge

    2012-01-01

    Herpes simplex virus 1 (HSV-1) invades the nervous system and causes pathological changes. In this study, we defined the remodeling of F-actin and its possible mechanisms during HSV-1 infection of neuronal cells. HSV-1 infection enhanced the formation of F-actin-based structures in the early stage of infection, which was followed by a continuous decrease in F-actin during the later stages of infection. The disruption of F-actin dynamics by chemical inhibitors significantly reduced the efficiency of viral infection and intracellular HSV-1 replication. The active form of the actin-depolymerizing factor cofilin 1 was found to increase at an early stage of infection and then to continuously decrease in a manner that corresponded to the remodeling pattern of F-actin, suggesting that cofilin 1 may be involved in the biphasic F-actin dynamics induced by HSV-1 infection. Knockdown of cofilin 1 impaired HSV-1-induced F-actin assembly during early infection and inhibited viral entry; however, overexpression of cofilin 1 did not affect F-actin assembly or viral entry during early infection but decreased intracellular viral reproduction efficiently. Our results, for the first time, demonstrated the biphasic F-actin dynamics in HSV-1 neuronal infection and confirmed the association of F-actin with the changes in the expression and activity of cofilin 1. These results may provide insight into the mechanism by which HSV-1 productively infects neuronal cells and causes pathogenesis. PMID:22623803

  19. L-H transition and pedestal studies on MAST

    NASA Astrophysics Data System (ADS)

    Meyer, H.; De Bock, M. F. M.; Conway, N. J.; Freethy, S. J.; Gibson, K.; Hiratsuka, J.; Kirk, A.; Michael, C. A.; Morgan, T.; Scannell, R.; Naylor, G.; Saarelma, S.; Saveliev, A. N.; Shevchenko, V. F.; Suttrop, W.; Temple, D.; Vann, R. G. L.; MAST, the; NBI Teams

    2011-11-01

    On MAST studies of the profile evolution of the electron temperature (Te), electron density (ne), radial electric field (Er) as well as novel measurements of the ion temperature (Ti) and toroidal current density (jphi) in the pedestal region allow further insight into the processes forming and defining the pedestal such as the H-mode access conditions and MHD stability. This includes studies of fast evolution of Te, ne and Er with Δt = 0.2 ms time resolution and the evolution of pe and jphi through an edge-localized mode (ELM) cycle. Measurements of the H-mode power threshold, PL-H revealed that about 40% more power is required to access H-mode in 4He than in D and that a change in the Z-position of the X-point can change PL-H significantly in single and double null configurations. The profile measurements in the L-mode phase prior to H-mode suggest that neither the gradient nor the value of the mean Te or Er at the plasma edge play a major role in triggering the L-H transition. After the transitions, first the fluctuations are suppressed, then the Er shear layer and the ne pedestal develops followed by the Te pedestal. In the banana regime at low collisionality (νsstarf) ∇Ti ≈ 0 leading to Ti > Te in the pedestal region with Ti ~ 0.3 keV close to the separatrix. A clear correlation of ∇Ti with νsstarf is observed. The measured jphi (using the motional Stark effect) Te and ne are in broad agreement with the common peeling-ballooning stability picture for ELMs and neoclassical calculations of the bootstrap current. The jphi and ∇pe evolution Δt ≈ 2 ms as well as profiles in discharges with counter current neutral beam injection raise questions with respect to this edge stability picture.

  20. Kinetic neoclassical transport in the H-mode pedestal

    DOE PAGES

    Battaglia, D. J.; Burrell, K. H.; Chang, C. S.; ...

    2014-07-16

    Multi-species kinetic neoclassical transport through the QH-mode pedestal and scrapeoff layer on DIII-D is calculated using XGC0, a 5D full-f particle-in-cell drift-kinetic solver with self-consistent neutral recycling and sheath potentials. We achieved quantitative agreement between the fluxdriven simulation and the experimental electron density, impurity density and orthogonal measurements of impurity temperature and flow profiles by adding random-walk particle diffusion to the guiding-center drift motion. Furthermore, we computed the radial electric field (Er) that maintains ambipolar transport across flux surfaces and to the wall self-consistently on closed and open magnetic field lines, and is in excellent agreement with experiment. The Ermore » inside the separatrix is the unique solution that balances the outward flux of thermal tail deuterium ions against the outward neoclassical electron flux and inward pinch of impurity and colder deuterium ions. Particle transport in the pedestal is primarily due to anomalous transport, while the ion heat and momentum transport is primarily due to the neoclassical transport. The full-f treatment quantifies the non-Maxwellian energy distributions that describe a number of experimental observations in low-collisionallity pedestals on DIII-D, including intrinsic co-Ip parallel flows in the pedestal, ion temperature anisotropy and large impurity temperatures in the scrape-off layer.« less

  1. Effect of pedestal fluctuation on ELM frequency in the EAST tokamak

    NASA Astrophysics Data System (ADS)

    Zhong, F. B.; Zhang, T.; Liu, Z. X.; Qu, H.; Liu, H. Q.; Li, G. Q.; Liu, Y.; Gao, W.; Duan, Y. M.; Yang, Y.; Zeng, L.; Xiang, H. M.; Geng, K. N.; Wen, F.; Zhang, S. B.; Gao, X.; the EAST Team

    2018-05-01

    The dependence of ELM frequency on heating power has been studied on the Experimental Advanced Superconducting Tokamak (EAST). It is found that the ELM frequency (f ELM) generally increases with the power through separatrix (P sep), indicating type-I ELM in these plasmas. However, there are two data points, named ‘abnormal ELM’ in the present paper, which have much lower f ELM than the ‘normal ELM’, while both types of ELM have similar ELM energy losses. The ‘abnormal ELM’ occurs at a phase with increased radiation power due to metal impurity influx events. The increased radiation power cannot explain the much lower f ELM for ‘abnormal ELM’, since the reduction of P sep is weaker than proportional to the observed reduction of the ELM frequency. The ‘abnormal ELM’ feature can be attributed to the enhanced amplitude of a coherent mode in the pedestal region. Comparing the pedestal evolutions for the two types of ELM with similar separatrix power P sep, it is actually found that the more pronounced pedestal coherent mode in the plasma with ‘abnormal ELM’ leads to a slower pressure pedestal recovery during the inter-ELM phase. This experimental result implies that the physical mechanism for ‘abnormal ELM’ is that the more pronounced pedestal fluctuation induces larger outward transport, slows down the pedestal evolution and leads to longer inter-ELM phase, i.e. lower ELM frequency.

  2. Actin, actin-binding proteins, and actin-related proteins in the nucleus.

    PubMed

    Kristó, Ildikó; Bajusz, Izabella; Bajusz, Csaba; Borkúti, Péter; Vilmos, Péter

    2016-04-01

    Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.

  3. Gyrokinetic Simulations of JET Carbon and ITER-Like Wall Pedestals

    NASA Astrophysics Data System (ADS)

    Hatch, David; Kotschenreuther, Mike; Mahajan, Swadesh; Liu, Xing; Blackmon, Austin; Giroud, Carine; Hillesheim, Jon; Maggi, Costanza; Saarelma, Samuli; JET Contributors Team

    2017-10-01

    Gyrokinetic simulations using the GENE code are presented, which target a fundamental understanding of JET pedestal transport and, in particular, its modification after installation of an ITER like wall (ILW). A representative pre-ILW (carbon wall) discharge is analyzed as a base case. In this discharge, magnetic diagnostics observe washboard modes, which preferentially affect the temperature pedestal and have frequencies (accounting for Doppler shift) consistent with microtearing modes and inconsistent with kinetic ballooning modes. A similar ILW discharge is examined, which recovers a similar value of H98, albeit at reduced pedestal temperature. This discharge is distinguished by a much higher value of eta, which produces strong ITG and ETG driven instabilities in gyrokinetic simulations. Experimental observations provide several targets for comparisons with simulation data, including the toroidal mode number and frequency of magnetic fluctuations, heat fluxes, and inter-ELM profile evolution. Strategies for optimizing pedestal performance will also be discussed. This work was supported by U.S. DOE Contract No. DE-FG02-04ER54742 and by EUROfusion under Grant No. 633053.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-03-21

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

  6. Quantitative Analysis of Statics and Dynamics of Actin Cables in Fission Yeast

    NASA Astrophysics Data System (ADS)

    Yusuf, Eddy; Wu, Jian-Qiu; Vavylonis, Dimitrios

    2010-03-01

    The assembly of actin and tubulin proteins into long filaments and bundles, i.e. closely-packed filaments, underlies important cellular processes such as cell motility, intracellular transport, and cell division. Recent theoretical and experimental work has addressed the nonequilibrium dynamics of single microtubules within live cells [1]. Actin filaments usually form dense networks that prevents microscopic imaging of individual filaments or bundles. Here, we studied actin dynamics using fission yeast that has low-density actin cytoskeleton consisting of actin cables (actin bundles aligned along the long axis of the cell) and ``actin patches.'' Yeast cells expressing GFP-CHD were imaged by 3D confocal microscopy. Stretching open active contours [2] were used to segment and track individual actin cables. We analyzed their curvature distribution, the tangent correlation, and the temporal bending amplitude fluctuations. We contrast our findings to equilibrium fluctuating semiflexible polymers and to microtubules in cells. We calculate the important time and length scales for the actin cables. We also discuss our findings within the broad context of understanding actin assembly in cells. [1] C. P. Brangwynne et. al., Phys. Rev. Lett. 100, 118104 (2008) [2] H. Li et. al., Proc. of the IEEE Int'l Symposium on Biomedical Imaging: From Nano to Macro, ISBI'09

  7. Regulation of intracellular trafficking and secretion of adiponectin by myosin II.

    PubMed

    Bedi, Deepa; Dennis, John C; Morrison, Edward E; Braden, Tim D; Judd, Robert L

    2017-08-19

    Adiponectin is a protein secreted by white adipocytes that plays an important role in insulin action, energy homeostasis and the development of atherosclerosis. The intracellular localization and trafficking of GLUT4 and leptin in adipocytes has been well studied, but little is known regarding the intracellular trafficking of adiponectin. Recent studies have demonstrated that constitutive adiponectin secretion is dependent on PIP2 levels and the integrity of cortical F-actin. Non-muscle myosin II is an actin-based motor that is associated with membrane vesicles and participates in vesicular trafficking in mammalian cells. Therefore, we investigated the role of myosin II in the trafficking and secretion of adiponectin in 3T3-L1 adipocytes. Confocal microscopy revealed that myosin IIA and IIB were dispersed throughout the cytoplasm of the adipocyte. Both myosin isoforms were localized in the Golgi/TGN region as evidenced by colocalization with the cis-Golgi marker, p115 and the trans-Golgi marker, γ-adaptin. Inhibition of myosin II activity by blebbistatin or actin depolymerization by latrunculin B dispersed myosin IIA and IIB towards the periphery while significantly inhibiting adiponectin secretion. Therefore, the constitutive trafficking and secretion of adiponectin in 3T3-L1 adipocytes occurs by an actin-dependent mechanism that involves the actin-based motors, myosin IIA and IIB. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Flux tube gyrokinetic simulations of the edge pedestal

    NASA Astrophysics Data System (ADS)

    Parker, Scott; Wan, Weigang; Chen, Yang

    2011-10-01

    The linear instabilities of DIII-D H-mode pedestal are studied with gyrokinetic micro-turbulence simulations. The simulation code GEM is an electromagnetic δf code with global tokamak geometry in the form of Miller equilibrium. Local flux tube simulations are carried out for multiple positions of two DIII-D profiles: shot #98889 and shot #131997. Near the top of the pedestal, the instability is clearly ITG. The dominant instability of the pedestal appears at the steep gradient region, and it is identified as a low frequency mode mostly driven by electron temperature gradient. The mode propagates along the electron diamagnetic direction for low n and may propagate along the ion direction for high n. At some positions near the steep gradient region, an ion instability is found which shows some characteristics of kinetic ballooning mode (KBM). These results will be compared to the results of E. Wang et al. and D. Fulton et al. in the same session. We thank R. Groebner and P. Snyder for providing experimental profiles and helpful discussions.

  9. Integrated fusion simulation with self-consistent core-pedestal coupling

    DOE PAGES

    Meneghini, O.; Snyder, P. B.; Smith, S. P.; ...

    2016-04-20

    In this study, accurate prediction of fusion performance in present and future tokamaks requires taking into account the strong interplay between core transport, pedestal structure, current profile and plasma equilibrium. An integrated modeling workflow capable of calculating the steady-state self- consistent solution to this strongly-coupled problem has been developed. The workflow leverages state-of-the-art components for collisional and turbulent core transport, equilibrium and pedestal stability. Validation against DIII-D discharges shows that the workflow is capable of robustly pre- dicting the kinetic profiles (electron and ion temperature and electron density) from the axis to the separatrix in good agreement with the experiments.more » An example application is presented, showing self-consistent optimization for the fusion performance of the 15 MA D-T ITER baseline scenario as functions of the pedestal density and ion effective charge Z eff.« less

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

    PubMed

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

    2009-02-10

    Asymmetric intracellular signals enable cells to migrate in response to external cues. The multiprotein WAVE (also known as SCAR or 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 establishment of neutrophil polarity. Earlier models proposed that spatially biased generation [7] or selection of protrusions [8] enables chemotaxis. These models require existing morphological polarity to control protrusions. We show that spatially biased generation and selection of WAVE complex recruitment also occur in morphologically unpolarized neutrophils during development of their first protrusions. Additionally, several mechanisms limit WAVE complex recruitment during polarization and movement: Intrinsic cues restrict WAVE complex distribution during establishment of polarity, and asymmetric intracellular signals constrain it in morphologically polarized cells. External gradients can overcome both intrinsic biases and control WAVE complex localization. After 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.

  11. System-wide organization of actin cytoskeleton determines organelle transport in hypocotyl plant cells

    PubMed Central

    Nowak, Jacqueline; Ivakov, Alexander; Somssich, Marc; Persson, Staffan; Nikoloski, Zoran

    2017-01-01

    The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms. PMID:28655850

  12. Control of edge localized modes by pedestal deposited impurity in the HL-2A tokamak

    NASA Astrophysics Data System (ADS)

    Zhang, Y. P.; Mazon, D.; Zou, X. L.; Zhong, W. L.; Gao, J. M.; Zhang, K.; Sun, P.; Dong, C. F.; Cui, Z. Y.; Liu, Yi; Shi, Z. B.; Yu, D. L.; Cheng, J.; Jiang, M.; Xu, J. Q.; Isobe, M.; Xiao, G. L.; Chen, W.; Song, S. D.; Bai, X. Y.; Zhang, P. F.; Yuan, G. L.; Ji, X. Q.; Li, Y. G.; Zhou, Y.; Delpech, L.; Ekedahl, A.; Giruzzi, G.; Hoang, T.; Peysson, Y.; Song, X. M.; Song, X. Y.; Li, X.; Ding, X. T.; Dong, J. Q.; Yang, Q. W.; Xu, M.; Duan, X. R.; Liu, Y.; the HL-2A Team

    2018-04-01

    Effect of the pedestal deposited impurity on the edge-localized mode (ELM) behaviour has been observed and intensively investigated in the HL-2A tokamak. Impurities have been externally seeded by a newly developed laser blow-off (LBO) system. Both mitigation and suppression of ELMs have been realized by LBO-seeded impurity. Measurements have shown that the LBO-seeded impurity particles are mainly deposited in the pedestal region. During the ELM mitigation phase, the pedestal density fluctuation is significantly increased, indicating that the ELM mitigation may be achieved by the enhancement of the pedestal transport. The transition from ELM mitigation to ELM suppression was triggered when the number of the LBO-seeded impurity exceeds a threshold value. During the ELM suppression phase, a harmonic coherent mode (HCM) is excited by the LBO-seeded impurity, and the pedestal density fluctuation is significantly decreased, the electron density is continuously increased, implying that HCM may reduce the pedestal turbulence, suppress ELMs, increase the pedestal pressure, thus extending the Peeling-Ballooning instability limit. It has been found that the occurance of the ELM mitigation and ELM suppression closely depends on the LBO laser spot diameter.

  13. Actinous enigma or enigmatic actin

    PubMed Central

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

    2014-01-01

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

  14. TetR-dependent gene regulation in intracellular Listeria monocytogenes demonstrates the spatiotemporal surface distribution of ActA.

    PubMed

    Schmitter, Sibylle; Fieseler, Lars; Klumpp, Jochen; Bertram, Ralph; Loessner, Martin J

    2017-08-01

    To enable specific and tightly controlled gene expression both in vitro and during the intracellular lifecycle of the pathogen Listeria monocytogenes, a TetR-dependent genetic induction system was developed. Highest concentration of cytoplasmic TetR and best repression of tetO-controlled genes was obtained by tetR expression from the synthetic promoter Pt 17 . Anhydrotetracycline (ATc) as inducer permitted concentration-dependent, fine-tuned expression of genes under control of the tetO operator and a suitable promoter. The actin-polymerizing ActA protein represents a major virulence factor of L. monocytogenes, required for actin-based motility and cell-to-cell spread in infected host cells. To be able to observe its spatial and temporal distribution on intracellular L. monocytogenes cells, conditional mutants featuring actA placed under TetR control were used to infect PtK2 epithelial cells. Following induction at different time intervals, the subsequent recruitment of actin by L. monocytogenes could be monitored. We found that cells displayed functional ActA after approximately 15 min, while formation of polarized actin tail was complete after 90-120 min. At this point, intracellular motility of the induced mutants was indistinguishable from wild-type bacteria. Interestingly, de novo ActA synthesis in intracellular Listeria also demonstrated the temporal, asymmetric redistribution of the membrane-anchored proteins from the lateral walls toward the cell poles. © 2017 John Wiley & Sons Ltd.

  15. Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types

    PubMed Central

    Yamashiro, Sawako; Gokhin, David S.; Kimura, Sumiko; Nowak, Roberta B.; Fowler, Velia M.

    2012-01-01

    Tropomodulins are a family of four proteins (Tmods 1–4) that cap the pointed ends of actin filaments in actin cytoskeletal structures in a developmentally regulated and tissue-specific manner. Unique among capping proteins, Tmods also bind tropomyosins (TMs), which greatly enhance the actin filament pointed-end capping activity of Tmods. Tmods are defined by a tropomyosin (TM)-regulated/Pointed-End Actin Capping (TM-Cap) domain in their unstructured N-terminal portion, followed by a compact, folded Leucine-Rich Repeat/Pointed-End Actin Capping (LRR-Cap) domain. By inhibiting actin monomer association and dissociation from pointed ends, Tmods regulate regulate actin dynamics and turnover, stabilizing actin filament lengths and cytoskeletal architecture. In this review, we summarize the genes, structural features, molecular and biochemical properties, actin regulatory mechanisms, expression patterns, and cell and tissue functions of Tmods. By understanding Tmods’ functions in the context of their molecular structure, actin regulation, binding partners, and related variants (leiomodins 1–3), we can draw broad conclusions that can explain the diverse morphological and functional phenotypes that arise from Tmod perturbation experiments in vitro and in vivo. Tmod-based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology. PMID:22488942

  16. Direct Transmembrane Interaction between Actin and the Pore-Competent, Cholesterol-Dependent Cytolysin Pneumolysin

    PubMed Central

    Hupp, Sabrina; Förtsch, Christina; Wippel, Carolin; Ma, Jiangtao; Mitchell, Timothy J.; Iliev, Asparouh I.

    2013-01-01

    The eukaryotic actin cytoskeleton is an evolutionarily well-established pathogen target, as a large number of bacterial factors disturb its dynamics to alter the function of the host cells. These pathogenic factors modulate or mimic actin effector proteins or they modify actin directly, leading to an imbalance of the precisely regulated actin turnover. Here, we show that the pore-forming, cholesterol-dependent cytolysin pneumolysin (PLY), a major neurotoxin of Streptococcus pneumoniae, has the capacity to bind actin directly and to enhance actin polymerisation in vitro. In cells, the toxin co-localised with F-actin shortly after exposure, and this direct interaction was verified by Förster resonance energy transfer. PLY was capable of exerting its effect on actin through the lipid bilayer of giant unilamellar vesicles, but only when its pore competence was preserved. The dissociation constant of G-actin binding to PLY in a biochemical environment was 170–190 nM, which is indicative of a high-affinity interaction, comparable to the affinity of other intracellular actin-binding factors. Our results demonstrate the first example of a direct interaction of a pore-forming toxin with cytoskeletal components, suggesting that the cross talk between pore-forming cytolysins and cells is more complex than previously thought. PMID:23219469

  17. Mechanical coupling between transsynaptic N-cadherin adhesions and actin flow stabilizes dendritic spines

    PubMed Central

    Chazeau, Anaël; Garcia, Mikael; Czöndör, Katalin; Perrais, David; Tessier, Béatrice; Giannone, Grégory; Thoumine, Olivier

    2015-01-01

    The morphology of neuronal dendritic spines is a critical indicator of synaptic function. It is regulated by several factors, including the intracellular actin/myosin cytoskeleton and transcellular N-cadherin adhesions. To examine the mechanical relationship between these molecular components, we performed quantitative live-imaging experiments in primary hippocampal neurons. We found that actin turnover and structural motility were lower in dendritic spines than in immature filopodia and increased upon expression of a nonadhesive N-cadherin mutant, resulting in an inverse relationship between spine motility and actin enrichment. Furthermore, the pharmacological stimulation of myosin II induced the rearward motion of actin structures in spines, showing that myosin II exerts tension on the actin network. Strikingly, the formation of stable, spine-like structures enriched in actin was induced at contacts between dendritic filopodia and N-cadherin–coated beads or micropatterns. Finally, computer simulations of actin dynamics mimicked various experimental conditions, pointing to the actin flow rate as an important parameter controlling actin enrichment in dendritic spines. Together these data demonstrate that a clutch-like mechanism between N-cadherin adhesions and the actin flow underlies the stabilization of dendritic filopodia into mature spines, a mechanism that may have important implications in synapse initiation, maturation, and plasticity in the developing brain. PMID:25568337

  18. Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.

    PubMed Central

    Lopez, I; Anthony, R G; Maciver, S K; Jiang, C J; Khan, S; Weeds, A G; Hussey, P J

    1996-01-01

    In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8693008

  19. Pedestal turbulence simulations using GENE

    NASA Astrophysics Data System (ADS)

    Liu, Xing; Kotschenreuther, M.; Hatch, D. R.; Zheng, L. J.; Mahajan, S.; Diallo, A.; Groebner, R. J.; Hubbard, A. E.; Hughes, J. W.; Maggi, C. F.; Saarelma, S.; JET Contributors

    2017-10-01

    We match frequencies, power balance, and other transport characteristics of several pedestals-two DIIID ELMy H-modes and a C-Mod I-mode, and attempt this for a C-Mod ELMy H-mode. Observed quasi-coherent fluctuations (QCFs) on the DIIID shots are identified as MTMs. The MTMs match frequency and power balance (with slight adjustment of temperature profile), and cause low transport in the density, ion heat and impurity channels- consistent with observed inter-ELM evolution of ion and electron temperature, electron and impurity density, or transport analysis of those channels. KBM can be ruled out as the dominant agent for heat transport. We find the Weakly Coherent Mode on C-Mod I-mode may be an electrostatic heavy particle/ITG mode. Analysis is ongoing for the C-Mod ELMy H-mode QCF. Pedestal density profiles in JET-ILW are consistent with ITG induced particle pinch. Work supported by US DOE under DE-FC02-04ER54698, DE-FG02-04ER54742 and DE-FC02-99ER54512 and by Eurofusion under Grant No. 633053.

  20. Intracellular pathways following uptake of bevacizumab in RPE cells.

    PubMed

    Aboul Naga, Shereen Hassan; Dithmer, Michaela; Chitadze, Guranda; Kabelitz, Dieter; Lucius, Ralph; Roider, Johann; Klettner, Alexa

    2015-02-01

    The anti-VEGF antibody bevacizumab is widely used off-label for the treatment of various ocular diseases, most commonly in age-related macular degeneration and diabetic macular edema. Bevacizumab is able to penetrate the retina and is found in the choroid after intravitreal injection in a time dependent manner. It has previously been shown to be taken up by the retinal pigment epithelium (RPE). In this study, we have investigated the intracellular pathway following uptake of bevacizumab in RPE cells, tested both in primary porcine RPE cells and in the human cell line ARPE19. Bevacizumab displays a characteristic, time-dependent pattern of intracellular distribution, as detected by immunofluorescence and pulse chase experiments. In both primary cells and the cell line, intracellular bevacizumab can be found after seven days, as detected by immunofluorescence and Western blotting. Immediately after application, bevacizumab partially colocalizes with Rab5, indicating some uptake in early endosomes. Intracellularly, bevacizumab is detected in the cytoskeletal fraction, aligning with actin filaments, as revealed by subcellular fractioning and immunofluorescence. Bevacizumab seems to travel along actin filaments by myosin7a, as determined by triple staining immunofluorescence. Interestingly, over a period of seven days, bevacizumab seems to accumulate in certain storage areas, as observed by immunofluorescence. Furthermore, results obtained with immunocytochemistry, Western blotting and flow cytometry indicate that bevacizumab may be released from the RPE cells via exosomes. In conclusion, bevacizumab is taken up by and transported in the retinal pigment epithelial cells in a characteristic, time-dependent manner, where it seems to move along actin filaments by myosin7a and seem to be partially released from the cells via exosomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Actin-based motility allows Listeria monocytogenes to avoid autophagy in the macrophage cytosol.

    PubMed

    Cheng, Mandy I; Chen, Chen; Engström, Patrik; Portnoy, Daniel A; Mitchell, Gabriel

    2018-05-03

    Listeria monocytogenes grows in the host cytosol and uses the surface protein ActA to promote actin polymerisation and mediate actin-based motility. ActA, along with two secreted bacterial phospholipases C, also mediates avoidance from autophagy, a degradative process that targets intracellular microbes. Although it is known that ActA prevents autophagic recognition of L. monocytogenes in epithelial cells by masking the bacterial surface with host factors, the relative roles of actin polymerisation and actin-based motility in autophagy avoidance are unclear in macrophages. Using pharmacological inhibition of actin polymerisation and a collection of actA mutants, we found that actin polymerisation prevented the colocalisation of L. monocytogenes with polyubiquitin, the autophagy receptor p62, and the autophagy protein LC3 during macrophage infection. In addition, the ability of L. monocytogenes to stimulate actin polymerisation promoted autophagy avoidance and growth in macrophages in the absence of phospholipases C. Time-lapse microscopy using green fluorescent protein-LC3 macrophages and a probe for filamentous actin showed that bacteria undergoing actin-based motility moved away from LC3-positive membranes. Collectively, these results suggested that although actin polymerisation protects the bacterial surface from autophagic recognition, actin-based motility allows escape of L. monocytogenes from autophagic membranes in the macrophage cytosol. © 2018 John Wiley & Sons Ltd.

  2. In Vivo, Villin Is Required for Ca2+-Dependent F-Actin Disruption in Intestinal Brush Borders

    PubMed Central

    Ferrary, Evelyne; Cohen-Tannoudji, Michel; Pehau-Arnaudet, Gérard; Lapillonne, Alexandre; Athman, Rafika; Ruiz, Tereza; Boulouha, Lilia; El Marjou, Fatima; Doye, Anne; Fontaine, Jean-Jacques; Antony, Claude; Babinet, Charles; Louvard, Daniel; Jaisser, Frédéric; Robine, Sylvie

    1999-01-01

    Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca2+-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca2+ differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca2+, whereas Ca2+ had no effect in villin-null isolates. Moreover, increase in intracellular Ca2+ by serosal carbachol or mucosal Ca2+ ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 ± 9.6%, compared with wild-type mice, 70 ± 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury. PMID:10459016

  3. Validation of a coupled core-transport, pedestal-structure, current-profile and equilibrium model

    NASA Astrophysics Data System (ADS)

    Meneghini, O.

    2015-11-01

    The first workflow capable of predicting the self-consistent solution to the coupled core-transport, pedestal structure, and equilibrium problems from first-principles and its experimental tests are presented. Validation with DIII-D discharges in high confinement regimes shows that the workflow is capable of robustly predicting the kinetic profiles from on axis to the separatrix and matching the experimental measurements to within their uncertainty, with no prior knowledge of the pedestal height nor of any measurement of the temperature or pressure. Self-consistent coupling has proven to be essential to match the experimental results, and capture the non-linear physics that governs the core and pedestal solutions. In particular, clear stabilization of the pedestal peeling ballooning instabilities by the global Shafranov shift and destabilization by additional edge bootstrap current, and subsequent effect on the core plasma profiles, have been clearly observed and documented. In our model, self-consistency is achieved by iterating between the TGYRO core transport solver (with NEO and TGLF for neoclassical and turbulent flux), and the pedestal structure predicted by the EPED model. A self-consistent equilibrium is calculated by EFIT, while the ONETWO transport package evolves the current profile and calculates the particle and energy sources. The capabilities of such workflow are shown to be critical for the design of future experiments such as ITER and FNSF, which operate in a regime where the equilibrium, the pedestal, and the core transport problems are strongly coupled, and for which none of these quantities can be assumed to be known. Self-consistent core-pedestal predictions for ITER, as well as initial optimizations, will be presented. Supported by the US Department of Energy under DE-FC02-04ER54698, DE-SC0012652.

  4. Actin-induced dimerization of palladin promotes actin-bundling

    PubMed Central

    Vattepu, Ravi; Yadav, Rahul; Beck, Moriah R

    2015-01-01

    A subset of actin binding proteins is able to form crosslinks between two or more actin filaments, thus producing structures of parallel or networked bundles. These actin crosslinking proteins interact with actin through either bivalent binding or dimerization. We recently identified two binding sites within the actin binding domain of palladin, an actin crosslinking protein that plays an important role in normal cell adhesion and motility during wound healing and embryonic development. In this study, we show that actin induces dimerization of palladin. Furthermore, the extent of dimerization reflects earlier comparisons of actin binding and bundling between different domains of palladin. On the basis of these results we hypothesized that actin binding may promote a conformational change that results in dimerization of palladin, which in turn may drive the crosslinking of actin filaments. The proximal distance between two actin binding sites on crosslinking proteins determines the ultrastructural properties of the filament network, therefore we also explored interdomain interactions using a combination of chemical crosslinking experiments and actin cosedimentation assays. Limited proteolysis data reveals that palladin is less susceptible to enzyme digestion after actin binding. Our results suggest that domain movements in palladin are necessary for interactions with actin and are induced by interactions with actin filaments. Accordingly, we put forth a model linking the structural changes to functional dynamics. PMID:25307943

  5. Poisons, ruffles and rockets: bacterial pathogens and the host cell cytoskeleton.

    PubMed

    Steele-Mortimer, O; Knodler, L A; Finlay, B B

    2000-02-01

    The cytoskeleton of eukaryotic cells is affected by a number of bacterial and viral pathogens. In this review we consider three recurring themes of cytoskeletal involvement in bacterial pathogenesis: 1) the effect of bacterial toxins on actin-regulating small GTP-binding proteins; 2) the invasion of non-phagocytic cells by the bacterial induction of ruffles at the plasma membrane; 3) the formation of actin tails and pedestals by intracellular and extracellular bacteria, respectively. Considerable progress has been made recently in the characterization of these processes. It is becoming clear that bacterial pathogens have developed a variety of sophisticated mechanisms for utilizing the complex cytoskeletal system of host cells. These bacterially-induced processes are now providing unique insights into the regulation of fundamental eukaryotic mechanisms.

  6. Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast

    PubMed Central

    Huckaba, Thomas M.; Lipkin, Thomas; Pon, Liza A.

    2006-01-01

    Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport. PMID:17178912

  7. Anomalous transport in the H-mode pedestal of Alcator C-Mod discharges

    NASA Astrophysics Data System (ADS)

    Pankin, A. Y.; Hughes, J. W.; Greenwald, M. J.; Kritz, A. H.; Rafiq, T.

    2017-02-01

    Anomalous transport in the H-mode pedestal region of five Alcator C-Mod discharges, representing a collisionality scan is analyzed. The understanding of anomalous transport in the pedestal region is important for the development of a comprehensive model for the H-mode pedestal slope. In this research, a possible role of the drift resistive inertial ballooning modes (Rafiq et al 2010 Phys. Plasmas 17 082511) in the edge of Alcator C-Mod discharges is analyzed. The stability analysis, carried out using the TRANSP code, indicates that the DRIBM modes are strongly unstable in Alcator C-Mod discharges with large electron collisionality. An improved interpretive analysis of H-mode pedestal experimental data is carried out utilizing the additive flux minimization technique (Pankin et al 2013 Phys. Plasmas 20 102501) together with the guiding-center neoclassical kinetic XGC0 code. The neoclassical and neutral physics are simulated in the XGC0 code and the anomalous fluxes are computed using the additive flux minimization technique. The anomalous fluxes are reconstructed and compared with each other for the collisionality scan Alcator C-Mod discharges. It is found that the electron thermal anomalous diffusivities at the pedestal top increase with the electron collisionality. This dependence can also point to the drift resistive inertial ballooning modes as the modes that drive the anomalous transport in the plasma edge of highly collisional discharges.

  8. Mechanics of biomimetic systems propelled by actin comet tails

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

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

  9. Control of bootstrap current in the pedestal region of tokamaks

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shaing, K. C.; Department of Engineering Physics, University of Wisconsin, Madison, Wisconsin 53796; Lai, A. L.

    2013-12-15

    The high confinement mode (H-mode) plasmas in the pedestal region of tokamaks are characterized by steep gradient of the radial electric field, and sonic poloidal U{sub p,m} flow that consists of poloidal components of the E×B flow and the plasma flow velocity that is parallel to the magnetic field B. Here, E is the electric field. The bootstrap current that is important for the equilibrium, and stability of the pedestal of H-mode plasmas is shown to have an expression different from that in the conventional theory. In the limit where ‖U{sub p,m}‖≫ 1, the bootstrap current is driven by themore » electron temperature gradient and inductive electric field fundamentally different from that in the conventional theory. The bootstrap current in the pedestal region can be controlled through manipulating U{sub p,m} and the gradient of the radial electric. This, in turn, can control plasma stability such as edge-localized modes. Quantitative evaluations of various coefficients are shown to illustrate that the bootstrap current remains finite when ‖U{sub p,m}‖ approaches infinite and to provide indications how to control the bootstrap current. Approximate analytic expressions for viscous coefficients that join results in the banana and plateau-Pfirsch-Schluter regimes are presented to facilitate bootstrap and neoclassical transport simulations in the pedestal region.« less

  10. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

    PubMed Central

    Navarro-Garcia, Fernando; Serapio-Palacios, Antonio; Ugalde-Silva, Paul; Tapia-Pastrana, Gabriela; Chavez-Dueñas, Lucia

    2013-01-01

    The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. PMID:23509714

  11. Real-Time Dynamics of Emerging Actin Networks in Cell-Mimicking Compartments

    PubMed Central

    Deshpande, Siddharth; Pfohl, Thomas

    2015-01-01

    Understanding the cytoskeletal functionality and its relation to other cellular components and properties is a prominent question in biophysics. The dynamics of actin cytoskeleton and its polymorphic nature are indispensable for the proper functioning of living cells. Actin bundles are involved in cell motility, environmental exploration, intracellular transport and mechanical stability. Though the viscoelastic properties of actin-based structures have been extensively probed, the underlying microstructure dynamics, especially their disassembly, is not fully understood. In this article, we explore the rich dynamics and emergent properties exhibited by actin bundles within flow-free confinements using a microfluidic set-up and epifluorescence microscopy. After forming entangled actin filaments within cell-sized quasi two-dimensional confinements, we induce their bundling using three different fundamental mechanisms: counterion condensation, depletion interactions and specific protein-protein interactions. Intriguingly, long actin filaments form emerging networks of actin bundles via percolation leading to remarkable properties such as stress generation and spindle-like intermediate structures. Simultaneous sharing of filaments in different links of the network is an important parameter, as short filaments do not form networks but segregated clusters of bundles instead. We encounter a hierarchical process of bundling and its subsequent disassembly. Additionally, our study suggests that such percolated networks are likely to exist within living cells in a dynamic fashion. These observations render a perspective about differential cytoskeletal responses towards numerous stimuli. PMID:25785606

  12. Investigation of the plasma shaping effects on the H-mode pedestal structure using coupled kinetic neoclassical/MHD stability simulations

    NASA Astrophysics Data System (ADS)

    Pankin, A. Y.; Rafiq, T.; Kritz, A. H.; Park, G. Y.; Snyder, P. B.; Chang, C. S.

    2017-06-01

    The effects of plasma shaping on the H-mode pedestal structure are investigated. High fidelity kinetic simulations of the neoclassical pedestal dynamics are combined with the magnetohydrodynamic (MHD) stability conditions for triggering edge localized mode (ELM) instabilities that limit the pedestal width and height in H-mode plasmas. The neoclassical kinetic XGC0 code [Chang et al., Phys. Plasmas 11, 2649 (2004)] is used in carrying out a scan over plasma elongation and triangularity. As plasma profiles evolve, the MHD stability limits of these profiles are analyzed with the ideal MHD ELITE code [Snyder et al., Phys. Plasmas 9, 2037 (2002)]. Simulations with the XGC0 code, which includes coupled ion-electron dynamics, yield predictions for both ion and electron pedestal profiles. The differences in the predicted H-mode pedestal width and height for the DIII-D discharges with different elongation and triangularities are discussed. For the discharges with higher elongation, it is found that the gradients of the plasma profiles in the H-mode pedestal reach semi-steady states. In these simulations, the pedestal slowly continues to evolve to higher pedestal pressures and bootstrap currents until the peeling-ballooning stability conditions are satisfied. The discharges with lower elongation do not reach the semi-steady state, and ELM crashes are triggered at earlier times. The plasma elongation is found to have a stronger stabilizing effect than the plasma triangularity. For the discharges with lower elongation and lower triangularity, the ELM frequency is large, and the H-mode pedestal evolves rapidly. It is found that the temperature of neutrals in the scrape-off-layer (SOL) region can affect the dynamics of the H-mode pedestal buildup. However, the final pedestal profiles are nearly independent of the neutral temperature. The elongation and triangularity affect the pedestal widths of plasma density and electron temperature profiles differently. This provides a new

  13. Investigation of the plasma shaping effects on the H-mode pedestal structure using coupled kinetic neoclassical/MHD stability simulations

    DOE PAGES

    Pankin, A. Y.; Rafiq, T.; Kritz, A. H.; ...

    2017-06-08

    The effects of plasma shaping on the H-mode pedestal structure are investigated. High fidelity kinetic simulations of the neoclassical pedestal dynamics are combined with the magnetohydrodynamic (MHD) stability conditions for triggering edge localized mode (ELM) instabilities that limit the pedestal width and height in H-mode plasmas. We use the neoclassical kinetic XGC0 code [Chang et al., Phys. Plasmas 11, 2649 (2004)] to carry out a scan over plasma elongation and triangularity. As plasma profiles evolve, the MHD stability limits of these profiles are analyzed with the ideal MHD ELITE code [Snyder et al., Phys. Plasmas 9, 2037 (2002)]. In simulationsmore » with the XGC0 code, which includes coupled ion-electron dynamics, yield predictions for both ion and electron pedestal profiles. The differences in the predicted H-mode pedestal width and height for the DIII-D discharges with different elongation and triangularities are discussed. For the discharges with higher elongation, it is found that the gradients of the plasma profiles in the H-mode pedestal reach semi-steady states. In these simulations, the pedestal slowly continues to evolve to higher pedestal pressures and bootstrap currents until the peeling-ballooning stability conditions are satisfied. The discharges with lower elongation do not reach the semi-steady state, and ELM crashes are triggered at earlier times. The plasma elongation is found to have a stronger stabilizing effect than the plasma triangularity. For the discharges with lower elongation and lower triangularity, the ELM frequency is large, and the H-mode pedestal evolves rapidly. It is found that the temperature of neutrals in the scrape-off-layer (SOL) region can affect the dynamics of the H-mode pedestal buildup. But the final pedestal profiles are nearly independent of the neutral temperature. The elongation and triangularity affect the pedestal widths of plasma density and electron temperature profiles differently. This provides a new

  14. Investigation of the plasma shaping effects on the H-mode pedestal structure using coupled kinetic neoclassical/MHD stability simulations

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pankin, A. Y.; Rafiq, T.; Kritz, A. H.

    The effects of plasma shaping on the H-mode pedestal structure are investigated. High fidelity kinetic simulations of the neoclassical pedestal dynamics are combined with the magnetohydrodynamic (MHD) stability conditions for triggering edge localized mode (ELM) instabilities that limit the pedestal width and height in H-mode plasmas. We use the neoclassical kinetic XGC0 code [Chang et al., Phys. Plasmas 11, 2649 (2004)] to carry out a scan over plasma elongation and triangularity. As plasma profiles evolve, the MHD stability limits of these profiles are analyzed with the ideal MHD ELITE code [Snyder et al., Phys. Plasmas 9, 2037 (2002)]. In simulationsmore » with the XGC0 code, which includes coupled ion-electron dynamics, yield predictions for both ion and electron pedestal profiles. The differences in the predicted H-mode pedestal width and height for the DIII-D discharges with different elongation and triangularities are discussed. For the discharges with higher elongation, it is found that the gradients of the plasma profiles in the H-mode pedestal reach semi-steady states. In these simulations, the pedestal slowly continues to evolve to higher pedestal pressures and bootstrap currents until the peeling-ballooning stability conditions are satisfied. The discharges with lower elongation do not reach the semi-steady state, and ELM crashes are triggered at earlier times. The plasma elongation is found to have a stronger stabilizing effect than the plasma triangularity. For the discharges with lower elongation and lower triangularity, the ELM frequency is large, and the H-mode pedestal evolves rapidly. It is found that the temperature of neutrals in the scrape-off-layer (SOL) region can affect the dynamics of the H-mode pedestal buildup. But the final pedestal profiles are nearly independent of the neutral temperature. The elongation and triangularity affect the pedestal widths of plasma density and electron temperature profiles differently. This provides a new

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

    PubMed Central

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

    2001-01-01

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

  16. Divertor, scrape-off layer and pedestal particle dynamics in the ELM cycle on ASDEX Upgrade

    NASA Astrophysics Data System (ADS)

    Laggner, F. M.; Keerl, S.; Gnilsen, J.; Wolfrum, E.; Bernert, M.; Carralero, D.; Guimarais, L.; Nikolaeva, V.; Potzel, S.; Cavedon, M.; Mink, F.; Dunne, M. G.; Birkenmeier, G.; Fischer, R.; Viezzer, E.; Willensdorfer, M.; Wischmeier, M.; Aumayr, F.; the EUROfusion MST1 Team; the ASDEX Upgrade Team

    2018-02-01

    In addition to the relaxation of the pedestal, edge localised modes (ELMs) introduce changes to the divertor and scrape-off layer (SOL) conditions. Their impact on the inter-ELM pedestal recovery is investigated, with emphasis on the electron density (n e) evolution. The typical ELM cycle occurring in an exemplary ASDEX Upgrade discharge interval at moderate applied gas puff and heating power is characterised, utilising several divertor, SOL and pedestal diagnostics. In the studied discharge interval the inner divertor target is detached before the ELM crash, while the outer target is attached. The particles and power expelled by the ELM crash lead to a re-attachment of the inner target plasma. After the ELM crash, the outer divertor target moves into a high recycling regime with large n e in front of the plate, which is accompanied by high main chamber neutral fluxes. On similar timescales, the inner target fully detaches and the high field side high density region (HFSHD) is formed reaching up to the high field side midplane. This state evolves again to the pre-ELM state, when the main chamber neutral fluxes are reduced later in the ELM cycle. Neither the timescale of the appearance of the HFSHD nor the increase of the main chamber neutral fluxes fit the timescale of the n e pedestal, which is faster. It is found that during the n e pedestal recovery, the magnetic activity at the low field side midplane is strongly reduced indicating a lower level of fluctuations. A rough estimation of the particle flux across the pedestal suggests that the particle flux is reduced in this period. In conclusion, the evolution of the n e pedestal is determined by a combination of neutral fluxes, HFSHD and reduced particle flux across the pedestal. A reduced particle flux explains the fast, experimentally observed re-establishment of the n e pedestal best, whereas neutrals and HFSHD impact on the evolution of the SOL and separatrix conditions.

  17. 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.

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

    PubMed

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

    1998-06-01

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

  19. Evolution of Edge Pedestal Profiles Between ELMs

    NASA Astrophysics Data System (ADS)

    Floyd, J. P.; Stacey, W. M.; Groebner, R. J.

    2012-10-01

    The measured edge profile evolution in DIII-D discharges is analyzed in terms of the implied thermal diffusivities, ion diffusion coefficients and pinch velocities, using the momentum-balance methodology of Ref. [1], extended to take into account ion orbit loss and X-point loss. The evolution of the density, temperature, rotation and radial electric field profiles in the edge pedestal between edge localized modes (ELMs) provides information of these diffusive and non-diffusive transport processes in the pedestal of H-mode plasmas. This methodology is incorporated in the GTEDGE code developed for DIII-D data interpretation. Using a smaller integration time for the charge exchange recombination measurements than in Ref. [1] allows a more detailed examination of the time evolution of the ion temperature and rotation profiles. 6pt [1] W.M. Stacey and R.J. Groebner, Nucl. Fusion 51, 063024 (2011).

  20. Erbium Distribution in Single Crystal YAG Fibers Grown by Laser-Heated Pedestal Growth Technique

    DTIC Science & Technology

    2015-08-28

    single crystal YAG fibers grown by laser - heated pedestal growth technique Single crystal (SC) yttrium aluminum garnet (YAG, Y3Al5O12) as a host...inserted into a SC YAG tube. This rod-in-tube was used as a preform in our laser -heated pedestal growth (LHPG) apparatus to grow a fiber with a radial...fibers grown by laser -heated pedestal growth technique Report Title Single crystal (SC) yttrium aluminum garnet (YAG, Y3Al5O12) as a host material has

  1. Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor

    PubMed Central

    Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J. K.; Schubert, Dirk W.; Stockinger, Hannes; Herrmann, Christian

    2014-01-01

    Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies. PMID:24190970

  2. Gamma interferon-induced guanylate binding protein 1 is a novel actin cytoskeleton remodeling factor.

    PubMed

    Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J K; Schubert, Dirk W; Stockinger, Hannes; Herrmann, Christian; Stürzl, Michael

    2014-01-01

    Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies.

  3. Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

    PubMed Central

    Kang, Hyeran; Bradley, Michael J.; McCullough, Brannon R.; Pierre, Anaëlle; Grintsevich, Elena E.; Reisler, Emil; De La Cruz, Enrique M.

    2012-01-01

    The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments. PMID:23027950

  4. Streamer formation and transport for parameters characteristic of H-mode pedestals

    NASA Astrophysics Data System (ADS)

    Blackmon, Austin; Hatch, D. R.; Kotschenreuther, M.; Mahajan, S.; Hazeltine, R. D.

    2017-10-01

    We investigate, through gyrokinetic simulations, the formation of streamers as a consequence of electron temperature gradient driven, electron scale instabilities. We also study the interaction of velocity shear with streamers for parameters typical of H-mode pedestals, exploring both the higher as well as lower temperature gradient regions. Without ExB shear, the streamers form at the pedestal top causing large heat fluxes; the modes, however, did not saturate. When ExB shear was turned on, the streamers dissipated, and heat flux was lowered, though still of significant magnitude. In the middle of the pedestal, with high temperature gradient, heat flux was insignificant. There was no evidence of streamers in this region, leading to a conclusion that streamers have a strong influence on heat flux. Work supported by US DOE under DE-FG02-04ER54742.

  5. Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

    PubMed Central

    Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

    2018-01-01

    Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

  6. Actin-Based Adhesion Modules Mediate Cell Interactions with the Extracellular Matrix and Neighboring Cells.

    PubMed

    Bachir, Alexia I; Horwitz, Alan Rick; Nelson, W James; Bianchini, Julie M

    2017-07-05

    Cell adhesions link cells to the extracellular matrix (ECM) and to each other and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping, functional modules. These modules establish physical associations with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as to sense and translate the mechanical properties of the cellular environment into changes in cell organization and behavior. Here, we review the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions and how adhesion molecules mediate cross talk between cell-ECM and cell-cell adhesion sites. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  7. Prediction and realization of ITER-like pedestal pressure in the high- B tokamak Alcator C-Mod

    NASA Astrophysics Data System (ADS)

    Hughes, Jerry

    2017-10-01

    Fusion power in a burning plasma will scale as the square of the plasma pressure, which is increased in a straightforward way by increasing magnetic field: Pfus p2 B4 . Experiments on Alcator C-Mod, a compact high- B tokamak, have tested predictive capability for pedestal pressure, at toroidal field BT up to 8T , and poloidal field BP up to 1T . These reactor-like fields enable C-Mod to approach an ITER predicted value of 90kPa . This is expected if, as in the EPED model, the pedestal is constrained by onset of kinetic ballooning modes (KBMs) and peeling-ballooning modes (PMB), yielding a pressure pedestal approximately as pped BT ×BP . One successful path to high confinement on C-Mod is the high-density (ne > 3 ×1020m-3) approach, pursued using enhanced D-alpha (EDAs) H-mode. In EDA H-mode, transport regulates both the pedestal profiles and the core impurity content, holding the pedestal stationary, at just below the PBM stability boundary. We have extended this stationary ELM-suppressed regime to the highest magnetic fields achievable on C-Mod, and used it to approach the maximum pedestal predicted by EPED at high density: pped 60kPa . Another approach to high pressure utilizes a pedestal limited by PBMs at low collisionality, where pressure increases with density and EPED predicts access to a higher ``Super H'' solution for pped. Experiments at reduced density (ne < 2 ×1020m-3) and strong plasma shaping (δ > 0.5) accessed these regimes on C-Mod, producing pedestals with world record pped 80kPa , at Tped 2keV . In both the high and low density approaches, the impact of the pedestal on core performance is substantial. Our exploration of high pedestal regimes yielded a volume-averaged pressure 〈 p 〉 > 2atm , a world record value for a magnetic fusion device. The results hold promise for the projection of pedestal pressure and overall performance of high field burning plasma devices. Supported by U.S. Department of Energy awards DE-FC02-99ER54512, DE-FG02

  8. Stationary QH-mode plasmas with high and wide pedestal at low rotation on DIII-D

    DOE PAGES

    Chen, Xi; Burrell, K. H.; Osborne, T. H.; ...

    2016-09-30

    A stationary, quiescent H-mode (QH-mode) regime with a wide pedestal and improved confinement at low rotation has been discovered on DIII-D with reactor relevant edge parameters and no ELMs. As the injected neutral beam torque is ramped down and the edge ExB rotation shear reduces, the transition from standard QH to the wide pedestal QH-mode occurs. And at the transition, the coherent edge harmonic oscillations (EHO) that usually regulate the standard QH edge cease and broadband edge MHD modes appear along with a rapid increase in the pedestal pressure height (by ≤60%) and width (by ≤50%). We posit that themore » enhanced edge turbulence-driven transport, enabled by the lower edge ExB flow shear due to lower torque reduces the pedestal gradient and, combined with the high edge instability limit provided by the balanced double-null plasma shape, permits the development of a broader and thus higher pedestal that is turbulence-transport-limited. Even with the significantly enhanced pedestal pressure, the edge operating point is below the peeling ballooning mode stability boundary and thus without ELMs. Improved transport in the outer core region (0.8≤ρ≤0.9) owing to increased ExB flow shear in that region and the enhanced pedestal boost the overall confinement by up to 45%. Our findings advance the physics basis for developing stationary ELM-free high-confinement operation at low rotation for future burning plasma where similar collisionality and rotation levels are expected.« less

  9. Plasma shaping and its impact on the pedestal of ASDEX Upgrade: edge stability and inter-ELM dynamics at varied triangularity

    NASA Astrophysics Data System (ADS)

    Laggner, F. M.; Wolfrum, E.; Cavedon, M.; Dunne, M. G.; Birkenmeier, G.; Fischer, R.; Willensdorfer, M.; Aumayr, F.; The EUROfusion MST1 Team; The ASDEX Upgrade Team

    2018-04-01

    The plasma shape, in particular the triangularity (δ), impacts on the pedestal stability. A scan of δ including a variation of heating power (P heat) and gas puff was performed to study the behaviour of edge localised modes (ELMs) and the pre-ELM pedestal stability for different plasma shapes. Generally, at higher δ the pedestal top electron density (n e) is enhanced and the ELM repetition frequency (f ELM) is reduced. For all δ, the pedestal top n e is already fully established to its pre-ELM value during the initial recovery phase of the n e pedestal, which takes place immediately after the ELM crash. The lowering of the f ELM with increasing δ is related to longer pedestal recovery phases, especially the last pre-ELM phase with clamped pedestal gradients (after the recovery phases of the n e and electron temperature (T e) pedestal) is extended. In all investigated discharge intervals, the pre-ELM pedestal profiles are in agreement with peeling-ballooning (PB) theory. Over the investigated range of δ, two well-separated f ELM bands are observed in several discharge intervals. Their occurrence is linked to the inter-ELM pedestal stability. In both kinds of ELM cycles the pedestal evolves similarly, however, the ‘fast’ ELM cycle occurs before the global plasma stored energy (W MHD) increases, which then provides a stabilising effect on the pedestal, extending the inter-ELM period in the case of the ‘slow’ ELM cycle. At the end of a ‘fast’ ELM cycle the n e profile is radially shifted inwards relative to the n e profile at the end of a ‘slow’ ELM cycle, leading to a reduced pressure gradient. The appearance of two f ELM bands suggests that the pedestal becomes more likely PB unstable in certain phases of the inter-ELM evolution. Such a behaviour is possible because the evolution of the global plasma is not rigidly coupled to the evolution of the pedestal structure on the timescales of an ELM cycle.

  10. Actin-related protein 2/3 complex-based actin polymerization is critical for male fertility.

    PubMed

    Lee, J S; Kwon, W S; Rahman, M S; Yoon, S J; Park, Y J; Pang, M G

    2015-09-01

    The actin-related protein 2/3 (Arp2/3) complex is critical for regulation of actin polymerization, which is associated with sperm motility and capacitation status. However, the function of the Arp2/3 complex in male fertility has not yet been fully elucidated. Therefore, this study was designed to investigate the role of the Arp2/3 complex in different processes in spermatozoa and its consequences on fertilization and early embryonic development. In this in vitro study, mouse spermatozoa were incubated with different concentrations (10, 100, and 500 μm) of CK-636, an Arp2/3 complex antagonist. Our results demonstrated that inhibition of the Arp2/3 complex by high concentrations (100 and 500 μm) of CK-636 induced hyper-activated motility and acrosomal reaction, whereas intracellular calcium and tyrosine phosphorylation levels in spermatozoa were inhibited. Moreover, exposure of spermatozoa to the highest concentration of CK-636 reduced fertilization and embryo development. Interestingly, fertilization was significantly increased after treatment with 100 μm CK-636, whereas embryonic development was significantly decreased. Therefore, we conclude that the Arp2/3 complex plays a decisive role in regulation of sperm function and male fertility via actin polymerization. We anticipate that the Arp2/3 complex may have clinical application as marker for male fertility and male contraceptive targeting. © 2015 American Society of Andrology and European Academy of Andrology.

  11. Critical forces for actin filament buckling and force transmission influence transport in actomyosin networks

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Gardel, Margaret

    Viscoelastic networks of biopolymers coordinate the motion of intracellular objects during transport. These networks have nonlinear mechanical properties due to events such as filament buckling or breaking of cross-links. The influence of such nonlinear properties on the time and length scales of transport is not understood. Here, we use in vitro networks of actin and the motor protein myosin II to clarify how intracellular forces regulate active diffusion. We observe two transitions in the mean-squared displacement of cross-linked actin with increasing motor concentration. The first is a sharp transition from initially subdiffusive to diffusive-like motion that requires filament buckling but does not cause net contraction of the network. Further increase of the motor density produces a second transition to network rupture and ballistic actin transport. This corresponds with an increase in the correlation of motion and thus may be caused when forces propagate far enough for global motion. We conclude that filament buckling and overall network contraction require different amounts of force and produce distinct transport properties. These nonlinear transitions may act as mechanical switches that can be turned on to produce observed motion within cells.

  12. Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

    PubMed Central

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

    2016-01-01

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

  13. Microcompartmentation of cytosolic aldolase by interaction with the actin cytoskeleton in Arabidopsis.

    PubMed

    Garagounis, Constantine; Kostaki, Kalliopi-Ioanna; Hawkins, Tim J; Cummins, Ian; Fricker, Mark D; Hussey, Patrick J; Hetherington, Alistair M; Sweetlove, Lee J

    2017-02-01

    Evidence is accumulating for molecular microcompartments formed when proteins interact in localized domains with the cytoskeleton, organelle surfaces, and intracellular membranes. To understand the potential functional significance of protein microcompartmentation in plants, we studied the interaction of the glycolytic enzyme fructose bisphosphate aldolase with actin in Arabidopsis thaliana. Homology modelling of a major cytosolic isozyme of aldolase, FBA8, suggested that the tetrameric holoenzyme has two actin binding sites and could therefore act as an actin-bundling protein, as was reported for animal aldolases. This was confirmed by in vitro measurements of an increase in viscosity of F-actin polymerized in the presence of recombinant FBA8. Simultaneously, interaction with F-actin caused non-competitive inhibition of aldolase activity. We did not detect co-localization of an FBA8-RFP fusion protein, expressed in an fba8-knockout background, with the actin cytoskeleton using confocal laser-scanning microscopy. However, we did find evidence for a low level of interaction using FRET-FLIM analysis of FBA8-RFP co-expressed with the actin-binding protein GFP-Lifeact. Furthermore, knockout of FBA8 caused minor alterations of guard cell actin cytoskeleton morphology and resulted in a reduced rate of stomatal closure in response to decreased humidity. We conclude that cytosolic aldolase can be microcompartmented in vivo by interaction with the actin cytoskeleton and may subtly modulate guard cell behaviour as a result. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  14. Stationary QH-mode plasmas with high and wide pedestal at low rotation on DIII-D

    NASA Astrophysics Data System (ADS)

    Chen, Xi; Burrell, K. H.; Osborne, T. H.; Solomon, W. M.; Barada, K.; Garofalo, A. M.; Groebner, R. J.; Luhmann, N. C.; McKee, G. R.; Muscatello, C. M.; Ono, M.; Petty, C. C.; Porkolab, M.; Rhodes, T. L.; Rost, J. C.; Snyder, P. B.; Staebler, G. M.; Tobias, B. J.; Yan, Z.; the DIII-D Team

    2017-02-01

    A stationary, quiescent H-mode (QH-mode) regime with a wide pedestal and improved confinement at low rotation has been discovered on DIII-D with reactor relevant edge parameters and no ELMs. As the injected neutral beam torque is ramped down and the edge E  ×  B rotation shear reduces, the transition from standard QH to the wide pedestal QH-mode occurs. At the transition, the coherent edge harmonic oscillations (EHO) that usually regulate the standard QH edge cease and broadband edge MHD modes appear along with a rapid increase in the pedestal pressure height (by  ⩽60%) and width (by  ⩽50%). We posit that the enhanced edge turbulence-driven transport, enabled by the lower edge E  ×  B flow shear due to lower torque reduces the pedestal gradient and, combined with the high edge instability limit provided by the balanced double-null plasma shape, permits the development of a broader and thus higher pedestal that is turbulence-transport-limited. Even with the significantly enhanced pedestal pressure, the edge operating point is below the peeling ballooning mode stability boundary and thus without ELMs. Improved transport in the outer core region (0.8  ⩽  ρ  ⩽0.9) owing to increased E  ×  B flow shear in that region and the enhanced pedestal boost the overall confinement by up to 45%. These findings advance the physics basis for developing stationary ELM-free high-confinement operation at low rotation for future burning plasma where similar collisionality and rotation levels are expected.

  15. Actin Interacting Protein1 and Actin Depolymerizing Factor Drive Rapid Actin Dynamics in Physcomitrella patens[W

    PubMed Central

    Augustine, Robert C.; Pattavina, Kelli A.; Tüzel, Erkan; Vidali, Luis; Bezanilla, Magdalena

    2011-01-01

    The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics. PMID:22003077

  16. Multi-device studies of pedestal physics and confinement in the I-mode regime

    DOE PAGES

    Hubbard, A. E.; Osborne, T.; Ryter, F.; ...

    2016-07-05

    This paper describes joint ITPA studies of the I-mode regime, which features an edge thermal barrier together with L-mode-like particle and impurity transport and no Edge Localized Modes (ELMs). The regime has been demonstrated on the Alcator C-Mod, ASDEX Upgrade and DIII-D tokamaks, over a wide range of device parameters and pedestal conditions. Dimensionless parameters at the pedestal show overlap across devices and extend to low collisionality. When they are matched, pedestal temperature profiles are also similar. Pedestals are stable to peeling ballooning modes, consistent with lack of ELMs. Access to Imode is independent of heating method (neutral beam injection,more » Ion Cyclotron and/or Electron Cyclotron Resonance Heating). Normalized energy confinement H 98,y2 ≥ 1 has been achieved for a range of 3 ≤ q 95 ≤ 4.9 and scales favourably with power. Changes in turbulence in the pedestal region accompany the transition from L-mode to I-mode. The L-I threshold increases with plasma density and current, and with device size, but has a weak dependence on toroidal magnetic field B T. The upper limit of power for I-modes, which is set by I-H transitions, increases with B T and the power range is largest on Alcator C-Mod at B > 5 T. Finally, issues for extrapolation to ITER and other future fusion devices are discussed.« less

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

    PubMed Central

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

    2014-01-01

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

  18. F-actin distribution at nodes of Ranvier and Schmidt-Lanterman incisures in mammalian sciatic nerves.

    PubMed

    Kun, Alejandra; Canclini, Lucía; Rosso, Gonzalo; Bresque, Mariana; Romeo, Carlos; Hanusz, Alicia; Cal, Karina; Calliari, Aldo; Sotelo Silveira, José; Sotelo, José R

    2012-07-01

    Very little is known about the function of the F-actin cytoskeleton in the regeneration and pathology of peripheral nerve fibers. The actin cytoskeleton has been associated with maintenance of tissue structure, transmission of traction and contraction forces, and an involvement in cell motility. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and intracellular transport therein. In this work, we analyze the distribution of F-actin at Schmidt-Lanterman Incisures (SLI) and nodes of Ranvier (NR) domains in normal, regenerating and pathologic Trembler J (TrJ/+) sciatic nerve fibers, of rats and mice. F-actin was quantified and it was found increased in TrJ/+, both in SLI and NR. However, SLI and NR of regenerating rat sciatic nerve did not show significant differences in F-actin, as compared with normal nerves. Cytochalasin-D and Latrunculin-A were used to disrupt the F-actin network in normal and regenerating rat sciatic nerve fibers. Both drugs disrupt F-actin, but in different ways. Cytochalasin-D did not disrupt Schwann cell (SC) F-actin at the NR. Latrunculin-A did not disrupt F-actin at the boundary region between SC and axon at the NR domain. We surmise that the rearrangement of F-actin in neurological disorders, as presented here, is an important feature of TrJ/+ pathology as a Charcot-Marie-Tooth (CMT) model. Copyright © 2012 Wiley Periodicals, Inc.

  19. Arabidopsis Formin3 Directs the Formation of Actin Cables and Polarized Growth in Pollen Tubes[W

    PubMed Central

    Ye, Jianrong; Zheng, Yiyan; Yan, An; Chen, Naizhi; Wang, Zhangkui; Huang, Shanjin; Yang, Zhenbiao

    2009-01-01

    Cytoplasmic actin cables are the most prominent actin structures in plant cells, but the molecular mechanism underlying their formation is unknown. The function of these actin cables, which are proposed to modulate cytoplasmic streaming and intracellular movement of many organelles in plants, has not been studied by genetic means. Here, we show that Arabidopsis thaliana formin3 (AFH3) is an actin nucleation factor responsible for the formation of longitudinal actin cables in pollen tubes. The Arabidopsis AFH3 gene encodes a 785–amino acid polypeptide, which contains a formin homology 1 (FH1) and a FH2 domain. In vitro analysis revealed that the AFH3 FH1FH2 domains interact with the barbed end of actin filaments and have actin nucleation activity in the presence of G-actin or G actin-profilin. Overexpression of AFH3 in tobacco (Nicotiana tabacum) pollen tubes induced excessive actin cables, which extended into the tubes' apices. Specific downregulation of AFH3 eliminated actin cables in Arabidopsis pollen tubes and reduced the level of actin polymers in pollen grains. This led to the disruption of the reverse fountain streaming pattern in pollen tubes, confirming a role for actin cables in the regulation of cytoplasmic streaming. Furthermore, these tubes became wide and short and swelled at their tips, suggesting that actin cables may regulate growth polarity in pollen tubes. Thus, AFH3 regulates the formation of actin cables, which are important for cytoplasmic streaming and polarized growth in pollen tubes. PMID:20023198

  20. Bacterial spread from cell to cell: beyond actin-based motility.

    PubMed

    Kuehl, Carole J; Dragoi, Ana-Maria; Talman, Arthur; Agaisse, Hervé

    2015-09-01

    Several intracellular pathogens display the ability to propagate within host tissues by displaying actin-based motility in the cytosol of infected cells. As motile bacteria reach cell-cell contacts they form plasma membrane protrusions that project into adjacent cells and resolve into vacuoles from which the pathogen escapes, thereby achieving spread from cell to cell. Seminal studies have defined the bacterial and cellular factors that support actin-based motility. By contrast, the mechanisms supporting the formation of protrusions and their resolution into vacuoles have remained elusive. Here, we review recent advances in the field showing that Listeria monocytogenes and Shigella flexneri have evolved pathogen-specific mechanisms of bacterial spread from cell to cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Diffusion rate limitations in actin-based propulsion of hard and deformable particles.

    PubMed

    Dickinson, Richard B; Purich, Daniel L

    2006-08-15

    The mechanism by which actin polymerization propels intracellular vesicles and invasive microorganisms remains an open question. Several recent quantitative studies have examined propulsion of biomimetic particles such as polystyrene microspheres, phospholipid vesicles, and oil droplets. In addition to allowing quantitative measurement of parameters such as the dependence of particle speed on its size, these systems have also revealed characteristic behaviors such a saltatory motion of hard particles and oscillatory deformation of soft particles. Such measurements and observations provide tests for proposed mechanisms of actin-based motility. In the actoclampin filament end-tracking motor model, particle-surface-bound filament end-tracking proteins are involved in load-insensitive processive insertion of actin subunits onto elongating filament plus-ends that are persistently tethered to the surface. In contrast, the tethered-ratchet model assumes working filaments are untethered and the free-ended filaments grow as thermal ratchets in a load-sensitive manner. This article presents a model for the diffusion and consumption of actin monomers during actin-based particle propulsion to predict the monomer concentration field around motile particles. The results suggest that the various behaviors of biomimetic particles, including dynamic saltatory motion of hard particles and oscillatory vesicle deformations, can be quantitatively and self-consistently explained by load-insensitive, diffusion-limited elongation of (+)-end-tethered actin filaments, consistent with predictions of the actoclampin filament-end tracking mechanism.

  2. Actin Bodies in Yeast Quiescent Cells: An Immediately Available Actin Reserve?

    PubMed Central

    Pinson, Benoît; Salin, Bénédicte; Daignan-Fornier, Bertrand

    2006-01-01

    Most eukaryotic cells spend most of their life in a quiescent state, poised to respond to specific signals to proliferate. In Saccharomyces cerevisiae, entry into and exit from quiescence are dependent only on the availability of nutrients in the environment. The transition from quiescence to proliferation requires not only drastic metabolic changes but also a complete remodeling of various cellular structures. Here, we describe an actin cytoskeleton organization specific of the yeast quiescent state. When cells cease to divide, actin is reorganized into structures that we named “actin bodies.” We show that actin bodies contain F-actin and several actin-binding proteins such as fimbrin and capping protein. Furthermore, by contrast to actin patches or cables, actin bodies are mostly immobile, and we could not detect any actin filament turnover. Finally, we show that upon cells refeeding, actin bodies rapidly disappear and actin cables and patches can be assembled in the absence of de novo protein synthesis. This led us to propose that actin bodies are a reserve of actin that can be immediately mobilized for actin cables and patches formation upon reentry into a proliferation cycle. PMID:16914523

  3. Pedestal and edge electrostatic turbulence characteristics from an XGC1 gyrokinetic simulation

    DOE PAGES

    Churchill, R. M.; Chang, C. S.; Ku, S.; ...

    2017-08-30

    Understanding the multi-scale neoclassical and turbulence physics in the edge region (pedestal + scrape-off layer (SOL)) is required in order to reliably predict performance in future fusion devices. We explore turbulent characteristics in the edge region from a multi-scale neoclassical and turbulent XGC1 gyrokinetic simulation in a DIII-D like tokamak geometry, here excluding neutrals and collisions. For an H-mode type plasma with steep pedestal, it is found that the electron density fluctuations increase towards the separatrix, and stay high well into the SOL, reaching a maximum value ofmore » $$\\delta {n}_{e}/{\\bar{n}}_{e}\\sim 0.18$$. Blobs are observed, born around the magnetic separatrix surface and propagate radially outward with velocities generally less than 1 km s –1. Strong poloidal motion of the blobs is also present, near 20 km s –1, consistent with E × B rotation. The electron density fluctuations show a negative skewness in the closed field-line pedestal region, consistent with the presence of 'holes', followed by a transition to strong positive skewness across the separatrix and into the SOL. These simulations indicate that not only neoclassical phenomena, but also turbulence, including the blob-generation mechanism, can remain important in the steep H-mode pedestal and SOL. Lastly, qualitative comparisons will be made to experimental observations.« less

  4. Pedestal and edge electrostatic turbulence characteristics from an XGC1 gyrokinetic simulation

    NASA Astrophysics Data System (ADS)

    Churchill, R. M.; Chang, C. S.; Ku, S.; Dominski, J.

    2017-10-01

    Understanding the multi-scale neoclassical and turbulence physics in the edge region (pedestal + scrape-off layer (SOL)) is required in order to reliably predict performance in future fusion devices. We explore turbulent characteristics in the edge region from a multi-scale neoclassical and turbulent XGC1 gyrokinetic simulation in a DIII-D like tokamak geometry, here excluding neutrals and collisions. For an H-mode type plasma with steep pedestal, it is found that the electron density fluctuations increase towards the separatrix, and stay high well into the SOL, reaching a maximum value of δ {n}e/{\\bar{n}}e˜ 0.18. Blobs are observed, born around the magnetic separatrix surface and propagate radially outward with velocities generally less than 1 km s-1. Strong poloidal motion of the blobs is also present, near 20 km s-1, consistent with E × B rotation. The electron density fluctuations show a negative skewness in the closed field-line pedestal region, consistent with the presence of ‘holes’, followed by a transition to strong positive skewness across the separatrix and into the SOL. These simulations indicate that not only neoclassical phenomena, but also turbulence, including the blob-generation mechanism, can remain important in the steep H-mode pedestal and SOL. Qualitative comparisons will be made to experimental observations.

  5. Self-consistent core-pedestal transport simulations with neural network accelerated models

    DOE PAGES

    Meneghini, Orso; Smith, Sterling P.; Snyder, Philip B.; ...

    2017-07-12

    Fusion whole device modeling simulations require comprehensive models that are simultaneously physically accurate, fast, robust, and predictive. In this paper we describe the development of two neural-network (NN) based models as a means to perform a snon-linear multivariate regression of theory-based models for the core turbulent transport fluxes, and the pedestal structure. Specifically, we find that a NN-based approach can be used to consistently reproduce the results of the TGLF and EPED1 theory-based models over a broad range of plasma regimes, and with a computational speedup of several orders of magnitudes. These models are then integrated into a predictive workflowmore » that allows prediction with self-consistent core-pedestal coupling of the kinetic profiles within the last closed flux surface of the plasma. Finally, the NN paradigm is capable of breaking the speed-accuracy trade-off that is expected of traditional numerical physics models, and can provide the missing link towards self-consistent coupled core-pedestal whole device modeling simulations that are physically accurate and yet take only seconds to run.« less

  6. Self-consistent core-pedestal transport simulations with neural network accelerated models

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Meneghini, Orso; Smith, Sterling P.; Snyder, Philip B.

    Fusion whole device modeling simulations require comprehensive models that are simultaneously physically accurate, fast, robust, and predictive. In this paper we describe the development of two neural-network (NN) based models as a means to perform a snon-linear multivariate regression of theory-based models for the core turbulent transport fluxes, and the pedestal structure. Specifically, we find that a NN-based approach can be used to consistently reproduce the results of the TGLF and EPED1 theory-based models over a broad range of plasma regimes, and with a computational speedup of several orders of magnitudes. These models are then integrated into a predictive workflowmore » that allows prediction with self-consistent core-pedestal coupling of the kinetic profiles within the last closed flux surface of the plasma. Finally, the NN paradigm is capable of breaking the speed-accuracy trade-off that is expected of traditional numerical physics models, and can provide the missing link towards self-consistent coupled core-pedestal whole device modeling simulations that are physically accurate and yet take only seconds to run.« less

  7. Self-consistent core-pedestal transport simulations with neural network accelerated models

    NASA Astrophysics Data System (ADS)

    Meneghini, O.; Smith, S. P.; Snyder, P. B.; Staebler, G. M.; Candy, J.; Belli, E.; Lao, L.; Kostuk, M.; Luce, T.; Luda, T.; Park, J. M.; Poli, F.

    2017-08-01

    Fusion whole device modeling simulations require comprehensive models that are simultaneously physically accurate, fast, robust, and predictive. In this paper we describe the development of two neural-network (NN) based models as a means to perform a snon-linear multivariate regression of theory-based models for the core turbulent transport fluxes, and the pedestal structure. Specifically, we find that a NN-based approach can be used to consistently reproduce the results of the TGLF and EPED1 theory-based models over a broad range of plasma regimes, and with a computational speedup of several orders of magnitudes. These models are then integrated into a predictive workflow that allows prediction with self-consistent core-pedestal coupling of the kinetic profiles within the last closed flux surface of the plasma. The NN paradigm is capable of breaking the speed-accuracy trade-off that is expected of traditional numerical physics models, and can provide the missing link towards self-consistent coupled core-pedestal whole device modeling simulations that are physically accurate and yet take only seconds to run.

  8. Branched actin networks push against each other at adherens junctions to maintain cell-cell adhesion.

    PubMed

    Efimova, Nadia; Svitkina, Tatyana M

    2018-05-07

    Adherens junctions (AJs) are mechanosensitive cadherin-based intercellular adhesions that interact with the actin cytoskeleton and carry most of the mechanical load at cell-cell junctions. Both Arp2/3 complex-dependent actin polymerization generating pushing force and nonmuscle myosin II (NMII)-dependent contraction producing pulling force are necessary for AJ morphogenesis. Which actin system directly interacts with AJs is unknown. Using platinum replica electron microscopy of endothelial cells, we show that vascular endothelial (VE)-cadherin colocalizes with Arp2/3 complex-positive actin networks at different AJ types and is positioned at the interface between two oppositely oriented branched networks from adjacent cells. In contrast, actin-NMII bundles are located more distally from the VE-cadherin-rich zone. After Arp2/3 complex inhibition, linear AJs split, leaving gaps between cells with detergent-insoluble VE-cadherin transiently associated with the gap edges. After NMII inhibition, VE-cadherin is lost from gap edges. We propose that the actin cytoskeleton at AJs acts as a dynamic push-pull system, wherein pushing forces maintain extracellular VE-cadherin transinteraction and pulling forces stabilize intracellular adhesion complexes. © 2018 Efimova and Svitkina.

  9. Navigating the plant cell: intracellular transport logistics in the green kingdom

    PubMed Central

    Geitmann, Anja; Nebenführ, Andreas

    2015-01-01

    Intracellular transport in plant cells occurs on microtubular and actin arrays. Cytoplasmic streaming, the rapid motion of plant cell organelles, is mostly driven by an actin–myosin mechanism, whereas specialized functions, such as the transport of large cargo or the assembly of a new cell wall during cell division, are performed by the microtubules. Different modes of transport are used, fast and slow, to either haul cargo over long distances or ascertain high-precision targeting, respectively. Various forms of the actin-specific motor protein myosin XI exist in plant cells and might be involved in different cellular functions. PMID:26416952

  10. Repair of the DSS-14 Pedestal Concrete

    NASA Technical Reports Server (NTRS)

    Mcclure, D.

    1985-01-01

    About three years after the Goldstone Deep Space Station antenna was dedicated, grout under the hydrostatic bearing runner was found to be interacting with the runner, causing rust to form between the runner and the sole plates upon which it rests. The rust formed unevenly and the runner could not be kept flat so in 1969 the grout was removed and replaced with a Portland cement and sand dry pack grout that was less likely to produce rust. In the years that followed, oil leaking from the runner assembly caused progressive deterioration of the drypack grout. In 1982 over one thousand hours of spacecraft tracking time were lost due to this deterioration. A plan was developed to rehabilitate the bearing. The plan called for raising the rotating structure free from the concrete pedestal and placing it on three pairs of external support columns. With the weight of the structure transferred to the columns, the pads and runner could be removed and the repair started. The very successful repair included the replacement of a significant portion of the antenna pedestal.

  11. 50. (no plate) Lens, lens pedestal, mercury float, drawing # ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    50. (no plate) Lens, lens pedestal, mercury float, drawing # 3101, sheet 1 of 2. Approved April 6, 1928. - Block Island Southeast Light, Spring Street & Mohegan Trail at Mohegan Bluffs, New Shoreham, Washington County, RI

  12. Oscillatory Increases in Alkalinity Anticipate Growth and May Regulate Actin Dynamics in Pollen Tubes of Lily[W][OA

    PubMed Central

    Lovy-Wheeler, Alenka; Kunkel, Joseph G.; Allwood, Ellen G.; Hussey, Patrick J.; Hepler, Peter K.

    2006-01-01

    Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events. PMID:16920777

  13. Insulin-induced cortical actin remodeling promotes GLUT4 insertion at muscle cell membrane ruffles

    PubMed Central

    Tong, Peter; Khayat, Zayna A.; Huang, Carol; Patel, Nish; Ueyama, Atsunori; Klip, Amira

    2001-01-01

    Insulin stimulates glucose uptake by recruiting glucose transporter 4 (GLUT4) from an intracellular compartment to the cell surface; this phenomenon is defective in type 2 diabetes. Here we examine the involvement of actin filaments in GLUT4 translocation and their possible defects in insulin resistance, using L6 myotubes expressing myc-tagged GLUT4. Insulin caused membrane ruffling, a dynamic distortion of the myotube dorsal surface. Fluorescence microscopy and immunogold staining of surface GLUT4myc coupled to backscatter electron microscopy revealed a high density of this protein in membrane ruffles. The t-SNAREs syntaxin4 and SNAP-23 were also abundant in these regions. Below the membrane, GLUT4 and the vesicular protein VAMP2, but not VAMP3, colocalized with the actin structures supporting the membrane ruffles. GLUT4myc externalization and membrane ruffles were reduced by jasplakinolide and by swinholide-A, drugs that affect actin filament stability and prevent actin branching, respectively. Insulin resistance generated by prolonged (24 hours) exposure of myotubes to high glucose and insulin diminished the acute insulin-dependent remodeling of cortical actin and GLUT4myc translocation, reminiscent of the effect of swinholide-A. We propose that GLUT4 vesicle incorporation into the plasma membrane involves insulin-dependent cortical actin remodeling and that defective actin remodeling contributes to insulin resistance. PMID:11489930

  14. Actin filaments regulate the adhesion between the plasma membrane and the cell wall of tobacco guard cells.

    PubMed

    Yu, Qin; Ren, Jing-Jing; Kong, Lan-Jing; Wang, Xiu-Ling

    2018-01-01

    During the opening and closing of stomata, guard cells undergo rapid and reversible changes in their volume and shape, which affects the adhesion of the plasma membrane (PM) to the cell wall (CW). The dynamics of actin filaments in guard cells are involved in stomatal movement by regulating structural changes and intracellular signaling. However, it is unclear whether actin dynamics regulate the adhesion of the PM to the CW. In this study, we investigated the relationship between actin dynamics and PM-CW adhesion by the hyperosmotic-induced plasmolysis of tobacco guard cells. We found that actin filaments in guard cells were depolymerized during mannitol-induced plasmolysis. The inhibition of actin dynamics by treatment with latrunculin B or jasplakinolide and the disruption of the adhesion between the PM and the CW by treatment with RGDS peptide (Arg-Gly-Asp-Ser) enhanced guard cell plasmolysis. However, treatment with latrunculin B alleviated the RGDS peptide-induced plasmolysis and endocytosis. Our results reveal that the actin depolymerization is involved in the regulation of the PW-CW adhesion during hyperosmotic-induced plasmolysis in tobacco guard cells.

  15. 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.

  16. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography.

    PubMed

    Hu, S; Brady, S R; Kovar, D R; Staiger, C J; Clark, G B; Roux, S J; Muday, G K

    2000-10-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  17. The role of actin networks in cellular mechanosensing

    NASA Astrophysics Data System (ADS)

    Azatov, Mikheil

    behavior as in cancer metastasis. In addition to stiffness, the local geometry or topography of the surface has been shown to modulate the movement, morphology, and cytoskeletal organization of cells. However, the effect of topography on fluctuations of intracellular structures, which arise from motor driven activity on a viscoelastic actin network are not known. I have used nanofabricated substrates with parallel ridges to show that the cell shape, the actin cytoskeleton and focal adhesions all align along the direction of the ridges, exhibiting a biphasic dependence on the spacing between ridges. I further demonstrated that palladin bands along actin stress fibers undergo a complex diffusive motion with velocities aligned along the direction of ridges. These results provide insight into the mechanisms of cellular mechanosensing of the environment, suggesting a complex interplay between the actin cytoskeleton and cellular adhesions in coordinating cellular response to surface topography. Overall, this work has advanced our understanding of mechanisms that govern cellular responses to their physical environment.

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

    PubMed

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

    2013-10-01

    A number of studies in the last decade have irrevocably promoted actin into a fully fledged member of the nuclear compartment, where it, among other crucial tasks, facilitates transcription and chromatin remodeling. Changes in nuclear actin levels have been linked to different cellular processes: decreased nuclear actin to quiescence and increased nuclear actin to differentiation. Importin 9 and exportin 6 transport factors are responsible for the continuous nucleocytoplasmic shuttling of actin, but the mechanisms, which result in modulated actin levels, have not been characterized. We find that in cells growing under normal growth conditions, the levels of nuclear actin vary considerably from cell to cell. To understand the basis for this, we have extensively quantified several cellular parameters while at the same time recording the import and export rates of green fluorescent protein (GFP)-tagged actin. Surprisingly, our dataset shows that the ratio of nuclear to cytoplasmic fluorescence intensity, but not nuclear shape, size, cytoplasm size, or their ratio, correlates negatively with both import and export rate of actin. This suggests that high-nuclear actin content is maintained by both diminished import and export. The high nuclear actin containing cells still show high mobility of actin, but it is not export competent, suggesting increased binding of actin to nuclear complexes. Creation of such export incompetent actin pool would ensure enough actin is retained in the nucleus and make it available for the various nuclear functions described for actin. Copyright © 2013 Wiley Periodicals, Inc.

  19. Pedestal evolution physics in low triangularity JET tokamak discharges with ITER-like wall

    NASA Astrophysics Data System (ADS)

    Bowman, C.; Dickinson, D.; Horvath, L.; Lunniss, A. E.; Wilson, H. R.; Cziegler, I.; Frassinetti, L.; Gibson, K.; Kirk, A.; Lipschultz, B.; Maggi, C. F.; Roach, C. M.; Saarelma, S.; Snyder, P. B.; Thornton, A.; Wynn, A.; Contributors, JET

    2018-01-01

    The pressure gradient of the high confinement pedestal region at the edge of tokamak plasmas rapidly collapses during plasma eruptions called edge localised modes (ELMs), and then re-builds over a longer time scale before the next ELM. The physics that controls the evolution of the JET pedestal between ELMs is analysed for 1.4 MA, 1.7 T, low triangularity, δ  =  0.2, discharges with the ITER-like wall, finding that the pressure gradient typically tracks the ideal magneto-hydrodynamic ballooning limit, consistent with a role for the kinetic ballooning mode. Furthermore, the pedestal width is often influenced by the region of plasma that has second stability access to the ballooning mode, which can explain its sometimes complex evolution between ELMs. A local gyrokinetic analysis of a second stable flux surface reveals stability to kinetic ballooning modes; global effects are expected to provide a destabilising mechanism and need to be retained in such second stable situations. As well as an electron-scale electron temperature gradient mode, ion scale instabilities associated with this flux surface include an electro-magnetic trapped electron branch and two electrostatic branches propagating in the ion direction, one with high radial wavenumber. In these second stability situations, the ELM is triggered by a peeling-ballooning mode; otherwise the pedestal is somewhat below the peeling-ballooning mode marginal stability boundary at ELM onset. In this latter situation, there is evidence that higher frequency ELMs are paced by an oscillation in the plasma, causing a crash in the pedestal before the peeling-ballooning boundary is reached. A model is proposed in which the oscillation is associated with hot plasma filaments that are pushed out towards the plasma edge by a ballooning mode, draining their free energy into the cooler plasma there, and then relaxing back to repeat the process. The results suggest that avoiding the oscillation and maximising the region

  20. Lights, camera, actin.

    PubMed

    Rubenstein, Peter A; Wen, Kuo-Kuang

    2005-10-01

    Actin participates in many important biological processes. Currently, intensive investigation is being carried out in a number of laboratories concerning the function of actin in these processes and the molecular basis of its functions. We present a glimpse into four of these areas: actin-like proteins in bacterial cells, actin in the eukaryotic nucleus, the conformational plasticity of the actin filament, and finally, Arp2/3-dependent regulation of actin filament branching and creation of new filament barbed ends. IUBMB Life, 57: 683-687, 2005.

  1. Actin-binding proteins sensitively mediate F-actin bundle stiffness

    NASA Astrophysics Data System (ADS)

    Claessens, Mireille M. A. E.; Bathe, Mark; Frey, Erwin; Bausch, Andreas R.

    2006-09-01

    Bundles of filamentous actin (F-actin) form primary structural components of a broad range of cytoskeletal processes including filopodia, sensory hair cell bristles and microvilli. Actin-binding proteins (ABPs) allow the cell to tailor the dimensions and mechanical properties of the bundles to suit specific biological functions. Therefore, it is important to obtain quantitative knowledge on the effect of ABPs on the mechanical properties of F-actin bundles. Here we measure the bending stiffness of F-actin bundles crosslinked by three ABPs that are ubiquitous in eukaryotes. We observe distinct regimes of bundle bending stiffness that differ by orders of magnitude depending on ABP type, concentration and bundle size. The behaviour observed experimentally is reproduced quantitatively by a molecular-based mechanical model in which ABP shearing competes with F-actin extension/compression. Our results shed new light on the biomechanical function of ABPs and demonstrate how single-molecule properties determine mesoscopic behaviour. The bending mechanics of F-actin fibre bundles are general and have implications for cytoskeletal mechanics and for the rational design of functional materials.

  2. The Plant Actin Cytoskeleton Responds to Signals from Microbe-Associated Molecular Patterns

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Henty-Ridilla, Jessica L.; Shimono, Masaki; Li, Jiejie

    2013-04-04

    Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence thatmore » the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.« less

  3. Interior view of bath 2 showing original pedestal lavatory and ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Interior view of bath 2 showing original pedestal lavatory and tub, facing south. - Albrook Air Force Station, Company Officer's Quarters, East side of Canfield Avenue, Balboa, Former Panama Canal Zone, CZ

  4. Covalent and non-covalent chemical engineering of actin for biotechnological applications.

    PubMed

    Kumar, Saroj; Mansson, Alf

    2017-11-15

    The cytoskeletal filaments are self-assembled protein polymers with 8-25nm diameters and up to several tens of micrometres length. They have a range of pivotal roles in eukaryotic cells, including transportation of intracellular cargoes (primarily microtubules with dynein and kinesin motors) and cell motility (primarily actin and myosin) where muscle contraction is one example. For two decades, the cytoskeletal filaments and their associated motor systems have been explored for nanotechnological applications including miniaturized sensor systems and lab-on-a-chip devices. Several developments have also revolved around possible exploitation of the filaments alone without their motor partners. Efforts to use the cytoskeletal filaments for applications often require chemical or genetic engineering of the filaments such as specific conjugation with fluorophores, antibodies, oligonucleotides or various macromolecular complexes e.g. nanoparticles. Similar conjugation methods are also instrumental for a range of fundamental biophysical studies. Here we review methods for non-covalent and covalent chemical modifications of actin filaments with focus on critical advantages and challenges of different methods as well as critical steps in the conjugation procedures. We also review potential uses of the engineered actin filaments in nanotechnological applications and in some key fundamental studies of actin and myosin function. Finally, we consider possible future lines of investigation that may be addressed by applying chemical conjugation of actin in new ways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Parameter dependence of ELM loss reduction by magnetic perturbations at low pedestal density and collisionality in ASDEX upgrade

    NASA Astrophysics Data System (ADS)

    Leuthold, N.; Suttrop, W.; Fischer, R.; Kappatou, A.; Kirk, A.; McDermott, R.; Mlynek, A.; Valovič, M.; Willensdorfer, M.; the ASDEX Upgrade Team; the EUROfusion MST1 Team

    2017-05-01

    ELM mitigation by magnetic perturbations is studied at low pedestal collisionalities down to ITER-like values ({ν }{e,{PED}}* =0.1) in ASDEX Upgrade. A comprehensive database of ELM energy losses for varying plasma density, heating power, edge safety factor and magnetic perturbation structure has been assembled to investigate parameter dependencies of ELM mitigation. It is found that magnetic perturbations with a toroidal mode number n = 2 can reduce the ELM energy loss normalized to the energy stored in the plasma pedestal from about 30% to less than 5%, i.e. by a factor of six, below an electron pedestal collisionality of {ν }{e,{PED}}* =0.4. At this level of ELM mitigation a significant reduction of the pedestal pressure and, therefore, global plasma confinement occurs. This pedestal pressure reduction is mostly due to a reduction of plasma density, the so-called ‘pump-out’ effect. Refueling by neutral beams and in particular by pellet injection is possible and can re-establish confinement, however, the ELM energy loss increases as well with increasing density.

  6. Enhanced H-mode pedestals with lithium injection in DIII-D

    DOE PAGES

    Osborne, Thomas H.; Jackson, Gary L.; Yan, Zheng; ...

    2015-05-08

    Periods of edge localized mode (ELM)-free H-mode with increased pedestal pressure and width were observed in the DIII-D tokamak when density fluctuations localized to the region near the separatrix were present. Injection of a powder of 45 μm diameter lithium particles increased the duration of the enhanced pedestal phases to up to 350 ms, and also increased the likelihood of a transition to the enhanced phase. Lithium injection at a level sufficient for triggering the extended enhanced phases resulted in significant lithium in the plasma core, but carbon and other higher Z impurities as well as radiated power levels weremore » reduced. Recycling of the working deuterium gas appeared unaffected by this level of lithium injection. The ion scale, k θ ρ s ~ 0.1–0.2, density fluctuations propagated in the electron drift direction with f ~ 80 kHz and occurred in bursts every ~1 ms. The fluctuation bursts correlated with plasma loss resulting in a flattening of the pressure profile in a region near the separatrix. This localized flattening 2 allowed higher overall pedestal pressure at the peeling-ballooning stability limit and higher pressure than expected under the EPED model due to reduction of the pressure gradient below the “ballooning critical profile”. Furthermore, reduction of the ion pressure by lithium dilution may contribute to the long ELM-free periods.« less

  7. Isotope and mixture effects on neoclassical transport in the pedestal

    NASA Astrophysics Data System (ADS)

    Pusztai, Istvan; Buller, Stefan; Omotani, John T.; Newton, Sarah L.

    2017-10-01

    The isotope mass scaling of the energy confinement time in tokamak plasmas differs from gyro-Bohm estimates, with implications for the extrapolation from current experiments to D-T reactors. Differences in mass scaling in L-mode and various H-mode regimes suggest that the isotope effect may originate from the pedestal. In the pedestal, sharp gradients render local diffusive estimates invalid, and global effects due to orbit-width scale profile variations have to be taken into account. We calculate neoclassical cross-field fluxes from a radially global drift-kinetic equation using the PERFECT code, to study isotope composition effects in density pedestals. The relative reduction to the peak heat flux due to global effects as a function of the density scale length is found to saturate at an isotope-dependent value that is larger for heavier ions. We also consider D-T and H-D mixtures with a focus on isotope separation. The ability to reproduce the mixture results via single-species simulations with artificial ``DT'' and ``HD'' species has been considered. These computationally convenient single ion simulations give a good estimate of the total ion heat flux in corresponding mixtures. Funding received from the International Career Grant of Vetenskapsradet (VR) (330-2014-6313) with Marie Sklodowska Curie Actions, Cofund, Project INCA 600398, and Framework Grant for Strategic Energy Research of VR (2014-5392).

  8. Inter-ELM evolution of the pedestal structures in type-I ELMy H-mode plasmas with LHW and NBI heating on EAST

    NASA Astrophysics Data System (ADS)

    Han, X.; Zang, Q.; Xiao, S.; Wang, T.; Hu, A.; Tian, B.; Li, D.; Zhou, H.; Zhao, J.; Hsieh, C.; Li, M.; Yan, N.; Gong, X.; Hu, L.; Xu, G.; Gao, X.; the EAST Team

    2017-04-01

    The evolution characteristics of type-I ELMy high-confinement mode pedestal are examined in EAST based on the recently developed Thomson scattering system. The influence of the plasma current on pedestal evolvement has been confirmed experimentally. In the higher I p case (500 kA) the pedestal height shows an increase trend until the onset of next ELM and in the lower I p cases (300 and 400 kA), however, this buildup saturates at the first ˜30% of the ELM cycle. In contrast, the width increases only during the first ˜70% of the ELM cycle and then keeps almost stable in three I p cases, but resulting in different widening size of ˜1.5, 1 and 0.5 cm for 300, 400 and 500 kA respectively. Experimental results show that the pedestal pressure width has good correlation with poloidal beta as {{{Δ }}}{{p}{{e}},\\psi }=0.16\\sqrt{{{β }}{{p}{{o}}{{l}}}}, where the fitting coefficient 0.16 is not changed with different plasma currents but a little larger than that of other machines. For each current level, the pedestal density increases while the pedestal temperature decreases. But with increasing {I}{{p}} platforms, the pedestal height prior to the ELM onset shows a near quadratic (within error bars) increase. Experimental measurements demonstrate that the decrease of {{Δ }}{W}{{E}{{L}}{{M}}} with increasing {ν }{{p}{{e}}{{d}}}* comes mostly from the reduction of the plasma temperature drop, while the pedestal density height keeps relatively stable. Additional injection of LHW has been proved to modify the pedestal structure which should be responsible for the remaining scatter of the experimental data.

  9. Nuclear Functions of Actin

    PubMed Central

    Visa, Neus; Percipalle, Piergiorgio

    2010-01-01

    Actin participates in several essential processes in the cell nucleus. Even though the presence of actin in the nucleus was proposed more than 30 years ago, nuclear processes that require actin have been only recently identified. Actin is part of chromatin remodeling complexes; it is associated with the transcription machineries; it becomes incorporated into newly synthesized ribonucleoproteins; and it influences long-range chromatin organization. As in the cytoplasm, nuclear actin works in conjunction with different types of actin-binding proteins that regulate actin function and bridge interactions between actin and other nuclear components. PMID:20452941

  10. How actin network dynamics control the onset of actin-based motility

    PubMed Central

    Kawska, Agnieszka; Carvalho, Kévin; Manzi, John; Boujemaa-Paterski, Rajaa; Blanchoin, Laurent; Martiel, Jean-Louis; Sykes, Cécile

    2012-01-01

    Cells use their dynamic actin network to control their mechanics and motility. These networks are made of branched actin filaments generated by the Arp2/3 complex. Here we study under which conditions the microscopic organization of branched actin networks builds up a sufficient stress to trigger sustained motility. In our experimental setup, dynamic actin networks or “gels” are grown on a hard bead in a controlled minimal protein system containing actin monomers, profilin, the Arp2/3 complex and capping protein. We vary protein concentrations and follow experimentally and through simulations the shape and mechanical properties of the actin gel growing around beads. Actin gel morphology is controlled by elementary steps including “primer” contact, growth of the network, entanglement, mechanical interaction and force production. We show that varying the biochemical orchestration of these steps can lead to the loss of network cohesion and the lack of effective force production. We propose a predictive phase diagram of actin gel fate as a function of protein concentrations. This work unveils how, in growing actin networks, a tight biochemical and physical coupling smoothens initial primer-caused heterogeneities and governs force buildup and cell motility. PMID:22908255

  11. Cryptococcus neoformans is internalized by receptor-mediated or 'triggered' phagocytosis, dependent on actin recruitment.

    PubMed

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both 'zipper' (receptor-mediated) and 'trigger' (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells.

  12. Superdiffusion dominates intracellular particle motion in the supercrowded cytoplasm of pathogenic Acanthamoeba castellanii

    NASA Astrophysics Data System (ADS)

    Reverey, Julia F.; Jeon, Jae-Hyung; Bao, Han; Leippe, Matthias; Metzler, Ralf; Selhuber-Unkel, Christine

    2015-06-01

    Acanthamoebae are free-living protists and human pathogens, whose cellular functions and pathogenicity strongly depend on the transport of intracellular vesicles and granules through the cytosol. Using high-speed live cell imaging in combination with single-particle tracking analysis, we show here that the motion of endogenous intracellular particles in the size range from a few hundred nanometers to several micrometers in Acanthamoeba castellanii is strongly superdiffusive and influenced by cell locomotion, cytoskeletal elements, and myosin II. We demonstrate that cell locomotion significantly contributes to intracellular particle motion, but is clearly not the only origin of superdiffusivity. By analyzing the contribution of microtubules, actin, and myosin II motors we show that myosin II is a major driving force of intracellular motion in A. castellanii. The cytoplasm of A. castellanii is supercrowded with intracellular vesicles and granules, such that significant intracellular motion can only be achieved by actively driven motion, while purely thermally driven diffusion is negligible.

  13. Plant actin cytoskeleton re-modeling by plant parasitic nematodes.

    PubMed

    Engler, Janice de Almeida; Rodiuc, Natalia; Smertenko, Andrei; Abad, Pierre

    2010-03-01

    The cytoskeleton is an important component of the plant's defense mechanism against the attack of pathogenic organisms. Plants however, are defenseless against parasitic root-knot and cyst nematodes and respond to the invasion by the development of a special feeding site that supplies the parasite with nutrients required for the completion of its life cycle. Recent studies of nematode invasion under treatment with cytoskeletal drugs and in mutant plants where normal functions of the cytoskeleton have been affected, demonstrate the importance of the cytoskeleton in the establishment of a feeding site and successful nematode reproduction. It appears that in the case of microfilaments, nematodes hijack the intracellular machinery that regulates actin dynamics and modulate the organization and properties of the actin filament network. Intervening with this process reduces the nematode infection efficiency and inhibits its life cycle. This discovery uncovers a new pathway that can be exploited for the protection of plants against nematodes.

  14. Placed on a Pedestal: Famous Faces in Clay

    ERIC Educational Resources Information Center

    Walkup, Nancy

    2010-01-01

    Artists have created portraits of people for thousands of years. In sculpture, a portrait of a person's face often includes the neck and part of the shoulders and chest. These artworks are called portrait busts. In this article, the author describes how her fifth-grade students created clay portrait busts on pedestal columns. The objectives are…

  15. 51. (no plate) Lens, lens pedestal, mercury float, shade holder ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    51. (no plate) Lens, lens pedestal, mercury float, shade holder installation, drawing # 3101, sheet 2 of 2. Approved April 6, 1928. - Block Island Southeast Light, Spring Street & Mohegan Trail at Mohegan Bluffs, New Shoreham, Washington County, RI

  16. Linear gyrokinetic simulations of microinstabilities within the pedestal region of H-mode NSTX discharges in a highly shaped geometry

    DOE PAGES

    Coury, M.; Guttenfelder, W.; Mikkelsen, D. R.; ...

    2016-06-30

    Linear (local) gyrokinetic predictions of edge microinstabilities in highly shaped, lithiated and non-lithiated NSTX discharges are reported using the gyrokinetic code GS2. Microtearing modes dominate the non-lithiated pedestal top. The stabilization of these modes at the lithiated pedestal top enables the electron temperature pedestal to extend further inwards, as observed experimentally. Kinetic ballooning modes are found to be unstable mainly at the mid-pedestal of both types of discharges, with un- stable trapped electron modes nearer the separatrix region. At electron wavelengths, ETG modes are found to be unstable from mid-pedestal outwards for η e, exp ~2.2 with higher growth ratesmore » for the lithiated discharge. Near the separatrix, the critical temperature gradient for driving ETG modes is reduced in the presence of lithium, re ecting the reduction of the lithiated density gradients observed experimentally. A preliminary linear study in the edge of non-lithiated discharges shows that the equilibrium shaping alters the electrostatic modes stability, found more unstable at high plasma shaping.« less

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

    PubMed Central

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

    2009-01-01

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

  18. Rab7b at the intersection of intracellular trafficking and cell migration.

    PubMed

    Distefano, Marita Borg; Kjos, Ingrid; Bakke, Oddmund; Progida, Cinzia

    2015-01-01

    Rab proteins are small GTPases essential for controlling and coordinating intracellular traffic. The small GTPase Rab7b regulates the retrograde transport from late endosomes toward the Trans-Golgi Network (TGN), and is important for the proper trafficking of several receptors such as Toll-like receptors (TLRs) and sorting receptors. We recently identified the actin motor protein myosin II as a new interaction partner for Rab7b, and found that Rab7b transport is dependent on myosin II. Interestingly, we also discovered that Rab7b influences the phosphorylation state of myosin II by controlling the activation status of the small GTPase RhoA. Consequently, Rab7b is important for the remodeling of actin filaments in processes such as stress fiber formation, cell adhesion, polarization and cell migration. Our finding that Rab7b can control actomyosin reorganization reveals yet another important role for Rab proteins, in addition to their already established role as master regulators of intracellular transport. Here we discuss our findings and speculate how they can explain the importance of Rab7b in dendritic cells (DCs).

  19. Diadenosine tetraphosphate-gating of cardiac K(ATP) channels requires intact actin cytoskeleton.

    PubMed

    Jovanović, S; Jovanović, A

    2001-09-01

    Diadenosine polyphosphates (ApnA) have been recently discovered in the heart, and their levels found to be regulated by ischemia. These signaling molecules are believed to regulate cellular processes that alarm a cell to metabolic stress. In particular, changes in cardiac diadenosine polyphosphates (ApnA) levels may contribute to the regulation of ATP-sensitive K+ (K(ATP)) channel activity, an ion channel that couples the cellular metabolic state with membrane excitability. A feature of myocardial ischemia is the disruption of the actin cytoskeleton which critically regulates the behavior of K(ATP) channels. Whether the integrity of actin microfilaments regulates the interaction of ApnA with K(ATP) channels is not known. The inside-out configuration of the patch-clamp technique was applied to cardiomyocytes isolated from guinea-pig heart. Following patch excision, the prototype dinucleotide, diadenosine tetraphosphate (Ap4A), inhibited K(ATP) channel opening. Treatment of the internal side of membrane patches with either cytochalasin B or DNase I, disrupters of the actin cytoskeleton, prevented Ap4A-induced inhibition of K(ATP) channel opening. Application of purified actin to DNase-treated membrane patches restored the ability of Ap4A to close K(ATP) channels. This study shows that inhibition of cardiac K(ATP) channel by Ap4A, a putative alarmone, requires intact subsarcolemmal actin network. Such interaction between K(ATP) channels, the cardiomyocyte cytoskeleton and intracellular Ap4A could affect different channel-dependent functions.

  20. F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF)

    PubMed Central

    Jiang, Chang-Jie; Weeds, Alan G.; Khan, Safina; Hussey, Patrick J.

    1997-01-01

    Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3–1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3–2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and Acanthamoeba actophorin, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long α-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 Å, respectively, with corresponding bond angles of 99.5° and 113°, close to the optimum for a strong hydrogen bond. PMID:9275236

  1. Navigating the plant cell: intracellular transport logistics in the green kingdom.

    PubMed

    Geitmann, Anja; Nebenführ, Andreas

    2015-10-01

    Intracellular transport in plant cells occurs on microtubular and actin arrays. Cytoplasmic streaming, the rapid motion of plant cell organelles, is mostly driven by an actin-myosin mechanism, whereas specialized functions, such as the transport of large cargo or the assembly of a new cell wall during cell division, are performed by the microtubules. Different modes of transport are used, fast and slow, to either haul cargo over long distances or ascertain high-precision targeting, respectively. Various forms of the actin-specific motor protein myosin XI exist in plant cells and might be involved in different cellular functions. © 2015 Geitmann and Nebenführ. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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

    PubMed

    Bittig, Arne T; Matschegewski, Claudia; Nebe, J Barbara; Stählke, Susanne; Uhrmacher, Adelinde M

    2014-09-09

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

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

    PubMed Central

    2014-01-01

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

  4. Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion

    PubMed Central

    Volceanov, Larisa; Herbst, Katharina; Biniossek, Martin; Schilling, Oliver; Haller, Dirk; Nölke, Thilo; Subbarayal, Prema; Rudel, Thomas; Zieger, Barbara

    2014-01-01

    ABSTRACT Chlamydia trachomatis is an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle of C. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress of C. trachomatis from human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens. PMID:25293760

  5. The myosin passenger protein Smy1 controls actin cable structure and dynamics by acting as a formin damper

    PubMed Central

    Chesarone-Cataldo, Melissa; Guérin, Christophe; Yu, Jerry H.; Wedlich-Soldner, Roland; Blanchoin, Laurent; Goode, Bruce L.

    2011-01-01

    Summary Formins are a conserved family of proteins with robust effects in promoting actin nucleation and elongation. However, the mechanisms restraining formin activities in cells to generate actin networks with particular dynamics and architectures are not well understood. In S. cerevisiae, formins assemble actin cables, which serve as tracks for myosin-dependent intracellular transport. Here, we show that the kinesin-like myosin passenger-protein Smy1 interacts with the FH2 domain of the formin Bnr1 to decrease rates of actin filament elongation, which is distinct from the formin displacement activity of Bud14. In vivo analysis of smy1Δ mutants demonstrates that this ‘damper’ mechanism is critical for maintaining proper actin cable architecture, dynamics, and function. We directly observe Smy1–3GFP being transported by myosin V and transiently pausing at the neck in a manner dependent on Bnr1. These observations suggest that Smy1 is part of a negative feedback mechanism that detects cable length and prevents overgrowth. PMID:21839918

  6. Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction

    PubMed Central

    Palani, Saravanan; Sommese, Ruth; Kamnev, Anton; Hatano, Tomoyuki; Sivaramakrishnan, Sivaraj

    2017-01-01

    Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into ∼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially. PMID:28655757

  7. DSS 13 phase 2 pedestal room microwave layout

    NASA Technical Reports Server (NTRS)

    Cwik, T.; Chen, J. C.

    1991-01-01

    The design and predicted performance is described of the microwave layout for three band operation of the beam waveguide antenna Deep Space Station 13. Three pedestal room microwave candidate layout designs were produced for simultaneous X/S and X/Ka band operation. One of the three designs was chosen based on given constraints, and for this design the microwave performance was estimated.

  8. Synthetic peptides that cause F-actin bundling and block actin depolymerization

    DOEpatents

    Sederoff, Heike [Raleigh, NC; Huber, Steven C [Savoy, IL; Larabell, Carolyn A [Berkeley, CA

    2011-10-18

    Synthetic peptides derived from sucrose synthase, and having homology to actin and actin-related proteins, sharing a common motif, useful for causing acting bundling and preventing actin depolymerization. Peptides exhibiting the common motif are described, as well as specific synthetic peptides which caused bundled actin and inhibit actin depolymerization. These peptides can be useful for treating a subject suffering from a disease characterized by cells having neoplastic growth, for anti-cancer therapeutics, delivered to subjects solely, or concomitantly or sequentially with other known cancer therapeutics. These peptides can also be used for stabilizing microfilaments in living cells and inhibiting growth of cells.

  9. MOLA Topography and Morphometry of Rampart and Pedestal Craters, Mars

    NASA Technical Reports Server (NTRS)

    Mitchell, D. E.; Sakimoto, S. E. H.; Garvin, J. B.

    2002-01-01

    Martian rampart and pedestal craters have characteristic geometric parameter ranges that are significantly different than fresh craters. Combined MOLA geometric measurements and MOC analyses can be used to constrain their modification. Additional information is contained in the original extended abstract.

  10. Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

    PubMed Central

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells. PMID:24586631

  11. Somatostatin, acting at receptor subtype 1, inhibits Rho activity, the assembly of actin stress fibers, and cell migration.

    PubMed

    Buchan, Alison M J; Lin, Chin-Yu; Choi, Jimmy; Barber, Diane L

    2002-08-09

    Somatostatin regulates multiple biological functions by acting through a family of five G protein-coupled receptors, somatostatin receptors (SSTRs) 1-5. Although all five receptor subtypes inhibit adenylate cyclase activity and decrease intracellular cAMP levels, specific receptor subtypes also couple to additional signaling pathways. In CCL39 fibroblasts expressing either human SSTR1 or SSTR2, we demonstrate that activation of SSTR1 (but not SSTR2) attenuated both thrombin- and integrin-stimulated Rho-GTP complex formation. The reduction in Rho-GTP formation in the presence of somatostatin was associated with decreased translocation of Rho and LIM kinase to the plasma membrane and fewer focal contacts. Activation of Rho resulted in the formation of intracellular actin stress fibers and cell migration. In CCL39-R1 cells, somatostatin treatment prevented actin stress fiber assembly and attenuated thrombin-stimulated cell migration through Transwell membranes to basal levels. To show that native SSTR1 shares the ability to inhibit Rho activation, we demonstrated that somatostatin treatment of human umbilical vein endothelial cells attenuated thrombin-stimulated Rho-GTP accumulation. These data show for the first time that a G protein-coupled receptor, SSTR1, inhibits the activation of Rho, the assembly of focal adhesions and actin stress fibers, and cell migration.

  12. Accumulation of actin in subsets of pioneer growth cone filopodia in response to neural and epithelial guidance cues in situ

    PubMed Central

    1993-01-01

    Directed outgrowth of neural processes must involve transmission of signals from the tips of filopodia to the central region of the growth cone. Here, we report on the distribution and dynamics of one possible element in this process, actin, in live growth cones which are reorienting in response to in situ guidance cues. In grasshopper embryonic limbs, pioneer growth cones respond to at least three types of guidance cues: a limb axis cue, intermediate target cells, and a circumferential band of epithelial cells. With time-lapse imaging of intracellularly injected rhodamine-phalloidin and rhodamine-actin, we monitored the distribution of actin during growth cone responses to these cues. In distal limb regions, accumulation of actin in filopodia and growth cone branches accompanies continued growth, while reduction of actin accompanies withdrawal. Where growth cones are reorienting to intermediate target cells, or along the circumferential epithelial band, actin selectively accumulates in the proximal regions of those filopodia that have contacted target cells or are extending along the band. Actin accumulations can be retrogradely transported along filopodia, and can extend into the central region of the growth cone. These results suggest that regulation and translocation of actin may be a significant element in growth cone steering. PMID:8227150

  13. Neoclassical Toroidal Viscosity Torque Induced by Plasma Response in a Low- β Tokamak with Edge Pedestal

    NASA Astrophysics Data System (ADS)

    Yan, Xingting; Zhu, Ping; Sun, Youwen

    2016-10-01

    The characteristic profile and magnitude are predicted in theory for the neoclassical toroidal viscosity (NTV) torque induced by the plasma response to the resonant magnetic perturbation (RMP) in a tokamak with an edge pedestal, using the newly developed module coupling the NIMROD and the NTVTOK codes. For a low β equilibrium, the NTV torque is mainly induced by the dominant toroidal mode of plasma response. The NTV torque profile is radially localized and peaked, which is determined by profiles of both the equilibrium temperature and the plasma response fields. In general, the peak of NTV torque profile is found to trace the pedestal location. The magnitude of NTV torque is extremely sensitive to the β of pedestal top; for a given plasma response, the peak value of NTV torque can increase by three orders of magnitude, when the pedestal β increases by only one order of magnitude. This suggests a more significant role of NTV torque in higher plasma β regimes. Supported by the National Magnetic Confinement Fusion Program of China under Grant Nos. 2014GB124002 and 2015GB101004, and the 100 Talent Program of the Chinese Academy of Sciences.

  14. High level of reactive oxygen species impaired mesenchymal stem cell migration via overpolymerization of F-actin cytoskeleton in systemic lupus erythematosus.

    PubMed

    Shi, D; Li, X; Chen, H; Che, N; Zhou, S; Lu, Z; Shi, S; Sun, L

    2014-12-01

    Some lines of evidence have demonstrated abnormalities of bone marrow mesenchymal stem cells (MSCs) in systemic lupus erythematosus (SLE) patients, characterized by defective phenotype of MSCs and slower growth with enhanced apoptosis and senescence. However, whether SLE MSCs demonstrate aberrant migration capacity or abnormalities in cytoskeleton are issues that remain poorly understood. In this study, we found that MSCs from SLE patients did show impairment in migration capacity as well as abnormalities in F-actin cytoskeleton, accompanied by a high level of intracellular reactive oxygen species (ROS). When normal MSCs were treated in vitro with H2O2, which increases intracellular ROS level as an oxidant, both reorganization of F-actin cytoskeleton and impairment of migration capability were observed. On the other hand, treatment with N-acetylcysteine (NAC), as an exogenous antioxidant, made F-actin more orderly and increased migration ratio in SLE MSCs. In addition, oral administration of NAC markedly reduced serum autoantibody levels and ameliorated lupus nephritis (LN) in MRL/lpr mice, partially reversing the abnormalities of MSCs. These results indicate that overpolymerization of F-actin cytoskeleton, which may be associated with high levels of ROS, causes impairment in the migration capacity of SLE MSCs and that oral administration of NAC may have potential therapeutic effects on MRL/lpr mice. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  15. Correlations between quasi-coherent fluctuations and the pedestal evolution during the inter-edge localized modes phase on DIII-D

    DOE PAGES

    Diallo, A.; Groebner, R. J.; Rhodes, T. L.; ...

    2015-05-15

    Direct measurements of the pedestal recovery during an edge-localized mode cycle provide evidence that quasi-coherent fluctuations (QCFs) play a role in the inter-ELM pedestal dynamics. When using fast Thomson scattering measurements, we found that the pedestal density and temperature evolutions are probed on sub-millisecond time scales to show a fast recovery of the density gradient compared to the temperature gradient. The temperature gradient appears to provide a drive for the onset of quasi-coherent fluctuations (as measured with the magnetic probe and the density diagnostics) localized in the pedestal. The amplitude evolution of these QCFs tracks the temperature gradient evolution includingmore » its saturation. Such correlation suggests that these QCFs play a key role in limiting the pedestal temperature gradient. Moreover, the saturation of the QCFs coincides with the pressure gradient reaching the kinetic-ballooning mode (KBM) critical gradient as predicted by EPED1. Furthermore, linear microinstability analysis using GS2 indicates that the steep gradient is near the KBM threshold. Finally, the modeling and the observations together suggest that QCFs are consistent with dominant KBMs, although microtearing cannot be excluded as subdominant.« less

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

    PubMed

    Thomasson, Maggie S; Macnaughtan, Megan A

    2013-12-15

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

  17. The WAVE2 complex regulates actin cytoskeletal reorganization and CRAC-mediated calcium entry during T cell activation.

    PubMed

    Nolz, Jeffrey C; Gomez, Timothy S; Zhu, Peimin; Li, Shuixing; Medeiros, Ricardo B; Shimizu, Yoji; Burkhardt, Janis K; Freedman, Bruce D; Billadeau, Daniel D

    2006-01-10

    The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and beta-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCgamma1 activation and IP(3)-mediated store release. These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

  18. The WAVE2 Complex Regulates Actin Cytoskeletal Reorganization and CRAC-Mediated Calcium Entry during T Cell Activation

    PubMed Central

    Nolz, Jeffrey C.; Gomez, Timothy S.; Zhu, Peimin; Li, Shuixing; Medeiros, Ricardo B.; Shimizu, Yoji; Burkhardt, Janis K.; Freedman, Bruce D.; Billadeau, Daniel D.

    2007-01-01

    Summary Background The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. Results By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and β-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCγ1 activation and IP3-mediated store release. Conclusions These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation. PMID:16401421

  19. Molecular architecture of the Spire-actin nucleus and its implication for actin filament assembly.

    PubMed

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

    2011-12-06

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

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

    PubMed Central

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

    2011-01-01

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

  1. Molecular Characterization of abLIM, a Novel Actin-binding and Double Zinc Finger Protein

    PubMed Central

    Roof, Dorothy J.; Hayes, Annmarie; Adamian, Michael; Chishti, Athar H.; Li, Tiansen

    1997-01-01

    Molecules that couple the actin-based cytoskeleton to intracellular signaling pathways are central to the processes of cellular morphogenesis and differentiation. We have characterized a novel protein, the actin-binding LIM (abLIM) protein, which could mediate such interactions between actin filaments and cytoplasmic targets. abLIM protein consists of a COOH-terminal cytoskeletal domain that is fused to an NH2-terminal domain consisting of four double zinc finger motifs. The cytoskeletal domain is ∼50% identical to erythrocyte dematin, an actin-bundling protein of the red cell membrane skeleton, while the zinc finger domains conform to the LIM motif consensus sequence. In vitro expression studies demonstrate that abLIM protein can bind to F-actin through the dematin-like domain. Transcripts corresponding to three distinct isoforms have a widespread tissue distribution. However, a polypeptide corresponding to the full-length isoform is found exclusively in the retina and is enriched in biochemical extracts of retinal rod inner segments. abLIM protein also undergoes extensive phosphorylation in light-adapted retinas in vivo, and its developmental expression in the retina coincides with the elaboration of photoreceptor inner and outer segments. Based on the composite primary structure of abLIM protein, actin-binding capacity, potential regulation via phosphorylation, and isoform expression pattern, we speculate that abLIM may play a general role in bridging the actin-based cytoskeleton with an array of potential LIM protein-binding partners. The developmental time course of abLIM expression in the retina suggests that the retina-specific isoform may have a specialized role in the development or elaboration of photoreceptor inner and outer segments. PMID:9245787

  2. Coupling of the hydration water dynamics and the internal dynamics of actin detected by quasielastic neutron scattering

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fujiwara, Satoru, E-mail: fujiwara.satoru@jaea.go.jp; Plazanet, Marie; Oda, Toshiro

    2013-02-15

    Highlights: ► Quasielastic neutron scattering spectra of F-actin and G-actin were measured. ► Analysis of the samples in D{sub 2}O and H{sub 2}O provided the spectra of hydration water. ► The first layer hydration water around F-actin is less mobile than around G-actin. ► This difference in hydration water is in concert with the internal dynamics of actin. ► Water outside the first layer behaves bulk-like but influenced by the first layer. -- Abstract: In order to characterize dynamics of water molecules around F-actin and G-actin, quasielastic neutron scattering experiments were performed on powder samples of F-actin and G-actin, hydratedmore » either with D{sub 2}O or H{sub 2}O, at hydration ratios of 0.4 and 1.0. By combined analysis of the quasielastic neutron scattering spectra, the parameter values characterizing the dynamics of the water molecules in the first hydration layer and those of the water molecules outside of the first layer were obtained. The translational diffusion coefficients (D{sub T}) of the hydration water in the first layer were found to be 1.2 × 10{sup −5} cm{sup 2}/s and 1.7 × 10{sup −5} cm{sup 2}/s for F-actin and G-actin, respectively, while that for bulk water was 2.8 × 10{sup −5} cm{sup 2}/s. The residence times were 6.6 ps and 5.0 ps for F-actin and G-actin, respectively, while that for bulk water was 0.62 ps. These differences between F-actin and G-actin, indicating that the hydration water around G-actin is more mobile than that around F-actin, are in concert with the results of the internal dynamics of F-actin and G-actin, showing that G-actin fluctuates more rapidly than F-actin. This implies that the dynamics of the hydration water is coupled to the internal dynamics of the actin molecules. The D{sub T} values of the water molecules outside of the first hydration layer were found to be similar to that of bulk water though the residence times are strongly affected by the first hydration layer. This supports

  3. 24. A SECTION OF OPEN BALUSTRADE AND AN INTERMEDIATE PEDESTAL ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    24. A SECTION OF OPEN BALUSTRADE AND AN INTERMEDIATE PEDESTAL (CENTERED ON A PYLON) OVER THE CENTRAL PORTIONS OF THE BRIDGE. THE WALKS ARE CONCRETE, WITH AN INTEGRAL CURB. THE ROADWAY IS ASPHALT. - Main Street Bridge, Spanning East Fork Whitewater River, Richmond, Wayne County, IN

  4. Correlated waves of actin filaments and PIP3 in Dictyostelium cells.

    PubMed

    Asano, Yukako; Nagasaki, Akira; Uyeda, Taro Q P

    2008-12-01

    Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct movements: (1) they extend protrusions randomly without net displacements; (2) they migrate persistently and unidirectionally in a keratocyte-like manner. Here, we monitored the intracellular distribution of phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)) to gain insight into roles PIP(3) plays in those spontaneous motilities. In keratocyte-like cells, PIP(3) showed convex distribution over the basal membrane, with no anterior enrichment. In stalled cells, as well as in wild type cells, PIP(3) repeated wave-like changes, including emergence, expansion and disappearance, on the basal membrane. The waves induced lamellipodia when they approached the cell edge, and the advancing speed of the waves was comparable to the migration speed of the keratocyte-like cells. LY294002, an inhibitor of PI3 kinase, abolished PIP(3) waves in stalled cells and stopped keratocyte-like cells. These results together suggested that keratocyte-like cells are "surfing" on the PIP(3) waves by coupling steady lamellipodial protrusions to the PIP(3) waves. Simultaneous live observation of actin filaments and PIP(3) in wild type or stalled amiB(-) cells indicated that the PIP(3) waves were correlated with wave-like distributions of actin filaments. Most notably, PIP(3) waves often followed actin waves, suggesting that PIP(3) induces local depolymerization of actin filaments. Consistent with this idea, cortical accumulation of PIP(3) was often correlated with local retraction of the periphery. We propose that the waves of PIP(3) and actin filaments are loosely coupled with each other and play important roles in generating spontaneous cell polarity. Copyright 2008 Wiley-Liss, Inc.

  5. Characterization of broadband fluctuations in wide-pedestal QH-mode plasmas on DIII-D

    NASA Astrophysics Data System (ADS)

    Muscatello, C. M.; Burrell, K. H.; Luhmann, N. C., Jr.; McKee, G. R.; Tobias, B.

    2016-10-01

    Edge broadband fluctuations observed in wide pedestal quiescent H-mode plasmas may play an important role in driving transport necessary for stabilizing the edge to kink-peeling modes, thought to lead to ELMs. Density fluctuation measurements from BES and MIR independently observe periodic bursts in the pedestal that show up spectrally as broadband fluctuations. The period of the fluctuation bursts correlate with the period of enhanced bicoherence in the frequency range of the fluctuations, suggesting nonlinear coupling of turbulence. Time-delay estimation analysis of the 2D BES data shows strong evidence of a low-frequency zonal flow in the pedestal with a period matching that of the bursts. The carbon pressure gradient and E × B velocity, determined from CER, and ECE emission also oscillate with the same period. This behavior can be described as a quasi-stationary, limit-cycle oscillation and modeled by a set of predator-prey equations relating the zonal flow, equilibrium flow, and turbulence amplitude. Supported by the US DOE under DE-FC02-04ER54698, DE-FG02-99ER54531, DE-AC02-09CH11466.

  6. Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function

    PubMed Central

    McGough, Amy; Pope, Brian; Chiu, Wah; Weeds, Alan

    1997-01-01

    Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter ‘actin crossovers' (∼75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled. PMID:9265645

  7. Intracellular protein determination using droplet-based immunoassays.

    PubMed

    Martino, Chiara; Zagnoni, Michele; Sandison, Mairi E; Chanasakulniyom, Mayuree; Pitt, Andrew R; Cooper, Jonathan M

    2011-07-01

    This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells.

  8. Multiple essential functions of Plasmodium falciparum actin-1 during malaria blood-stage development.

    PubMed

    Das, Sujaan; Lemgruber, Leandro; Tay, Chwen L; Baum, Jake; Meissner, Markus

    2017-08-15

    The phylum Apicomplexa includes intracellular parasites causing immense global disease burden, the deadliest of them being the human malaria parasite Plasmodium falciparum, which invades and replicates within erythrocytes. The cytoskeletal protein actin is well conserved within apicomplexans but divergent from mammalian actins, and was primarily reported to function during host cell invasion. However, novel invasion mechanisms have been described for several apicomplexans, and specific functions of the acto-myosin system are being reinvestigated. Of the two actin genes in P. falciparum, actin-1 (pfact1) is ubiquitously expressed in all life-cycle stages and is thought to be required for erythrocyte invasion, although its functions during parasite development are unknown, and definitive in vivo characterisation during invasion is lacking. Here we have used a conditional Cre-lox system to investigate the functions of PfACT1 during P. falciparum blood-stage development and host cell invasion. We demonstrate that PfACT1 is crucially required for segregation of the plastid-like organelle, the apicoplast, and for efficient daughter cell separation during the final stages of cytokinesis. Surprisingly, we observe that egress from the host cell is not an actin-dependent process. Finally, we show that parasites lacking PfACT1 are capable of microneme secretion, attachment and formation of a junction with the erythrocyte, but are incapable of host cell invasion. This study provides important mechanistic insights into the definitive essential functions of PfACT1 in P. falciparum, which are not only of biological interest, but owing to functional divergence from mammalian actins, could also form the basis for the development of novel therapeutics against apicomplexans.

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

    PubMed

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

    2016-03-04

    Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Torsional Rigidity of Single Actin Filaments and Actin-Actin Bond Breaking Force under Torsion Measured Directly by in vitro Micromanipulation

    NASA Astrophysics Data System (ADS)

    Tsuda, Yuri; Yasutake, Hironori; Ishijima, Akihiko; Yanagida, Toshio

    1996-11-01

    Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular fluctuations of filaments supplied the torsional rigidity as (8.0 ± 1.2) × 10-26 Nm2. This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin-actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600-320 pN when filaments were turned through 90 degrees, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable flexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.

  11. There is more than one way to model an elephant. Experiment-driven modeling of the actin cytoskeleton.

    PubMed

    Ditlev, Jonathon A; Mayer, Bruce J; Loew, Leslie M

    2013-02-05

    Mathematical modeling has established its value for investigating the interplay of biochemical and mechanical mechanisms underlying actin-based motility. Because of the complex nature of actin dynamics and its regulation, many of these models are phenomenological or conceptual, providing a general understanding of the physics at play. But the wealth of carefully measured kinetic data on the interactions of many of the players in actin biochemistry cries out for the creation of more detailed and accurate models that could permit investigators to dissect interdependent roles of individual molecular components. Moreover, no human mind can assimilate all of the mechanisms underlying complex protein networks; so an additional benefit of a detailed kinetic model is that the numerous binding proteins, signaling mechanisms, and biochemical reactions can be computationally organized in a fully explicit, accessible, visualizable, and reusable structure. In this review, we will focus on how comprehensive and adaptable modeling allows investigators to explain experimental observations and develop testable hypotheses on the intracellular dynamics of the actin cytoskeleton. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. There is More Than One Way to Model an Elephant. Experiment-Driven Modeling of the Actin Cytoskeleton

    PubMed Central

    Ditlev, Jonathon A.; Mayer, Bruce J.; Loew, Leslie M.

    2013-01-01

    Mathematical modeling has established its value for investigating the interplay of biochemical and mechanical mechanisms underlying actin-based motility. Because of the complex nature of actin dynamics and its regulation, many of these models are phenomenological or conceptual, providing a general understanding of the physics at play. But the wealth of carefully measured kinetic data on the interactions of many of the players in actin biochemistry cries out for the creation of more detailed and accurate models that could permit investigators to dissect interdependent roles of individual molecular components. Moreover, no human mind can assimilate all of the mechanisms underlying complex protein networks; so an additional benefit of a detailed kinetic model is that the numerous binding proteins, signaling mechanisms, and biochemical reactions can be computationally organized in a fully explicit, accessible, visualizable, and reusable structure. In this review, we will focus on how comprehensive and adaptable modeling allows investigators to explain experimental observations and develop testable hypotheses on the intracellular dynamics of the actin cytoskeleton. PMID:23442903

  13. Test and model correlation of the atmospheric emission photometric imager fiberglass pedestal

    NASA Technical Reports Server (NTRS)

    Lee, H. M., III; Barker, L. A.

    1990-01-01

    The correlation is presented of the static loads testing and finite element modeling for the fiberglass pedestal used on the Atmospheric Emission Photometric Imaging (AEPI) experiment. This payload is to be launched in the space shuttle as part of the ATLAS-1 experiment. Strain gauge data from rosettes around the highly loaded base are compared to the same load case run for the Spacelab 1 testing done in 1981. Correlation of the model and test data was accomplished through comparison of the composite stress invariant using the expected flight loads for the ATLAS-1 mission. Where appropriate, the Tsai-Wu failure criteria was used in the development of the key margins of safety. Margins of safety are all positive for the pedestal and are reported.

  14. Synaptic Vesicle Mobility and Presynaptic F-Actin Are Disrupted in a N-ethylmaleimide–sensitive Factor Allele of Drosophila

    PubMed Central

    Nunes, Paula; Haines, Nicola; Kuppuswamy, Venkat; Fleet, David J.

    2006-01-01

    N-ethylmaleimide sensitive factor (NSF) can dissociate the soluble NSF attachment receptor (SNARE) complex, but NSF also participates in other intracellular trafficking functions by virtue of SNARE-independent activity. Drosophila that express a neural transgene encoding a dominant-negative form of NSF2 show an 80% reduction in the size of releasable synaptic vesicle pool, but no change in the number of vesicles in nerve terminal boutons. Here we tested the hypothesis that vesicles in the NSF2 mutant terminal are less mobile. Using a combination of genetics, pharmacology, and imaging we find a substantial reduction in vesicle mobility within the nerve terminal boutons of Drosophila NSF2 mutant larvae. Subsequent analysis revealed a decrease of filamentous actin in both NSF2 dominant-negative and loss-of-function mutants. Lastly, actin-filament disrupting drugs also decrease vesicle movement. We conclude that a factor contributing to the NSF mutant phenotype is a reduction in vesicle mobility, which is associated with decreased presynaptic F-actin. Our data are consistent with a model in which actin filaments promote vesicle mobility and suggest that NSF participates in establishing or maintaining this population of actin. PMID:16914524

  15. Observations of highly sheared turbulence in the H-mode pedestal using Phase Contrast Imaging on DIII-D

    NASA Astrophysics Data System (ADS)

    Rost, J. C.; Marinoni, A.; Davis, E. M.; Porkolab, M.; Burrell, K. H.

    2017-10-01

    Highly sheared turbulence with short radial correlation lengths has been measured near the top of the H-mode pedestal, in addition to the previously measured highly-sheared turbulence measured in the Er well. Turbulence in regions of large velocity shear is characterized by radial correlation lengths shorter than the poloidal wavelength (L < λ 2 cm) and large Doppler-shifted frequencies (f > 200 kHz). The phase contrast imaging (PCI) diagnostic on DIII-D is ideally suited to measuring this density turbulence due to the measurement geometry and high frequency bandwidth. Radial localization is achieved by optical filtering, varying the ExB profile, and shifting the plasma position. Reconfiguration of the Er well, such as at the L-H transition or the transition to wide pedestal QH-mode, shows a near-instantaneous change (t < 1 ms) to the sheared turbulence in the Er well ( 1 cm inside the separatrix). In contrast, the sheared turbulence near the top of the pedestal ( 2 cm inside the separatrix) varies over times scales of tens of ms, consistent with pedestal evolution. Work supported by the US Department of Energy under DE-FG02-94ER54235 and DE-FC02-04ER54698.

  16. Myopodin is an F-actin bundling protein with multiple independent actin-binding regions.

    PubMed

    Linnemann, Anja; Vakeel, Padmanabhan; Bezerra, Eduardo; Orfanos, Zacharias; Djinović-Carugo, Kristina; van der Ven, Peter F M; Kirfel, Gregor; Fürst, Dieter O

    2013-02-01

    The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric α-actinin, as well as its binding to actin, α-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of α-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin's ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.

  17. 49. (no plate) 1,000 poind Mercury Float Pedestal and clock ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    49. (no plate) 1,000 poind Mercury Float Pedestal and clock case sheet no. 1 of 4 drawing # 5765. Approved 2-3-1916. - Block Island Southeast Light, Spring Street & Mohegan Trail at Mohegan Bluffs, New Shoreham, Washington County, RI

  18. Using LGI experiments to achieve better understanding of pedestal-edge coupling in NSTX-U

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Zhehui

    2015-02-23

    PowerPoint presentation. Latest advances in granule or dust injection technologies, fast and high-resolution imaging, together with micro-/nano-structured material fabrication, provide new opportunities to examine plasma-material interaction (PMI) in magnetic fusion environment. Some of our previous work in these areas is summarized. The upcoming LGI experiments in NSTX-U will shed new light on granular matter transport in the pedestal-edge region. In addition to particle control, these results can also be used for code validation and achieving better understanding of pedestal-edge coupling in fusion plasmas in both NSTX-U and others.

  19. Nucleation of actin polymerization by gelsolin.

    PubMed

    Ditsch, A; Wegner, A

    1994-08-15

    The time-course of assembly of actin with gelsolin was measured by the fluorescence increase of a fluorescent label covalently linked to actin. The actin concentrations ranged from values far below the critical concentration to values above the critical concentration of the pointed ends of actin filaments. If the concentration of actin was in the range of the critical monomer concentration (0.64 microM), the time-course of the concentration of actin assembled with gelsolin revealed a sigmoidal shape. At higher actin concentrations the time-course of association of actin with gelsolin approximated an exponential curve. The measured time-courses of assembly were quantitatively interpreted by kinetic rate equations. A poor fit was obtained if two actin molecules were assumed to bind to gelsolin to form a 1:2 gelsolin-actin complex and subsequently further actin molecules were assumed to polymerize onto the 1:2 gelsolin-actin complex toward the pointed end. A considerably better agreement between calculated and measured time-courses was achieved if additional creation of actin filaments by fast fragmentation of newly formed actin filaments by not yet consumed gelsolin was assumed to occur. This suggests that both polymerization of actin onto gelsolin and fragmentation of actin filaments contribute to formation of new actin filaments by gelsolin. Furthermore it could be demonstrated that below the critical monomer concentration appreciable amounts of actin are incorporated into gelsolin-actin oligomers.

  20. PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

    PubMed Central

    Hong, Nan Hyung; Qi, Aidong

    2015-01-01

    Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover. PMID:26323691

  1. Actin stress in cell reprogramming

    PubMed Central

    Guo, Jun; Wang, Yuexiu; Sachs, Frederick; Meng, Fanjie

    2014-01-01

    Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals. PMID:25422450

  2. Treatment of Actinic Purpura

    PubMed Central

    2017-01-01

    Mature skin is prone to bruising, resulting in a condition known as actinic purpura, characterized by unsightly ecchymosis and purple patches. Similar to other skin conditions, the incidence of actinic purpura increases with advancing age and occurs with equal frequency among men and women. The unsightly appearance of actinic purpura may be a source of emotional distress among the elderly. A new product has been formulated specifically for the treatment of actinic purpura. This product contains retinol, α-hydroxy acids, arnica oil, ceramides, niacinamide, and phytonadione, which effectively treat actinic purpura by improving local circulation, thickening the skin, and repairing the skin barrier. The objective of this paper is to review the beneficial properties of these ingredients and their respective roles in the treatment of actinic purpura. PMID:28979656

  3. Actin assembly factors regulate the gelation kinetics and architecture of F-actin networks.

    PubMed

    Falzone, Tobias T; Oakes, Patrick W; Sees, Jennifer; Kovar, David R; Gardel, Margaret L

    2013-04-16

    Dynamic regulation of the actin cytoskeleton is required for diverse cellular processes. Proteins regulating the assembly kinetics of the cytoskeletal biopolymer F-actin are known to impact the architecture of actin cytoskeletal networks in vivo, but the underlying mechanisms are not well understood. Here, we demonstrate that changes to actin assembly kinetics with physiologically relevant proteins profilin and formin (mDia1 and Cdc12) have dramatic consequences on the architecture and gelation kinetics of otherwise biochemically identical cross-linked F-actin networks. Reduced F-actin nucleation rates promote the formation of a sparse network of thick bundles, whereas increased nucleation rates result in a denser network of thinner bundles. Changes to F-actin elongation rates also have marked consequences. At low elongation rates, gelation ceases and a solution of rigid bundles is formed. By contrast, rapid filament elongation accelerates dynamic arrest and promotes gelation with minimal F-actin density. These results are consistent with a recently developed model of how kinetic constraints regulate network architecture and underscore how molecular control of polymer assembly is exploited to modulate cytoskeletal architecture and material properties. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

    PubMed Central

    Spracklen, Andrew J.; Fagan, Tiffany N.; Lovander, Kaylee E.; Tootle, Tina L.

    2015-01-01

    Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling

  5. Erk regulation of actin capping and bundling by Eps8 promotes cortex tension and leader bleb-based migration

    PubMed Central

    Logue, Jeremy S; Cartagena-Rivera, Alexander X; Baird, Michelle A; Davidson, Michael W; Chadwick, Richard S; Waterman, Clare M

    2015-01-01

    Within the confines of tissues, cancer cells can use blebs to migrate. Eps8 is an actin bundling and capping protein whose capping activity is inhibited by Erk, a key MAP kinase that is activated by oncogenic signaling. We tested the hypothesis that Eps8 acts as an Erk effector to modulate actin cortex mechanics and thereby mediate bleb-based migration of cancer cells. Cells confined in a non-adhesive environment migrate in the direction of a very large ‘leader bleb.’ Eps8 bundling activity promotes cortex tension and intracellular pressure to drive leader bleb formation. Eps8 capping and bundling activities act antagonistically to organize actin within leader blebs, and Erk mediates this effect. An Erk biosensor reveals concentrated kinase activity within leader blebs. Bleb contents are trapped by the narrow neck that separates the leader bleb from the cell body. Thus, Erk activity promotes actin bundling by Eps8 to enhance cortex tension and drive the bleb-based migration of cancer cells under non-adhesive confinement. DOI: http://dx.doi.org/10.7554/eLife.08314.001 PMID:26163656

  6. The Relationships Between ELM Suppression, Pedestal Profiles, and Lithium Wall Coatings in NSTX

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    D.P. Boyle, R. Maingi, P.B. Snyder, J. Manickam, T.H. Osborne, R.E. Bell, B.P. LeBlanc, and the NSTX Team

    2012-08-17

    Recently in the National Spherical Torus Experiment (NSTX), increasing lithium wall coatings suppressed edge localized modes (ELMs), gradually but not quite monotonically. This work details profile and stability analysis as ELMs disappeared throughout the lithium scan. While the quantity of lithium deposited between discharges did not uniquely determine the presence of ELMs, profile analysis demonstrated that lithium was correlated to wider density and pressure pedestals with peak gradients farther from the separatrix. Moreover, the ELMy and ELM-free discharges were cleanly separated by their density and pedestal widths and peak gradient locations. Ultimately, ELMs were only suppressed when lithium caused themore » density pedestal to widen and shift inward. These changes in the density gradient were directly reflected in the pressure gradient and calculated bootstrap current. This supports the theory that ELMs in NSTX are caused by peeling and/or ballooning modes, as kink/peeling modes are stabilized when the edge current and pressure gradient shift away from the separatrix. Edge stability analysis using ELITE corroborated this picture, as reconstructed equilibria from ELM-free discharges were generally farther from their kink/peeling stability boundaries than ELMy discharges. We conclude that density profile control provided by lithium is the key first step to ELM suppression in NSTX« less

  7. The yeast actin cytoskeleton.

    PubMed

    Mishra, Mithilesh; Huang, Junqi; Balasubramanian, Mohan K

    2014-03-01

    The actin cytoskeleton is a complex network of dynamic polymers, which plays an important role in various fundamental cellular processes, including maintenance of cell shape, polarity, cell division, cell migration, endocytosis, vesicular trafficking, and mechanosensation. Precise spatiotemporal assembly and disassembly of actin structures is regulated by the coordinated activity of about 100 highly conserved accessory proteins, which nucleate, elongate, cross-link, and sever actin filaments. Both in vivo studies in a wide range of organisms from yeast to metazoans and in vitro studies of purified proteins have helped shape the current understanding of actin dynamics and function. Molecular genetics, genome-wide functional analysis, sophisticated real-time imaging, and ultrastructural studies in concert with biochemical analysis have made yeast an attractive model to understand the actin cytoskeleton, its molecular dynamics, and physiological function. Studies of the yeast actin cytoskeleton have contributed substantially in defining the universal mechanism regulating actin assembly and disassembly in eukaryotes. Here, we review some of the important insights generated by the study of actin cytoskeleton in two important yeast models the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  8. Helical variation of density profiles and fluctuations in the tokamak pedestal with applied 3D fields and implications for confinement

    NASA Astrophysics Data System (ADS)

    Wilcox, R. S.; Rhodes, T. L.; Shafer, M. W.; Sugiyama, L. E.; Ferraro, N. M.; Lyons, B. C.; McKee, G. R.; Paz-Soldan, C.; Wingen, A.; Zeng, L.

    2018-05-01

    Small 3D perturbations to the magnetic field in DIII-D ( δB /B ˜2 ×10-4 ) result in large modulations of density fluctuation amplitudes in the pedestal, which are shown using Doppler backscattering measurements to vary by a factor of 2. Helical perturbations of equilibrium density within flux surfaces have previously been observed in the pedestal of DIII-D plasmas when 3D fields are applied and were correlated with density fluctuation asymmetries in the pedestal. These intra-surface density and pressure variations are shown through two fluid MHD modeling studies using the M3D-C1 code to be due to the misalignment of the density and temperature equilibrium iso-surfaces in the pedestal region. This modeling demonstrates that the phase shift between the two iso-surfaces corresponds to the diamagnetic direction of the two species, with the mass density surfaces shifted in the ion diamagnetic direction relative to the temperature and magnetic flux iso-surfaces. The resulting pedestal density, potential, and turbulence asymmetries within flux surfaces near the separatrix may be at least partially responsible for several poorly understood phenomena that occur with the application of 3D fields in tokamaks, including density pump out and the increase in power required to transition from L- to H-mode.

  9. Detection of a weak maser emission pedestal associated with the SiO maser. [in variable late stars

    NASA Technical Reports Server (NTRS)

    Snyder, L. E.; Dickinson, D. F.; Brown, L. W.; Buhl, D.

    1978-01-01

    Results are reported for high-spectral-resolution observations of the v = 1, J = 1-0 SiO maser sources at 43,122.027 MHz (6.95 mm wavelength) associated with the variable stars Omega Cet, NML Tau, VY CMa, R Leo, W Hya, VX Sgr, NML Cyg, and R Cas. A weak underlying maser emission pedestal is clearly observed in the spectra of all but NML Cyg and R Cas. The data indicate that the underlying pedestal of SiO emission appears to originate in a shell-like region around the star, has a thermal appearance even though it must be due to weak maser emission, and appears to be part of the spectral signature of SiO maser emission from late-type stars. It is found that the center velocities of the pedestals may be used to determine stellar radial velocities. Observations of large-scale time variations in the intensity of the Ori A SiO maser and the detection of weak maser pedestals associated with each of the two strong emission-feature groups in Orion are also discussed. It is suggested that the Orion molecular cloud might contain two late-type long-period variable stars that may be semiregular variables.

  10. Direct Interaction of CaVβ with Actin Up-regulates L-type Calcium Currents in HL-1 Cardiomyocytes*

    PubMed Central

    Stölting, Gabriel; de Oliveira, Regina Campos; Guzman, Raul E.; Miranda-Laferte, Erick; Conrad, Rachel; Jordan, Nadine; Schmidt, Silke; Hendriks, Johnny; Gensch, Thomas; Hidalgo, Patricia

    2015-01-01

    Expression of the β-subunit (CaVβ) is required for normal function of cardiac L-type calcium channels, and its up-regulation is associated with heart failure. CaVβ binds to the α1 pore-forming subunit of L-type channels and augments calcium current density by facilitating channel opening and increasing the number of channels in the plasma membrane, by a poorly understood mechanism. Actin, a key component of the intracellular trafficking machinery, interacts with Src homology 3 domains in different proteins. Although CaVβ encompasses a highly conserved Src homology 3 domain, association with actin has not yet been explored. Here, using co-sedimentation assays and FRET experiments, we uncover a direct interaction between CaVβ and actin filaments. Consistently, single-molecule localization analysis reveals streaklike structures composed by CaVβ2 that distribute over several micrometers along actin filaments in HL-1 cardiomyocytes. Overexpression of CaVβ2-N3 in HL-1 cells induces an increase in L-type current without altering voltage-dependent activation, thus reflecting an increased number of channels in the plasma membrane. CaVβ mediated L-type up-regulation, and CaVβ-actin association is prevented by disruption of the actin cytoskeleton with cytochalasin D. Our study reveals for the first time an interacting partner of CaVβ that is directly involved in vesicular trafficking. We propose a model in which CaVβ promotes anterograde trafficking of the L-type channels by anchoring them to actin filaments in their itinerary to the plasma membrane. PMID:25533460

  11. Building bridges: formin1 of Arabidopsis forms a connection between the cell wall and the actin cytoskeleton.

    PubMed

    Martinière, Alexandre; Gayral, Philippe; Hawes, Chris; Runions, John

    2011-04-01

    Actin microfilament (MF) organization and remodelling is critical to cell function. The formin family of actin binding proteins are involved in nucleating MFs in Arabidopsis thaliana. They all contain formin homology domains in the intracellular, C-terminal half of the protein that interacts with MFs. Formins in class I are usually targeted to the plasma membrane and this is true of Formin1 (AtFH1) of A. thaliana. In this study, we have investigated the extracellular domain of AtFH1 and we demonstrate that AtFH1 forms a bridge from the actin cytoskeleton, across the plasma membrane and is anchored within the cell wall. AtFH1 has a large, extracellular domain that is maintained by purifying selection and that contains four conserved regions, one of which is responsible for immobilising the protein. Protein anchoring within the cell wall is reduced in constructs that express truncations of the extracellular domain and in experiments in protoplasts without primary cell walls. The 18 amino acid proline-rich extracellular domain that is responsible for AtFH1 anchoring has homology with cell-wall extensins. We also have shown that anchoring of AtFH1 in the cell wall promotes actin bundling within the cell and that overexpression of AtFH1 has an inhibitory effect on organelle actin-dependant dynamics. Thus, the AtFH1 bridge provides stable anchor points for the actin cytoskeleton and is probably a crucial component of the signalling response and actin-remodelling mechanisms. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

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

    PubMed Central

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

    2016-01-01

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

  13. Co-transcriptional nuclear actin dynamics

    PubMed Central

    Percipalle, Piergiorgio

    2013-01-01

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

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

    PubMed Central

    Case, Lindsay B.; Waterman, Clare M.

    2011-01-01

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

  15. Filopodia-like Actin Cables Position Nuclei in Association with Perinuclear Actin in Drosophila Nurse Cells

    PubMed Central

    Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H.

    2013-01-01

    Summary Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery. PMID:24091012

  16. 21. View of endlift slide on pedestal and threaded shaft ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    21. View of end-lift slide on pedestal and threaded shaft with level gears. Curved deck joint at underside of roadway deck is seen, as well as submarine electrical cables resting on the masonry pier. (Nov. 30, 1988) - University Heights Bridge, Spanning Harlem River at 207th Street & West Harlem Road, New York County, NY

  17. Nucleus-associated actin in Amoeba proteus.

    PubMed

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

    2016-10-01

    The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms. Copyright © 2016 Elsevier GmbH. All rights reserved.

  18. Gyrokinetic neoclassical study of the bootstrap current in the tokamak edge pedestal with fully non-linear Coulomb collisions

    DOE PAGES

    Hager, Robert; Chang, C. S.

    2016-04-08

    As a follow-up on the drift-kinetic study of the non-local bootstrap current in the steep edge pedestal of tokamak plasma by Koh et al. [Phys. Plasmas 19, 072505 (2012)], a gyrokinetic neoclassical study is performed with gyrokinetic ions and drift-kinetic electrons. Besides the gyrokinetic improvement of ion physics from the drift-kinetic treatment, a fully non-linear Fokker-Planck collision operator—that conserves mass, momentum, and energy—is used instead of Koh et al.'s linearized collision operator in consideration of the possibility that the ion distribution function is non-Maxwellian in the steep pedestal. An inaccuracy in Koh et al.'s result is found in the steepmore » edge pedestal that originated from a small error in the collisional momentum conservation. The present study concludes that (1) the bootstrap current in the steep edge pedestal is generally smaller than what has been predicted from the small banana-width (local) approximation [e.g., Sauter et al., Phys. Plasmas 6, 2834 (1999) and Belli et al., Plasma Phys. Controlled Fusion 50, 095010 (2008)], (2) the plasma flow evaluated from the local approximation can significantly deviate from the non-local results, and (3) the bootstrap current in the edge pedestal, where the passing particle region is small, can be dominantly carried by the trapped particles in a broad trapped boundary layer. In conclusion, a new analytic formula based on numerous gyrokinetic simulations using various magnetic equilibria and plasma profiles with self-consistent Grad-Shafranov solutions is constructed.« less

  19. Gyrokinetic neoclassical study of the bootstrap current in the tokamak edge pedestal with fully non-linear Coulomb collisions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hager, Robert; Chang, C. S.

    As a follow-up on the drift-kinetic study of the non-local bootstrap current in the steep edge pedestal of tokamak plasma by Koh et al. [Phys. Plasmas 19, 072505 (2012)], a gyrokinetic neoclassical study is performed with gyrokinetic ions and drift-kinetic electrons. Besides the gyrokinetic improvement of ion physics from the drift-kinetic treatment, a fully non-linear Fokker-Planck collision operator—that conserves mass, momentum, and energy—is used instead of Koh et al.'s linearized collision operator in consideration of the possibility that the ion distribution function is non-Maxwellian in the steep pedestal. An inaccuracy in Koh et al.'s result is found in the steepmore » edge pedestal that originated from a small error in the collisional momentum conservation. The present study concludes that (1) the bootstrap current in the steep edge pedestal is generally smaller than what has been predicted from the small banana-width (local) approximation [e.g., Sauter et al., Phys. Plasmas 6, 2834 (1999) and Belli et al., Plasma Phys. Controlled Fusion 50, 095010 (2008)], (2) the plasma flow evaluated from the local approximation can significantly deviate from the non-local results, and (3) the bootstrap current in the edge pedestal, where the passing particle region is small, can be dominantly carried by the trapped particles in a broad trapped boundary layer. In conclusion, a new analytic formula based on numerous gyrokinetic simulations using various magnetic equilibria and plasma profiles with self-consistent Grad-Shafranov solutions is constructed.« less

  20. Gyrokinetic neoclassical study of the bootstrap current in the tokamak edge pedestal with fully non-linear Coulomb collisions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hager, Robert, E-mail: rhager@pppl.gov; Chang, C. S., E-mail: cschang@pppl.gov

    As a follow-up on the drift-kinetic study of the non-local bootstrap current in the steep edge pedestal of tokamak plasma by Koh et al. [Phys. Plasmas 19, 072505 (2012)], a gyrokinetic neoclassical study is performed with gyrokinetic ions and drift-kinetic electrons. Besides the gyrokinetic improvement of ion physics from the drift-kinetic treatment, a fully non-linear Fokker-Planck collision operator—that conserves mass, momentum, and energy—is used instead of Koh et al.'s linearized collision operator in consideration of the possibility that the ion distribution function is non-Maxwellian in the steep pedestal. An inaccuracy in Koh et al.'s result is found in the steepmore » edge pedestal that originated from a small error in the collisional momentum conservation. The present study concludes that (1) the bootstrap current in the steep edge pedestal is generally smaller than what has been predicted from the small banana-width (local) approximation [e.g., Sauter et al., Phys. Plasmas 6, 2834 (1999) and Belli et al., Plasma Phys. Controlled Fusion 50, 095010 (2008)], (2) the plasma flow evaluated from the local approximation can significantly deviate from the non-local results, and (3) the bootstrap current in the edge pedestal, where the passing particle region is small, can be dominantly carried by the trapped particles in a broad trapped boundary layer. A new analytic formula based on numerous gyrokinetic simulations using various magnetic equilibria and plasma profiles with self-consistent Grad-Shafranov solutions is constructed.« less

  1. Damage effects of protoporphyrin IX - sonodynamic therapy on the cytoskeletal F-actin of Ehrlich ascites carcinoma cells.

    PubMed

    Zhao, Xia; Liu, Quanhong; Tang, Wei; Wang, Xiaobing; Wang, Pan; Gong, Liyan; Wang, Yuan

    2009-01-01

    In this study, we report evidence of the damage effects of sonodynamic therapy (SDT) on a novel intracellular target, cytoskeletal F-actin, that has great importance for cancer treatment. Ehrlich ascites carcinoma (EAC) cells suspended in PBS were exposed to ultrasound at 1.34 MHz for up to 60s in the presence and absence of protoporphyrin IX (PPIX). To evaluate the polymeric state and distribution of actin filaments (AF) we employed FITC-Phalloidin staining. The percentage of cells with intact AF was decreased with 10-80 microM PPIX after ultrasonic exposure, while only few cells with disturbed F-actin were observed with 80 microM PPIX alone. The fluorescence intensity of FITC-Phalloidin labeled cells was detected by flow cytometry. The morphological changes of EAC cells were observed by scanning electron microscope (SEM). The nuclei were stained with Hoechst 33258 to determine apoptosis. Cytoskeletal F-actin and cell morphological changes were dependent on the time after SDT. Some cells suffered deformations of plasma membrane as blebs that reacted positively to FITC-Phalloidin at 2h after SDT treatment. Many of the cells showed the typically apoptotic chromatin fragmentation. The alterations were more significant 4h later. Our results showed that cytoskeletal F-actin might represent an important target for the SDT treatment and the observed effect on F-actin and the subsequent bleb formation mainly due to apoptosis formation due to the treatment.

  2. Discovery of functional interactions among actin regulators by analysis of image fluctuations in an unperturbed motile cell system.

    PubMed

    Isogai, Tadamoto; Danuser, Gaudenz

    2018-05-26

    Cell migration is driven by propulsive forces derived from polymerizing actin that pushes and extends the plasma membrane. The underlying actin network is constantly undergoing adaptation to new mechano-chemical environments and intracellular conditions. As such, mechanisms that regulate actin dynamics inherently contain multiple feedback loops and redundant pathways. Given the highly adaptable nature of such a system, studies that use only perturbation experiments (e.g. knockdowns, overexpression, pharmacological activation/inhibition, etc.) are challenged by the nonlinearity and redundancy of the pathway. In these pathway configurations, perturbation experiments at best describe the function(s) of a molecular component in an adapting (e.g. acutely drug-treated) or fully adapted (e.g. permanent gene silenced) cell system, where the targeted component now resides in a non-native equilibrium. Here, we propose how quantitative live-cell imaging and analysis of constitutive fluctuations of molecular activities can overcome these limitations. We highlight emerging actin filament barbed-end biology as a prime example of a complex, nonlinear molecular process that requires a fluctuation analytic approach, especially in an unperturbed cellular system, to decipher functional interactions of barbed-end regulators, actin polymerization and membrane protrusion.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

  3. Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin*

    PubMed Central

    Parker, William H.; Qu, Zhi-chao; May, James M.

    2015-01-01

    Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. PMID:26152729

  4. Loss of Actin-Based Motility Impairs Ectromelia Virus Release In Vitro but Is Not Critical to Spread In Vivo.

    PubMed

    Duncan, Melanie Laura; Horsington, Jacquelyn; Eldi, Preethi; Al Rumaih, Zahrah; Karupiah, Gunasegaran; Newsome, Timothy P

    2018-03-05

    Ectromelia virus (ECTV) is an orthopoxvirus and the causative agent of mousepox. Like other poxviruses such as variola virus (agent of smallpox), monkeypox virus and vaccinia virus (the live vaccine for smallpox), ECTV promotes actin-nucleation at the surface of infected cells during virus release. Homologs of the viral protein A36 mediate this function through phosphorylation of one or two tyrosine residues that ultimately recruit the cellular Arp2/3 actin-nucleating complex. A36 also functions in the intracellular trafficking of virus mediated by kinesin-1. Here, we describe the generation of a recombinant ECTV that is specifically disrupted in actin-based motility allowing us to examine the role of this transport step in vivo for the first time. We show that actin-based motility has a critical role in promoting the release of virus from infected cells in vitro but plays a minor role in virus spread in vivo. It is likely that loss of microtubule-dependent transport is a major factor for the attenuation observed when A36R is deleted.

  5. Loss of Actin-Based Motility Impairs Ectromelia Virus Release In Vitro but Is Not Critical to Spread In Vivo

    PubMed Central

    Duncan, Melanie Laura; Horsington, Jacquelyn; Eldi, Preethi; Al Rumaih, Zahrah; Karupiah, Gunasegaran

    2018-01-01

    Ectromelia virus (ECTV) is an orthopoxvirus and the causative agent of mousepox. Like other poxviruses such as variola virus (agent of smallpox), monkeypox virus and vaccinia virus (the live vaccine for smallpox), ECTV promotes actin-nucleation at the surface of infected cells during virus release. Homologs of the viral protein A36 mediate this function through phosphorylation of one or two tyrosine residues that ultimately recruit the cellular Arp2/3 actin-nucleating complex. A36 also functions in the intracellular trafficking of virus mediated by kinesin-1. Here, we describe the generation of a recombinant ECTV that is specifically disrupted in actin-based motility allowing us to examine the role of this transport step in vivo for the first time. We show that actin-based motility has a critical role in promoting the release of virus from infected cells in vitro but plays a minor role in virus spread in vivo. It is likely that loss of microtubule-dependent transport is a major factor for the attenuation observed when A36R is deleted. PMID:29510577

  6. Actin Assembly Factors Regulate the Gelation Kinetics and Architecture of F-actin Networks

    PubMed Central

    Falzone, Tobias T.; Oakes, Patrick W.; Sees, Jennifer; Kovar, David R.; Gardel, Margaret L.

    2013-01-01

    Dynamic regulation of the actin cytoskeleton is required for diverse cellular processes. Proteins regulating the assembly kinetics of the cytoskeletal biopolymer F-actin are known to impact the architecture of actin cytoskeletal networks in vivo, but the underlying mechanisms are not well understood. Here, we demonstrate that changes to actin assembly kinetics with physiologically relevant proteins profilin and formin (mDia1 and Cdc12) have dramatic consequences on the architecture and gelation kinetics of otherwise biochemically identical cross-linked F-actin networks. Reduced F-actin nucleation rates promote the formation of a sparse network of thick bundles, whereas increased nucleation rates result in a denser network of thinner bundles. Changes to F-actin elongation rates also have marked consequences. At low elongation rates, gelation ceases and a solution of rigid bundles is formed. By contrast, rapid filament elongation accelerates dynamic arrest and promotes gelation with minimal F-actin density. These results are consistent with a recently developed model of how kinetic constraints regulate network architecture and underscore how molecular control of polymer assembly is exploited to modulate cytoskeletal architecture and material properties. PMID:23601318

  7. Helical variation of density profiles and fluctuations in the tokamak pedestal with applied 3D fields and implications for confinement

    DOE PAGES

    Wilcox, R. S.; Rhodes, T. L.; Shafer, M. W.; ...

    2018-04-19

    Smore » mall 3D perturbations to the magnetic field in DIII-D ( δ B / B ~ 2 × 10 - 4 ) result in large modulations of density fluctuation amplitudes in the pedestal, which are shown using Doppler backscattering measurements to vary by a factor of 2. Helical perturbations of equilibrium density within flux surfaces have previously been observed in the pedestal of DIII-D plasmas when 3D fields are applied and were correlated with density fluctuation asymmetries in the pedestal. These intra-surface density and pressure variations are shown through two fluid MHD modeling studies using the M3D-C1 code to be due to the misalignment of the density and temperature equilibrium iso-surfaces in the pedestal region. This modeling demonstrates that the phase shift between the two iso-surfaces corresponds to the diamagnetic direction of the two species, with the mass density surfaces shifted in the ion diamagnetic direction relative to the temperature and magnetic flux iso-surfaces. Finally, the resulting pedestal density, potential, and turbulence asymmetries within flux surfaces near the separatrix may be at least partially responsible for several poorly understood phenomena that occur with the application of 3D fields in tokamaks, including density pump out and the increase in power required to transition from L- to H-mode.« less

  8. Helical variation of density profiles and fluctuations in the tokamak pedestal with applied 3D fields and implications for confinement

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wilcox, R. S.; Rhodes, T. L.; Shafer, M. W.

    Smore » mall 3D perturbations to the magnetic field in DIII-D ( δ B / B ~ 2 × 10 - 4 ) result in large modulations of density fluctuation amplitudes in the pedestal, which are shown using Doppler backscattering measurements to vary by a factor of 2. Helical perturbations of equilibrium density within flux surfaces have previously been observed in the pedestal of DIII-D plasmas when 3D fields are applied and were correlated with density fluctuation asymmetries in the pedestal. These intra-surface density and pressure variations are shown through two fluid MHD modeling studies using the M3D-C1 code to be due to the misalignment of the density and temperature equilibrium iso-surfaces in the pedestal region. This modeling demonstrates that the phase shift between the two iso-surfaces corresponds to the diamagnetic direction of the two species, with the mass density surfaces shifted in the ion diamagnetic direction relative to the temperature and magnetic flux iso-surfaces. Finally, the resulting pedestal density, potential, and turbulence asymmetries within flux surfaces near the separatrix may be at least partially responsible for several poorly understood phenomena that occur with the application of 3D fields in tokamaks, including density pump out and the increase in power required to transition from L- to H-mode.« less

  9. 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.

  10. 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

  11. The actin multigene family and livestock speciation using the polymerase chain reaction.

    PubMed

    Fairbrother, K S; Hopwood, A J; Lockley, A K; Bardsley, R G

    1998-01-01

    Actins constitute a family of highly-conserved multifunctional intracellular proteins, best known as myofibrillar components in striated muscle fibres. Most vertebrate genomes contain numerous actin genes with high sequence homology in protein coding regions but considerable variability in intron number and sizes. This genetic diversity can be utilised for livestock speciation purposes. The high sequence conservation has enabled a single pair of oligonucleotides to be used to prime the polymerase chain reaction (PCR) with DNA extracted from all animals so far studied. Multiple amplification products were obtained which on gel electrophoresis constituted characteristic species-specific 'fingerprints'. The patterns were reproducible, did not vary between individuals of the same breed or between different breeds within a species, and could be generated even from heat-processed muscle held at 120 degrees C for one hour. Given the capacity of PCR to amplify relatively short sequences in highly-degraded DNA, this approach may be suitable for authentication of processed meat products.

  12. High-frequency coherent edge fluctuations in a high-pedestal-pressure quiescent H-mode plasma.

    PubMed

    Yan, Z; McKee, G R; Groebner, R J; Snyder, P B; Osborne, T H; Burrell, K H

    2011-07-29

    A set of high frequency coherent (HFC) modes (f=80-250 kHz) is observed with beam emission spectroscopy measurements of density fluctuations in the pedestal of a strongly shaped quiescent H-mode plasma on DIII-D, with characteristics predicted for kinetic ballooning modes (KBM): propagation in the ion-diamagnetic drift direction; a frequency near 0.2-0.3 times the ion-diamagnetic frequency; inferred toroidal mode numbers of n∼10-25; poloidal wave numbers of k(θ)∼0.17-0.4 cm(-1); and high measured decorrelation rates (τ(c)(-1)∼ω(s)∼0.5×10(6) s(-1)). Their appearance correlates with saturation of the pedestal pressure. © 2011 American Physical Society

  13. Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

    PubMed Central

    Carlier, Marie-France; Laurent, Valérie; Santolini, Jérôme; Melki, Ronald; Didry, Dominique; Xia, Gui-Xian; Hong, Yan; Chua, Nam-Hai; Pantaloni, Dominique

    1997-01-01

    Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics. PMID:9087445

  14. Glutathione depletion triggers actin cytoskeleton changes via actin-binding proteins.

    PubMed

    Zepeta-Flores, Nahum; Valverde, Mahara; Lopez-Saavedra, Alejandro; Rojas, Emilio

    2018-06-04

    The importance of glutathione (GSH) in alternative cellular roles to the canonically proposed, were analyzed in a model unable to synthesize GSH. Gene expression analysis shows that the regulation of the actin cytoskeleton pathway is strongly impacted by the absence of GSH. To test this hypothesis, we evaluate the effect of GSH depletion via buthionine sulfoximine (5 and 12.5 mM) in human neuroblastoma MSN cells. In the present study, 70% of GSH reduction did not induce reactive oxygen species, lipoperoxidation, or cytotoxicity, which enabled us to evaluate the effect of glutathione in the absence of oxidative stress. The cells with decreasing GSH levels acquired morphology changes that depended on the actin cytoskeleton and not on tubulin. We evaluated the expression of three actin-binding proteins: thymosin β4, profilin and gelsolin, showing a reduced expression, both at gene and protein levels at 24 hours of treatment; however, this suppression disappears after 48 hours of treatment. These changes were sufficient to trigger the co-localization of the three proteins towards cytoplasmic projections. Our data confirm that a decrease in GSH in the absence of oxidative stress can transiently inhibit the actin binding proteins and that this stimulus is sufficient to induce changes in cellular morphology via the actin cytoskeleton.

  15. Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.

    PubMed

    Lopata, Anna; Hughes, Ruth; Tiede, Christian; Heissler, Sarah M; Sellers, James R; Knight, Peter J; Tomlinson, Darren; Peckham, Michelle

    2018-04-26

    Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.

  16. Direct interaction of CaVβ with actin up-regulates L-type calcium currents in HL-1 cardiomyocytes.

    PubMed

    Stölting, Gabriel; de Oliveira, Regina Campos; Guzman, Raul E; Miranda-Laferte, Erick; Conrad, Rachel; Jordan, Nadine; Schmidt, Silke; Hendriks, Johnny; Gensch, Thomas; Hidalgo, Patricia

    2015-02-20

    Expression of the β-subunit (CaVβ) is required for normal function of cardiac L-type calcium channels, and its up-regulation is associated with heart failure. CaVβ binds to the α1 pore-forming subunit of L-type channels and augments calcium current density by facilitating channel opening and increasing the number of channels in the plasma membrane, by a poorly understood mechanism. Actin, a key component of the intracellular trafficking machinery, interacts with Src homology 3 domains in different proteins. Although CaVβ encompasses a highly conserved Src homology 3 domain, association with actin has not yet been explored. Here, using co-sedimentation assays and FRET experiments, we uncover a direct interaction between CaVβ and actin filaments. Consistently, single-molecule localization analysis reveals streaklike structures composed by CaVβ2 that distribute over several micrometers along actin filaments in HL-1 cardiomyocytes. Overexpression of CaVβ2-N3 in HL-1 cells induces an increase in L-type current without altering voltage-dependent activation, thus reflecting an increased number of channels in the plasma membrane. CaVβ mediated L-type up-regulation, and CaVβ-actin association is prevented by disruption of the actin cytoskeleton with cytochalasin D. Our study reveals for the first time an interacting partner of CaVβ that is directly involved in vesicular trafficking. We propose a model in which CaVβ promotes anterograde trafficking of the L-type channels by anchoring them to actin filaments in their itinerary to the plasma membrane. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation

    PubMed Central

    Fritzsche, Marco; Fernandes, Ricardo A.; Chang, Veronica T.; Colin-York, Huw; Clausen, Mathias P.; Felce, James H.; Galiani, Silvia; Erlenkämper, Christoph; Santos, Ana M.; Heddleston, John M.; Pedroza-Pacheco, Isabela; Waithe, Dominic; de la Serna, Jorge Bernardino; Lagerholm, B. Christoffer; Liu, Tsung-li; Chew, Teng-Leong; Betzig, Eric; Davis, Simon J.; Eggeling, Christian

    2017-01-01

    T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse. This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament lengths of two differently sized filamentous actin populations, wherein formin-mediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact with activating surfaces and no cytoskeletal rearrangements. Our work suggests that incipient signaling in T cells initiates global cytoskeletal rearrangements across the whole cell, including a stiffening process for possibly mechanically supporting contact formation at the immunological synapse interface as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions. PMID:28691087

  18. Semi-analytical study of the tokamak pedestal density profile in a single-null diverted plasma with puffing-recycling gas sources

    NASA Astrophysics Data System (ADS)

    Shi, Bingren

    2010-10-01

    The tokamak pedestal density structure is generally studied using a diffusion-dominant model. Recent investigations (Stacey and Groebner 2009 Phys. Plasmas 16 102504) from first principle based physics have shown a plausible existence of large inward convection in the pedestal region. The diffusion-convection equation with rapidly varying convection and diffusion coefficients in the near edge region and model puffing-recycling neutral particles is studied in this paper. A peculiar property of its solution for the existence of the large convection case is that the pedestal width of the density profile, qualitatively different from the diffusion-dominant case, depends mainly on the width of the inward convection and only weakly on the neutral penetration length and its injection position.

  19. Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Ying; Li, Zhaohui; Liu, Misha

    Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeledmore » single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.« less

  20. Global gyrokinetic simulation of Tokamak edge pedestal instabilities.

    PubMed

    Wan, Weigang; Parker, Scott E; Chen, Yang; Yan, Zheng; Groebner, Richard J; Snyder, Philip B

    2012-11-02

    Global electromagnetic gyrokinetic simulations show the existence of near threshold conditions for both a high-n kinetic ballooning mode (KBM) and an intermediate-n kinetic version of peeling-ballooning mode (KPBM) in the edge pedestal of two DIII-D H-mode discharges. When the magnetic shear is reduced in a narrow region of steep pressure gradient, the KPBM is significantly stabilized, while the KBM is weakly destabilized and hence becomes the most-unstable mode. Collisions decrease the KBM's critical β and increase the growth rate.

  1. Role of Cyclic Nucleotide-Dependent Actin Cytoskeletal Dynamics: [Ca2+]i and Force Suppression in Forskolin-Pretreated Porcine Coronary Arteries

    PubMed Central

    Hocking, Kyle M.; Baudenbacher, Franz J.; Putumbaka, Gowthami; Venkatraman, Sneha; Cheung-Flynn, Joyce; Brophy, Colleen M.; Komalavilas, Padmini

    2013-01-01

    Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca2+]i) and phosphorylation of myosin light chains (MLC). However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA) prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca2+]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm. PMID:23593369

  2. Role of cyclic nucleotide-dependent actin cytoskeletal dynamics:Ca(2+)](i) and force suppression in forskolin-pretreated porcine coronary arteries.

    PubMed

    Hocking, Kyle M; Baudenbacher, Franz J; Putumbaka, Gowthami; Venkatraman, Sneha; Cheung-Flynn, Joyce; Brophy, Colleen M; Komalavilas, Padmini

    2013-01-01

    Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca(2+)]i) and phosphorylation of myosin light chains (MLC). However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA) prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca(2+)]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.

  3. Mechanical compression and hydrostatic pressure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose.

    PubMed

    Knight, M M; Toyoda, T; Lee, D A; Bader, D L

    2006-01-01

    In numerous cell types, the cytoskeleton has been widely implicated in mechanotransduction pathways involving stretch-activated ion channels, integrins and deformation of intracellular organelles. Studies have also demonstrated that the cytoskeleton can undergo remodelling in response to mechanical stimuli such as tensile strain or fluid flow. In articular chondrocytes, the mechanotransduction pathways are complex, inter-related and as yet, poorly understood. Furthermore, little is known of how the chondrocyte cytoskeleton responds to physiological mechanical loading. This study utilises the well-characterised chondrocyte-agarose model and an established confocal image-analysis technique to demonstrate that both static and cyclic, compressive strain and hydrostatic pressure all induce remodelling of actin microfilaments. This remodelling was characterised by a change from a uniform to a more punctate distribution of cortical actin around the cell periphery. For some loading regimes, this remodelling was reversed over a subsequent 1h unloaded period. This reversible remodelling of actin cytoskeleton may therefore represent a mechanism through which the chondrocyte alters its mechanical properties and mechanosensitivity in response to physiological mechanical loading.

  4. 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.

  5. Actin filaments as tension sensors.

    PubMed

    Galkin, Vitold E; Orlova, Albina; Egelman, Edward H

    2012-02-07

    The field of mechanobiology has witnessed an explosive growth over the past several years as interest has greatly increased in understanding how mechanical forces are transduced by cells and how cells migrate, adhere and generate traction. Actin, a highly abundant and anomalously conserved protein, plays a large role in forming the dynamic cytoskeleton that is so essential for cell form, motility and mechanosensitivity. While the actin filament (F-actin) has been viewed as dynamic in terms of polymerization and depolymerization, new results suggest that F-actin itself may function as a highly dynamic tension sensor. This property may help explain the unusual conservation of actin's sequence, as well as shed further light on actin's essential role in structures from sarcomeres to stress fibers. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Rac interacts with Abi-1 and WAVE2 to promote an Arp2/3-dependent actin recruitment during chlamydial invasion.

    PubMed

    Carabeo, Rey A; Dooley, Cheryl A; Grieshaber, Scott S; Hackstadt, Ted

    2007-09-01

    Chlamydiae are Gram-negative obligate intracellular pathogens to which access to an intracellular environment is fundamental to their development. Chlamydial attachment to host cells induces the activation of the Rac GTPase, which is required for the localization of WAVE2 at the sites of chlamydial entry. Co-immunoprecipitation experiments demonstrated that Chlamydia trachomatis infection promoted the interaction of Rac with WAVE2 and Abi-1, but not with IRSp53. siRNA depletion of WAVE2 and Abi-1 abrogated chlamydia-induced actin recruitment and significantly reduced the uptake of the pathogen by the depleted cells. Chlamydia invasion also requires the Arp2/3 complex as demonstrated by its localization to the sites of chlamydial attachment and the reduced efficiency of chlamydial invasion in cells overexpressing the VCA domain of the neural Wiskott-Aldrich syndrome protein. Thus, C. trachomatis activates Rac and promotes its interaction with WAVE2 and Abi-1 to activate the Arp2/3 complex resulting in the induction of actin cytoskeletal rearrangements that are required for invasion.

  7. Interactions between G-actin and myosin subfragment 1: immunochemical probing of the NH2-terminal segment on actin.

    PubMed

    DasGupta, G; White, J; Cheung, P; Reisler, E

    1990-09-11

    The role of the N-terminal segment of actin in myosin-induced polymerization of G-actin was studied by using peptide antibodies directed against the first seven N-terminal residues of alpha-skeletal actin. Light scattering, fluorescence, and analytical ultracentrifugation experiments showed that the Fab fragments of these antibodies inhibited the polymerization of G-actin by myosin subfragment 1 (S-1) by inhibiting the binding of these proteins to each other. Fluorescence measurements using actin labeled with pyrenyliodoacetamide revealed that Fab inhibited the initial step in the binding of S-1 to G-actin. It is deduced from these results and from other literature data that the initial contact between G-actin and S-1 involves residues 1-7 on actin and residues 633-642 on the S-1 heavy chain. This interaction appears to be of major importance for the binding of S-1 and G-actin. The presence of additional myosin contact sites on G-actin was indicated by concentration-dependent recovery of S-1 binding to G-actin without displacement of Fab. The reduced Fab inhibition of S-1 binding to polymerizing and polymerized actin is consistent with the tightening of acto-S-1 binding at these sites or the creation of new sites upon formation of F-actin.

  8. 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

  9. Excitation of high wavenumber fluctuations by externally-imposed helical fields in edge pedestal plasmas

    NASA Astrophysics Data System (ADS)

    Singh, R.; Kim, J.-H.; Jhang, Hogun; Das, S.

    2018-03-01

    Two-step mode coupling analyses for nonlinear excitation of the ballooning mode (BM) in pedestal plasma by external helical magnetic field perturbation [Resonant Magnetic Perturbations (RMP)] are presented. This technique allows calculating the effect of higher harmonic sidebands generated by interaction of long scale RMP pump and BM. It is shown that RMP field perturbations can modify the BM growth rate and frequency through nonlinear Reynolds stress and magnetic stress. In particular, it is shown that both stresses can efficiently excite high wavenumber BM fluctuations which, in turn, can enhance the transport in the pedestal. Another notable feature of this analysis is the existence of short scale (high- k y ) nonlinear instability at Alfven time scale near the ideal BM threshold boundary.

  10. Role of gelsolin interaction with actin in regulation and creation of actin nuclei in chemotactic peptide activated polymorphonuclear neutrophils.

    PubMed Central

    Deaton, J D; Guerrero, T; Howard, T H

    1992-01-01

    In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin

  11. Role of gelsolin interaction with actin in regulation and creation of actin nuclei in chemotactic peptide activated polymorphonuclear neutrophils.

    PubMed

    Deaton, J D; Guerrero, T; Howard, T H

    1992-12-01

    In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin

  12. Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.

    PubMed

    Parker, William H; Qu, Zhi-chao; May, James M

    2015-08-28

    Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Intracellular Pressure Dynamics in Blebbing Cells

    PubMed Central

    Strychalski, Wanda; Guy, Robert D.

    2016-01-01

    Blebs are pressure-driven protrusions that play an important role in cell migration, particularly in three-dimensional environments. A bleb is initiated when the cytoskeleton detaches from the cell membrane, resulting in the pressure-driven flow of cytosol toward the area of detachment and local expansion of the cell membrane. Recent experiments involving blebbing cells have led to conflicting hypotheses regarding the timescale of intracellular pressure propagation. The interpretation of one set of experiments supports a poroelastic model of the cytoplasm that leads to slow pressure equilibration when compared to the timescale of bleb expansion. A different study concludes that pressure equilibrates faster than the timescale of bleb expansion. To address this discrepancy, a dynamic computational model of the cell was developed that includes mechanics of and the interactions among the cytoplasm, the actin cortex, the cell membrane, and the cytoskeleton. The model results quantify the relationship among cytoplasmic rheology, pressure, and bleb expansion dynamics, and provide a more detailed picture of intracellular pressure dynamics. This study shows the elastic response of the cytoplasm relieves pressure and limits bleb size, and that both permeability and elasticity of the cytoplasm determine bleb expansion time. Our model with a poroelastic cytoplasm shows that pressure disturbances from bleb initiation propagate faster than the timescale of bleb expansion and that pressure equilibrates slower than the timescale of bleb expansion. The multiple timescales in intracellular pressure dynamics explain the apparent discrepancy in the interpretation of experimental results. PMID:26958893

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

    PubMed Central

    Yang, Qing; Zhang, Xiao-Feng; Pollard, Thomas D.

    2012-01-01

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

  15. Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells: the role of the actin cytoskeleton.

    PubMed

    Pritchard, Scott; Erickson, Geoffrey R; Guilak, Farshid

    2002-11-01

    Loading of the spine alters the osmotic environment in the intervertebral disk (IVD) as interstitial water is expressed from the tissue. Cells from the three zones of the IVD, the anulus fibrosus (AF), transition zone (TZ), and nucleus pulposus (NP), respond to osmotic stress with altered biosynthesis through a pathway that may involve calcium (Ca(2+)) as a second messenger. We examined the hypothesis that IVD cells respond to hyperosmotic stress by increasing the concentration of intracellular calcium ([Ca(2+)](i)) through a mechanism involving F-actin. In response to hyperosmotic stress, control cells from all zones decreased in volume and cells from the AF and TZ exhibited [Ca(2+)](i) transients, while cells from the NP did not. Extracellular Ca(2+) was necessary to initiate [Ca(2+)](i) transients. Stabilization of F-actin with phalloidin prevented the Ca(2+) response in AF and TZ cells and decreased the rate of volume change in cells from all zones, coupled with an increase in the elastic moduli and apparent viscosity. Conversely, actin breakdown with cytochalasin D facilitated Ca(2+) signaling while decreasing the elastic moduli and apparent viscosity for NP cells. These results suggest that hyperosmotic stress induces volume change in IVD cells and may initiate [Ca(2+)](i) transients through an actin-dependent mechanism.

  16. A Second Las17 Monomeric Actin-Binding Motif Functions in Arp2/3-Dependent Actin Polymerization During Endocytosis

    PubMed Central

    Feliciano, Daniel; Tolsma, Thomas O.; Farrell, Kristen B.; Aradi, Al; Di Pietro, Santiago M.

    2018-01-01

    During clathrin-mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G-actin) and a central-acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3-dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G-actin-binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G-actin-binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two-hybrid system, GST-pulldown, fluorescence polarization and pyrene-actin polymerization assays, we show that LGM binds G-actin and is necessary for normal Arp2/3-mediated actin polymerization in vitro. Live-cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G-actin-binding motif, WH2. These results establish a second G-actin-binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME. PMID:25615019

  17. Fascin regulates nuclear actin during Drosophila oogenesis

    PubMed Central

    Kelpsch, Daniel J.; Groen, Christopher M.; Fagan, Tiffany N.; Sudhir, Sweta; Tootle, Tina L.

    2016-01-01

    Drosophila oogenesis provides a developmental system with which to study nuclear actin. During Stages 5–9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, whereas overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin, but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates that Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. In addition, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin and the mechanisms regulating nuclear actin are widely conserved. PMID:27535426

  18. Intrinsic, Functional, and Structural Properties of β-Thymosins and β-Thymosin/WH2 Domains in the Regulation and Coordination of Actin Self-Assembly Dynamics and Cytoskeleton Remodeling.

    PubMed

    Renault, L

    2016-01-01

    β-Thymosins are a family of heat-stable multifunctional polypeptides that are expressed as small proteins of about 5kDa (~45 amino acids) almost exclusively in multicellular animals. They were first isolated from the thymus. As full-length or truncated polypeptides, they appear to stimulate a broad range of extracellular activities in various signaling pathways, including tissue repair and regeneration, inflammation, cell migration, and immune defense. However, their cell surface receptors and structural mechanisms of regulations in these multiple pathways remain still poorly understood. Besides their extracellular activities, they belong to a larger family of small, intrinsically disordered actin-binding domains called WH2/β-thymosin domains that have been identified in more than 1800 multidomain proteins found in different taxonomic domains of life and involved in various actin-based motile processes including cell morphogenesis, motility, adhesions, tissue development, intracellular trafficking, or pathogen infections. This review briefly surveys the main recent findings to understand how these small, intrinsically disordered but functional domains can interact with many unrelated partners and can thus integrate and coordinate various intracellular activities in actin self-assembly dynamics and cell signaling pathways linked to their cytoskeleton remodeling. © 2016 Elsevier Inc. All rights reserved.

  19. Geometrical Determinants of Neuronal Actin Waves.

    PubMed

    Tomba, Caterina; Braïni, Céline; Bugnicourt, Ghislain; Cohen, Floriane; Friedrich, Benjamin M; Gov, Nir S; Villard, Catherine

    2017-01-01

    Hippocampal neurons produce in their early stages of growth propagative, actin-rich dynamical structures called actin waves. The directional motion of actin waves from the soma to the tip of neuronal extensions has been associated with net forward growth, and ultimately with the specification of neurites into axon and dendrites. Here, geometrical cues are used to control actin wave dynamics by constraining neurons on adhesive stripes of various widths. A key observable, the average time between the production of consecutive actin waves, or mean inter-wave interval (IWI), was identified. It scales with the neurite width, and more precisely with the width of the proximal segment close to the soma. In addition, the IWI is independent of the total number of neurites. These two results suggest a mechanistic model of actin wave production, by which the material conveyed by actin waves is assembled in the soma until it reaches the threshold leading to the initiation and propagation of a new actin wave. Based on these observations, we formulate a predictive theoretical description of actin wave-driven neuronal growth and polarization, which consistently accounts for different sets of experiments.

  20. Geometrical Determinants of Neuronal Actin Waves

    PubMed Central

    Tomba, Caterina; Braïni, Céline; Bugnicourt, Ghislain; Cohen, Floriane; Friedrich, Benjamin M.; Gov, Nir S.; Villard, Catherine

    2017-01-01

    Hippocampal neurons produce in their early stages of growth propagative, actin-rich dynamical structures called actin waves. The directional motion of actin waves from the soma to the tip of neuronal extensions has been associated with net forward growth, and ultimately with the specification of neurites into axon and dendrites. Here, geometrical cues are used to control actin wave dynamics by constraining neurons on adhesive stripes of various widths. A key observable, the average time between the production of consecutive actin waves, or mean inter-wave interval (IWI), was identified. It scales with the neurite width, and more precisely with the width of the proximal segment close to the soma. In addition, the IWI is independent of the total number of neurites. These two results suggest a mechanistic model of actin wave production, by which the material conveyed by actin waves is assembled in the soma until it reaches the threshold leading to the initiation and propagation of a new actin wave. Based on these observations, we formulate a predictive theoretical description of actin wave-driven neuronal growth and polarization, which consistently accounts for different sets of experiments. PMID:28424590

  1. Actinic keratosis among seafarers.

    PubMed

    Oldenburg, M; Kuechmeister, B; Ohnemus, U; Baur, X; Moll, I

    2013-11-01

    The aim of this study was to assess the prevalence of UV-induced actinic keratosis and further skin lesions. A newly developed questionnaire about lifetime UV radiation exposure was completed by 514 seafarers. An experienced dermatologist inspected the whole-body skin status of all participants. The questionnaire revealed a pre-employment UV radiation exposure in 104 seafarers, sunbed use in 26 subjects and a median work-related UV radiation exposure at sea of 20 years. The diagnosis of actinic keratoses was made in 94 seafarers and the clinical diagnosis of skin cancers in 48 seafarers (28 basal cell carcinoma, 11 squamous cell carcinoma, 9 malignant melanoma). After age standardisation according to a European reference population, the male European seafarers in this study had a 1.80-fold increased risk of actinic keratosis. Actinic keratoses [OR 1.03 (1.01-1.05)] and squamous cell carcinoma [OR 1.07 (1.01-1.13)] were related to the duration of seafaring time in years. A significant association was also found between actinic keratosis/squamous cell carcinoma and sunlight exposure during home leave [OR 1.67 (1.03-2.81) and OR 6.19 (1.18-32.40)]. Furthermore, the engine room personnel-especially the technical officers-were at higher risk of developing actinic keratosis. Due to the high prevalence of actinic keratosis especially among older seafarers with fair skin, with longer duration of seafaring employment at sea and with higher UV exposure during home leave, more intensive advice should be given on sun protection both at sea and ashore.

  2. The relationships between edge localized modes suppression, pedestal profiles and lithium wall coatings in NSTX

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Boyle, D. P.; Maingi, R.; Snyder, P. B.

    2011-01-01

    Recently in the National Spherical Torus Experiment (NSTX), increasing lithium wall coatings suppressed edge localized modes (ELMs), gradually but not quite monotonically. This work details profile and stability analysis as ELMs disappeared throughout the lithium scan. While the quantity of lithium deposited between discharges did not uniquely determine the presence of ELMs, profile analysis demonstrated that lithium was correlated with wider density and pressure pedestals with peak gradients farther from the separatrix. Moreover, the ELMy and ELM-free discharges were cleanly separated by their density and pedestal widths and peak gradient locations. Ultimately, ELMs were only suppressed when lithium caused themore » density pedestal to widen and shift inward. These changes in the density gradient were directly reflected in the pressure gradient and calculated bootstrap current. This supports the theory that ELMs in NSTX are caused by peeling and/or ballooning modes, as kink/peeling modes are stabilized when the edge current and pressure gradient shift away from the separatrix. Edge stability analysis using ELITE corroborated this picture, as reconstructed equilibria from ELM-free discharges were generally farther from their kink/peeling stability boundaries than ELMy discharges. We conclude that density profile control provided by lithium is the key first step to ELM suppression in NSTX.« less

  3. A novel technique for real-time estimation of edge pedestal density gradients via reflectometer time delay data

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zeng, L., E-mail: zeng@fusion.gat.com; Doyle, E. J.; Rhodes, T. L.

    2016-11-15

    A new model-based technique for fast estimation of the pedestal electron density gradient has been developed. The technique uses ordinary mode polarization profile reflectometer time delay data and does not require direct profile inversion. Because of its simple data processing, the technique can be readily implemented via a Field-Programmable Gate Array, so as to provide a real-time density gradient estimate, suitable for use in plasma control systems such as envisioned for ITER, and possibly for DIII-D and Experimental Advanced Superconducting Tokamak. The method is based on a simple edge plasma model with a linear pedestal density gradient and low scrape-off-layermore » density. By measuring reflectometer time delays for three adjacent frequencies, the pedestal density gradient can be estimated analytically via the new approach. Using existing DIII-D profile reflectometer data, the estimated density gradients obtained from the new technique are found to be in good agreement with the actual density gradients for a number of dynamic DIII-D plasma conditions.« less

  4. Bootstrap Current for the Edge Pedestal Plasma in a Diverted Tokamak Geometry

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Koh, S.; Chang, C. S.; Ku, S.

    The edge bootstrap current plays a critical role in the equilibrium and stability of the steep edge pedestal plasma. The pedestal plasma has an unconventional and difficult neoclassical property, as compared with the core plasma. It has a narrow passing particle region in velocity space that can be easily modified or destroyed by Coulomb collisions. At the same time, the edge pedestal plasma has steep pressure and electrostatic potential gradients whose scale-lengths are comparable with the ion banana width, and includes a magnetic separatrix surface, across which the topological properties of the magnetic field and particle orbits change abruptly. Amore » driftkinetic particle code XGC0, equipped with a mass-momentum-energy conserving collision operator, is used to study the edge bootstrap current in a realistic diverted magnetic field geometry with a self-consistent radial electric field. When the edge electrons are in the weakly collisional banana regime, surprisingly, the present kinetic simulation confirms that the existing analytic expressions [represented by O. Sauter et al. , Phys. Plasmas 6 , 2834 (1999)] are still valid in this unconventional region, except in a thin radial layer in contact with the magnetic separatrix. The agreement arises from the dominance of the electron contribution to the bootstrap current compared with ion contribution and from a reasonable separation of the trapped-passing dynamics without a strong collisional mixing. However, when the pedestal electrons are in plateau-collisional regime, there is significant deviation of numerical results from the existing analytic formulas, mainly due to large effective collisionality of the passing and the boundary layer trapped particles in edge region. In a conventional aspect ratio tokamak, the edge bootstrap current from kinetic simulation can be significantly less than that from the Sauter formula if the electron collisionality is high. On the other hand, when the aspect ratio is close

  5. Bootstrap current for the edge pedestal plasma in a diverted tokamak geometry

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Koh, S.; Choe, W.; Chang, C. S.

    The edge bootstrap current plays a critical role in the equilibrium and stability of the steep edge pedestal plasma. The pedestal plasma has an unconventional and difficult neoclassical property, as compared with the core plasma. It has a narrow passing particle region in velocity space that can be easily modified or destroyed by Coulomb collisions. At the same time, the edge pedestal plasma has steep pressure and electrostatic potential gradients whose scale-lengths are comparable with the ion banana width, and includes a magnetic separatrix surface, across which the topological properties of the magnetic field and particle orbits change abruptly. Amore » drift-kinetic particle code XGC0, equipped with a mass-momentum-energy conserving collision operator, is used to study the edge bootstrap current in a realistic diverted magnetic field geometry with a self-consistent radial electric field. When the edge electrons are in the weakly collisional banana regime, surprisingly, the present kinetic simulation confirms that the existing analytic expressions [represented by O. Sauter et al., Phys. Plasmas 6, 2834 (1999)] are still valid in this unconventional region, except in a thin radial layer in contact with the magnetic separatrix. The agreement arises from the dominance of the electron contribution to the bootstrap current compared with ion contribution and from a reasonable separation of the trapped-passing dynamics without a strong collisional mixing. However, when the pedestal electrons are in plateau-collisional regime, there is significant deviation of numerical results from the existing analytic formulas, mainly due to large effective collisionality of the passing and the boundary layer trapped particles in edge region. In a conventional aspect ratio tokamak, the edge bootstrap current from kinetic simulation can be significantly less than that from the Sauter formula if the electron collisionality is high. On the other hand, when the aspect ratio is close to

  6. Regulation of Retinoschisin Secretion in Weri-Rb1 Cells by the F-Actin and Microtubule Cytoskeleton

    PubMed Central

    Kitamura, Eiko; Gribanova, Yekaterina E.; Farber, Debora B.

    2011-01-01

    Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process. PMID:21738583

  7. Surface-induced polymerization of actin.

    PubMed Central

    Renault, A; Lenne, P F; Zakri, C; Aradian, A; Vénien-Bryan, C; Amblard, F

    1999-01-01

    Living cells contain a very large amount of membrane surface area, which potentially influences the direction, the kinetics, and the localization of biochemical reactions. This paper quantitatively evaluates the possibility that a lipid monolayer can adsorb actin from a nonpolymerizing solution, induce its polymerization, and form a 2D network of individual actin filaments, in conditions that forbid bulk polymerization. G- and F-actin solutions were studied beneath saturated Langmuir monolayers containing phosphatidylcholine (PC, neutral) and stearylamine (SA, a positively charged surfactant) at PC:SA = 3:1 molar ratio. Ellipsometry, tensiometry, shear elastic measurements, electron microscopy, and dark-field light microscopy were used to characterize the adsorption kinetics and the interfacial polymerization of actin. In all cases studied, actin follows a monoexponential reaction-limited adsorption with similar time constants (approximately 10(3) s). At a longer time scale the shear elasticity of the monomeric actin adsorbate increases only in the presence of lipids, to a 2D shear elastic modulus of mu approximately 30 mN/m, indicating the formation of a structure coupled to the monolayer. Electron microscopy shows the formation of a 2D network of actin filaments at the PC:SA surface, and several arguments strongly suggest that this network is indeed causing the observed elasticity. Adsorption of F-actin to PC:SA leads more quickly to a slightly more rigid interface with a modulus of mu approximately 50 mN/m. PMID:10049338

  8. Gyrokinetic analysis of pedestal transport

    NASA Astrophysics Data System (ADS)

    Kotschenreuther, Mike; Liu, X.; Hatch, Dr; Zheng, Lj; Mahajan, S.; Diallo, A.; Groebner, Rj; Hubbard, Ae; Hughes, Jw; Maggi, Cf; Saarelma, S.; JET Contributors

    2017-10-01

    Surprisingly, basic considerations can determine which modes are responsible for pedestal energy transport (e.g., KBM, ETG, ITG, MTM etc.). Gyrokinetic simulations of experiments, and analysis of the Gyrokinetic-Maxwell equations, find that each mode type produces characteristic ratios of transport in the various channels: density, heat and impurities. This, together with the relative size of the driving sources of each channel, can strongly constrain or determine the dominant modes causing energy transport. MHD-like modes are not the dominant agent of energy transport - when the density source is weak as is often expected. Drift modes must fill this role. Detailed examination of experimental observations, including frequency and transport channel behavior, with simulations, demonstrates these points. Also see related posters by X. Liu, D.R. Hatch, and A. Blackmon. Work supported by US DOE under DE-FC02-04ER54698, DE-FG02-04ER54742 and DE-FC02-99ER54512 and by Eurofusion under Grant No. 633053.

  9. 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.

  10. Polycation induced actin bundles.

    PubMed

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

    2011-04-01

    Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4Å) MTS-1 (1,1-methanedyl bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Deuterium charge exchange recombination spectroscopy from the top of the pedestal to the scrape off layer in H-mode plasmas

    NASA Astrophysics Data System (ADS)

    Haskey, S. R.; Grierson, B. A.; Stagner, L.; Burrell, K. H.; Chrystal, C.; Groebner, R. J.; Ashourvan, A.; Pablant, N. A.

    2017-10-01

    Recent completion of the thirty two channel main-ion (deuterium) charge exchange recombination spectroscopy (CER) diagnostic on DIII-D [J.L. Luxon, Nucl. Fusion 42 (2002) 614] enables detailed comparisons between impurity and main-ion temperature, density, and toroidal rotation. Sixteen sightlines cover the core of the plasma and another sixteen are densely packed towards the edge, providing high resolution measurements of the pedestal and steep gradient edge region of H-mode plasmas. The complexities of the Dα spectrum require fitting with a comprehensive model, as well as using iterative collisional radiative modeling to determine the underlying thermal deuterium ion properties. Large differences in the structure and magnitude of impurity (C6+) and main-ion (D+) toroidal rotation profiles are seen in the H-mode pedestal. Additionally the D+ temperature can be half the value of the C6+ temperature at the separatrix and shows more of a pedestal structure. Typically only the impurity properties are measured and the main-ion properties are either assumed to be the same, or inferred using neoclassical models, which require validation in the steep gradient region. These measured differences have implications for transport model validation, intrinsic rotation studies, pedestal stability, and the boundary conditions for scrape off layer and plasma material interactions studies.

  12. Deuterium charge exchange recombination spectroscopy from the top of the pedestal to the scrape off layer in H-mode plasmas

    DOE PAGES

    Haskey, S. R.; Grierson, B. A.; Stagner, L.; ...

    2017-10-25

    Recent completion of the thirty two channel main-ion (deuterium) charge exchange recombination spectroscopy (CER) diagnostic on DIII-D enables detailed comparisons between impurity and main-ion temperature, density, and toroidal rotation. Sixteen sightlines cover the core of the plasma and another sixteen are densely packed towards the edge, providing high resolution measurements of the pedestal and steep gradient edge region of H-mode plasmas. The complexities of the D α spectrum require fitting with a comprehensive model, as well as using iterative collisional radiative modeling to determine the underlying thermal deuterium ion properties. Large differences in the structure and magnitude of impurity (Cmore » 6+) and main-ion (D +) toroidal rotation profiles are seen in the H-mode pedestal. Additionally the D + temperature can be half the value of the C 6+ temperature at the separatrix and shows more of a pedestal structure. Typically only the impurity properties are measured and the main-ion properties are either assumed to be the same, or inferred using neoclassical models, which require validation in the steep gradient region. Furthermore, these measured differences have implications for transport model validation, intrinsic rotation studies, pedestal stability, and the boundary conditions for scrape off layer and plasma material interactions studies.« less

  13. Feedback Regulation of Intracellular Hydrostatic Pressure in Surface Cells of the Lens

    PubMed Central

    Gao, Junyuan; Sun, Xiurong; White, Thomas W.; Delamere, Nicholas A.; Mathias, Richard T.

    2015-01-01

    In wild-type lenses from various species, an intracellular hydrostatic pressure gradient goes from ∼340 mmHg in central fiber cells to 0 mmHg in surface cells. This gradient drives a center-to-surface flow of intracellular fluid. In lenses in which gap-junction coupling is increased, the central pressure is lower, whereas if gap-junction coupling is reduced, the central pressure is higher but surface pressure is always zero. Recently, we found that surface cell pressure was elevated in PTEN null lenses. This suggested disruption of a feedback control system that normally maintained zero surface cell pressure. Our purpose in this study was to investigate and characterize this feedback control system. We measured intracellular hydrostatic pressures in mouse lenses using a microelectrode/manometer-based system. We found that all feedback went through transport by the Na/K ATPase, which adjusted surface cell osmolarity such that pressure was maintained at zero. We traced the regulation of Na/K ATPase activity back to either TRPV4, which sensed positive pressure and stimulated activity, or TRPV1, which sensed negative pressure and inhibited activity. The inhibitory effect of TRPV1 on Na/K pumps was shown to signal through activation of the PI3K/AKT axis. The stimulatory effect of TRPV4 was shown in previous studies to go through a different signal transduction path. Thus, there is a local two-legged feedback control system for pressure in lens surface cells. The surface pressure provides a pedestal on which the pressure gradient sits, so surface pressure determines the absolute value of pressure at each radial location. We speculate that the absolute value of intracellular pressure may set the radial gradient in the refractive index, which is essential for visual acuity. PMID:26536260

  14. Septins arrange F-actin-containing fibers on the Chlamydia trachomatis inclusion and are required for normal release of the inclusion by extrusion.

    PubMed

    Volceanov, Larisa; Herbst, Katharina; Biniossek, Martin; Schilling, Oliver; Haller, Dirk; Nölke, Thilo; Subbarayal, Prema; Rudel, Thomas; Zieger, Barbara; Häcker, Georg

    2014-10-07

    Chlamydia trachomatis is an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle of C. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress of C. trachomatis from human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens. Importance: Chlamydia trachomatis is a frequent bacterial pathogen throughout the world, causing mostly eye and genital infections. C. trachomatis can develop only inside host cells; it multiplies inside a membranous vacuole in the cytosol, termed an inclusion. The inclusion is covered by cytoskeletal "coats" or "cages," whose organization and function are poorly understood. We here report that a relatively little-characterized group of proteins, septins, is required to organize actin fibers on the

  15. Nuclear positioning by actin cables and perinuclear actin

    PubMed Central

    Huelsmann, Sven; Brown, Nicholas H

    2014-01-01

    Nuclear positioning is an important process during development and homeostasis. Depending on the affected tissue, mislocalized nuclei can alter cellular processes such as polarization, differentiation, or migration and lead ultimately to diseases. Many cells actively control the position of their nucleus using their cytoskeleton and motor proteins. We have recently shown that during Drosophila oogenesis, nurse cells employ cytoplasmic actin cables in association with perinuclear actin to position their nucleus. Here, we briefly summarize our work and discuss why nuclear positioning in nurse cells is specialized but the molecular mechanisms are likely to be more generally used. PMID:24905988

  16. Non-Straub type actin from molluscan catch muscle

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shelud'ko, Nikolay S., E-mail: sheludko@stl.ru; Girich, Ulyana V.; Lazarev, Stanislav S.

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

  17. Actin polymerization drives septation of Listeria monocytogenes namA hydrolase mutants, demonstrating host correction of a bacterial defect.

    PubMed

    Alonzo, Francis; McMullen, P David; Freitag, Nancy E

    2011-04-01

    The Gram-positive bacterial cell wall presents a structural barrier that requires modification for protein secretion and large-molecule transport as well as for bacterial growth and cell division. The Gram-positive bacterium Listeria monocytogenes adjusts cell wall architecture to promote its survival in diverse environments that include soil and the cytosol of mammalian cells. Here we provide evidence for the enzymatic flexibility of the murein hydrolase NamA and demonstrate that bacterial septation defects associated with a loss of NamA are functionally complemented by physical forces associated with actin polymerization within the host cell cytosol. L. monocytogenes ΔnamA mutants formed long bacterial chains during exponential growth in broth culture; however, normal septation could be restored if mutant cells were cocultured with wild-type L. monocytogenes bacteria or by the addition of exogenous NamA. Surprisingly, ΔnamA mutants were not significantly attenuated for virulence in mice despite the pronounced exponential growth septation defect. The physical force of L. monocytogenes-mediated actin polymerization within the cytosol was sufficient to sever ΔnamA mutant intracellular chains and thereby enable the process of bacterial cell-to-cell spread so critical for L. monocytogenes virulence. The inhibition of actin polymerization by cytochalasin D resulted in extended intracellular bacterial chains for which septation was restored following drug removal. Thus, despite the requirement for NamA for the normal septation of exponentially growing L. monocytogenes cells, the hydrolase is essentially dispensable once L. monocytogenes gains access to the host cell cytosol. This phenomenon represents a notable example of eukaryotic host cell complementation of a bacterial defect.

  18. The nuclear F-actin interactome of Xenopus oocytes reveals an actin-bundling kinesin that is essential for meiotic cytokinesis

    PubMed Central

    Samwer, Matthias; Dehne, Heinz-Jürgen; Spira, Felix; Kollmar, Martin; Gerlich, Daniel W; Urlaub, Henning; Görlich, Dirk

    2013-01-01

    Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis. PMID:23727888

  19. F-actin and microtubule suspensions as indeterminate fluids.

    PubMed

    Buxbaum, R E; Dennerll, T; Weiss, S; Heidemann, S R

    1987-03-20

    The viscosity of F-actin and microtubule suspensions has been measured as a function of shear rate with a Weissenberg rheogoniometer. At shear rates of less than 1.0 per second the viscosity of suspensions of these two structural proteins is inversely proportional to shear rate. These results are consistent with previous in vivo measurements of the viscosity of cytoplasm. This power law implies that shear stress is independent of shear rate; that is, shear stress is a constant at all shear rates less than 1.0 per second. Thus the flow profile of these fluids is indeterminate, or nearly so. This flow property may explain several aspects of intracellular motility in living cells. Possible explanations for this flow property are based on a recent model for semidilute suspensions of rigid rods or a classical friction model for liquid crystals.

  20. Electrostatic interactions between the Bni1p formin FH2 domain and actin influence actin filament nucleation

    DOE PAGES

    Baker, Joseph L.; Courtemanche, Naomi; Parton, Daniel L.; ...

    2014-12-04

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

  1. Plasma levels of F-actin and F:G-actin ratio as potential new biomarkers in patients with septic shock.

    PubMed

    Belsky, Justin B; Morris, Daniel C; Bouchebl, Ralph; Filbin, Michael R; Bobbitt, Kevin R; Jaehne, Anja K; Rivers, Emanuel P

    2016-01-01

    To compare plasma levels of F-actin, G-actin and thymosin beta 4 (TB4) in humans with septic shock, noninfectious systemic inflammatory response syndrome (SIRS) and healthy controls. F-actin was significantly elevated in septic shock as compared with noninfectious SIRS and healthy controls. G-actin levels were greatest in the noninfectious SIRS group but significantly elevated in septic shock as compared with healthy controls. TB4 was not detectable in the septic shock or noninfectious SIRS group above the assay's lowest detection range (78 ng/ml). F-actin is significantly elevated in patients with septic shock as compared with noninfectious SIRS. F-actin and the F:G-actin ratio are potential biomarkers for the diagnosis of septic shock.

  2. A systems-biology approach to yeast actin cables.

    PubMed

    Drake, Tyler; Yusuf, Eddy; Vavylonis, Dimitrios

    2012-01-01

    We focus on actin cables in yeast as a model system for understanding cytoskeletal organization and the workings of actin itself. In particular, we highlight quantitative approaches on the kinetics of actin-cable assembly and methods of measuring their morphology by image analysis. Actin cables described by these studies can span greater lengths than a thousand end-to-end actin-monomers. Because of this difference in length scales, control of the actin-cable system constitutes a junction between short-range interactions - among actin-monomers and nucleating, polymerization-facilitating, side-binding, severing, and cross-linking proteins - and the emergence of cell-scale physical form as embodied by the actin cables themselves.

  3. Leishmania infection inhibits macrophage motility by altering F-actin dynamics and the expression of adhesion complex proteins

    PubMed Central

    de Menezes, Juliana Perrone Bezerra; Koushik, Amrita; Das, Satarupa; Guven, Can; Siegel, Ariel; Laranjeira-Silva, Maria Fernanda; Losert, Wolfgang; Andrews, Norma W.

    2016-01-01

    Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function is still poorly understood. In this study we show that L. amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis-infected macrophages also show reduced directional migration in response to the chemokine MCP-1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin and phosphorylated FAK when compared to non-infected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F-actin turnover frequency in L. amazonensis-infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane-extracellular matrix interactions. PMID:27641840

  4. Stochastic Severing of Actin Filaments by Actin Depolymerizing Factor/Cofilin Controls the Emergence of a Steady Dynamical Regime

    PubMed Central

    Roland, Jeremy; Berro, Julien; Michelot, Alphée; Blanchoin, Laurent; Martiel, Jean-Louis

    2008-01-01

    Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-Pi versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo. PMID:18065447

  5. 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

  6. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

    Actin is a globular protein which forms long polar filaments in the eukaryotic cytoskeleton. Actin networks play a key role in cell mechanics and cell motility. They have also been implicated in information transmission and processing, memory and learning in neuronal cells. The actin filaments have been shown to support propagation of voltage pulses. Here we apply a coupled nonlinear transmission line model of actin filaments to study interactions between voltage pulses. To represent digital information we assign a logical TRUTH value to the presence of a voltage pulse in a given location of the actin filament, and FALSE to the pulse's absence, so that information flows along the filament with pulse transmission. When two pulses, representing Boolean values of input variables, interact, then they can facilitate or inhibit further propagation of each other. We explore this phenomenon to construct Boolean logical gates and a one-bit half-adder with interacting voltage pulses. We discuss implications of these findings on cellular process and technological applications.

  7. Actin filaments-A target for redox regulation.

    PubMed

    Wilson, Carlos; Terman, Jonathan R; González-Billault, Christian; Ahmed, Giasuddin

    2016-10-01

    Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through noncovalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates-the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL-and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Actin dynamics in Amoeba proteus motility.

    PubMed

    Pomorski, P; Krzemiński, P; Wasik, A; Wierzbicka, K; Barańska, J; Kłopocka, W

    2007-01-01

    We studied the distribution of the endogenous Arp2/3 complex in Amoeba proteus and visualised the ratio of filamentous (F-actin) to total actin in living cells. The presented results show that in the highly motile Amoeba proteus, Arp2/3 complex-dependent actin polymerisation is involved in the formation of the branching network of the contractile layer, adhesive structures, and perinuclear cytoskeleton. The aggregation of the Arp2/3 complex in the cortical network, with the exception of the uroid and advancing fronts, and the spatial orientation of microfilaments at the leading edge suggest that actin polymerisation in this area is not sufficient to provide the driving force for membrane displacement. The examined proteins were enriched in the pinocytotic pseudopodia and the perinuclear cytoskeleton in pinocytotic amoebae. In migrating amoebae, the course of changes in F-actin concentration corresponded with the distribution of tension in the cell cortex. The maximum level of F-actin in migrating amoebae was observed in the middle-posterior region and in the front of retracting pseudopodia. Arp2/3 complex-dependent actin polymerisation did not seem to influence F-actin concentration. The strongly condensed state of the microfilament system could be attributed to strong isometric contraction of the cortical layer accompanied by its retraction from distal cell regions. Isotonic contraction was limited to the uroid.

  9. A Systems-Biology Approach to Yeast Actin Cables

    PubMed Central

    Drake, Tyler; Yusuf, Eddy; Vavylonis, Dimitrios

    2011-01-01

    We focus on actin cables in yeast as a model system for understanding cytoskeletal organization and the workings of actin itself. In particular, we highlight quantitative approaches on the kinetics of actin cable assembly and methods of measuring their morphology by image analysis. Actin cables described by these studies can span greater lengths than a thousand end-to-end actin monomers. Because of this difference in length scales, control of the actin-cable system constitutes a junction between short-range interactions—among actin monomers and nucleating, polymerization-facilitating, side-binding, severing, and cross-linking proteins—and the emergence of cell-scale physical form as embodied by the actin cables themselves. PMID:22161338

  10. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    PubMed Central

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

    2015-01-01

    Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s−1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening. PMID:25615864

  11. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    DOE PAGES

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; ...

    2014-06-01

    Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s -1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away frommore » the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.« less

  12. Allele-specific Effects of Human Deafness γ-Actin Mutations (DFNA20/26) on the Actin/Cofilin Interaction*

    PubMed Central

    Bryan, Keith E.; Rubenstein, Peter A.

    2009-01-01

    Auditory hair cell function requires proper assembly and regulation of the nonmuscle gamma isoactin-rich cytoskeleton, and six point mutations in this isoactin cause a type of delayed onset autosomal dominant nonsyndromic progressive hearing loss, DFNA20/26. The molecular basis underlying this actin-dependent hearing loss is unknown. To address this problem, the mutations have been introduced into yeast actin, and their effects on actin function were assessed in vivo and in vitro. Because we previously showed that polymerization was unaffected in five of the six mutants, we have focused on proteins that regulate actin, in particular cofilin, which severs F-actin and sequesters actin monomers. The mutations do not affect the interaction of cofilin with G-actin. However, T89I and V370A mutant F-actins are much more susceptible to cofilin disassembly than WT filaments in vitro. Conversely, P332A filaments demonstrate enhanced resistance. Wild type actin solutions containing T89I, K118M, or P332A mutant actins at mole fractions similar to those found in the hair cell respond in vitro toward cofilin in a manner proportional to the level of the mutant present. Finally, depression of cofilin action in vivo by elimination of the cofilin-activating protein, Aip1p, rescues the inability to grow on glycerol caused by K118M, T278I, P332A, and V370A. These results suggest that a filament instability caused by these mutations can be balanced by decreasing a system in vivo that promotes increased filament turnover. Such mutant-dependent filament destabilization could easily result in hair cell malfunction leading to the late-onset hearing loss observed in these patients. PMID:19419963

  13. Support pedestals for interconnecting a cover and nozzle band wall in a gas turbine nozzle segment

    DOEpatents

    Yu, Yufeng Phillip; Itzel, Gary Michael; Webbon, Waylon Willard; Bagepalli, Radhakrishna; Burdgick, Steven Sebastian; Kellock, Iain Robertson

    2002-01-01

    A gas turbine nozzle segment has outer and inner band portions. Each band portion includes a nozzle wall, a cover and an impingement plate between the cover and nozzle wall defining two cavities on opposite sides of the impingement plate. Cooling steam is supplied to one cavity for flow through the apertures of the impingement plate to cool the nozzle wall. Structural pedestals interconnect the cover and nozzle wall and pass through holes in the impingement plate to reduce localized stress otherwise resulting from a difference in pressure within the chamber of the nozzle segment and the hot gas path and the fixed turbine casing surrounding the nozzle stage. The pedestals may be cast or welded to the cover and nozzle wall.

  14. NASTRAN structural model for the large ground antenna pedestal with applications to hydrostatic bearing of film

    NASA Technical Reports Server (NTRS)

    Chian, C. T.

    1986-01-01

    Investigations were conducted on the 64-meter antenna hydrostatic bearing oil film thickness under a variety of loads and elastic moduli. These parametric studies used a NASTRAN pedestal structural model to determine the deflections under the hydrostatic bearing pad. The deflections formed the input for a computer program to determine the hydrostratic bearing oil film thickness. For the future 64-meter to 70-meter antenna extension and for the 2.2-meter (86-in.) haunch concrete replacement cases, the program predicted safe oil film thickness (greater than 0.13 mm (0.005 in.) at the corners of the pad). The effects of varying moduli of elasticity for different sections of the pedestal and the film height under stressed runner conditions were also studied.

  15. Collisional Ion and Electron Scale Gyrokinetic Simulations in the Tokamak Pedestal

    NASA Astrophysics Data System (ADS)

    Belli, E. A.; Candy, J.; Snyder, P. B.

    2016-10-01

    A new gyrokinetic solver, CGYRO, has been developed for precise studies of high collisionality regimes, such as the H-mode pedestal and L-mode edge. Building on GYRO and NEO, CGYRO uses the same velocity-space coordinates as NEO to optimize the accuracy of the collision dynamics and allow for advanced operators beyond the standard Lorentz pitch-angle scattering model. These advanced operators include energy diffusion and finite-FLR collisional effects. The code is optimized for multiscale (coupled electron and ion turbulence scales) simulations, employing a new spatial discretization and array distribution scheme that targets scalability on next-generation (exascale) HPC systems. In this work, CGYRO is used to study the complex spectrum of modes in the pedestal region. The onset of the linear KBM with full collisional effects is assessed to develop an improved KBM/RBM model for EPED. The analysis is extended to high k to explore the role of electron-scale (ETG-range) physics. Comparisons with new analytic collisional theories are made. Inclusion of sonic toroidal rotation (including full centrifugal effects) for studies including heavy wall impurities is also reported. Work supported in part by the US DOE under DE-FC02-06ER54873 and DE-FC02-08ER54963.

  16. F-Actin Dynamics in Neurospora crassa ▿ †

    PubMed Central

    Berepiki, Adokiye; Lichius, Alexander; Shoji, Jun-Ya; Tilsner, Jens; Read, Nick D.

    2010-01-01

    This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth. PMID:20139238

  17. Extracellular and Intracellular Mechanisms Mediating Metastatic Activity of Exogenous Osteopontin

    PubMed Central

    Mandelin, Jami; Lin, Emme C. K.; Hu, Dana D.; Knowles, Susan K.; Do, Kim-Anh; Wang, Xuemei; Sage, E. Helene; Smith, Jeffrey W.; Arap, Wadih; Pasqualini, Renata

    2009-01-01

    BACKGROUND Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. We used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo designed to test extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS In the extracellular environment, we confirm that soluble osteopontin is required for its pro-metastatic effects; this phenomenon is specific, RGD-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induces rapid Tyr-418 dephosphorylation of c-Src, with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation is followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSION Our results reveal a complex molecular interaction as well as a dual role for osteopontin in metastasis that is dependent on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to anti-metastasis strategies. PMID:19224553

  18. Actinic comedonal plaque.

    PubMed

    Eastern, J S; Martin, S

    1980-12-01

    Solitary plaques developed on the sun-exposed and damaged skin of five elderly, fair-skinned individuals. The lesions, erythematous to bluish confluent nodules and plaques with a cribriform appearance and comedone-like structures, presented a distinctive histologic picture of dilated, keratin-filled follicles within a matrix of amorphous, damaged collagen. We believe these cases demonstrate a distinct entity within the realm of actinic dermatoses, for which the name "actinic comedonal plaque" seems appropriate.

  19. Spire, an actin nucleation factor, regulates cell division during Drosophila heart development.

    PubMed

    Xu, Peng; Johnson, Tamara L; Stoller-Conrad, Jessica R; Schulz, Robert A

    2012-01-01

    The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis.

  20. Yogi Berra, Forrest Gump, and the discovery of Listeria actin comet tails.

    PubMed

    Portnoy, Daniel A

    2012-04-01

    In 1988, eminent cell biologist Lew Tilney and newly appointed Assistant Professor of Microbiology Dan Portnoy met at a picnic and initiated a collaboration that led to a groundbreaking paper published in Journal of Cell Biology entitled "Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes." The paper has been cited more than 800 times, the most of any publication in the careers of both investigators. Using an electron microscope from the Sputnik era, they assembled a stunning collection of micrographs that illustrated how L. monocytogenes enters the host cell and exploits a host system of actin-based motility to move within cells and into neighboring cells without leaving the host cell cytosol. This research captured the imagination of cell biologists and microbiologists alike and led to novel insights into cytoskeletal dynamics. Here, Portnoy provides a retrospective that shares text from the original submission that was deleted at the time of publication, along with reviewers' comments ranging from "It is really just a show and tell paper and doesn';t have any meat" to "the finding will have major impact in cell biology and in medicine. Potentially, the paper will be a classic."

  1. Control of the actin cytoskeleton in root hair development.

    PubMed

    Pei, Weike; Du, Fei; Zhang, Yi; He, Tian; Ren, Haiyun

    2012-05-01

    The development of root hair includes four stages: bulge site selection, bulge formation, tip growth, and maturation. The actin cytoskeleton is involved in all of these stages and is organized into distinct arrangements in the different stages. In addition to the actin configuration, actin isoforms also play distinct roles in the different stages. The actin cytoskeleton is regulated by actin-binding proteins, such as formin, Arp2/3 complex, profilin, actin depolymerizing factor, and villin. Some upstream signals, i.e. calcium, phospholipids, and small GTPase regulate the activity of these actin-binding proteins to produce the proper actin configuration. We constructed a working model on how the actin cytoskeleton is controlled by actin-binding proteins and upstream signaling in root hair development based on the current literature: at the tip of hairs, actin polymerization appears to be facilitated by Arp2/3 complex that is activated by small GTPase, and profilin that is regulated by phosphatidylinositol 4,5-bisphosphate. Meanwhile, actin depolymerization and turnover are likely mediated by villin and actin depolymerizing factor, which are stimulated by calcium. At the shank, actin cables are produced by formin and villin. Under the complicated interaction, the actin cytoskeleton is controlled spatially and temporally during root hair development. © 2012 Elsevier Ireland Ltd. All rights reserved.

  2. Retired NASA F-18 being hoisted up by crane to pedestal mount at Lancaster California Municipal Base

    NASA Technical Reports Server (NTRS)

    1997-01-01

    An F/A-18 aircraft formerly flown by NASA's Dryden Flight Research Center, Edwards, California, is lifted by crane towards what has become its new home - a pedestal in front of the municipal baseball stadium in the city of Lancaster, California. The F/A-18 had been flown by NASA Dryden as a safety chase aircraft on research missions and for various other pilot proficiency and support duties prior to its recent retirement. The aircraft is now mounted nose skyward on the 28-foot-tall pedestal in front of the stadium, appropriately named 'The Hangar.' The stadium is the home field of the Lancaster Jethawks, a Class-A farm team of the Seattle Mariners.

  3. Retired NASA F-18 being hoisted up by crane to pedestal mount at Lancaster California Municipal Base

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Under the watchful eyes of news media and officials of the city of Lancaster, California, from a balcony, workers steady an F/A-18 Hornet airframe as it is gently lifted into the air prior to mounting on a pedestal in front of 'The Hangar,' the city's municipal stadium. The F/A-18 was formerly flown by NASA's Dryden Flight Research Center, Edwards, California, as a safety chase and support aircraft prior to its recent retirement. The aircraft is now mounted nose skyward on a 28-foot-tall pedestal on front of the stadium. The stadium is the home field of the Lancaster Jethawks, a Class-A farm team of the Seattle Mariners.

  4. Disrupting actin-myosin-actin connectivity in airway smooth muscle as a treatment for asthma?

    PubMed

    Lavoie, Tera L; Dowell, Maria L; Lakser, Oren J; Gerthoffer, William T; Fredberg, Jeffrey J; Seow, Chun Y; Mitchell, Richard W; Solway, Julian

    2009-05-01

    Breathing is known to functionally antagonize bronchoconstriction caused by airway muscle contraction. During breathing, tidal lung inflation generates force fluctuations that are transmitted to the contracted airway muscle. In vitro, experimental application of force fluctuations to contracted airway smooth muscle strips causes them to relengthen. Such force fluctuation-induced relengthening (FFIR) likely represents the mechanism by which breathing antagonizes bronchoconstriction. Thus, understanding the mechanisms that regulate FFIR of contracted airway muscle could suggest novel therapeutic interventions to increase FFIR, and so to enhance the beneficial effects of breathing in suppressing bronchoconstriction. Here we propose that the connectivity between actin filaments in contracting airway myocytes is a key determinant of FFIR, and suggest that disrupting actin-myosin-actin connectivity by interfering with actin polymerization or with myosin polymerization merits further evaluation as a potential novel approach for preventing prolonged bronchoconstriction in asthma.

  5. Effect of anomalous transport on kinetic simulations of the H-mode pedestal

    NASA Astrophysics Data System (ADS)

    Bateman, G.; Pankin, A. Y.; Kritz, A. H.; Rafiq, T.; Park, G. Y.; Ku, S.; Chang, C. S.

    2009-11-01

    The MMM08 and MMM95 Multi-Mode transport models [1,2], are used to investigate the effect of anomalous transport in XGC0 gyrokinetic simulations [3] of tokamak H-mode pedestal growth. Transport models are implemented in XGC0 using the Framework for Modernization and Componentization of Fusion Modules (FMCFM). Anomalous transport is driven by steep temperature and density gradients and is suppressed by high values of flow shear in the pedestal. The radial electric field, used to calculate the flow shear rate, is computed self-consistently in the XGC0 code with the anomalous transport, Lagrangian charged particle dynamics and neutral particle effects. XGC0 simulations are used to provide insight into how thermal and particle transport, together with the sources of heat and charged particles, determine the shape and growth rate of the temperature and density profiles. [1] F.D. Halpern et al., Phys. Plasmas 15 (2008) 065033; J.Weiland et al., Nucl. Fusion 49 (2009) 965933; A.Kritz et al., EPS (2009) [2] G. Bateman, et al, Phys. Plasmas 5 (1998) 1793 [3] C.S. Chang, S. Ku, H. Weitzner, Phys. Plasmas 11 (2004) 2649

  6. Structure of the Rigor Actin-Tropomyosin-Myosin Complex

    PubMed Central

    Behrmann, Elmar; Müller, Mirco; Penczek, Pawel A.; Mannherz, Hans Georg; Manstein, Dietmar J.; Raunser, Stefan

    2014-01-01

    The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. Myosin converts the chemical energy stored in ATP into force and movement along actin filaments. Myosin binding to actin induces conformational changes that are coupled to the nucleotide-binding pocket and amplified by a specialized region of the motor domain for efficient force generation. Tropomyosin plays a key role in regulating the productive interaction between myosins and actin. Here, we report the 8 Å resolution structure of the actin-tropomyosin-myosin complex determined by cryo electron microscopy. The pseudo-atomic model of the complex obtained from fitting crystal structures into the map defines the large actin-myosin-tropomyosin interface and the molecular interactions between the proteins in detail and allows us to propose a structural model for tropomyosin dependent myosin binding to actin and actin-induced nucleotide release from myosin. PMID:22817895

  7. Permeabilization Activated Reduction in Fluorescence (PARF): a novel method to measure kinetics of protein interactions with intracellular structures

    PubMed Central

    Singh, Pali P.; Hawthorne, Jenci L.; Davis, Christie A.; Quintero, Omar A.

    2016-01-01

    Understanding kinetic information is fundamental in understanding biological function. Advanced imaging technologies have fostered the development of kinetic analyses in cells. We have developed Permeabilization Activated Reduction in Fluorescence (PARF) analysis for determination of apparent t1/2 and immobile fraction, describing the dissociation of a protein of interest from intracellular structures. To create conditions where dissociation events are observable, cells expressing a fluorescently-tagged protein are permeabilized with digitonin, diluting the unbound protein into the extracellular media. As the media volume is much larger than the cytosolic volume, the concentration of the unbound pool decreases drastically, shifting the system out of equilibrium--favoring dissociation events. Loss of bound protein is observed as loss of fluorescence from intracellular structures and can be fit to an exponential decay. We compared PARF dissociation kinetics with previously published equilibrium kinetics as determined by FRAP. PARF dissociation rates agreed with the equilibrium-based FRAP analysis predictions of the magnitude of those rates. When used to investigate binding kinetics of a panel of cytoskeletal proteins, PARF analysis revealed that filament stabilization resulted in slower fluorescence loss. Additionally, commonly used “general” F-actin labels display differences in kinetic properties, suggesting that not all fluorescently-tagged actin labels interact with the actin network in the same way. We also observed differential dissociation kinetics for GFP-VASP depending on which cellular structure was being labeled. These results demonstrate that PARF analysis of non-equilibrium systems reveals kinetic information without the infrastructure investment required for other quantitative approaches such as FRAP, photoactivation, or in vitro reconstitution assays. PMID:27126922

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

    PubMed Central

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

    2015-01-01

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

  9. Actin expression in some Platyhelminthe species.

    PubMed

    Fagotti, A; Panara, F; Di Rosa, I; Simoncelli, F; Gabbiani, G; Pascolini, R

    1994-10-01

    Actin expression in some Platyhelminthe species was demonstrated by western-blotting and immunocytochemical analysis using two distinct anti-actin antibodies: the anti-total actin that reacts against all actin isoforms of higher vertebrates and the anti-alpha SM-1 that recognizes the alpha-smooth muscle (alpha SM) isotype of endothermic vertebrates (Skalli et al., 1986). Western-blotting experiments showed that all species tested, including some free-living Platyhelminthes (Tricladida and Rhabdocoela) and the parasitic Fasciola hepatica, were stained by anti-total actin antibody while only Dugesidae and Dendrocoelidae showed a positive immunoreactivity against anti-alpha SM-1. These results were confirmed by cytochemical immunolocalization using both avidin biotin conjugated peroxidase reaction on paraffin sections, and immunogold staining on Lowicryl 4KM embedded specimens. Our findings may contribute to the understanding of Platyhelminthes phylogeny.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gomibuchi, Yuki; Uyeda, Taro Q.P.; Wakabayashi, Takeyuki, E-mail: tw007@nasu.bio.teikyo-u.ac.jp

    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-143more » 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

  11. Actin growth profile in clathrin-mediated endocytosis

    NASA Astrophysics Data System (ADS)

    Tweten, D. J.; Bayly, P. V.; Carlsson, A. E.

    2017-05-01

    Clathrin-mediated endocytosis in yeast is driven by a protein patch containing close to 100 different types of proteins. Among the proteins are 5000 -10 000 copies of polymerized actin, and successful endocytosis requires growth of the actin network. Since it is not known exactly how actin network growth drives endocytosis, we calculate the spatial distribution of actin growth required to generate the force that drives the process. First, we establish the force distribution that must be supplied by actin growth, by combining membrane-bending profiles obtained via electron microscopy with established theories of membrane mechanics. Next, we determine the profile of actin growth, using a continuum mechanics approach and an iterative procedure starting with an actin growth profile obtained from a linear analysis. The profile has fairly constant growth outside a central hole of radius 45-50 nm, but very little growth in this hole. This growth profile can reproduce the required forces if the actin shear modulus exceeds 80 kPa, and the growing filaments can exert very large polymerization forces. The growth profile prediction could be tested via electron-microscopy or super-resolution experiments in which the turgor pressure is suddenly turned off.

  12. Interactions of histatin-3 and histatin-5 with actin.

    PubMed

    Blotnick, Edna; Sol, Asaf; Bachrach, Gilad; Muhlrad, Andras

    2017-03-06

    Histatins are histidine rich polypeptides produced in the parotid and submandibular gland and secreted into the saliva. Histatin-3 and -5 are the most important polycationic histatins. They possess antimicrobial activity against fungi such as Candida albicans. Histatin-5 has a higher antifungal activity than histatin-3 while histatin-3 is mostly involved in wound healing in the oral cavity. We found that these histatins, like other polycationic peptides and proteins, such as LL-37, lysozyme and histones, interact with extracellular actin. Histatin-3 and -5 polymerize globular actin (G-actin) to filamentous actin (F-actin) and bundle F-actin filaments. Both actin polymerization and bundling by histatins is pH sensitive due to the high histidine content of histatins. In spite of the equal number of net positive charges and histidine residues in histatin-3 and -5, less histatin-3 is needed than histatin-5 for polymerization and bundling of actin. The efficiency of actin polymerization and bundling by histatins greatly increases with decreasing pH. Histatin-3 and -5 induced actin bundles are dissociated by 100 and 50 mM NaCl, respectively. The relatively low NaCl concentration required to dissociate histatin-induced bundles implies that the actin-histatin filaments bind to each other mainly by electrostatic forces. The binding of histatin-3 to F-actin is stronger than that of histatin-5 showing that hydrophobic forces have also some role in histatin-3- actin interaction. Histatins affect the fluorescence of probes attached to the D-loop of G-actin indicating histatin induced changes in actin structure. Transglutaminase cross-links histatins to actin. Competition and limited proteolysis experiments indicate that the main histatin cross-linking site on actin is glutamine-49 on the D-loop of actin. Both histatin-3 and -5 interacts with actin, however, histatin 3 binds stronger to actin and affects actin structure at lower concentration than histatin-5 due to the extra 8

  13. 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

  14. Influence of gold nanoparticles on the 805 nm gain in Tm3+/Yb3+ codoped PbO-GeO2 pedestal waveguides

    NASA Astrophysics Data System (ADS)

    de Assumpção, T. A. A.; Camilo, M. E.; Alayo, M. I.; da Silva, D. M.; Kassab, L. R. P.

    2017-10-01

    The production and characterization of pedestal waveguides based on PbO-GeO2 amorphous thin films codoped with Tm3+/Yb3+, with and without gold nanoparticles (NPs), are reported. Pedestal structure was obtained by conventional photolithography and plasma etching. Tm3+/Yb3+ codoped PGO amorphous thin film was obtained by RF Magnetron Sputtering deposition and used as core layer in the pedestal optical waveguide. The minimum propagation losses in the waveguide were 3.6 dB/cm at 1068 nm. The internal gain at 805 nm was enhanced and increased to 8.67 dB due to the presence of gold NPs. These results demonstrate for the first time that Tm3+/Yb3+ codoped PbO-GeO2 waveguides are promising for first telecom window and integrated photonics, especially for applications on fiber network at short distances.

  15. Three’s company: The fission yeast actin cytoskeleton

    PubMed Central

    Kovar, David R.; Sirotkin, Vladimir; Lord, Matthew

    2010-01-01

    How the actin cytoskeleton assembles into different structures to drive diverse cellular processes is a fundamental cell biological question. In addition to orchestrating the appropriate combination of regulators and actin-binding proteins, different actin-based structures must insulate themselves from one another to maintain specificity within a crowded cytoplasm. Actin specification is particularly vexing in complex eukaryotes where a multitude of protein isoforms and actin structures operate within the same cell. Fission yeast Schizosaccharomyces pombe possesses a single actin isoform that functions in three distinct structures throughout the cell cycle. In this review, we explore recent studies in fission yeast that help unravel how different actin structures operate in cells. PMID:21145239

  16. The nature of the globular- to fibrous-actin transition.

    PubMed

    Oda, Toshiro; Iwasa, Mitsusada; Aihara, Tomoki; Maéda, Yuichiro; Narita, Akihiro

    2009-01-22

    Actin plays crucial parts in cell motility through a dynamic process driven by polymerization and depolymerization, that is, the globular (G) to fibrous (F) actin transition. Although our knowledge about the actin-based cellular functions and the molecules that regulate the G- to F-actin transition is growing, the structural aspects of the transition remain enigmatic. We created a model of F-actin using X-ray fibre diffraction intensities obtained from well oriented sols of rabbit skeletal muscle F-actin to 3.3 A in the radial direction and 5.6 A along the equator. Here we show that the G- to F-actin conformational transition is a simple relative rotation of the two major domains by about 20 degrees. As a result of the domain rotation, the actin molecule in the filament is flat. The flat form is essential for the formation of stable, helical F-actin. Our F-actin structure model provides the basis for understanding actin polymerization as well as its molecular interactions with actin-binding proteins.

  17. Liquid behavior of cross-linked actin bundles.

    PubMed

    Weirich, Kimberly L; Banerjee, Shiladitya; Dasbiswas, Kinjal; Witten, Thomas A; Vaikuntanathan, Suriyanarayanan; Gardel, Margaret L

    2017-02-28

    The actin cytoskeleton is a critical regulator of cytoplasmic architecture and mechanics, essential in a myriad of physiological processes. Here we demonstrate a liquid phase of actin filaments in the presence of the physiological cross-linker, filamin. Filamin condenses short actin filaments into spindle-shaped droplets, or tactoids, with shape dynamics consistent with a continuum model of anisotropic liquids. We find that cross-linker density controls the droplet shape and deformation timescales, consistent with a variable interfacial tension and viscosity. Near the liquid-solid transition, cross-linked actin bundles show behaviors reminiscent of fluid threads, including capillary instabilities and contraction. These data reveal a liquid droplet phase of actin, demixed from the surrounding solution and dominated by interfacial tension. These results suggest a mechanism to control organization, morphology, and dynamics of the actin cytoskeleton.

  18. Molecular cloning of actin genes in Trichomonas vaginalis and phylogeny inferred from actin sequences.

    PubMed

    Bricheux, G; Brugerolle, G

    1997-08-01

    The parasitic protozoan Trichomonas vaginalis is known to contain the ubiquitous and highly conserved protein actin. A genomic library and a cDNA library have been screened to identify and clone the actin gene(s) of T. vaginalis. The nucleotide sequence of one gene and its flanking regions have been determined. The open reading frame encodes a protein of 376 amino acids. The sequence is not interrupted by any introns and the promoter could be represented by a 10 bp motif close to a consensus motif also found upstream of most sequenced T. vaginalis genes. The five different clones isolated from the cDNA library have similar sequences and encode three actin proteins differing only by one or two amino acids. A phylogenetic analysis of 31 actin sequences by distance matrix and parsimony methods, using centractin as outgroup, gives congruent trees with Parabasala branching above Diplomonadida.

  19. Consequences of Molecular-Scale Non-Equilibrium Activity on the Dynamics and Mechanics of Self-Assembled Actin-Based Structures and Materials

    NASA Astrophysics Data System (ADS)

    Marshall Mccall, Patrick

    Living cells are hierarchically self-organized forms of active soft matter: molecules on the nanometer scale form functional structures and organelles on the micron scale, which then compose cells on the scale of 10s of microns. While the biological functions of intracellular organelles are defined by the composition and properties of the structures themselves, how those bulk properties emerge from the properties and interactions of individual molecules remains poorly understood. Actin, a globular protein which self-assembles into dynamic semi-flexible polymers, is the basic structural material of cells and the major component of many functional organelles. In this thesis, I have used purified actin as a model system to explore the interplay between molecular-scale dynamics and organelle-scale functionality, with particular focus on the role of molecular-scale non-equilibrium activity. One of the most canonical forms of molecular-scale non-equilibrium activity is that of mechanoenzymes, also called motor proteins. These proteins utilized the free energy liberated by hydrolysis of ATP to perform mechanical work, thereby introducing non-equilibrium "active" stresses on the molecular scale. Combining experiments with mathematical modeling, we demonstrate in this thesis that non-equilibrium motor activity is sufficient to drive self-organization and pattern formation of the multimeric actin-binding motor protein Myosin II on 1D reconstituted actomyosin bundles. Like myosin, actin is itself an ATPase. However, nono-equilibrium ATP hydrolysis on actin is known to regulate the stability and assembly kinetics of actin filaments rather than generate active stresses per se. At the level of single actin filaments, the inhomogeneous nucleotide composition generated along the filament length by hydrolysis directs binding of regulatory proteins like cofilin, which mediate filament disassembly and thereby accelerate actin filament turnover. The concequences of this non

  20. Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics.

    PubMed

    Ono, Shoichiro; Ono, Kanako

    2002-03-18

    Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B-induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.

  1. Progress in understanding the enhanced pedestal H-mode in NSTX

    DOE PAGES

    Gerhardt, S. P.; Canik, J. M.; Maingi, R.; ...

    2014-08-01

    The paper describes the enhanced pedestal (EP) H-mode observed in the National Spherical Torus Experiment (NSTX). The defining characteristics of EP H-mode are given, namely i)transition after the L- to H-mode transition, ii) region of very steep ion temperature gradient, and iii) associated region of strong rotational shear. A newly observed long-pulse EP H-mode example shows quiescent behavior for as long as the heating and current drive sources are maintained. Cases are shown where the region of steep ion temperature gradient is located at the very edge, and cases where it is shifted up to 10 cm inward from themore » plasma edge; these cases are united by a common dependence of the ion temperature gradient on the toroidal rotation frequency shear. EP H-mode examples have been observed across a wide range of q95 and pedestal collisionality. No strong changes in the fluctuation amplitudes have been observed following the eP H-mode transition, and transport analysis indicates that the ion t hermal transport is comparable to or less than anticipated from a simple neoclassical transport model. Cases are shown where EP H-modes were reliably generated, through these low-q95 examples were difficult to sustain. A case where an externally triggered ELM precipitates the transition to EP H-mode is also shown, though an initial experiment designed to trigger EP-H-modes in this fashion was successful.« less

  2. H-mode pedestal stability and ELMs in Alcator C-Mod

    NASA Astrophysics Data System (ADS)

    Mossessian, Dmitri

    2002-11-01

    For steady state H-mode operation, a relaxation mechanism is required to limit build-up of the edge gradient and impurity accumulation. The major relaxation mechanism seen on most of the existing tokamaks - large type I ELMs - drive high particle and energy fluxes that present a significant power load on the divertor plates. On Alcator C-Mod, however, type I ELMs are not observed. Instead, more benign mechanisms - EDA and small grassy ELMs - appear to drive enhanced particle transport at the edge of H-mode plasmas. Both have good energy confinement, no impurity accumulation, and are steady state. In EDA the edge relaxation mechanism is provided by a quasicoherent electromagnetic mode localized in the outer part of the pedestal. Non-linear gyrofluid and linear gyrokinetic simulations, as well as real geometry fluctuation modeling based on fluid equations show the presence of a coherent mode. Based on those results the observed mode is tentatively identified as resistive ballooning. At higher edge pressure gradient the mode is replaced by broadband fluctuations and small irregular ELMs are observed. Based on ideal MHD calculations that include effects of bootstrap current, these ELMs are identified as medium n coupled ideal peeling/ballooning modes. The stability threshold and modes structure of these modes are studied with recently developed linear MHD stability code ELITE and the results are compared with the observed dependence of the ELMs' character on pedestal parameters and plasma shape.

  3. Managing actinic keratosis in primary care.

    PubMed

    Salmon, Nicola; Tidman, Michael J

    2016-10-01

    Actinic, or solar, keratosis is caused by chronic ultraviolet-induced damage to the epidermis. In the UK, 15-23% of individuals have actinic keratosis lesions. Risk factors include: advanced age; male gender; cumulative sun exposure or phototherapy; Fitzpatrick skin phototypes I-II; long-term immuno-suppression and genetic syndromes e.g. xeroderma pigmentosum and albinism. Actinic keratoses are regarded by some authorities as premalignant lesions that may transform into invasive squamous cell carcinoma (SCC) and by others as in situ SCC that may progress to an invasive stage. The risk of malignant change appears low; up to 0.5% per lesion per year. Up to 20-30% of lesions may spontaneously regress but in the absence of any reliable prognostic clinical indicators regarding malignant potential active treatment is considered appropriate. Actinic keratosis lesions may present as discrete hyperkeratotic papules, cutaneous horns, or more subtle flat lesions on sun-exposed areas of skin. The single most helpful diagnostic sign is an irregularly roughened surface texture: a sandpaper-like feel almost always indicates actinic damage. Dermatoscopy can be helpful in excluding signs of basal cell carcinoma when actinic keratosis is non-keratotic. It is always important to consider the possibility of SCC. The principal indication for referral to secondary care is the possibility of cutaneous malignancy. However, widespread and severe actinic damage in patients who are immunosuppressed is also a reason for referral.

  4. Bioinformatics study of the mangrove actin genes

    NASA Astrophysics Data System (ADS)

    Basyuni, M.; Wasilah, M.; Sumardi

    2017-01-01

    This study describes the bioinformatics methods to analyze eight actin genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, subcellular localization, similarity, and phylogenetic. The physical and chemical properties of eight mangroves showed variation among the genes. The percentage of the secondary structure of eight mangrove actin genes followed the order of a helix > random coil > extended chain structure for BgActl, KcActl, RsActl, and A. corniculatum Act. In contrast to this observation, the remaining actin genes were random coil > extended chain structure > a helix. This study, therefore, shown the prediction of secondary structure was performed for necessary structural information. The values of chloroplast or signal peptide or mitochondrial target were too small, indicated that no chloroplast or mitochondrial transit peptide or signal peptide of secretion pathway in mangrove actin genes. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove actin genes. To clarify the relationship among the mangrove actin gene, a phylogenetic tree was constructed. Three groups of mangrove actin genes were formed, the first group contains B. gymnorrhiza BgAct and R. stylosa RsActl. The second cluster which consists of 5 actin genes the largest group, and the last branch consist of one gene, B. sexagula Act. The present study, therefore, supported the previous results that plant actin genes form distinct clusters in the tree.

  5. Cell adhesion in zebrafish myogenesis: distribution of intermediate filaments, microfilaments, intracellular adhesion structures and extracellular matrix.

    PubMed

    Costa, Manoel L; Escaleira, Roberta C; Jazenko, Fernanda; Mermelstein, Claudia S

    2008-10-01

    To overcome the limitations of in vitro studies, we have been studying myogenesis in situ in zebrafish embryos, at a sub-cellular level. While in previous works we focused on myofibrillogenesis and some aspects of adhesion structures, here we describe in more detail cell adhesion structures and interactions among cytoskeletal components, membrane and extracellular matrix during zebrafish muscle development. We studied the intermediate filaments, and we describe the full range of desmin distribution in zebrafish development, from perinuclear to striated, until its deposition around the intersomite septa of older somites. This adhesion structure, positive for desmin and actin, has not been previously observed in myogenesis in vitro. We also show that actin is initially located in the intersomite septum region whereas it is confined to the myofibrils later on. While actin localization changes during development, the adhesion complex proteins vinculin, paxillin, talin, dystrophin, laminin and fibronectin always appear exclusively at the intersomite septa, and appear to be co-distributed, even though the extracellular proteins accumulates before the intracellular ones. Contrary to the adhesion proteins, that are continuously distributed, desmin and sarcomeric actin form triangular aggregates among the septa and the cytoskeleton. We studied the cytoskeletal linker plectin as well, and we show that it has a distribution similar to desmin and not to actin. We conclude that the in situ adhesion structures differ from their in vitro counterparts, and that the actual zebrafish embryo myogenesis is quite different than that which occurs in in vitro systems. Copyright 2008 Wiley-Liss, Inc.

  6. Kinetics of Binding of Caldesmon to Actin*

    PubMed Central

    Chalovich, Joseph M.; Chen, Yi-der; Dudek, Ronald; Luo, Hai

    2005-01-01

    The time course of interaction of caldesmon with actin may be monitored by fluorescence changes that occur upon the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or to acrylodan actin. The concentration dependence of the observed rate of caldesmon-actin binding was analyzed to a first approximation as a single-step reaction using a Monte Carlo simulation. The derived association and dissociation rates were 107 m−1 s−1 and 18.2 s−1, respectively. Smooth muscle tropomyosin enhances the binding of caldesmon to actin, and this was found to be due to a reduction in the rate of dissociation to 6.3 s −1. There is no evidence from this study for a different mechanism of binding in the presence of tropomyosin. The fluorescence changes that occurred with the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl))-labeled caldesmon to actin or actin-tropomyosin were reversed by the addition of myosin subfragment 1 as predicted by a competitive binding mechanism. PMID:7730374

  7. Are non-muscle actin isoforms functionally equivalent?

    PubMed

    Simiczyjew, Aleksandra; Pietraszek-Gremplewicz, Katarzyna; Mazur, Antonina Joanna; Nowak, Dorota

    2017-11-01

    Actin is highly conserved and it is the most widespread protein in eukaryotic cells. One of the most important features of actin, which allows it to have many different functions, is its ability to polymerize and interact with many other proteins. Actins are the major constituent of the actin cytoskeleton, which is an important system that is involved in various aspects of cell function, including cell motility, structure, integrity, regulation of signal transduction and transcription. Six mammal actin isoforms are highly conserved and share common functions. Two of them, β and γ non-muscle actin isoforms, which differ only by four amino acids located at the N-terminus of the polypeptide chain, are required for survival and proper cell functioning. We also summarized data about actbl2, which is suggested to be a newly discovered isoactin. Here, we review the current knowledge about tissue-specific expression of the non-muscle actin isoforms and possible functional differences between them. We also discuss molecular tools, which in recent years have allowed for a better understanding of the role of these proteins in cell functioning.

  8. [Photodynamic therapy for actinic cheilitis].

    PubMed

    Castaño, E; Comunión, A; Arias, D; Miñano, R; Romero, A; Borbujo, J

    2009-12-01

    Actinic cheilitis is a subtype of actinic keratosis that mainly affects the lower lip and has a higher risk of malignant transformation. Its location on the labial mucosa influences the therapeutic approach. Vermilionectomy requires local or general anesthetic and is associated with a risk of an unsightly scar, and the treatment with 5-fluorouracil or imiquimod lasts for several weeks and the inflammatory reaction can be very intense. A number of authors have used photodynamic therapy as an alternative to the usual treatments. We present 3 patients with histologically confirmed actinic cheilitis treated using photodynamic therapy with methyl aminolevulinic acid as the photosensitizer and red light at 630 nm. The clinical response was good, with no recurrences after 3 to 6 months of follow-up. Our experience supports the use of photodynamic therapy as a good alternative for the treatment of actinic cheilitis.

  9. Demonstration of prominent actin filaments in the root columella

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  10. Actin cable dynamics in budding yeast

    PubMed Central

    Yang, Hyeong-Cheol; Pon, Liza A.

    2002-01-01

    Actin cables, bundles of actin filaments that align along the long axis of budding yeast, are crucial for establishment of cell polarity. We fused green fluorescent protein (GFP) to actin binding protein 140 (Abp140p) and visualized actin cable dynamics in living yeast. We detected two populations of actin cables: (i) bud-associated cables, which extend from the bud along the mother-bud axis, and (ii) randomly oriented cables, which are relatively short. Time-lapse imaging of Abp140p–GFP revealed an apparent increase in the length of bud-associated actin cables. Analysis of movement of Abp140p–GFP fiduciary marks on bud-associated cables and fluorescence loss in photobleaching experiments revealed that this apparent elongation occurs by assembly of new material at the end of the cable within the bud and movement of the opposite end of the cable toward the tip of the mother cell distal to the bud. The rate of extension of the tip of an elongating actin cable is 0.29 ± 0.08 μm/s. Latrunculin A (Lat-A) treatment completely blocked this process. We also observed movement of randomly oriented cables around the cortex of cells at a rate of 0.59 ± 0.14 μm/s. Mild treatment with Lat-A did not affect the velocity of movement of randomly oriented cables. However, Lat-A treatment did increase the number of randomly oriented, motile cables per cell. Our observations suggest that establishment of bud-associated actin cables during the cell cycle is accomplished not by realignment of existing cables but by assembly of new cables within the bud or bud neck, followed by elongation. PMID:11805329

  11. Nucleotides induce chemotaxis and actin polymerization in immature but not mature human dendritic cells via activation of pertussis toxin-sensitive P2y receptors.

    PubMed

    Idzko, Marco; Dichmann, Stefan; Ferrari, Davide; Di Virgilio, Francesco; la Sala, Andrea; Girolomoni, Giampiero; Panther, Elisabeth; Norgauer, Johannes

    2002-08-01

    Dendritic cells (DCs) are considered the principal initiators of immune response because of their ability to migrate into peripheral tissues and lymphoid organs, process antigens, and activate naive T cells. There is evidence that extracellular nucleotides regulate certain functions of DCs via G-protein-coupled P2Y receptors (P2YR) and ion-channel-gated P2X receptors (P2XR). Here we investigated the chemotactic activity and analyzed the migration-associated intracellular signaling events such as actin reorganization and Ca(++) transients induced by common P2R agonists such as adenosine 5'-triphosphate (ATP) and 2-methylthioadenosine triphosphate, the P2YR agonists UTP and adenosine 5'-diphosphate (ADP), or the P2XR agonists alphabeta-methylenadenosine-5'-triphosphate and 2',3'-(4-benzoyl)benzoyl-ATP. The common P2R agonists and the selective P2YR agonists turned out to be potent chemotactic stimuli for immature DCs, but not for mature DCs. In contrast, P2XR agonists had only marginal chemotactic activity in both DC types. Chemotaxis was paralleled by a rise in the intracellular Ca(++) concentration and by actin polymerization. Studies with pertussis toxin implicated that intracellular signaling events such as actin polymerization, mobilization of intracellular Ca(++), and migration induced by nucleotides was mediated via G(i/o) protein-coupled P2YR. Moreover, functional studies revealed selective down-regulation of this G(i/o) protein-coupled chemotactic P2YR responsiveness during maturation, although immature and mature DCs expressed similar amounts of mRNA for the P2R subtypes (P2Y(2)R, P2Y(4)R, P2Y(5)R, P2Y(7)R, P2Y(11)R and P2X(1)R, P2X(4)R, P2X(7)R), and no major differences in respect to the mRNA expression of these receptors could be observed by semiquantitative reverse transcription and polymerase chain reaction (RT-PCR). In summary, our data describe a differential chemotactic response of immature and mature DCs to nucleotides, and lend further support to

  12. From Cytoskeleton to Gene Expression: Actin in the Nucleus.

    PubMed

    Viita, Tiina; Vartiainen, Maria K

    2017-01-01

    Although most people still associate actin mainly with the cytoskeleton, several lines of evidence, with the earliest studies dating back to decades ago, have emphasized the importance of actin also inside the cell nucleus. Actin has been linked to many gene expression processes from gene activation to chromatin remodeling, but also to maintenance of genomic integrity and intranuclear movement of chromosomes and chromosomal loci. Recent advances in visualizing different forms and dynamic properties of nuclear actin have clearly advanced our understanding of the basic concepts by which actin operates in the nucleus. In this chapter we address the different breakthroughs in nuclear actin studies, as well as discuss the regulation nuclear actin and the importance of nuclear actin dynamics in relation to its different nuclear functions. Our aim is to highlight the fact that actin should be considered as an essential component of the cell nucleus, and its nuclear actions should be taken into account also in experiments on cytoplasmic actin networks.

  13. A Continuum Model of Actin Waves in Dictyostelium discoideum

    PubMed Central

    Khamviwath, Varunyu; Hu, Jifeng; Othmer, Hans G.

    2013-01-01

    Actin waves are complex dynamical patterns of the dendritic network of filamentous actin in eukaryotes. We developed a model of actin waves in PTEN-deficient Dictyostelium discoideum by deriving an approximation of the dynamics of discrete actin filaments and combining it with a signaling pathway that controls filament branching. This signaling pathway, together with the actin network, contains a positive feedback loop that drives the actin waves. Our model predicts the structure, composition, and dynamics of waves that are consistent with existing experimental evidence, as well as the biochemical dependence on various protein partners. Simulation suggests that actin waves are initiated when local actin network activity, caused by an independent process, exceeds a certain threshold. Moreover, diffusion of proteins that form a positive feedback loop with the actin network alone is sufficient for propagation of actin waves at the observed speed of . Decay of the wave back can be caused by scarcity of network components, and the shape of actin waves is highly dependent on the filament disassembly rate. The model allows retraction of actin waves and captures formation of new wave fronts in broken waves. Our results demonstrate that a delicate balance between a positive feedback, filament disassembly, and local availability of network components is essential for the complex dynamics of actin waves. PMID:23741312

  14. Actin filaments – a target for redox regulation

    PubMed Central

    Wilson, Carlos; Terman, Jonathan R.; González-Billault, Christian; Ahmed, Giasuddin

    2016-01-01

    Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through non-covalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates – the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL – and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. PMID:27309342

  15. Leishmania infection inhibits macrophage motility by altering F-actin dynamics and the expression of adhesion complex proteins.

    PubMed

    de Menezes, Juliana Perrone Bezerra; Koushik, Amrita; Das, Satarupa; Guven, Can; Siegel, Ariel; Laranjeira-Silva, Maria Fernanda; Losert, Wolfgang; Andrews, Norma W

    2017-03-01

    Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis-infected macrophages also show reduced directional migration in response to the chemokine MCP-1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F-actin turnover frequency in L. amazonensis-infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane-extracellular matrix interactions. © 2016 John Wiley & Sons Ltd.

  16. Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over

    PubMed Central

    Al Tanoury, Ziad; Schaffner-Reckinger, Elisabeth; Halavatyi, Aliaksandr; Hoffmann, Céline; Moes, Michèle; Hadzic, Ermin; Catillon, Marie; Yatskou, Mikalai; Friederich, Evelyne

    2010-01-01

    Background Initially detected in leukocytes and cancer cells derived from solid tissues, L-plastin/fimbrin belongs to a large family of actin crosslinkers and is considered as a marker for many cancers. Phosphorylation of L-plastin on residue Ser5 increases its F-actin binding activity and is required for L-plastin-mediated cell invasion. Methodology/Principal Findings To study the kinetics of L-plastin and the impact of L-plastin Ser5 phosphorylation on L-plastin dynamics and actin turn-over in live cells, simian Vero cells were transfected with GFP-coupled WT-L-plastin, Ser5 substitution variants (S5/A, S5/E) or actin and analyzed by fluorescence recovery after photobleaching (FRAP). FRAP data were explored by mathematical modeling to estimate steady-state reaction parameters. We demonstrate that in Vero cell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly, L-plastin affected actin turn-over by decreasing the actin dissociation rate by four-fold, increasing thereby the amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibition studies and siRNA knock-down of PKC isozymes pointed to the involvement of the novel PKC-δ isozyme in the PMA-elicited signaling pathway leading to L-plastin Ser5 phosphorylation. Furthermore, the L-plastin contribution to actin dynamics regulation was substantiated by its association with a protein complex comprising cortactin, which is known to be involved in this process. Conclusions/Significance Altogether these findings quantitatively

  17. Erbium laser resurfacing for actinic cheilitis.

    PubMed

    Cohen, Joel L

    2013-11-01

    Actinic cheilitis is a precancerous condition characterized by grayish-whitish area(s) of discoloration on the mucosal lip, often blunting the demarcation between mucosa and cutaneous lip. Actinic cheilitis is considered to be an early part of the spectrum of squamous cell carcinoma. Squamous cell carcinoma specifically of the lip has a high rate of recurrence and metastasis through the oral cavity leading to a poor overall survival. Risk factors for the development of actinic cheilitis include chronic solar irradiation, increasing age, male gender, light skin complexion, immunosuppression, and possibly tobacco and alcohol consumption. Treatment options include topical pharmacotherapy (eg, fluorouracil, imiquimod) or procedural interventions (eg, cryotherapy, electrosurgery, surgical vermillionectomy, laser resurfacing), each with their known advantages and disadvantages. There is little consensus as to which treatment options offer the most clinical utility given the paucity of comparative clinical data. In my practice, laser resurfacing has become an important tool for the treatment of actinic cheilitis owing to its ease of use and overall safety, tolerability, and cosmetic acceptability. Herein the use of erbium laser resurfacing is described for three actinic cheilitis presentations for which I find it particularly useful: clinically prominent actinic cheilitis, biopsy-proven actinic cheilitis, and treatment of the entire lip following complete tumor excision of squamous cell carcinoma. All patients were treated with a 2940-nm erbium laser (Sciton Profile Contour Tunable Resurfacing Laser [TRL], Sciton, Inc., Palo Alto, CA).

  18. Tyrosine kinases activate store-mediated Ca2+ entry in human platelets through the reorganization of the actin cytoskeleton.

    PubMed Central

    Rosado, J A; Graves, D; Sage, S O

    2000-01-01

    We have recently reported that store-mediated Ca(2+) entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed 'secretion-like coupling'. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca(2+) entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin- or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca(2+) entry were found to be entirely dependent on the actin cytoskeleton. PP1, an inhibitor of the Src family of proteins, partially inhibited store-mediated Ca(2+) entry. In addition, depletion of intracellular Ca(2+) stores stimulated cytoskeletal association of the cytoplasmic tyrosine kinase pp60(src), a process that was sensitive to treatment with cytochalasin D and PP1, but not to inhibition of Ras proteins using prenylcysteine analogues. Finally, combined inhibition of both Ras proteins and tyrosine kinases resulted in complete inhibition of Ca(2+) entry, suggesting that these two families of proteins have independent effects in the activation of store-mediated Ca(2+) entry in human platelets. PMID:11023829

  19. Reconstituting the motility of isolated intracellular cargoes.

    PubMed

    Hendricks, Adam G; Goldman, Yale E; Holzbaur, Erika L F

    2014-01-01

    Kinesin, dynein, and myosin transport intracellular cargoes including organelles, membrane-bound vesicles, and mRNA along the cytoskeleton. These motor proteins work collectively in teams to transport cargoes over long distances and navigate around obstacles in the cell. In addition, several types of motors often interact on the same cargo to allow bidirectional transport and switching between the actin and microtubule networks. To examine transport of native cargoes in a simplified in vitro system, techniques have been developed to isolate endogenous cargoes and reconstitute their motility. Isolated cargoes can be tracked and manipulated with high precision using total internal reflection fluorescence microscopy and optical trapping. Through use of native cargoes, we can examine vesicular transport in a minimal system while retaining endogenous motor stoichiometry and the biochemical and mechanical characteristics of both motor and cargo. © 2014 Elsevier Inc. All rights reserved.

  20. ECE-imaging of the H-mode pedestal (invited).

    PubMed

    Tobias, B J; Austin, M E; Boom, J E; Burrell, K H; Classen, I G J; Domier, C W; Luhmann, N C; Nazikian, R; Snyder, P B

    2012-10-01

    A synthetic diagnostic has been developed that reproduces the highly structured electron cyclotron emission (ECE) spectrum radiated from the edge region of H-mode discharges. The modeled dependence on local perturbations of the equilibrium plasma pressure allows for interpretation of ECE data for diagnosis of local quantities. Forward modeling of the diagnostic response in this region allows for improved mapping of the observed fluctuations to flux surfaces within the plasma, allowing for the poloidal mode number of coherent structures to be resolved. In addition, other spectral features that are dependent on both T(e) and n(e) contain information about pedestal structure and the electron energy distribution of localized phenomena, such as edge filaments arising during edge-localized mode (ELM) activity.

  1. Impact of the pedestal plasma density on dynamics of edge localized mode crashes and energy loss scaling

    DOE PAGES

    Xu, X. Q.; Ma, J. F.; Li, G. Q.

    2014-12-29

    The latest BOUT++ studies show an emerging understanding of dynamics of edge localized mode(ELM) crashes and the consistent collisionality scaling of ELMenergy losses with the world multi-tokamak database. A series of BOUT++ simulations are conducted to investigate the scaling characteristics of the ELMenergy losses vs collisionality via a density scan. Moreover, the linear results demonstrate that as the pedestal collisionality decreases, the growth rate of the peeling-ballooning modes decreases for high n but increases for low n (1 < n < 5), therefore the width of the growth rate spectrum γ(n) becomes narrower and the peak growth shifts to lowermore » n. For nonlinear BOUT++ simulations show a two-stage process of ELM crash evolution of (i) initial bursts of pressure blob and void creation and (ii) inward void propagation. The inward void propagation stirs the top of pedestal plasma and yields an increasing ELM size with decreasing collisionality after a series of micro-bursts. The pedestal plasma density plays a major role in determining the ELMenergy loss through its effect on the edge bootstrap current and ion diamagnetic stabilization. Finally, the critical trend emerges as a transition (1) linearly from ballooning-dominated states at high collisionality to peeling-dominated states at low collisionality with decreasing density and (2) nonlinearly from turbulence spreading dynamics at high collisionality into avalanche-like dynamics at low collisionality.« less

  2. Xenopus egg cytoplasm with intact actin.

    PubMed

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

  3. Capillary pericytes express α-smooth muscle actin, which requires prevention of filamentous-actin depolymerization for detection.

    PubMed

    Alarcon-Martinez, Luis; Yilmaz-Ozcan, Sinem; Yemisci, Muge; Schallek, Jesse; Kılıç, Kıvılcım; Can, Alp; Di Polo, Adriana; Dalkara, Turgay

    2018-03-21

    Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at -20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling. © 2018, Alarcon-Martinez et al.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  5. Resistance of Actin to Cleavage during Apoptosis

    NASA Astrophysics Data System (ADS)

    Song, Qizhong; Wei, Tie; Lees-Miller, Susan; Alnemri, Emad; Watters, Dianne; Lavin, Martin F.

    1997-01-01

    A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1β -converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.

  6. Live-cell imaging of G-actin dynamics using sequential FDAP

    PubMed Central

    Kiuchi, Tai; Nagai, Tomoaki; Ohashi, Kazumasa; Watanabe, Naoki; Mizuno, Kensaku

    2011-01-01

    Various microscopic techniques have been developed to understand the mechanisms that spatiotemporally control actin filament dynamics in live cells. Kinetic data on the processes of actin assembly and disassembly on F-actin have been accumulated. However, the kinetics of cytoplasmic G-actin, a key determinant for actin polymerization, has remained unclear because of a lack of appropriate methods to measure the G-actin concentration quantitatively. We have developed two new microscopic techniques based on the fluorescence decay after photoactivation (FDAP) time-lapse imaging of photoswitchable Dronpa-labeled actin. These techniques, sequential FDAP (s-FDAP) and multipoint FDAP, were used to measure the time-dependent changes in and spatial distribution of the G-actin concentration in live cells. Use of s-FDAP provided data on changes in the G-actin concentration with high temporal resolution; these data were useful for the model analysis of actin assembly processes in live cells. The s-FDAP analysis also provided evidence that the cytoplasmic G-actin concentration substantially decreases after cell stimulation and that the extent of stimulus-induced actin assembly and cell size extension are linearly correlated with the G-actin concentration before cell stimulation. The advantages of using s-FDAP and multipoint FDAP to measure spatiotemporal G-actin dynamics and the roles of G-actin concentration and ADF/cofilin in stimulus-induced actin assembly and lamellipodium extension in live cells are discussed. PMID:22754616

  7. Measured vs. Predicted Pedestal Pressure During RMP ELM Control in DIII-D

    NASA Astrophysics Data System (ADS)

    Zywicki, Bailey; Fenstermacher, Max; Groebner, Richard; Meneghini, Orso

    2017-10-01

    From database analysis of DIII-D plasmas with Resonant Magnetic Perturbations (RMPs) for ELM control, we will compare the experimental pedestal pressure (p_ped) to EPED code predictions and present the dependence of any p_ped differences from EPED on RMP parameters not included in the EPED model e.g. RMP field strength, toroidal and poloidal spectrum etc. The EPED code, based on Peeling-Ballooning and Kinetic Ballooning instability constraints, will also be used by ITER to predict the H-mode p_ped without RMPs. ITER plans to use RMPs as an effective ELM control method. The need to control ELMs in ITER is of the utmost priority, as it directly correlates to the lifetime of the plasma facing components. An accurate means of determining the impact of RMP ELM control on the p_ped is needed, because the device fusion power is strongly dependent on p_ped. With this new collection of data, we aim to provide guidance to predictions of the ITER pedestal during RMP ELM control that can be incorporated in a future predictive code. Work supported in part by US DoE under the Science Undergraduate Laboratory Internship (SULI) program and under DE-FC02-04ER54698, and DE-AC52-07NA27344.

  8. Role of cytoskeletal mechanics and cell membrane fluidity in the intracellular delivery of molecules mediated by laser-activated carbon nanoparticles.

    PubMed

    Holguin, Stefany Y; Anderson, Caleb F; Thadhani, Naresh N; Prausnitz, Mark R

    2017-10-01

    Exposure of cells and nanoparticles to near-infrared nanosecond pulsed laser light can lead to efficient intracellular delivery of molecules while maintaining high cell viability by a photoacoustic phenomenon known as transient nanoparticle energy transduction (TNET). Here, we examined the influence of cytoskeletal mechanics and plasma membrane fluidity on intracellular uptake of molecules and loss of cell viability due to TNET. We found that destabilization of actin filaments using latrunculin A led to greater uptake of molecules and less viability loss caused by TNET. Stabilization of actin filaments using jasplakinolide had no significant effect on uptake or viability loss caused by TNET. To study the role of plasma membrane fluidity, we increased fluidity by depletion of membrane cholesterol using methyl-β-cyclodextrin and decreased fluidity by enrichment of the membrane with cholesterol using water-soluble cholesterol. Neither of these membrane fluidity changes significantly altered cellular uptake or viability loss caused by TNET. We conclude that weakening mechanical integrity of the cytoskeleton can increase intracellular uptake and decrease loss of cell viability, while plasma membrane fluidity does not appear to play a significant role in uptake or viability loss caused by TNET. The positive effects of cytoskeletal weakening may be due to an enhanced ability of the cell to recover from the effects of TNET and maintain viability. Biotechnol. Bioeng. 2017;114: 2390-2399. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. 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.

  10. Mechanics model for actin-based motility.

    PubMed

    Lin, Yuan

    2009-02-01

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

  11. Diverse roles of actin in C. elegans early embryogenesis

    PubMed Central

    Velarde, Nathalie; Gunsalus, Kristin C; Piano, Fabio

    2007-01-01

    Background The actin cytoskeleton plays critical roles in early development in Caenorhabditis elegans. To further understand the complex roles of actin in early embryogenesis we use RNAi and in vivo imaging of filamentous actin (F-actin) dynamics. Results Using RNAi, we found processes that are differentially sensitive to levels of actin during early embryogenesis. Mild actin depletion shows defects in cortical ruffling, pseudocleavage, and establishment of polarity, while more severe depletion shows defects in polar body extrusion, cytokinesis, chromosome segregation, and eventually, egg production. These defects indicate that actin is required for proper oocyte development, fertilization, and a wide range of important events during early embryogenesis, including proper chromosome segregation. In vivo visualization of the cortical actin cytoskeleton shows dynamics that parallel but are distinct from the previously described myosin dynamics. Two distinct types of actin organization are observed at the cortex. During asymmetric polarization to the anterior, or the establishment phase (Phase I), actin forms a meshwork of microfilaments and focal accumulations throughout the cortex, while during the anterior maintenance phase (Phase II) it undergoes a morphological transition to asymmetrically localized puncta. The proper asymmetric redistribution is dependent on the PAR proteins, while both asymmetric redistribution and morphological transitions are dependent upon PFN-1 and NMY-2. Just before cytokinesis, actin disappears from most of the cortex and is only found around the presumptive cytokinetic furrow. Finally, we describe dynamic actin-enriched comets in the early embryo. Conclusion During early C. elegans embryogenesis actin plays more roles and its organization is more dynamic than previously described. Morphological transitions of F-actin, from meshwork to puncta, as well as asymmetric redistribution, are regulated by the PAR proteins. Results from this study

  12. Myosin Vs organize actin cables in fission yeast

    PubMed Central

    Lo Presti, Libera; Chang, Fred; Martin, Sophie G.

    2012-01-01

    Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV∆ defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7–Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces. PMID:23051734

  13. Myosin Vs organize actin cables in fission yeast.

    PubMed

    Lo Presti, Libera; Chang, Fred; Martin, Sophie G

    2012-12-01

    Myosin V motors are believed to contribute to cell polarization by carrying cargoes along actin tracks. In Schizosaccharomyces pombe, Myosin Vs transport secretory vesicles along actin cables, which are dynamic actin bundles assembled by the formin For3 at cell poles. How these flexible structures are able to extend longitudinally in the cell through the dense cytoplasm is unknown. Here we show that in myosin V (myo52 myo51) null cells, actin cables are curled, bundled, and fail to extend into the cell interior. They also exhibit reduced retrograde flow, suggesting that formin-mediated actin assembly is impaired. Myo52 may contribute to actin cable organization by delivering actin regulators to cell poles, as myoV defects are partially suppressed by diverting cargoes toward cell tips onto microtubules with a kinesin 7-Myo52 tail chimera. In addition, Myo52 motor activity may pull on cables to provide the tension necessary for their extension and efficient assembly, as artificially tethering actin cables to the nuclear envelope via a Myo52 motor domain restores actin cable extension and retrograde flow in myoV mutants. Together these in vivo data reveal elements of a self-organizing system in which the motors shape their own tracks by transporting cargoes and exerting physical pulling forces.

  14. Actin filament curvature biases branching direction

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  15. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. PMID:23923011

  16. The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux.

    PubMed

    Joseph, Noah; Reicher, Barak; Barda-Saad, Mira

    2014-02-01

    During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca(2+)) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca(2+) signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca(2+) from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca(2+) across the plasma membrane in a process termed store-operated Ca(2+) entry (SOCE). Because CRAC channels are themselves inhibited by Ca(2+) ions, additional factors are suggested to enable the sustained Ca(2+) influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca(2+) evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca(2+) flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca(2+) signaling by controlling the spatial and temporal distribution of Ca(2+) sources and sinks, modulating TCR-dependent Ca(2+) signals, which are required for an appropriate T cell response. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters

  17. The candidate tumor suppressor SASH1 interacts with the actin cytoskeleton and stimulates cell-matrix adhesion.

    PubMed

    Martini, Melanie; Gnann, Alexandra; Scheikl, Daniela; Holzmann, Bernhard; Janssen, Klaus-Peter

    2011-11-01

    SASH1, a member of the SLY-family of signal adapter proteins, is a candidate tumor suppressor in breast and colon cancer. Reduced expression of SASH1 is correlated with aggressive tumor growth, metastasis formation, and inferior prognosis. However, the biological role of SASH1 remains largely unknown. To unravel the function of SASH1, we have analyzed the intracellular localization of endogenous SASH1, and have generated structural SASH1 mutants. SASH1 localized to the nucleus as well as to the cytoplasm in epithelial cells. In addition, SASH1 was enriched in lamellipodia and membrane ruffles, where it co-distributed with the actin cytoskeleton. Moreover, we demonstrate a novel interaction of SASH1 with the oncoprotein cortactin, a known regulator of actin polymerization in lamellipodia. Enhanced SASH1 expression significantly increased the content of filamentous actin, leading to the formation of cell protrusions and elongated cell shape. This activity was mapped to the central, evolutionarily conserved domain of SASH1. Furthermore, expression of SASH1 inhibited cell migration and lead to increased cell adhesion to fibronectin and laminin, whereas knock-down of endogenous SASH1 resulted in significantly reduced cell-matrix adhesion. Taken together, our findings unravel for the first time a mechanistic role for SASH1 in tumor formation by regulating the adhesive and migratory behaviour of cancer cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Characterizing Intracellular Ice Formation of Lymphoblasts Using Low-Temperature Raman Spectroscopy.

    PubMed

    Yu, Guanglin; Yap, Yan Rou; Pollock, Kathryn; Hubel, Allison

    2017-06-20

    Raman microspectroscopy was used to quantify freezing response of cells to various cooling rates and solution compositions. The distribution pattern of cytochrome c in individual cells was used as a measure of cell viability in the frozen state and this metric agreed well with the population-averaged viability and trypan blue staining experiments. Raman imaging of cells demonstrated that intracellular ice formation (IIF) was common and did not necessarily result in cell death. The amount of intracellular ice as well as ice crystal size played a role in determining whether or not ice inside the cell was a lethal event. Intracellular ice crystals were colocated to the sections of cell membrane in close proximity to extracellular ice. Increasing the distance between extracellular ice and cell membrane decreased the incidence of IIF. Reducing the effective stiffness of the cell membrane by disrupting the actin cytoskeleton using cytochalasin D increased the amount of IIF. Strong intracellular osmotic gradients were observed when IIF was present. These observations support the hypothesis that interactions between the cell membrane and extracellular ice result in IIF. Raman spectromicroscopy provides a powerful tool for observing IIF and understanding its role in cell death during freezing, and enables the development, to our knowledge, of new and improved cell preservation protocols. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. The Dynamic Actin Cytoskeleton in Smooth Muscle.

    PubMed

    Tang, Dale D

    2018-01-01

    Smooth muscle contraction requires both myosin activation and actin cytoskeletal remodeling. Actin cytoskeletal reorganization facilitates smooth muscle contraction by promoting force transmission between the contractile unit and the extracellular matrix (ECM), and by enhancing intercellular mechanical transduction. Myosin may be viewed to serve as an "engine" for smooth muscle contraction whereas the actin cytoskeleton may function as a "transmission system" in smooth muscle. The actin cytoskeleton in smooth muscle also undergoes restructuring upon activation with growth factors or the ECM, which controls smooth muscle cell proliferation and migration. Abnormal smooth muscle contraction, cell proliferation, and motility contribute to the development of vascular and pulmonary diseases. A number of actin-regulatory proteins including protein kinases have been discovered to orchestrate actin dynamics in smooth muscle. In particular, Abelson tyrosine kinase (c-Abl) is an important molecule that controls actin dynamics, contraction, growth, and motility in smooth muscle. Moreover, c-Abl coordinates the regulation of blood pressure and contributes to the pathogenesis of airway hyperresponsiveness and vascular/airway remodeling in vivo. Thus, c-Abl may be a novel pharmacological target for the development of new therapy to treat smooth muscle diseases such as hypertension and asthma. © 2018 Elsevier Inc. All rights reserved.

  20. The Bacterial Actin MamK

    PubMed Central

    Ozyamak, Ertan; Kollman, Justin; Agard, David A.; Komeili, Arash

    2013-01-01

    It is now recognized that actin-like proteins are widespread in bacteria and, in contrast to eukaryotic actins, are highly diverse in sequence and function. The bacterial actin, MamK, represents a clade, primarily found in magnetotactic bacteria, that is involved in the proper organization of subcellular organelles, termed magnetosomes. We have previously shown that MamK from Magnetospirillum magneticum AMB-1 (AMB-1) forms dynamic filaments in vivo. To gain further insights into the molecular mechanisms that underlie MamK dynamics and function, we have now studied the in vitro properties of MamK. We demonstrate that MamK is an ATPase that, in the presence of ATP, assembles rapidly into filaments that disassemble once ATP is depleted. The mutation of a conserved active site residue (E143A) abolishes ATPase activity of MamK but not its ability to form filaments. Filament disassembly depends on both ATPase activity and potassium levels, the latter of which results in the organization of MamK filaments into bundles. These data are consistent with observations indicating that accessory factors are required to promote filament disassembly and for spatial organization of filaments in vivo. We also used cryo-electron microscopy to obtain a high resolution structure of MamK filaments. MamK adopts a two-stranded helical filament architecture, but unlike eukaryotic actin and other actin-like filaments, subunits in MamK strands are unstaggered giving rise to a unique filament architecture. Beyond extending our knowledge of the properties and function of MamK in magnetotactic bacteria, this study emphasizes the functional and structural diversity of bacterial actins in general. PMID:23204522

  1. Probing actin polymerization by intermolecular cross-linking.

    PubMed

    Millonig, R; Salvo, H; Aebi, U

    1988-03-01

    We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant

  2. NASTRAN Structural Model for the Large 64-meter Antenna Pedestal. Part 3: Applications to Hydrostatic Bearing Oil Film

    NASA Technical Reports Server (NTRS)

    Chian, C. T.; Schonfeld, D.

    1984-01-01

    Investigations are conducted on the 64-meter antenna hydrostatic bearing oil film thickness under a variety of loads and elastic moduli. These parametric studies use a NASTRAN pedestal structural model to determine the deflections under the hydrostatic bearing pad. The deflections form the input for a computer program to determine the hydrostatic bearing oil film thickness. For the future 64-meter to 70-meter antenna extension and for the 2.2-meter (86-in.) haunch concrete replacement cases, safe oil film thickness (greater than 0.13 mm (0.005 in.) at the corners of the pad) are predicted. The effects of varying moduli of elasticity for different sections of the pedestal and the film height under distressed runner conditions are also studied.

  3. Eukaryotic chaperonin containing T-complex polypeptide 1 interacts with filamentous actin and reduces the initial rate of actin polymerization in vitro

    PubMed Central

    Grantham, Julie; Ruddock, Lloyd W.; Roobol, Anne; Carden, Martin J.

    2002-01-01

    We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament “seeds” and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament (+) ends, beyond its already well-established role in producing new actin monomers. PMID:12482199

  4. Formin' actin in the nucleus.

    PubMed

    Baarlink, Christian; Grosse, Robert

    2014-01-01

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

  5. Liquid droplets of cross-linked actin filaments

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Banerjee, Shiladitya; Dasbiswas, Kinjal; Vaikuntanathan, Suriyanarayan; Gardel, Margaret

    Soft materials constructed from biomolecules self-assemble into a myriad of structures that work in concert to support cell physiology. One critical soft material is the actin cytoskeleton, a viscoelastic gel composed of cross-linked actin filaments. Although actin networks are primarily known for their elastic properties, which are crucial to regulating cell mechanics, the viscous behavior has been theorized to enable shape changes and flows. We experimentally demonstrate a fluid phase of cross-linked actin, where cross-linker condenses dilute short actin filaments into spindle-shaped droplets, or tactoids. Tactoids have shape dynamics consistent with a continuum model of liquid crystal droplets. The cross-linker, which acts as a long range attractive interaction, analogous to molecular cohesion, controls the tactoid shape and dynamics, which reports on the liquid's interfacial tension and viscosity. We investigate how the cross-linker properties and filament length influence the liquid properties. These results demonstrate a novel mechanism to control organization of the actin cytoskeleton and provide insight into design principles for complex, macromolecular liquid phases.

  6. Sarcomeric Pattern Formation by Actin Cluster Coalescence

    PubMed Central

    Friedrich, Benjamin M.; Fischer-Friedrich, Elisabeth; Gov, Nir S.; Safran, Samuel A.

    2012-01-01

    Contractile function of striated muscle cells depends crucially on the almost crystalline order of actin and myosin filaments in myofibrils, but the physical mechanisms that lead to myofibril assembly remains ill-defined. Passive diffusive sorting of actin filaments into sarcomeric order is kinetically impossible, suggesting a pivotal role of active processes in sarcomeric pattern formation. Using a one-dimensional computational model of an initially unstriated actin bundle, we show that actin filament treadmilling in the presence of processive plus-end crosslinking provides a simple and robust mechanism for the polarity sorting of actin filaments as well as for the correct localization of myosin filaments. We propose that the coalescence of crosslinked actin clusters could be key for sarcomeric pattern formation. In our simulations, sarcomere spacing is set by filament length prompting tight length control already at early stages of pattern formation. The proposed mechanism could be generic and apply both to premyofibrils and nascent myofibrils in developing muscle cells as well as possibly to striated stress-fibers in non-muscle cells. PMID:22685394

  7. The role of the actin cytoskeleton in calcium signaling in starfish oocytes.

    PubMed

    Santella, Luigia; Puppo, Agostina; Chun, Jong Tai

    2008-01-01

    Ca2+ is the most universal second messenger in cells from the very first moment of fertilization. In all animal species, fertilized eggs exhibit massive mobilization of intracellular Ca2+ to orchestrate the initial events of development. Echinoderm eggs have been an excellent model system for studying fertilization and the cell cycle due to their large size and abundance. In preparation for fertilization, the cell cycle-arrested oocytes must undergo meiotic maturation. Studies of starfish oocytes have shown that Ca2+ signaling is intimately involved in this process. Our knowledge of the molecular mechanism of meiotic maturation and fertilization has expanded greatly in the past two decades due to the discovery of cell cycle-related kinases and Ca2+-mobilizing second messengers. However, the molecular details of their actions await elucidation of other cellular elements that assist in the creation and transduction of Ca2+ signals. In this regard, the actin cytoskeleton, the receptors for second messengers and the Ca2+-binding proteins also require more attention. This article reviews the physiological significance and the mechanism of intracellular Ca2+ mobilization in starfish oocytes during maturation and fertilization.

  8. Measurement and Analysis of in vitro Actin Polymerization

    PubMed Central

    Doolittle, Lynda K.; Rosen, Michael K.; Padrick, Shae B.

    2014-01-01

    Summary The polymerization of actin underlies force generation in numerous cellular processes. While actin polymerization can occur spontaneously, cells maintain control over this important process by preventing actin filament nucleation and then allowing stimulated polymerization and elongation by several regulated factors. Actin polymerization, regulated nucleation and controlled elongation activities can be reconstituted in vitro, and used to probe the signaling cascades cells use to control when and where actin polymerization occurs. Introducing a pyrene fluorophore allows detection of filament formation by an increase in pyrene fluorescence. This method has been used for many years and continues to be broadly used, owing to its simplicity and flexibility. Here we describe how to perform and analyze these in vitro actin polymerization assays, with an emphasis on extracting useful descriptive parameters from kinetic data. PMID:23868594

  9. Hypertrophic Stimulation Increases β-actin Dynamics in Adult Feline Cardiomyocytes

    PubMed Central

    Balasubramanian, Sundaravadivel; Mani, Santhosh K.; Kasiganesan, Harinath; Baicu, Catalin C.; Kuppuswamy, Dhandapani

    2010-01-01

    The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While α-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of β-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, β-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of β-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of β-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of β-actin. To determine the localization and dynamics of β-actin, we adenovirally expressed GFP-tagged β-actin in isolated adult cardiomyocytes. The ectopically expressed β-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of β-actin dynamics revealed that β-actin at the Z-discs is constantly being exchanged with β-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while β-actin overexpression improved cardiomyocyte contractility, immunoneutralization of β-actin resulted in a reduced contractility suggesting that β-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of β-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during

  10. Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.

    PubMed

    Balasubramanian, Sundaravadivel; Mani, Santhosh K; Kasiganesan, Harinath; Baicu, Catalin C; Kuppuswamy, Dhandapani

    2010-07-12

    The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac

  11. Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 in Purkinje cells is regulated by calcium and actin filaments.

    PubMed

    Fukatsu, Kazumi; Bannai, Hiroko; Inoue, Takafumi; Mikoshiba, Katsuhiko

    2010-09-01

    Inositol 1,4,5-trisphosphate receptor type 1 (IP(3) R1) is an intracellular Ca(2+) release channel that plays crucial roles in the functions of Purkinje cells. The dynamics of IP(3) R1 on the endoplasmic reticulum membrane and the distribution of IP(3) R1 in neurons are thought to be important for the spatial regulation of Ca(2+) release. In this study, we analyzed the lateral diffusion of IP(3) R1 in Purkinje cells in cerebellar slice cultures using fluorescence recovery after photobleaching. In the dendrites of Purkinje cells, IP(3) R1 showed lateral diffusion with an effective diffusion constant of approximately 0.30 μm(2) /s, and the diffusion of IP(3) R1 was negatively regulated by actin filaments. We found that actin filaments were also involved in the regulation of IP(3) R1 diffusion in the spine of Purkinje cells. Glutamate or quisqualic acid stimulation, which activates glutamate receptors and leads to a Ca(2+) transient in Purkinje cells, decreased the diffusion of IP(3) R1 and increased the density of actin in spines. These findings indicate that the neuronal activity-dependent augmentation of actin contributes to the stabilization of IP(3) R1 in spines. © 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.

  12. Stability and dynamics of the edge pedestal in the low collisionality regime: physics mechanisms for steady-state ELM-free operation

    NASA Astrophysics Data System (ADS)

    Snyder, P. B.; Burrell, K. H.; Wilson, H. R.; Chu, M. S.; Fenstermacher, M. E.; Leonard, A. W.; Moyer, R. A.; Osborne, T. H.; Umansky, M.; West, W. P.; Xu, X. Q.

    2007-08-01

    Understanding the physics of the edge pedestal and edge localized modes (ELMs) is of great importance for ITER and the optimization of the tokamak concept. The peeling-ballooning model has quantitatively explained many observations, including ELM onset and pedestal constraints, in the standard H-mode regime. The ELITE code has been developed to efficiently evaluate peeling-ballooning stability for comparison with observation and predictions for future devices. We briefly review recent progress in the peeling-ballooning model, including experimental validation of ELM onset and pedestal height predictions, and nonlinear 3D simulations of ELM dynamics, which together lead to an emerging understanding of the physics of the onset and dynamics of ELMs in the standard intermediate to high collisionality regime. We also discuss new studies of the apparent power dependence of the pedestal, and studies of the impact of sheared toroidal flow. Recently, highly promising low collisionality regimes without ELMs have been discovered, including the quiescent H-mode (QH) and resonant magnetic perturbation (RMP) regimes. We present recent observations from the DIII-D tokamak of the density, shape and rotation dependence of QH discharges, and studies of the peeling-ballooning stability in this regime. We propose a model of the QH-mode in which the observed edge harmonic oscillation (EHO) is a saturated kink/peeling mode which is destabilized by current and rotation, and drives significant transport, allowing a near steady-state edge plasma. The model quantitatively predicts the observed density dependence and qualitatively predicts observed mode structure, rotation dependence and outer gap dependence. Low density RMP discharges are found to operate in a similar regime, but with the EHO replaced by an applied magnetic perturbation.

  13. 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

  14. Nuclear positioning by actin cables and perinuclear actin: Special and general?

    PubMed

    Huelsmann, Sven; Brown, Nicholas H

    2014-01-01

    Nuclear positioning is an important process during development and homeostasis. Depending on the affected tissue, mislocalized nuclei can alter cellular processes such as polarization, differentiation, or migration and lead ultimately to diseases. Many cells actively control the position of their nucleus using their cytoskeleton and motor proteins. We have recently shown that during Drosophila oogenesis, nurse cells employ cytoplasmic actin cables in association with perinuclear actin to position their nucleus. Here, we briefly summarize our work and discuss why nuclear positioning in nurse cells is specialized but the molecular mechanisms are likely to be more generally used.

  15. IFT88 influences chondrocyte actin organization and biomechanics.

    PubMed

    Wang, Z; Wann, A K T; Thompson, C L; Hassen, A; Wang, W; Knight, M M

    2016-03-01

    Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. IFT88(orpk) cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Biphasic interactions between a cationic dendrimer and actin.

    PubMed

    Ruenraroengsak, Pakatip; Florence, Alexander T

    2010-12-01

    Gene delivery systems face the problem not only of the route toward the cell and tissues in question, but also of the molecularly crowded environment of both the cytoplasm and the nucleus itself. One of the physical barriers in the cytoplasm for diffusing nanoparticles is an actin network. Here, we describe the finding that a self-fluorescent sixth generation cationic dendrimer (6 nm in diameter) interacts reversibly and possibly electrostatically with actin filaments in vitro. Not only does this interaction slow the diffusion of the dendrimer but it also affects actin polymerization in a biphasic manner. At low concentrations the dendrimer behaves like a G-binding actin protein, retarding actin polymerization, whereas at high concentrations the dendrimer acts as a nucleating protein accelerating the polymerization. Thus in vivo the diffusion of a dendrimer carrier such as this has both physical and chemical elements: by decreasing polymerization it might accelerate its own transport, and by enhancing actin polymerization retard it. This finding suggests that such a dendrimer may have a role as an anticancer agent through its inhibitory effect on actin polymerization.

  17. Actin genes and their expression in pacific white shrimp, Litopenaeus vannamei.

    PubMed

    Zhang, Xiaoxi; Zhang, Xiaojun; Yuan, Jianbo; Du, Jiangli; Li, Fuhua; Xiang, Jianhai

    2018-04-01

    Actin is a multi-functional gene family that can be divided into muscle-type actins and non-muscle-type actins. In this study, 37 unigenes encoding actins were identified from RNA-Seq data of Pacific white shrimp, Litopenaeus vannamei. According to phylogenetic analysis, four and three cDNAs belong to cytoplasmic- and heart-type actins and were named LvActinCT and LvActinHT, respectively. 10 cDNAs belong to the slow-type skeletal muscle actins, and 18 belong to the fast-type skeletal muscle actins; they were designated LvActinSSK and LvActinFSK, respectively. Some muscle actin genes formed gene clusters in the genome. Multiple alternative transcription starts sites (ATSSs) were found for LvActinCT1. Based on the early developmental expression profile, almost all LvActins were highly expressed between the early limb bud and post-larval stages. Using LvActinSSK5 as probes, slow-type muscle was localized in pleopod muscle and superficial ventral muscle. We also found three actin genes that were down-regulated in the hemocytes of white spot syndrome virus (WSSV)- and Vibrio parahaemolyticus-infected L. vannamei. This study provides valuable information on the actin gene structure of shrimp, furthers our understanding of the shrimp muscle system and helps us develop strategies for disease control and sustainable shrimp farming.

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

    PubMed

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

    2004-06-01

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

  19. Actin Hydrophobic Loop (262-274) and Filament Nucleation and Elongation

    PubMed Central

    Shvetsov, Alexander; Galkin, Vitold E.; Orlova, Albina; Phillips, Martin; Bergeron, Sarah E.; Rubenstein, Peter A.; Egelman, Edward H.; Reisler, Emil

    2014-01-01

    Summary The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently using a yeast actin mutant, L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked G-actin does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin - to assist with actin nucleation - and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not alone, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin the helical twist of F-actin changes by ~ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin, and a change of twist by ~ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or a competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics to both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors. PMID:18037437

  20. Bacterial Actins? An Evolutionary Perspective

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.; York, Amanda L.

    2003-01-01

    According to the conventional wisdom, the existence of a cytoskeleton in eukaryotes and its absence in prokaryotes constitute a fundamental divide between the two domains of life. An integral part of the dogma is that a cytoskeleton enabled an early eukaryote to feed upon prokaryotes, a consequence of which was the occasional endosymbiosis and the eventual evolution of organelles. Two recent papers present compelling evidence that actin, one of the principal components of a cytoskeleton, has a homolog in Bacteria that behaves in many ways like eukaryotic actin. Sequence comparisons reveml that eukaryotic actin and the bacterial homolog (mreB protein), unlike many other proteins common to eukaryotes and Bacteria, have very different and more highly extended evolutionary histories.

  1. Correlative nanoscale imaging of actin filaments and their complexes

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  2. LIPS database with LIPService: a microscopic image database of intracellular structures in Arabidopsis guard cells.

    PubMed

    Higaki, Takumi; Kutsuna, Natsumaro; Hasezawa, Seiichiro

    2013-05-16

    Intracellular configuration is an important feature of cell status. Recent advances in microscopic imaging techniques allow us to easily obtain a large number of microscopic images of intracellular structures. In this circumstance, automated microscopic image recognition techniques are of extreme importance to future phenomics/visible screening approaches. However, there was no benchmark microscopic image dataset for intracellular organelles in a specified plant cell type. We previously established the Live Images of Plant Stomata (LIPS) database, a publicly available collection of optical-section images of various intracellular structures of plant guard cells, as a model system of environmental signal perception and transduction. Here we report recent updates to the LIPS database and the establishment of a database table, LIPService. We updated the LIPS dataset and established a new interface named LIPService to promote efficient inspection of intracellular structure configurations. Cell nuclei, microtubules, actin microfilaments, mitochondria, chloroplasts, endoplasmic reticulum, peroxisomes, endosomes, Golgi bodies, and vacuoles can be filtered using probe names or morphometric parameters such as stomatal aperture. In addition to the serial optical sectional images of the original LIPS database, new volume-rendering data for easy web browsing of three-dimensional intracellular structures have been released to allow easy inspection of their configurations or relationships with cell status/morphology. We also demonstrated the utility of the new LIPS image database for automated organelle recognition of images from another plant cell image database with image clustering analyses. The updated LIPS database provides a benchmark image dataset for representative intracellular structures in Arabidopsis guard cells. The newly released LIPService allows users to inspect the relationship between organellar three-dimensional configurations and morphometrical parameters.

  3. Global simulation of edge pedestal micro-instabilities

    NASA Astrophysics Data System (ADS)

    Wan, Weigang; Parker, Scott; Chen, Yang

    2011-10-01

    We study micro turbulence of the tokamak edge pedestal with global gyrokinetic particle simulations. The simulation code GEM is an electromagnetic δf code. Two sets of DIII-D experimental profiles, shot #131997 and shot #136051 are used. The dominant instabilities appear to be two kinds of modes both propagating in the electron diamagnetic direction, with comparable linear growth rates. The low n mode is at the Alfven frequency range and driven by density and ion temperature gradients. The high n mode is driven by electron temperature gradient and has a low real frequency. A β scan shows that the low n mode is electromagnetic. Frequency analysis shows that the high n mode is sometimes mixed with an ion instability. Experimental radial electric field is applied and its effects studied. We will also show some preliminary nonlinear results. We thank R. Groebner, P. Snyder and Y. Zheng for providing experimental profiles and helpful discussions.

  4. Actin expression in trypanosomatids (Euglenozoa: Kinetoplastea)

    PubMed Central

    Souza, Ligia Cristina Kalb; Pinho, Rosana Elisa Gonçalves Gonçalves; Lima, Carla Vanessa de Paula; Fragoso, Stênio Perdigão; Soares, Maurilio José

    2013-01-01

    Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids. PMID:23903980

  5. Functional adaptation between yeast actin and its cognate myosin motors.

    PubMed

    Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

    2011-09-02

    We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins.

  6. Oligomerization of coronin: Implication on actin filament length in Leishmania.

    PubMed

    Srivastava, Rashmi; Prasadareddy Kajuluri, Lova; Pathak, Neelam; Gupta, Chhitar M; Sahasrabuddhe, Amogh A

    2015-12-01

    Coronin proteins bind with actin filaments and participate in regulation of actin-dependent processes. These proteins contain a coiled-coil domain at their C-terminus, which is responsible for their dimeric or trimeric forms. However, the functional significance of these oligomeric configurations in organizing the actin cytoskeleton is obscure. Here, we report that the Leishmania coronin exists in a higher oligomeric form through its coiled-coil domain, the truncation of which ablates the ability of Leishmania coronin to assist actin-filament formation. F-actin co-sedimentation assay using purified proteins shows that the coiled-coil domain does not interact with actin-filaments and its absence does not abrogate actin-coronin interaction. Furthermore, it was shown that unlike other coronins, Leishmania coronin interacts with actin-filaments through its unique region. These results provided important insights into the role of coronin oligomerization in modulating actin-network. © 2015 Wiley Periodicals, Inc.

  7. Coactosin accelerates cell dynamism by promoting actin polymerization.

    PubMed

    Hou, Xubin; Katahira, Tatsuya; Ohashi, Kazumasa; Mizuno, Kensaku; Sugiyama, Sayaka; Nakamura, Harukazu

    2013-07-01

    During development, cells dynamically move or extend their processes, which are achieved by actin dynamics. In the present study, we paid attention to Coactosin, an actin binding protein, and studied its role in actin dynamics. Coactosin was associated with actin and Capping protein in neural crest cells and N1E-115 neuroblastoma cells. Accumulation of Coactosin to cellular processes and its association with actin filaments prompted us to reveal the effect of Coactosin on cell migration. Coactosin overexpression induced cellular processes in cultured neural crest cells. In contrast, knock-down of Coactosin resulted in disruption of actin polymerization and of neural crest cell migration. Importantly, Coactosin was recruited to lamellipodia and filopodia in response to Rac signaling, and mutated Coactosin that cannot bind to F-actin did not react to Rac signaling, nor support neural crest cell migration. It was also shown that deprivation of Rac signaling from neural crest cells by dominant negative Rac1 (DN-Rac1) interfered with neural crest cell migration, and that co-transfection of DN-Rac1 and Coactosin restored neural crest cell migration. From these results we have concluded that Coactosin functions downstream of Rac signaling and that it is involved in neurite extension and neural crest cell migration by actively participating in actin polymerization. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Transduction of skin-migrating dendritic cells by human adenovirus 5 occurs via an actin-dependent phagocytic pathway.

    PubMed

    Guzman, Efrain; Taylor, Geraldine; Hope, Jayne; Herbert, Rebecca; Cubillos-Zapata, Carolina; Charleston, Bryan

    2016-10-01

    Dendritic cells (DC) are central to the initiation of immune responses, and various approaches have been used to target vaccines to DC in order to improve immunogenicity. Cannulation of lymphatic vessels allows for the collection of DC that migrate from the skin. These migrating DC are involved in antigen uptake and presentation following vaccination. Human replication-deficient adenovirus (AdV) 5 is a promising vaccine vector for delivery of recombinant antigens. Although the mechanism of AdV attachment and penetration has been extensively studied in permissive cell lines, few studies have addressed the interaction of AdV with DC. In this study, we investigated the interaction of bovine skin-migrating DC and replication-deficient AdV-based vaccine vectors. We found that, despite lack of expression of Coxsackie B-Adenovirus Receptor and other known adenovirus receptors, AdV readily enters skin-draining DC via an actin-dependent endocytosis. Virus exit from endosomes was pH independent, and neutralizing antibodies did not prevent virus entry but did prevent virus translocation to the nucleus. We also show that combining adenovirus with adjuvant increases the absolute number of intracellular virus particles per DC but not the number of DC containing intracellular virus. This results in increased trans-gene expression and antigen presentation. We propose that, in the absence of Coxsackie B-Adenovirus Receptor and other known receptors, AdV5-based vectors enter skin-migrating DC using actin-dependent endocytosis which occurs in skin-migrating DC, and its relevance to vaccination strategies and vaccine vector targeting is discussed.

  9. 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

  10. How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

    PubMed Central

    1988-01-01

    We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross- linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell. PMID:3392099

  11. Microtubule-Actin Cross-Linking Factor 1: Domains, Interaction Partners, and Tissue-Specific Functions.

    PubMed

    Goryunov, Dmitry; Liem, Ronald K H

    2016-01-01

    The cytoskeleton of most eukaryotic cells is composed of three principal filamentous components: actin filaments, microtubules (MTs), and intermediate filaments. It is a highly dynamic system that plays crucial roles in a wide range of cellular processes, including migration, adhesion, cytokinesis, morphogenesis, intracellular traffic and signaling, and structural flexibility. Among the large number of cytoskeleton-associated proteins characterized to date, microtubule-actin cross-linking factor 1 (MACF1) is arguably the most versatile integrator and modulator of cytoskeleton-related processes. MACF1 belongs to the plakin family of proteins, and within it, to the spectraplakin subfamily. These proteins are characterized by the ability to bridge MT and actin cytoskeletal networks in a dynamic fashion, which underlies their involvement in the regulation of cell migration, axonal extension, and vesicular traffic. Studying MACF1 functions has provided insights not only into the regulation of the cytoskeleton but also into molecular mechanisms of both normal cellular physiology and cellular pathology. Multiple MACF1 isoforms exist, composed of a large variety of alternatively spliced domains. Each of these domains mediates a specific set of interactions and functions. These functions are manifested in tissue and cell-specific phenotypes observed in conditional MACF1 knockout mice. The conditional models described to date reveal critical roles of MACF1 in mammalian skin, nervous system, heart muscle, and intestinal epithelia. Complete elimination of MACF1 is early embryonic lethal, indicating an essential role for MACF1 in early development. Further studies of MACF1 domains and their interactions will likely reveal multiple new roles of this protein in various tissues. © 2016 Elsevier Inc. All rights reserved.

  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. Thymosin-beta(4) changes the conformation and dynamics of actin monomers.

    PubMed Central

    De La Cruz, E M; Ostap, E M; Brundage, R A; Reddy, K S; Sweeney, H L; Safer, D

    2000-01-01

    Thymosin-beta(4) (Tbeta(4)) binds actin monomers stoichiometrically and maintains the bulk of the actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers. The K(d) of the actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation. At 25 degrees C, the free energy of Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange. PMID:10777749

  14. Treatment options for actinic keratoses.

    PubMed

    McIntyre, William J; Downs, Michael R; Bedwell, Sondra A

    2007-09-01

    Actinic keratoses are rough, scaly lesions that commonly occur on sun-exposed areas of the skin. The prevalence of the condition increases with age. Actinic keratoses are thought to be carcinomas in situ, which can progress to squamous cell carcinomas. The decision to treat can be based on cosmetic reasons; symptom relief; or, most importantly, the prevention of malignancy and metastasis. Treatment options include ablative (destructive) therapies such as cryosurgery, curettage with electrosurgery, and photodynamic therapy. Topical therapies are used in patients with multiple lesions. Fluorouracil has been the traditional topical treatment for actinic keratoses, although imiquimod 5% cream and diclofenac 3% gel are effective alternative therapies. There are too few controlled trials comparing treatment modalities for physicians to make sound, evidence-based treatment decisions.

  15. Side-binding proteins modulate actin filament dynamics

    PubMed Central

    Crevenna, Alvaro H; Arciniega, Marcelino; Dupont, Aurélie; Mizuno, Naoko; Kowalska, Kaja; Lange, Oliver F; Wedlich-Söldner, Roland; Lamb, Don C

    2015-01-01

    Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics. DOI: http://dx.doi.org/10.7554/eLife.04599.001 PMID:25706231

  16. 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

  17. Actin Cytoskeleton-Based Plant Synapse as Gravitransducer in the Transition Zone of the Root Apex

    NASA Astrophysics Data System (ADS)

    Baluska, Frantisek; Barlow, Peter; Volkmann, Dieter; Mancuso, Stefano

    The actin cytoskeleton was originally proposed to act as the signal transducer in the plant gravity sensory-motoric circuit. Surprisingly, however, several studies have documented that roots perfom gravisensing and gravitropism more effectively if exposed to diverse anti-F-actin drugs. Our study, using decapped maize root apices, has revealed that depolymerization of F-actin stimulates gravity perception in cells of the transition zone where root gravitropism is initiated (Mancuso et al. 2006). It has been proposed (Balǔka et al. 2005, 2009a) that s the non-growing adhesive end-poles, enriched with F-actin and myosin VIII, and active in endocytic recycling of both PIN transporters and cell wall pectins cross-linked with calcium and boron, act as the gravisensing domains, and that these impinge directly upon the root motoric responses via control of polar auxin transport. This model suggests that mechanical asymmetry at these plant synapses determines vectorial gravity-controlled auxin transport. Due to the gravity-imposed mechanical load upon the protoplast, a tensional stress is also imposed upon the plasma membrane of the physically lower synaptic cell pole. This stress is then relieved by shifting the endocytosis-exocytosis balance towards exocytosis (Balǔka et al. s 2005, 2009a,b). This `Synaptic Auxin Secretion' hypothesis does not conflict with the `Starch Statolith' hypothesis, which is based on amyloplast sedimentation. In fact, the `Synaptic Auxin Secretion' hypothesis has many elements which allow its unification with the Starch-Statolith model (Balǔka et al. 2005, 2009a,b). s References Balǔka F, Volkmann D, Menzel D (2005) Plant synapses: actin-based adhesion s domains for cell-to-cell communication. Trends Plant Sci 10: 106-111 Balǔka F, Schlicht M, s Wan Y-L, Burbach C, Volkmann D (2009a) Intracellular domains and polarity in root apices: from synaptic domains to plant neurobiology. Nova Acta Leopoldina 96: 103-122 Balǔka s F, Mancuso S

  18. 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

  19. Nucleotide-dependent conformational states of actin

    PubMed Central

    Pfaendtner, Jim; Branduardi, Davide; Parrinello, Michele; Pollard, Thomas D.; Voth, Gregory A.

    2009-01-01

    The influence of the state of the bound nucleotide (ATP, ADP-Pi, or ADP) on the conformational free-energy landscape of actin is investigated. Nucleotide-dependent folding of the DNase-I binding (DB) loop in monomeric actin and the actin trimer is carried out using all-atom molecular dynamics (MD) calculations accelerated with a multiscale implementation of the metadynamics algorithm. Additionally, an investigation of the opening and closing of the actin nucleotide binding cleft is performed. Nucleotide-dependent free-energy profiles for all of these conformational changes are calculated within the framework of metadynamics. We find that in ADP-bound monomer, the folded and unfolded states of the DB loop have similar relative free-energy. This result helps explain the experimental difficulty in obtaining an ordered crystal structure for this region of monomeric actin. However, we find that in the ADP-bound actin trimer, the folded DB loop is stable and in a free-energy minimum. It is also demonstrated that the nucleotide binding cleft favors a closed conformation for the bound nucleotide in the ATP and ADP-Pi states, whereas the ADP state favors an open confirmation, both in the monomer and trimer. These results suggest a mechanism of allosteric interactions between the nucleotide binding cleft and the DB loop. This behavior is confirmed by an additional simulation that shows the folding free-energy as a function of the nucleotide cleft width, which demonstrates that the barrier for folding changes significantly depending on the value of the cleft width. PMID:19620726

  20. Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

    PubMed Central

    1991-01-01

    The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide- stimulated cells was examined. F-actin was quantified by the TRITC- labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar. PMID:1918158

  1. Actinic cheilitis in dental practice.

    PubMed

    Savage, N W; McKay, C; Faulkner, C

    2010-06-01

    Actinic cheilitis is a potentially premalignant condition involving predominantly the vermilion of the lower lip. The aim of the current paper was to review the clinical presentation of actinic cheilitis and demonstrate the development of management plans using a series of cases. These are designed to provide immediate treatment where required but also to address the medium and long-term requirements of the patient. The authors suggest that the clinical examination of lips and the assessment of actinic cheilitis and other lip pathology become a regular part of the routine soft tissue examination undertaken as a part of the periodic examination of dental patients. Early recognition of actinic cheilitis can allow the development of strategies for individual patients that prevent progression. These are based on past sun exposure, future lifestyle changes and the daily use of emollient sunscreens, broad-brimmed hats and avoidance of sun exposure during the middle of the day. This is a service that is not undertaken as a matter of routine in general medical practice as patients are not seen with the regularity of dental patients and generally not under the ideal examination conditions available in the dental surgery.

  2. Rheology of Membrane-Attached Minimal Actin Cortices.

    PubMed

    Nöding, Helen; Schön, Markus; Reinermann, Corinna; Dörrer, Nils; Kürschner, Aileen; Geil, Burkhard; Mey, Ingo; Heussinger, Claus; Janshoff, Andreas; Steinem, Claudia

    2018-04-26

    The actin cortex is a thin cross-linked network attached to the plasma membrane, which is responsible for the cell's shape during migration, division, and growth. In a reductionist approach, we created a minimal actin cortex (MAC) attached to a lipid membrane to correlate the filamentous actin architecture with its viscoelastic properties. The system is composed of a supported 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayer doped with the receptor lipid phosphatidylinositol(4,5)-bisphosphate (PtdIns(4,5)P 2 ) to which a constitutively active mutant of ezrin, which is a direct membrane-cytoskeleton linker, is bound. The formation of the MAC on the supported lipid bilayer is analyzed as a function of increasing PtdIns(4,5)P 2 /ezrin pinning points, revealing an increase in the intersections between actin filaments, that is, the node density of the MAC. Bead tracking microrheology on the membrane-attached actin network provides information about its viscoelastic properties. The results show that ezrin serves as a dynamic cross-linker for the actin cortex attached to the lipid bilayer and that the stiffness of the network is influenced by the pinning point density, relating the plateau storage modulus G 0 to the node density of the MAC.

  3. Regulation of the Pollen-Specific Actin-Depolymerizing Factor LlADF1

    PubMed Central

    Allwood, Ellen G.; Anthony, Richard G.; Smertenko, Andrei P.; Reichelt, Stefanie; Drobak, Bjorn K.; Doonan, John H.; Weeds, Alan G.; Hussey, Patrick J.

    2002-01-01

    Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved. We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LlADF1. Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation. Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains. This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth. We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LlADF1 by ∼60%. Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes. Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy. PMID:12417710

  4. Membrane-associated actin from the microvillar membranes of ascites tumor cells

    PubMed Central

    1982-01-01

    A membrane fraction (MF2) has been purified from isolated microvilli of the MAT-C1 subline of the 13762 rat mammary ascites adenocarcinoma under conditions which cause F-actin depolymerization. This membrane preparation contains actin as a major component, although no filamentous structures are observed by transmission electron microscopy. Membranes were extracted with a Triton X-100-containing actin-stabilizing buffer (S buffer) or actin-destabilizing buffer (D buffer). In D buffer greater than 90% of metabolically labeled protein and glycoprotein was extracted, and 80-90% of these labeled species was extracted in S buffer. When S buffer extracts of MF2 were fractionated by either gel filtration on Sepharose 6 B or rate-zonal sucrose density gradient centrifugation, most of the actin was found to be intermediate in size between G- and F-actin. In D buffer most of the MF2 actin behaved as G-actin. Extraction and gel filtration of intact microvilli in S buffer also showed the presence of the intermediate form of actin, indicating that it did not arise during membrane preparation. When [35S]methionine-labeled G-actin from ascites cells was added to S buffer extracts of MF2 and chromatographed, all of the radioactivity chromatographed as G-actin, indicating that the intermediate form of actin did not result from an association of G-actin molecules during extraction or chromatography. The results of this study suggest that the microvillar membrane fraction is enriched in an intermediate form of actin smaller than F-actin and larger than G-actin. PMID:6890066

  5. Membrane-associated actin from the microvillar membranes of ascites tumor cells.

    PubMed

    Carraway, K L; Cerra, R F; Jung, G; Carraway, C A

    1982-09-01

    A membrane fraction (MF2) has been purified from isolated microvilli of the MAT-C1 subline of the 13762 rat mammary ascites adenocarcinoma under conditions which cause F-actin depolymerization. This membrane preparation contains actin as a major component, although no filamentous structures are observed by transmission electron microscopy. Membranes were extracted with a Triton X-100-containing actin-stabilizing buffer (S buffer) or actin-destabilizing buffer (D buffer). In D buffer greater than 90% of metabolically labeled protein and glycoprotein was extracted, and 80-90% of these labeled species was extracted in S buffer. When S buffer extracts of MF2 were fractionated by either gel filtration on Sepharose 6 B or rate-zonal sucrose density gradient centrifugation, most of the actin was found to be intermediate in size between G- and F-actin. In D buffer most of the MF2 actin behaved as G-actin. Extraction and gel filtration of intact microvilli in S buffer also showed the presence of the intermediate form of actin, indicating that it did not arise during membrane preparation. When [35S]methionine-labeled G-actin from ascites cells was added to S buffer extracts of MF2 and chromatographed, all of the radioactivity chromatographed as G-actin, indicating that the intermediate form of actin did not result from an association of G-actin molecules during extraction or chromatography. The results of this study suggest that the microvillar membrane fraction is enriched in an intermediate form of actin smaller than F-actin and larger than G-actin.

  6. Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

    NASA Astrophysics Data System (ADS)

    Kumar, Nitin; Gardel, Margaret

    Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

  7. Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy.

    PubMed

    Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O

    2010-12-22

    The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.

  8. 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

  9. Mechanisms of the cytopathic action of actin-ADP-ribosylating toxins.

    PubMed

    Aktories, K; Wegner, A

    1992-10-01

    Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP-ribosylate actin monomers. Toxin-induced ADP-ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP-ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins.

  10. The evolution of compositionally and functionally distinct actin filaments.

    PubMed

    Gunning, Peter W; Ghoshdastider, Umesh; Whitaker, Shane; Popp, David; Robinson, Robert C

    2015-06-01

    The actin filament is astonishingly well conserved across a diverse set of eukaryotic species. It has essentially remained unchanged in the billion years that separate yeast, Arabidopsis and man. In contrast, bacterial actin-like proteins have diverged to the extreme, and many of them are not readily identified from sequence-based homology searches. Here, we present phylogenetic analyses that point to an evolutionary drive to diversify actin filament composition across kingdoms. Bacteria use a one-filament-one-function system to create distinct filament systems within a single cell. In contrast, eukaryotic actin is a universal force provider in a wide range of processes. In plants, there has been an expansion of the number of closely related actin genes, whereas in fungi and metazoa diversification in tropomyosins has increased the compositional variety in actin filament systems. Both mechanisms dictate the subset of actin-binding proteins that interact with each filament type, leading to specialization in function. In this Hypothesis, we thus propose that different mechanisms were selected in bacteria, plants and metazoa, which achieved actin filament compositional variation leading to the expansion of their functional diversity. © 2015. Published by The Company of Biologists Ltd.

  11. Modeling the effect of lithium-induced pedestal profiles on scrape-off-layer turbulence and the heat flux width

    DOE PAGES

    Russell, David A.; D'Ippolito, Daniel A.; Myra, James R.; ...

    2015-09-01

    The effect of lithium (Li) wall coatings on scrape-off-layer (SOL) turbulence in the National Spherical Torus Experiment (NSTX) is modeled with the Lodestar SOLT (“SOL Turbulence”) code. Specifically, the implications for the SOL heat flux width of experimentally observed, Li-induced changes in the pedestal profiles are considered. The SOLT code used in the modeling has been expanded recently to include ion temperature evolution and ion diamagnetic drift effects. This work focuses on two NSTX discharges occurring pre- and with-Li deposition. The simulation density and temperature profiles are constrained, inside the last closed flux surface only, to match those measured inmore » the two experiments, and the resulting drift-interchange-driven turbulence is explored. The effect of Li enters the simulation only through the pedestal profile constraint: Li modifies the experimental density and temperature profiles in the pedestal, and these profiles affect the simulated SOL turbulence. The power entering the SOL measured in the experiments is matched in the simulations by adjusting “free” dissipation parameters (e.g., diffusion coefficients) that are not measured directly in the experiments. With power-matching, (a) the heat flux SOL width is smaller, as observed experimentally by infra-red thermography, and (b) the simulated density fluctuation amplitudes are reduced with Li, as inferred for the experiments as well from reflectometry analysis. The instabilities and saturation mechanisms that underlie the SOLT model equilibria are also discussed.« less

  12. Cell-cycle regulation of formin-mediated actin cable assembly

    PubMed Central

    Miao, Yansong; Wong, Catherine C. L.; Mennella, Vito; Michelot, Alphée; Agard, David A.; Holt, Liam J.; Yates, John R.; Drubin, David G.

    2013-01-01

    Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation. PMID:24133141

  13. Mechanism of Cdc42-induced actin polymerization in neutrophil extracts.

    PubMed

    Zigmond, S H; Joyce, M; Yang, C; Brown, K; Huang, M; Pring, M

    1998-08-24

    Cdc42, activated with GTPgammaS, induces actin polymerization in supernatants of lysed neutrophils. This polymerization, like that induced by agonists, requires elongation at filament barbed ends. To determine if creation of free barbed ends was sufficient to induce actin polymerization, free barbed ends in the form of spectrin-actin seeds or sheared F-actin filaments were added to cell supernatants. Neither induced polymerization. Furthermore, the presence of spectrin-actin seeds did not increase the rate of Cdc42-induced polymerization, suggesting that the presence of Cdc42 did not facilitate polymerization from spectrin-actin seeds such as might have been the case if Cdc42 inhibited capping or released G-actin from a sequestered pool. Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 micron in 15 s. The mean length of filaments formed from spectrin-actin seeds was <0.4 micron. Had spectrin-actin seeds elongated at comparable rates before they were capped, they would have induced longer filaments. There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number. These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends.

  14. 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.

  15. 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

  16. Evaluation of DSS-14 pedestal-review of top surface repair procedures

    NASA Technical Reports Server (NTRS)

    Oesterle, R. G.; Musser, D. W.; Salse, E. A. B.

    1983-01-01

    Proposed repair procedures for the top surface of the pedestal supporting the hydrostatic bearing runner for the 64m Antenna are presented. These procedures included: (1) removal of existing grout and concrete to approximately 8 in. below original concrete surface using a presplitting technique with expansive cement followed by secondary breaking; (2) preparation of exposed concrete surface including an epoxy bonding agent; and (3) replacement of material removed with 8 in. of new concrete surface including an epoxy bonding agent; and (4) replacement of material removed with 8 in. of new concrete and 4 in. of new grout.

  17. eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1

    PubMed Central

    García-Ortiz, Almudena; Martín-Cofreces, Noa B.; Ibiza, Sales; Ortega, Ángel; Izquierdo-Álvarez, Alicia; Trullo, Antonio; Victor, Víctor M.; Calvo, Enrique; Sot, Begoña; Martínez-Ruiz, Antonio; Vázquez, Jesús; Sánchez-Madrid, Francisco

    2017-01-01

    The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS. PMID:28394935

  18. Cations Modulate Actin Bundle Mechanics, Assembly Dynamics, and Structure.

    PubMed

    Castaneda, Nicholas; Zheng, Tianyu; Rivera-Jacquez, Hector J; Lee, Hyun-Ju; Hyun, Jaekyung; Balaeff, Alexander; Huo, Qun; Kang, Hyeran

    2018-04-12

    Actin bundles are key factors in the mechanical support and dynamic reorganization of the cytoskeleton. High concentrations of multivalent counterions promote bundle formation through electrostatic attraction between actin filaments that are negatively charged polyelectrolytes. In this study, we evaluate how physiologically relevant divalent cations affect the mechanical, dynamic, and structural properties of actin bundles. Using a combination of total internal reflection fluorescence microscopy, transmission electron microscopy, and dynamic light scattering, we demonstrate that divalent cations modulate bundle stiffness, length distribution, and lateral growth. Molecular dynamics simulations of an all-atom model of the actin bundle reveal specific actin residues coordinate cation-binding sites that promote the bundle formation. Our work suggests that specific cation interactions may play a fundamental role in the assembly, structure, and mechanical properties of actin bundles.

  19. Structural basis for profilin-mediated actin nucleotide exchange

    PubMed Central

    Porta, Jason C.; Borgstahl, Gloria E.O.

    2015-01-01

    Actin is a ubiquitous eukaryotic protein that is responsible for cellular scaffolding, motility and division. The ability of actin to form a helical filament is the driving force behind these cellular activities. Formation of a filament is dependent the successful exchange of actin’s ADP for ATP. Mammalian profilin is a small actin binding protein that catalyzes the exchange of nucleotide and facilitates the addition of an actin monomer to a growing filament. Here, crystal structures of profilin:actin have been determined showing an actively exchanging ATP. The structural analysis shows how the binding of profilin to the barbed end of actin causes a rotation of the small domain relative to the large domain. This conformational change is propagated to the ATP site and causes a shift in the nucleotide loops which in turn causes a repositioning of Ca2+ to its canonical position as the cleft closes around ATP. Reversing the solvent exposure of Trp-356 is also involved in cleft closure. In addition, secondary calcium binding sites were identified. PMID:22366544

  20. Actin motility: formin a SCAry tail.

    PubMed

    Alberts, Art; Way, Michael

    2011-01-11

    A new biochemical analysis has revealed that the Rickettsia bacterial protein Sca2--recently shown to be essential for virulence and actin-dependent motility--assembles actin filaments using a mechanism that functionally resembles the processive elongation tactics used by formins. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Bacterial metabolite S-equol modulates glucagon-like peptide-1 secretion from enteroendocrine L cell line GLUTag cells via actin polymerization.

    PubMed

    Harada, Kazuki; Sada, Shoko; Sakaguchi, Hidekazu; Takizawa, Mai; Ishida, Rika; Tsuboi, Takashi

    2018-07-02

    S-equol is one of gut bacterial metabolites produced from soybean isoflavone daizein. While S-equol is known to promote glucose-induced insulin secretion from pancreatic β cells, whether S-equol affects glucagon-like peptide-1 (GLP-1) secretion from enteroendoceine L cells remains unclear. Here we assessed the effect of S-equol on GLP-1 secretion from mouse enteroendocrine L cell line GLUTag cells. GLUTag cells expressed GPR30 and estrogen receptors, which are putative S-equol receptors. Application of S-equol induced an increase in intracellular Ca 2+ levels via GPR30. However, S-equol did not enhance GLP-1 exocytosis, and long-term treatment of S-equol suppressed GLP-1 secretion. Moreover, immunocytochemistry revealed that S-equol increased the density of cortical actin filaments via G 12/13 signaling under GPR30. These data suggest that S-equol prevents GLP-1 secretion as a result of competing regulation between Ca 2+ mobilization and actin reorganization. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Improved kinetic neoclassical transport calculation for a low-collisionality QH-mode pedestal

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Battaglia, D. J.; Burrell, K. H.; Chang, C. S.

    The role of neoclassical, anomalous and neutral transport to the overall H-mode pedestal and scrape-off layer (SOL) structure in an ELM-free QH-mode discharge on DIII-D is explored using XGC0, a 5D full-f multi-species particle-in-cell drift-kinetic solver with self-consistent neutral recycling and sheath potentials. The work in this paper builds on previous work aimed at achieving quantitative agreement between the flux-driven simulation and the experimental electron density, impurity density and orthogonal measurements of impurity temperature and flow profiles. Improved quantitative agreement is achieved by performing the calculations with a more realistic electron mass, larger neutral density and including finite-Larmor-radius corrections self-consistentlymore » in the drift-kinetic motion of the particles. Consequently, the simulations provide stronger evidence that the radial electric field (E r) in the pedestal is primarily established by the required balance between the loss of high-energy tail main ions against a pinch of colder main ions and impurities. The kinetic loss of a small population of ions carrying a large proportion of energy and momentum leads to a separation of the particle and energy transport rates and introduces a source of intrinsic edge torque. Ion orbit loss and finite orbit width effects drive the energy distributions away from Maxwellian, and describe the anisotropy, poloidal asymmetry and local minimum near the separatrix observed in the T i profile.« less

  3. Improved kinetic neoclassical transport calculation for a low-collisionality QH-mode pedestal

    DOE PAGES

    Battaglia, D. J.; Burrell, K. H.; Chang, C. S.; ...

    2016-07-15

    The role of neoclassical, anomalous and neutral transport to the overall H-mode pedestal and scrape-off layer (SOL) structure in an ELM-free QH-mode discharge on DIII-D is explored using XGC0, a 5D full-f multi-species particle-in-cell drift-kinetic solver with self-consistent neutral recycling and sheath potentials. The work in this paper builds on previous work aimed at achieving quantitative agreement between the flux-driven simulation and the experimental electron density, impurity density and orthogonal measurements of impurity temperature and flow profiles. Improved quantitative agreement is achieved by performing the calculations with a more realistic electron mass, larger neutral density and including finite-Larmor-radius corrections self-consistentlymore » in the drift-kinetic motion of the particles. Consequently, the simulations provide stronger evidence that the radial electric field (E r) in the pedestal is primarily established by the required balance between the loss of high-energy tail main ions against a pinch of colder main ions and impurities. The kinetic loss of a small population of ions carrying a large proportion of energy and momentum leads to a separation of the particle and energy transport rates and introduces a source of intrinsic edge torque. Ion orbit loss and finite orbit width effects drive the energy distributions away from Maxwellian, and describe the anisotropy, poloidal asymmetry and local minimum near the separatrix observed in the T i profile.« less

  4. Mechanism of Cdc42-induced Actin Polymerization in Neutrophil Extracts

    PubMed Central

    Zigmond, Sally H.; Joyce, Michael; Yang, Changsong; Brown, Kevin; Huang, Minzhou; Pring, Martin

    1998-01-01

    Cdc42, activated with GTPγS, induces actin polymerization in supernatants of lysed neutrophils. This polymerization, like that induced by agonists, requires elongation at filament barbed ends. To determine if creation of free barbed ends was sufficient to induce actin polymerization, free barbed ends in the form of spectrin-actin seeds or sheared F-actin filaments were added to cell supernatants. Neither induced polymerization. Furthermore, the presence of spectrin-actin seeds did not increase the rate of Cdc42-induced polymerization, suggesting that the presence of Cdc42 did not facilitate polymerization from spectrin-actin seeds such as might have been the case if Cdc42 inhibited capping or released G-actin from a sequestered pool. Electron microscopy revealed that Cdc42-induced filaments elongated rapidly, achieving a mean length greater than 1 μm in 15 s. The mean length of filaments formed from spectrin-actin seeds was <0.4 μm. Had spectrin-actin seeds elongated at comparable rates before they were capped, they would have induced longer filaments. There was little change in mean length of Cdc42-induced filaments between 15 s and 5 min, suggesting that the increase in F-actin over this time was due to an increase in filament number. These data suggest that Cdc42 induction of actin polymerization requires both creation of free barbed ends and facilitated elongation at these ends. PMID:9722612

  5. Actin-based propulsion of a microswimmer.

    PubMed

    Leshansky, A M

    2006-07-01

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

  6. Traveling waves in actin dynamics and cell motility

    PubMed Central

    Allard, Jun; Mogilner, Alex

    2012-01-01

    Much of current understanding of cell motility arose from studying steady treadmilling of actin arrays. Recently, there have been a growing number of observations of a more complex, non-steady, actin behavior, including self-organized waves. It is becoming clear that these waves result from activation and inhibition feedbacks in actin dynamics acting on different scales, but the exact molecular nature of these feedbacks and respective roles of biomechanics and biochemistry are still unclear. Here, we review recent advances achieved in experimental and theoretical studies of actin waves and discuss mechanisms and physiological significance of wavy protrusions. PMID:22985541

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  8. Lateral Membrane Diffusion Modulated by a Minimal Actin Cortex

    PubMed Central

    Heinemann, Fabian; Vogel, Sven K.; Schwille, Petra

    2013-01-01

    Diffusion of lipids and proteins within the cell membrane is essential for numerous membrane-dependent processes including signaling and molecular interactions. It is assumed that the membrane-associated cytoskeleton modulates lateral diffusion. Here, we use a minimal actin cortex to directly study proposed effects of an actin meshwork on the diffusion in a well-defined system. The lateral diffusion of a lipid and a protein probe at varying densities of membrane-bound actin was characterized by fluorescence correlation spectroscopy (FCS). A clear correlation of actin density and reduction in mobility was observed for both the lipid and the protein probe. At high actin densities, the effect on the protein probe was ∼3.5-fold stronger compared to the lipid. Moreover, addition of myosin filaments, which contract the actin mesh, allowed switching between fast and slow diffusion in the minimal system. Spot variation FCS was in accordance with a model of fast microscopic diffusion and slower macroscopic diffusion. Complementing Monte Carlo simulations support the analysis of the experimental FCS data. Our results suggest a stronger interaction of the actin mesh with the larger protein probe compared to the lipid. This might point toward a mechanism where cortical actin controls membrane diffusion in a strong size-dependent manner. PMID:23561523

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

    PubMed Central

    1991-01-01

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

  10. Ion-dependent Polymerization Differences between Mammalian β- and γ-Nonmuscle Actin Isoforms*

    PubMed Central

    Bergeron, Sarah E.; Zhu, Mei; Thiem, Suzanne M.; Friderici, Karen H.; Rubenstein, Peter A.

    2010-01-01

    β- and γ-nonmuscle actins differ by 4 amino acids at or near the N terminus and distant from polymerization interfaces. β-Actin contains an Asp1-Asp2-Asp3 and Val10 whereas γ-actin has a Glu1-Glu2-Glu3 and Ile10. Despite these small changes, conserved across mammals, fish, and birds, their differential localization in the same cell suggests they may play different roles reflecting differences in their biochemical properties. To test this hypothesis, we established a baculovirus-driven expression system for producing these actins in isoform-pure populations although contaminated with 20–25% insect actin. Surprisingly, Ca-γ-actin exhibits a slower monomeric nucleotide exchange rate, a much longer nucleation phase, and a somewhat slower elongation rate than β-actin. In the Mg-form, this difference between the two is much smaller. Ca-γ-actin depolymerizes half as fast as does β-actin. Mixing experiments with Ca-actins reveal the two will readily co-polymerize. In the Ca-form, phosphate release from polymerizing β-actin occurs much more rapidly and extensively than polymerization, whereas phosphate release lags behind polymerization with γ-actin. Phosphate release during treadmilling is twice as fast with β- as with γ-actin. With Mg-actin in the initial stages, phosphate release for both actins correlates much more closely with polymerization. Calcium bound in the high affinity binding site of γ-actin may cause a selective energy barrier relative to β-actin that retards the equilibration between G- and F-monomer conformations resulting in a slower polymerizing actin with greater filament stability. This difference may be particularly important in sites such as the γ-actin-rich cochlear hair cell stereocilium where local mm calcium concentrations may exist. PMID:20308063

  11. Density and magnetic fluctuations in type III-ELM pedestal evolution in JET: experimental and numerical characterization

    NASA Astrophysics Data System (ADS)

    De Masi, G.; Predebon, I.; Spagnolo, S.; Meneses, L.; Delabie, E.; Lupelli, I.; Hillesheim, J. C.; Maggi, C.; Contributors, JET

    2018-04-01

    Density and magnetic fluctuation measurements in low-β type-III ELM discharges are obtained in the Joint European Torus (JET). They are observed during the inter-ELM pedestal evolution, after the LH transition phase, at about 60-70 kHz. Density fluctuations are measured with a correlation reflectometer system installed on the low-field side and they are localized at the pedestal top. Magnetic fluctuations with a spatial scale k_yρ_i˜ 0.1 are measured through a high resolution coil array. The main features and the relations with local plasma parameters are presented. The nature of these fluctuations is discussed along with linear gyrokinetic simulations. Ion temperature gradient (ITG) modes are the dominant instabilities in the frequency range of interest. In terms of radial localization, typical oscillation frequency and qualitative relation with the possible linear drive, ITG modes are consistent with the experimental density fluctuations measurements. Micro-tearing modes (MTMs), found unstable with a lower growth rate, appear a possible explanation for magnetic fluctuations in terms of typical wavenumbers and direction of propagation.

  12. Mechanics of composite actin networks: in vitro and cellular perspectives

    NASA Astrophysics Data System (ADS)

    Upadhyaya, Arpita

    2014-03-01

    Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

  13. Microheterogeneity of actin gels formed under controlled linear shear.

    PubMed

    Cortese, J D; Frieden, C

    1988-10-01

    The diffusion coefficients and fluorescence polarization properties of actin subjected to a known shear have been determined both during and after polymerization, using a modification of a cone-plate Wells-Brookfield rheometer that allows monitoring of samples with an epifluorescence microscope. Fluorescence polarization and fluorescence photobleaching recovery experiments using rhodamine-labeled actin as a tracer showed that under conditions of low shear (shear rates of 0.05 s-1), a spatial heterogeneity of polymerized actin was observed with respect to fluorescence intensity and the diffusion coefficients with actin mobility becoming quite variable in different regions of the sample. In addition, complex changes in fluorescence polarization were noted after stopping the shear. Actin filaments of controlled length were obtained using plasma gelsolin (gelsolin/actin molar ratios of 1:50 to 1:300). At ratios of 1:50, neither spatial heterogeneity nor changes in polarization were observed on subjecting the polymerized actin to shear. At ratios of approximately 1:100, a decrease on the intensity of fluorescence polarization occurs on stopping the shear. Longer filaments exhibit spatial micro-heterogeneity and complex changes in fluorescence polarization. In addition, at ratios of 1:100 or 1:300, the diffusion coefficient decreases as the total applied shear increased. This behavior is interpreted as bundling of filaments aligned under shear. We also find that the F-actin translational diffusion coefficients decrease as the total applied shear increases (shear rates between 0.05 and 12.66 s-1), as expected for a cumulative process. When chicken gizzard filamin was added to gelsolin-actin filaments (at filamin/actin molar ratios of 1:300 to 1:10), a similar decrease in the diffusion coefficients was observed for unsheared samples. Spatial microheterogeneity might be related to the effects of the shear field in the alignment of filaments, and the balance between a three

  14. Actin kinetics shapes cortical network structure and mechanics

    PubMed Central

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

    2016-01-01

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

  15. Actin kinetics shapes cortical network structure and mechanics.

    PubMed

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

    2016-04-01

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

  16. Structural dynamics of F-actin: I. Changes in the C terminus.

    PubMed

    Orlova, A; Egelman, E H

    1995-02-03

    The biochemical properties of G-actin, and the kinetics of polymerization of G-actin into F-actin, are dependent upon whether Mg2+ or Ca2+ is bound at the high-affinity metal-binding site in actin. Three-dimensional reconstructions from electron micrographs show that a bridge of density, that we interpret as arising from a major shift of the C terminus, exists between the two strands of the filament in Ca(2+)-actin that is absent in Mg(2+)-actin. This bridge is also absent in models of F-actin built from an atomic structure of G-Ca(2+)-actin. The cleavage of the DNase I-binding loop in actin between residues 42 and 43, with the non-covalent association of the 42 cleaved residues with the remainder of the actin, induces an even larger bridge of density between the two strands. When the bridge is absent, the two C-terminal residues in F-actin are easily cleaved by trypsin, while these residues become increasingly resistant to tryptic cleavage as the bridge becomes more prominent. Conversely, cleavage of the two C-terminal residues leads to a conformational change in the DNase I-binding loop. Since both the DNase I-binding loop and the metal-binding site are quite distant from the C terminus, large allosteric effects must exist in F-actin. The conformational change in F-actin that results from the creation of this bridge may be induced by myosin binding, since this movement generates changes in actin's diffraction that are very similar to the changes in the muscle X-ray pattern during activation that are associated with the binding of myosin to the thin filament.

  17. Actin dynamics, architecture, and mechanics in cell motility.

    PubMed

    Blanchoin, Laurent; Boujemaa-Paterski, Rajaa; Sykes, Cécile; Plastino, Julie

    2014-01-01

    Tight coupling between biochemical and mechanical properties of the actin cytoskeleton drives a large range of cellular processes including polarity establishment, morphogenesis, and motility. This is possible because actin filaments are semi-flexible polymers that, in conjunction with the molecular motor myosin, can act as biological active springs or "dashpots" (in laymen's terms, shock absorbers or fluidizers) able to exert or resist against force in a cellular environment. To modulate their mechanical properties, actin filaments can organize into a variety of architectures generating a diversity of cellular organizations including branched or crosslinked networks in the lamellipodium, parallel bundles in filopodia, and antiparallel structures in contractile fibers. In this review we describe the feedback loop between biochemical and mechanical properties of actin organization at the molecular level in vitro, then we integrate this knowledge into our current understanding of cellular actin organization and its physiological roles.

  18. Maleimidobenzoyl-G-actin: Structural properties and interaction with skeletal myosin subfragment-1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bettache, N.; Bertrand, R.; Kassab, R.

    1990-09-25

    The authors have investigated various structural and interaction properties of maleimidobenzoyl-G-actin (MBS-actin), a new, internally cross-linked G-actin derivative that does not exhibit, at moderate protein concentration, the salt-and myosin subfragment 1 (S-1)--induced polymerizations of G-actin and reacts reversibly and covalently in solution with S-1 at or near the F-actin binding region of the heavy chain. The far-ultraviolet CD spectrum and {alpha}-helix content of the MBS-actin were identical with those displayed by native G-actin. {sup 45}Ca{sup 2+} measurements showed the same content of tightly bound Ca{sup 2+} in MBS-actin as in G-actin and the EDTA treatment of the modified protein promotedmore » the same red shift of the intrinsic fluorescence spectrum as observed with native G-actin. Incubation of concentrated MBS-actin solutions with 100 mM KCl+5 mM MgCl{sub 2} led to the polymerization of the actin derivative when the critical monomer concentration reached 1.6mg/mL, at 25{degree}C, pH 8.0. The MBS-F-actin formed activated the Mg{sup 2+}-ATPase of S-1 to the same extent as native F-actin. The MBS-G-actin exhibited a DNase I inhibitor activity very close to that found with native G-actin and was to be at all affected by its specific covalent conjugation to S-1. This finding led them to isolate, for the first time, by gel filtration, a ternary complex comprising DNase I tightly bound to MBS-actin cross-linked to the S-1 heavy chain, demonstrating that S-1 and DNase I bind at distinct sites on G-actin. Collectively, the data illustrate further the nativeness of the MBS-G-actin and its potential use in solution studies of the actin-myosin head interactions.« less

  19. Load Adaptation of Lamellipodial Actin Networks.

    PubMed

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

    2017-09-21

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

  20. Effect of phosphorylation of myelin basic protein by MAPK on its interactions with actin and actin binding to a lipid membrane in vitro.

    PubMed

    Boggs, Joan M; Rangaraj, Godha; Gao, Wen; Heng, Yew-Meng

    2006-01-17

    Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is most likely responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also polymerize actin, bundle F-actin filaments, and bind actin filaments to lipid bilayers through electrostatic interactions. MBP consists of a number of posttranslationally modified isomers of varying charge, some resulting from phosphorylation at several sites by different kinases, including mitogen-activated protein kinase (MAPK). Phosphorylation of MBP in oligodendrocytes occurs in response to various extracellular stimuli. Phosphorylation/dephosphorylation of MBP also occurs in the myelin sheath in response to electrical activity in the brain. Here we investigate the effect of phosphorylation of MBP on its interaction with actin in vitro by phosphorylating the most highly charged unmodified isomer, C1, at two sites with MAPK. Phosphorylation decreased the ability of MBP to polymerize actin and to bundle actin filaments but had no effect on the dissociation constant of the MBP-actin complex or on the ability of Ca2+-calmodulin to dissociate the complex. The most significant effect of phosphorylation on the MBP-actin complex was a dramatic reduction in its ability to bind to negatively charged lipid bilayers. The effect was much greater than that reported earlier for another charge isomer of MBP, C8, in which six arginines were deiminated to citrulline, resulting in a reduction of net positive charge of 6. These results indicate that although average electrostatic forces are the primary determinant of the interaction of MBP with actin, phosphorylation may have an additional effect due to a site-specific electrostatic effect or to a conformational change. Thus, phosphorylation of MBP, which occurs in response to various extracellular signals in both myelin and oligodendrocytes, attenuates the ability of MBP to polymerize and bundle actin and to

  1. Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

    PubMed Central

    Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

    2012-01-01

    Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

  2. Binding of actin to lens alpha crystallins

    NASA Technical Reports Server (NTRS)

    Gopalakrishnan, S.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Actin has been coupled to a cyanogen bromide-activated Sepharose 4B column, then tested for binding to alpha, beta, and gamma crystallin preparations from the bovine lens. Alpha, but not beta or gamma, crystallins bound to the actin affinity column in a time dependent and saturable manner. Subfractionation of the alpha crystallin preparation into the alpha-A and alpha-B species, followed by incubation with the affinity column, demonstrated that both species bound approximately the same. Together, these studies demonstrate a specific and saturable binding of lens alpha-A and alpha-B with actin.

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

    PubMed

    Mohammadinejad, Sarah; Ghamkhari, Behnoush; Abdolmaleki, Sarah

    2016-08-21

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

  4. Organization and function of the actin cytoskeleton in developing root cells.

    PubMed

    Blancaflor, Elison B; Wang, Yuh-Shuh; Motes, Christy M

    2006-01-01

    The actin cytoskeleton is a highly dynamic structure, which mediates various cellular functions in large part through accessory proteins that tilt the balance between monomeric G-actin and filamentous actin (F-actin) or by facilitating interactions between actin and the plasma membrane, microtubules, and other organelles. Roots have become an attractive model to study actin in plant development because of their simple anatomy and accessibility of some root cell types such as root hairs for microscopic analyses. Roots also exhibit a remarkable developmental plasticity and possess a delicate sensory system that is easily manipulated, so that one can design experiments addressing a range of important biological questions. Many facets of root development can be regulated by the diverse actin network found in the various root developmental regions. Various molecules impinge on this actin scaffold to define how a particular root cell type grows or responds to a specific environmental signal. Although advances in genomics are leading the way toward elucidating actin function in roots, more significant strides will be realized when such tools are combined with improved methodologies for accurately depicting how actin is organized in plant cells.

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

    PubMed

    Yokota, Etsuo; Vidali, Luis; Tominaga, Motoki; Tahara, Hiroshi; Orii, Hidefumi; Morizane, Yosuke; Hepler, Peter K; Shimmen, Teruo

    2003-10-01

    In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin

  6. Actinic prurigo of the lip: Two case reports

    PubMed Central

    Miranda, Ana MO; Ferrari, Thiago M; Werneck, Juliana T; Junior, Arley Silva; Cunha, Karin S; Dias, Eliane P

    2014-01-01

    Actinic prurigo is a photodermatosis that can affect the skin, conjunctiva and lips. It is caused by an abnormal reaction to sunlight and is more common in high-altitude living people, mainly in indigenous descendants. The diagnosis of actinic prurigo can be challenging, mainly when lip lesions are the only manifestation, which is not a common clinical presentation. The aim of this article is to report two cases of actinic prurigo showing only lip lesions. The patients were Afro-American and were unaware of possible Indian ancestry. Clinical exam, photographs, videoroscopy examination and biopsy were performed, and the diagnosis of actinic prurigo was established. Topical corticosteroid and lip balm with ultraviolet protection were prescribed with excellent results. The relevance of this report is to show that although some patients may not demonstrate the classical clinical presentation of actinic prurigo, the associated clinical and histological exams are determinants for the correct diagnosis and successful treatment of this disease. PMID:25133153

  7. Triggering signaling pathways using F-actin self-organization.

    PubMed

    Colin, A; Bonnemay, L; Gayrard, C; Gautier, J; Gueroui, Z

    2016-10-04

    The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity.

  8. Triggering signaling pathways using F-actin self-organization

    PubMed Central

    Colin, A.; Bonnemay, L.; Gayrard, C.; Gautier, J.; Gueroui, Z.

    2016-01-01

    The spatiotemporal organization of proteins within cells is essential for cell fate behavior. Although it is known that the cytoskeleton is vital for numerous cellular functions, it remains unclear how cytoskeletal activity can shape and control signaling pathways in space and time throughout the cell cytoplasm. Here we show that F-actin self-organization can trigger signaling pathways by engineering two novel properties of the microfilament self-organization: (1) the confinement of signaling proteins and (2) their scaffolding along actin polymers. Using in vitro reconstitutions of cellular functions, we found that both the confinement of nanoparticle-based signaling platforms powered by F-actin contractility and the scaffolding of engineered signaling proteins along actin microfilaments can drive a signaling switch. Using Ran-dependent microtubule nucleation, we found that F-actin dynamics promotes the robust assembly of microtubules. Our in vitro assay is a first step towards the development of novel bottom-up strategies to decipher the interplay between cytoskeleton spatial organization and signaling pathway activity. PMID:27698406

  9. Bacterial subversion of host actin dynamics at the plasma membrane.

    PubMed

    Carabeo, Rey

    2011-10-01

    Invasion of non-phagocytic cells by a number of bacterial pathogens involves the subversion of the actin cytoskeletal remodelling machinery to produce actin-rich cell surface projections designed to engulf the bacteria. The signalling that occurs to induce these actin-rich structures has considerable overlap among a diverse group of bacteria. The molecular organization within these structures act in concert to internalize the invading pathogen. This dynamic process could be subdivided into three acts - actin recruitment, engulfment, and finally, actin disassembly/internalization. This review will present the current state of knowledge of the molecular processes involved in each stage of bacterial invasion, and provide a perspective that highlights the temporal and spatial control of actin remodelling that occurs during bacterial invasion. © 2011 Blackwell Publishing Ltd.

  10. A nucleator arms race: cellular control of actin assembly.

    PubMed

    Campellone, Kenneth G; Welch, Matthew D

    2010-04-01

    For over a decade, the actin-related protein 2/3 (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 seen a surge in the discovery of mammalian proteins with roles in actin nucleation and dynamics. Newly recognized nucleation-promoting factors, such as WASP and SCAR homologue (WASH), WASP homologue associated with actin, membranes and microtubules (WHAMM), and junction-mediating regulatory protein (JMY), stimulate ARP2/3 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.

  11. Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation.

    PubMed

    Zenker, Jennifer; White, Melanie D; Gasnier, Maxime; Alvarez, Yanina D; Lim, Hui Yi Grace; Bissiere, Stephanie; Biro, Maté; Plachta, Nicolas

    2018-04-19

    Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation. Copyright © 2018 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2013-01-01

    Previous reports have indicated that parasite actins are short and inherently unstable, despite being required for motility. Here, we re-examine the polymerization properties of actin in Toxoplasma gondii (TgACTI), unexpectedly finding that it exhibits isodesmic polymerization in contrast to the conventional nucleation-elongation process of all previously studied actins from both eukaryotes and bacteria. TgACTI polymerization kinetics lacks both a lag phase and critical concentration, normally characteristic of actins. Unique among actins, the kinetics of assembly can be fit with a single set of rate constants for all subunit interactions, without need for separate nucleation and elongation rates. This isodesmic model accurately predicts the assembly, disassembly, and the size distribution of TgACTI filaments in vitro, providing a mechanistic explanation for actin dynamics in vivo. Our findings expand the repertoire of mechanisms by which actin polymerization is governed and offer clues about the evolution of self-assembling, stabilized protein polymers. PMID:23921463

  13. Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides.

    PubMed

    Saarikangas, Juha; Zhao, Hongxia; Lappalainen, Pekka

    2010-01-01

    The plasma membrane and the underlying cortical actin cytoskeleton undergo continuous dynamic interplay that is responsible for many essential aspects of cell physiology. Polymerization of actin filaments against cellular membranes provides the force for a number of cellular processes such as migration, morphogenesis, and endocytosis. Plasma membrane phosphoinositides (especially phosphatidylinositol bis- and trisphosphates) play a central role in regulating the organization and dynamics of the actin cytoskeleton by acting as platforms for protein recruitment, by triggering signaling cascades, and by directly regulating the activities of actin-binding proteins. Furthermore, a number of actin-associated proteins, such as BAR domain proteins, are capable of directly deforming phosphoinositide-rich membranes to induce plasma membrane protrusions or invaginations. Recent studies have also provided evidence that the actin cytoskeleton-plasma membrane interactions are misregulated in a number of pathological conditions such as cancer and during pathogen invasion. Here, we summarize the wealth of knowledge on how the cortical actin cytoskeleton is regulated by phosphoinositides during various cell biological processes. We also discuss the mechanisms by which interplay between actin dynamics and certain membrane deforming proteins regulate the morphology of the plasma membrane.

  14. Filamentous actin organization in the unfertilized sea urchin egg cortex.

    PubMed

    Henson, J H; Begg, D A

    1988-06-01

    We have investigated the organization of filamentous actin in the cortex of unfertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus. Rhodamine phalloidin and anti-actin immunofluorescent staining of isolated cortices reveal a punctate pattern of fluorescent sources. Comparison of this pattern with SEM images of microvillar morphology and distribution indicates that filamentous actin in the cortex is predominantly localized in the microvilli. Thin-section TEM and quick-freeze deep-etch ultrastructure of isolated cortices demonstrates that this microvillar-associated actin is in a novel organizational state composed of very short filaments arranged in a tight network and that these filament networks form mounds that extend beyond the plane of the plasma membrane. Actin filaments within the networks do not exhibit free ends and make end-on attachments with the membrane only within the region of the evaginating microvilli. Myosin S-1 dissociable crosslinks, 2-3 nm in diameter, are observed between network filaments and between network filaments and the membrane. A second population of long, individual actin filaments is observed in close lateral association with the plasma membrane and frequently complexes with the microvillar actin networks. The filamentous actin of the unfertilized egg cortex may participate in establishing the mechanical properties of the egg surface and may function in nucleating the assembly of cortical actin following fertilization.

  15. Management of actinic keratosis.

    PubMed

    2013-07-01

    Actinic keratoses are common, often multiple, epidermal lesions found mainly on the sun-exposed skin of fair-skinned middle-aged and older people.(1) Over time, lesions may remain unchanged or may proliferate, regress, reappear or develop into squamous cell carcinoma (SCC).(2) Detectable (spot) lesions are often associated with alteration of the surrounding skin (field) where subclinical lesions might be present.(2) Interventions may target individual or multiple lesions or a whole field.(2) Here, we update our previous review(3) on the prevention and treatment of actinic keratoses, focusing on the licensed treatments most commonly used in the UK and recommended in UK guidelines.

  16. Nonequilibrium stabilization of an RNA/protein droplet emulsion by nuclear actin

    NASA Astrophysics Data System (ADS)

    Brangwynne, Clifford

    2013-03-01

    Actin plays a structural role in the cytoplasm. However, actin takes on new functions and structures in the nucleus that are poorly understood. The nuclei of the large oocytes of the frog X. laevisspecifically accumulate actin to reach high concentrations; however, it remains unclear if this actin polymerizes into a network, and what, if any, structural role such an actin network might play. Here, we use microrheological and confocal imaging techniques to probe the local architecture and mechanics of the nucleus. Our data show that actin forms a weak network that spatially organizes the nucleus by kinetically stabilizing embedded liquid-like RNA/protein bodies which are important for cell growth. In actin-disrupted nuclei this RNA/protein droplet emulsion is destabilized leading to homotypic coalescence into single large droplets. Our data provide intriguing new insights into why large cell nuclei require an actin-based structural scaffold.

  17. The cell wall of Arabidopsis thaliana influences actin network dynamics.

    PubMed

    Tolmie, Frances; Poulet, Axel; McKenna, Joseph; Sassmann, Stefan; Graumann, Katja; Deeks, Michael; Runions, John

    2017-07-20

    In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. Intracellular Domain Fragment of CD44 Alters CD44 Function in Chondrocytes*

    PubMed Central

    Mellor, Liliana; Knudson, Cheryl B.; Hida, Daisuke; Askew, Emily B.; Knudson, Warren

    2013-01-01

    The hyaluronan receptor CD44 undergoes sequential proteolytic cleavage at the cell surface. The initial cleavage of the CD44 extracellular domain is followed by a second intramembranous cleavage of the residual CD44 fragment, liberating the C-terminal cytoplasmic tail of CD44. In this study conditions that promote CD44 cleavage resulted in a diminished capacity to assemble and retain pericellular matrices even though sufficient non-degraded full-length CD44 remained. Using stable and transient overexpression of the cytoplasmic domain of CD44, we determined that the intracellular domain interfered with anchoring of the full-length CD44 to the cytoskeleton and disrupted the ability of the cells to bind hyaluronan and assemble a pericellular matrix. Co-immunoprecipitation assays were used to determine whether the mechanism of this interference was due to competition with actin adaptor proteins. CD44 of control chondrocytes was found to interact and co-immunoprecipitate with both the 65- and 130-kDa isoforms of ankyrin-3. Moreover, this interaction with ankyrin-3 proteins was diminished in cells overexpressing the CD44 intracellular domain. Mutating the putative ankyrin binding site of the transiently transfected CD44 intracellular domain diminished the inhibitory effects of this protein on matrix retention. Although CD44 in other cells types has been shown to interact with members of the ezrin/radixin/moesin (ERM) family of adaptor proteins, only modest interactions between CD44 and moesin could be demonstrated in chondrocytes. The data suggest that release of the CD44 intracellular domain into the cytoplasm of cells such as chondrocytes exerts a competitive or dominant-negative effect on the function of full-length CD44. PMID:23884413

  19. Surfing pathogens and the lessons learned for actin polymerization.

    PubMed

    Frischknecht, F; Way, M

    2001-01-01

    A number of unrelated bacterial species as well as vaccinia virus (ab)use the process of actin polymerization to facilitate and enhance their infection cycle. Studies into the mechanism by which these pathogens hijack and control the actin cytoskeleton have provided many interesting insights into the regulation of actin polymerization in migrating cells. This review focuses on what we have learnt from the actin-based motilities of Listeria, Shigella and vaccinia and discusses what we would still like to learn from our nasty friends, including enteropathogenic Escherichia coli and Rickettsia

  20. 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.

  1. Actin Engine in Immunological Synapse

    PubMed Central

    Piragyte, Indre

    2012-01-01

    T cell activation and function require physical contact with antigen presenting cells at a specialized junctional structure known as the immunological synapse. Once formed, the immunological synapse leads to sustained T cell receptor-mediated signalling and stabilized adhesion. High resolution microscopy indeed had a great impact in understanding the function and dynamic structure of immunological synapse. Trends of recent research are now moving towards understanding the mechanical part of immune system, expanding our knowledge in mechanosensitivity, force generation, and biophysics of cell-cell interaction. Actin cytoskeleton plays inevitable role in adaptive immune system, allowing it to bear dynamic and precise characteristics at the same time. The regulation of mechanical engine seems very complicated and overlapping, but it enables cells to be very sensitive to external signals such as surface rigidity. In this review, we focus on actin regulators and how immune cells regulate dynamic actin rearrangement process to drive the formation of immunological synapse. PMID:22916042

  2. Actin in Mung Bean Mitochondria and Implications for Its Function[W][OA

    PubMed Central

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

    2011-01-01

    Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin–green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography–tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed. PMID:21984697

  3. A peek into tropomyosin binding and unfolding on the actin filament.

    PubMed

    Singh, Abhishek; Hitchcock-Degregori, Sarah E

    2009-07-24

    Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism). This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding

  4. Diversification of caldesmon-linked actin cytoskeleton in cell motility

    PubMed Central

    Mayanagi, Taira

    2011-01-01

    The actin cytoskeleton plays a key role in regulating cell motility. Caldesmon (CaD) is an actin-linked regulatory protein found in smooth muscle and non-muscle cells that is conserved among a variety of vertebrates. It binds and stabilizes actin filaments, as well as regulating actin-myosin interaction in a calcium (Ca2+)/calmodulin (CaM)- and/or phosphorylation-dependent manner. CaD function is regulated qualitatively by Ca2+/CaM and by its phosphorylation state and quantitatively at the mRNA level, by three different transcriptional regulation of the CALD1 gene. CaD has numerous functions in cell motility, such as migration, invasion and proliferation, exerted via the reorganization of the actin cytoskeleton. Here we will outline recent findings regarding CaD's structural features and functions. PMID:21350330

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

    PubMed Central

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

    2000-01-01

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

  6. Single-Molecule Studies of Actin Assembly and Disassembly Factors

    PubMed Central

    Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.

    2014-01-01

    The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103

  7. 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

  8. Automated Detection of Actinic Keratoses in Clinical Photographs

    PubMed Central

    Hames, Samuel C.; Sinnya, Sudipta; Tan, Jean-Marie; Morze, Conrad; Sahebian, Azadeh; Soyer, H. Peter; Prow, Tarl W.

    2015-01-01

    Background Clinical diagnosis of actinic keratosis is known to have intra- and inter-observer variability, and there is currently no non-invasive and objective measure to diagnose these lesions. Objective The aim of this pilot study was to determine if automatically detecting and circumscribing actinic keratoses in clinical photographs is feasible. Methods Photographs of the face and dorsal forearms were acquired in 20 volunteers from two groups: the first with at least on actinic keratosis present on the face and each arm, the second with no actinic keratoses. The photographs were automatically analysed using colour space transforms and morphological features to detect erythema. The automated output was compared with a senior consultant dermatologist’s assessment of the photographs, including the intra-observer variability. Performance was assessed by the correlation between total lesions detected by automated method and dermatologist, and whether the individual lesions detected were in the same location as the dermatologist identified lesions. Additionally, the ability to limit false positives was assessed by automatic assessment of the photographs from the no actinic keratosis group in comparison to the high actinic keratosis group. Results The correlation between the automatic and dermatologist counts was 0.62 on the face and 0.51 on the arms, compared to the dermatologist’s intra-observer variation of 0.83 and 0.93 for the same. Sensitivity of automatic detection was 39.5% on the face, 53.1% on the arms. Positive predictive values were 13.9% on the face and 39.8% on the arms. Significantly more lesions (p<0.0001) were detected in the high actinic keratosis group compared to the no actinic keratosis group. Conclusions The proposed method was inferior to assessment by the dermatologist in terms of sensitivity and positive predictive value. However, this pilot study used only a single simple feature and was still able to achieve sensitivity of detection of 53

  9. Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting.

    PubMed

    Staquicini, Daniela I; Rangel, Roberto; Guzman-Rojas, Liliana; Staquicini, Fernanda I; Dobroff, Andrey S; Tarleton, Christy A; Ozbun, Michelle A; Kolonin, Mikhail G; Gelovani, Juri G; Marchiò, Serena; Sidman, Richard L; Hajjar, Katherine A; Arap, Wadih; Pasqualini, Renata

    2017-06-26

    Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression. To identify functional protein networks and potential inhibitors, we screened an internalizing phage (iPhage) display library in tumor cells, and selected LGRFYAASG as a cytosol-targeting peptide. By affinity purification and mass spectrometry, intracellular annexin A2 was identified as the corresponding binding protein. Consistently, annexin A2 and a cell-internalizing, penetratin-fused version of the selected peptide (LGRFYAASG-pen) co-localized and specifically accumulated in the cytoplasm at the cell edges and cell-cell contacts. Functionally, tumor cells incubated with LGRFYAASG-pen showed disruption of filamentous actin, focal adhesions and caveolae-mediated membrane trafficking, resulting in impaired cell adhesion and migration in vitro. These effects were paralleled by a decrease in the phosphorylation of both focal adhesion kinase (Fak) and protein kinase B (Akt). Likewise, tumor cells pretreated with LGRFYAASG-pen exhibited an impaired capacity to colonize the lungs in vivo in several mouse models. Together, our findings demonstrate an unrecognized functional link between intracellular annexin A2 and tumor cell adhesion, migration and in vivo grafting. Moreover, this work uncovers a new peptide motif that binds to and inhibits intracellular annexin A2 as a candidate therapeutic lead for potential translation into clinical applications.

  10. Altered Actin Centripetal Retrograde Flow in Physically Restricted Immunological Synapses

    PubMed Central

    Yu, Cheng-han; Wu, Hung-Jen; Kaizuka, Yoshihisa; Vale, Ronald D.; Groves, Jay T.

    2010-01-01

    Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3ε on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network. PMID:20686692

  11. Actin Filament Polymerization Regulates Gliding Motility by Apicomplexan ParasitesV⃞

    PubMed Central

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

    2003-01-01

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

  12. The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains.

    PubMed

    Beer, Lara-Antonia; Tatge, Helma; Schneider, Carmen; Ruschig, Maximilian; Hust, Michael; Barton, Jessica; Thiemann, Stefan; Fühner, Viola; Russo, Giulio; Gerhard, Ralf

    2018-06-01

    Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum , the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile . All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N -terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains.

  13. Multiple forms of Spire-actin complexes and their functional consequences.

    PubMed

    Chen, Christine K; Sawaya, Michael R; Phillips, Martin L; Reisler, Emil; Quinlan, Margot E

    2012-03-23

    Spire is a WH2 domain-containing actin nucleator essential for establishing an actin mesh during oogenesis. In vitro, in addition to nucleating filaments, Spire can sever them and sequester actin monomers. Understanding how Spire is capable of these disparate functions and which are physiologically relevant is an important goal. To study severing, we examined the effect of Drosophila Spire on preformed filaments in bulk and single filament assays. We observed rapid depolymerization of actin filaments by Spire, which we conclude is largely due to its sequestration activity and enhanced by its weak severing activity. We also studied the solution and crystal structures of Spire-actin complexes. We find structural and functional differences between constructs containing four WH2 domains (Spir-ABCD) and two WH2 domains (Spir-CD) that may provide insight into the mechanisms of nucleation and sequestration. Intriguingly, we observed lateral interactions between actin monomers associated with Spir-ABCD, suggesting that the structures built by these four tandem WH2 domains are more complex than originally imagined. Finally, we propose that Spire-actin mixtures contain both nuclei and sequestration structures.

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

    PubMed

    Wang, Ruizhe; Carlsson, Anders E

    2015-11-25

    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.

  15. k-space image correlation to probe the intracellular dynamics of gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Bouzin, M.; Sironi, L.; Chirico, G.; D'Alfonso, L.; Inverso, D.; Pallavicini, P.; Collini, M.

    2016-04-01

    The collective action of dynein, kinesin and myosin molecular motors is responsible for the intracellular active transport of cargoes, vesicles and organelles along the semi-flexible oriented filaments of the cytoskeleton. The overall mobility of the cargoes upon binding and unbinding to motor proteins can be modeled as an intermittency between Brownian diffusion in the cell cytoplasm and active ballistic excursions along actin filaments or microtubules. Such an intermittent intracellular active transport, exhibited by star-shaped gold nanoparticles (GNSs, Gold Nanostars) upon internalization in HeLa cancer cells, is investigated here by combining live-cell time-lapse confocal reflectance microscopy and the spatio-temporal correlation, in the reciprocal Fourier space, of the acquired image sequences. At first, the analytical theoretical framework for the investigation of a two-state intermittent dynamics is presented for Fourier-space Image Correlation Spectroscopy (kICS). Then simulated kICS correlation functions are employed to evaluate the influence of, and sensitivity to, all the kinetic and dynamic parameters the model involves (the transition rates between the diffusive and the active transport states, the diffusion coefficient and drift velocity of the imaged particles). The optimal procedure for the analysis of the experimental data is outlined and finally exploited to derive whole-cell maps for the parameters underlying the GNSs super-diffusive dynamics. Applied here to the GNSs subcellular trafficking, the proposed kICS analysis can be adopted for the characterization of the intracellular (super-) diffusive dynamics of any fluorescent or scattering biological macromolecule.

  16. STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release.

    PubMed

    Darbellay, Basile; Arnaudeau, Serge; Bader, Charles R; Konig, Stephane; Bernheim, Laurent

    2011-07-25

    Cytosolic Ca(2+) signals encoded by repetitive Ca(2+) releases rely on two processes to refill Ca(2+) stores: Ca(2+) reuptake from the cytosol and activation of a Ca(2+) influx via store-operated Ca(2+) entry (SOCE). However, SOCE activation is a slow process. It is delayed by >30 s after store depletion because stromal interaction molecule 1 (STIM1), the Ca(2+) sensor of the intracellular stores, must form clusters and migrate to the membrane before being able to open Orai1, the plasma membrane Ca(2+) channel. In this paper, we identify a new protein, STIM1L, that colocalizes with Orai1 Ca(2+) channels and interacts with actin to form permanent clusters. This property allowed the immediate activation of SOCE, a characteristic required for generating repetitive Ca(2+) signals with frequencies within seconds such as those frequently observed in excitable cells. STIM1L was expressed in several mammalian tissues, suggesting that many cell types rely on this Ca(2+) sensor for their Ca(2+) homeostasis and intracellular signaling.

  17. STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release

    PubMed Central

    Arnaudeau, Serge; Bader, Charles R.; Bernheim, Laurent

    2011-01-01

    Cytosolic Ca2+ signals encoded by repetitive Ca2+ releases rely on two processes to refill Ca2+ stores: Ca2+ reuptake from the cytosol and activation of a Ca2+ influx via store-operated Ca2+ entry (SOCE). However, SOCE activation is a slow process. It is delayed by >30 s after store depletion because stromal interaction molecule 1 (STIM1), the Ca2+ sensor of the intracellular stores, must form clusters and migrate to the membrane before being able to open Orai1, the plasma membrane Ca2+ channel. In this paper, we identify a new protein, STIM1L, that colocalizes with Orai1 Ca2+ channels and interacts with actin to form permanent clusters. This property allowed the immediate activation of SOCE, a characteristic required for generating repetitive Ca2+ signals with frequencies within seconds such as those frequently observed in excitable cells. STIM1L was expressed in several mammalian tissues, suggesting that many cell types rely on this Ca2+ sensor for their Ca2+ homeostasis and intracellular signaling. PMID:21788372

  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. Toward the Structure of Dynamic Membrane-Anchored Actin Networks

    PubMed Central

    Weber, Igor

    2007-01-01

    In the cortex of a motile cell, membrane-anchored actin filaments assemble into structures of varying shape and function. Filopodia are distinguished by a core of bundled actin filaments within finger-like extensions of the membrane. In a recent paper by Medalia et al1 cryo-electron tomography has been used to reconstruct, from filopodia of Dictyostelium cells, the 3-dimensional organization of actin filaments in connection with the plasma membrane. A special arrangement of short filaments converging toward the filopod's tip has been called a “terminal cone”. In this region force is applied for protrusion of the membrane. Here we discuss actin organization in the filopodia of Dictyostelium in the light of current views on forces that are generated by polymerizing actin filaments, and on the resistance of membranes against deformation that counteracts these forces. PMID:19262130

  20. Monoubiquitination Inhibits the Actin Bundling Activity of Fascin*

    PubMed Central

    Lin, Shengchen; Lu, Shuang; Mulaj, Mentor; Fang, Bin; Keeley, Tyler; Wan, Lixin; Hao, Jihui; Muschol, Martin; Sun, Jianwei; Yang, Shengyu

    2016-01-01

    Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys247 and Lys250, two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC50, delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions. PMID:27879315

  1. Monoubiquitination Inhibits the Actin Bundling Activity of Fascin.

    PubMed

    Lin, Shengchen; Lu, Shuang; Mulaj, Mentor; Fang, Bin; Keeley, Tyler; Wan, Lixin; Hao, Jihui; Muschol, Martin; Sun, Jianwei; Yang, Shengyu

    2016-12-30

    Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys 247 and Lys 250 , two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC 50 , delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. A single charge in the actin binding domain of fascin can independently tune the linear and non-linear response of an actin bundle network.

    PubMed

    Maier, M; Müller, K W; Heussinger, C; Köhler, S; Wall, W A; Bausch, A R; Lieleg, O

    2015-05-01

    Actin binding proteins (ABPs) not only set the structure of actin filament assemblies but also mediate the frequency-dependent viscoelastic moduli of cross-linked and bundled actin networks. Point mutations in the actin binding domain of those ABPs can tune the association and dissociation dynamics of the actin/ABP bond and thus modulate the network mechanics both in the linear and non-linear response regime. We here demonstrate how the exchange of a single charged amino acid in the actin binding domain of the ABP fascin triggers such a modulation of the network rheology. Whereas the overall structure of the bundle networks is conserved, the transition point from strain-hardening to strain-weakening sensitively depends on the cross-linker off-rate and the applied shear rate. Our experimental results are consistent both with numerical simulations of a cross-linked bundle network and a theoretical description of the bundle network mechanics which is based on non-affine bending deformations and force-dependent cross-link dynamics.

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

    PubMed

    Naresh, S; Atreja, S K

    2015-12-01

    In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa. © 2015 Blackwell Verlag GmbH.

  4. Method of pedestal and common-mode noise correction for switched-capacitor analog memories

    DOEpatents

    Britton, C.L.

    1997-09-23

    A method and apparatus are disclosed for correcting common-mode noise and pedestal noise in a multichannel array of switched-capacitor analog memories wherein each analog memory is connected to an associated analog-to-digital converter. The apparatus comprises a single differential element in two different embodiments. In a first embodiment, the differential element is a reference analog memory connected to a buffer. In the second embodiment, the differential dement is a reference analog memory connected to a reference analog-to-digital connected to an array of digital summing circuits. 4 figs.

  5. Method of pedestal and common-mode noise correction for switched-capacitor analog memories

    DOEpatents

    Britton, C.L.

    1996-12-31

    A method and apparatus are disclosed for correcting common-mode noise and pedestal noise in a multichannel array of switched-capacitor analog memories wherein each analog memory is connected to an associated analog-to-digital converter. The apparatus comprises a single differential element in two different embodiments. In a first embodiment, the differential element is a reference analog memory connected to a buffer. In the second embodiment, the differential element is a reference analog memory connected to a reference analog-to-digital connected to an array of digital summing circuits. 4 figs.

  6. Method of pedestal and common-mode noise correction for switched-capacitor analog memories

    DOEpatents

    Britton, Charles L.

    1997-01-01

    A method and apparatus for correcting common-mode noise and pedestal noise in a multichannel array of switched-capacitor analog memories wherein each analog memory is connected to an associated analog-to-digital converter. The apparatus comprises a single differential element in two different embodiments. In a first embodiment, the differential element is a reference analog memory connected to a buffer. In the second embodiment, the differential dement is a reference analog memory connected to a reference analog-to-digital connected to an array of digital summing circuits.

  7. Method of pedestal and common-mode noise correction for switched-capacitor analog memories

    DOEpatents

    Britton, Charles L.

    1996-01-01

    A method and apparatus for correcting common-mode noise and pedestal noise in a multichannel array of switched-capacitor analog memories wherein each analog memory is connected to an associated analog-to-digital converter. The apparatus comprises a single differential element in two different embodiments. In a first embodiment, the differential element is a reference analog memory connected to a buffer. In the second embodiment, the differential element is a reference analog memory connected to a reference analog-to-digital connected to an array of digital summing circuits.

  8. Human spire interacts with the barbed end of the actin filament.

    PubMed

    Ito, Takuto; Narita, Akihiro; Hirayama, Tasuku; Taki, Masayasu; Iyoshi, Shohei; Yamamoto, Yukio; Maéda, Yuichiro; Oda, Toshiro

    2011-04-22

    Spire is an actin nucleator that initiates actin polymerization at a specific place in the cell. Similar to the Arp2/3 complex, spire was initially considered to bind to the pointed end of the actin filament when it generates a new actin filament. Subsequently, spire was reported to be associated with the barbed end (B-end); thus, there is still no consensus regarding the end with which spire interacts. Here, we report direct evidence that spire binds to the B-end of the actin filament, under conditions where spire accelerates actin polymerization. Using electron microscopy, we visualized the location of spire bound to the filament by gold nanoparticle labeling of the histidine-tagged spire, and the polarity of the actin filament was determined by image analysis. In addition, our results suggest that multiple spires, linked through one gold nanoparticle, enhance the acceleration of actin polymerization. The B-end binding of spire provides the basis for understanding its functional mechanism in the cell. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. 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.

  10. Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry*

    PubMed Central

    Stokasimov, Ema; Rubenstein, Peter A.

    2009-01-01

    Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G- and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts. PMID:19605362

  11. Z-disc-associated, Alternatively Spliced, PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy*

    PubMed Central

    Lin, Xiaoyan; Ruiz, Janelle; Bajraktari, Ilda; Ohman, Rachel; Banerjee, Soojay; Gribble, Katherine; Kaufman, Joshua D.; Wingfield, Paul T.; Griggs, Robert C.; Fischbeck, Kenneth H.; Mankodi, Ami

    2014-01-01

    The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle. PMID:24668811

  12. F-actin distribution and function during sexual development in Eimeria maxima.

    PubMed

    Frölich, Sonja; Wallach, Michael

    2015-06-01

    To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.

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

    PubMed

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

    2015-02-27

    The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. The role of septins in infections with vacuole-dwelling intracellular bacteria.

    PubMed

    Häcker, Georg

    2017-07-29

    Septins are a relatively little understood group of GTPases that form large assemblies in cells from all eukaryotes other than plants. Septins were first identified in cell division but have also been implicated in microbial infections. Septins often associate with cytoskeletal proteins - most often described for filamentous (F-) actin - and are considered cytoskeletal components themselves. Septins have increasingly been found to partake in processes that are linked to intracellular membranes, from mitochondria to phagosomes, and evidence is accumulating that septins specifically bind to membranes. Since a number of microorganisms have specialized to live and grow inside membranous vacuoles in the cytosol of mammalian cells, this membrane-association of septins suggests that septins may also be involved in the membranous, vacuolar structures that develop around these microbes. However, data are limited on this issue: septins have been identified by proteome analysis on some microbe-bearing vacuoles, but more extensive experimental data are only available for infections with the obligate intracellular bacterium Chlamydia trachomatis. In this review article I will discuss the available data and speculate about the mechanisms of recruitment and potential functions of septins for vacuole-dwelling microorganisms, which may be peculiar to Chlamydia or may pertain more generally to this class of microbes. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. The effects of near-UV radiation on elasmobranch lens cytoskeletal actin.

    PubMed

    Zigman, S; Rafferty, N S; Scholz, D L; Lowe, K

    1992-08-01

    The role of near-UV radiation as a cytoskeletal actin-damaging agent was investigated. Two procedures were used to analyse fresh smooth dogfish (Mustelus canis) eye lenses that were incubated for up to 22 hr in vitro, with elasmobranch Ringer's medium, and with or without exposure to a near-UV lamp (emission principally at 365 nm; irradiance of 2.5 mW cm-2). These were observed histologically using phalloidin-rhodamine specific staining and by transmission electron microscopy. In addition, solutions of purified polymerized rabbit muscle actin were exposed to the same UV conditions and depolymerization was assayed by ultracentrifugation and high-pressure liquid chromatography. While the two actins studied do differ very slightly in some amino acid sequences, they would react physically nearly identically. The results showed that dogfish lenses developed superficial opacities due to near-UV exposure. Whole mounts of lens epithelium exhibited breakdown of actin filaments in the basal region of the cells within 18 hr of UV exposure. TEM confirmed the breakdown of actin filaments due to UV exposure. SDS-PAGE and immunoblotting positively identified actin in these cells. Direct exposure of purified polymerized muscle actin in polymerizing buffer led to an increase in actin monomer of approximately 25% in the UV-exposed solutions within 3-18 hr, whether assayed by ultracentrifugation or HPLC. The above indicates that elasmobranch lens epithelial cells contain UV-labile actin filaments, and that near-UV radiation, as is present in the sunlit environment, can break down the actin structure in these cells. Furthermore, breakdown of purified polymerized muscle actin does occur due to near-UV light exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Rab8 Regulates the Actin-based Movement of MelanosomesV⃞

    PubMed Central

    Chabrillat, Marion L.; Wilhelm, Claire; Wasmeier, Christina; Sviderskaya, Elena V.; Louvard, Daniel; Coudrier, Evelyne

    2005-01-01

    Rab GTPases have been implicated in the regulation of specific microtubule- and actin-based motor proteins. We devised an in vitro motility assay reconstituting the movement of melanosomes on actin bundles in the presence of ATP to investigate the role of Rab proteins in the actin-dependent movement of melanosomes. Using this assay, we confirmed that Rab27 is required for the actin-dependent movement of melanosomes, and we showed that a second Rab protein, Rab8, also regulates this movement. Rab8 was partially associated with mature melanosomes. Expression of Rab8Q67L perturbed the cellular distribution and increased the frequency of microtubule-independent movement of melanosomes in vivo. Furthermore, anti-Rab8 antibodies decreased the number of melanosomes moving in vitro on actin bundles, whereas melanosomes isolated from cells expressing Rab8Q67L exhibited 70% more movements than wild-type melanosomes. Together, our observations suggest that Rab8 is involved in regulating the actin-dependent movement of melanosomes. PMID:15673612

  17. Theory-based model for the pedestal, edge stability and ELMs in tokamaks

    NASA Astrophysics Data System (ADS)

    Pankin, A. Y.; Bateman, G.; Brennan, D. P.; Schnack, D. D.; Snyder, P. B.; Voitsekhovitch, I.; Kritz, A. H.; Janeschitz, G.; Kruger, S.; Onjun, T.; Pacher, G. W.; Pacher, H. D.

    2006-04-01

    An improved model for triggering edge localized mode (ELM) crashes is developed for use within integrated modelling simulations of the pedestal and ELM cycles at the edge of H-mode tokamak plasmas. The new model is developed by using the BALOO, DCON and ELITE ideal MHD stability codes to derive parametric expressions for the ELM triggering threshold. The whole toroidal mode number spectrum is studied with these codes. The DCON code applies to low mode numbers, while the BALOO code applies to only high mode numbers and the ELITE code applies to intermediate and high mode numbers. The variables used in the parametric stability expressions are the normalized pressure gradient and the parallel current density, which drive ballooning and peeling modes. Two equilibria motivated by DIII-D geometry with different plasma triangularities are studied. It is found that the stable region in the high triangularity discharge covers a much larger region of parameter space than the corresponding stability region in the low triangularity discharge. The new ELM trigger model is used together with a previously developed model for pedestal formation and ELM crashes in the ASTRA integrated modelling code to follow the time evolution of the temperature profiles during ELM cycles. The ELM frequencies obtained in the simulations of low and high triangularity discharges are observed to increase with increasing heating power. There is a transition from second stability to first ballooning mode stability as the heating power is increased in the high triangularity simulations. The results from the ideal MHD stability codes are compared with results from the resistive MHD stability code NIMROD.

  18. Cloning and characterization of an abalone (Haliotis discus hannai) actin gene

    NASA Astrophysics Data System (ADS)

    Ma, Hongming; Xu, Wei; Mai, Kangsen; Liufu, Zhiguo; Chen, Hong

    2004-10-01

    An actin encoding gene was cloned by using RT-PCR, 3‧ RACE and 5‧ RACE from abalone Haliotis discus hannai. The full length of the gene is 1532 base pairs, which contains a long 3‧ untranslated region of 307 base pairs and 79 base pairs of 5‧ untranslated sequence. The open reading frame encodes 376 amino acid residues. Sequence comparison with those of human and other mollusks showed high conservation among species at amino acid level. The identities was 96%, 97% and 96% respectively compared with Aplysia californica, Biomphalaria glabrata and Homo sapience β-actin. It is also indicated that this actin is more similar to the human cytoplasmic actin (β-actin) than to human muscle actin.

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

    PubMed Central

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

    2014-01-01

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

  20. Retinoids for prevention and treatment of actinic keratosis*

    PubMed Central

    Ianhez, Mayra; Fleury, Luiz Fernando Fróes; Miot, Hélio Amante; Bagatin, Edileia

    2013-01-01

    Actinic keratosis is a common cause of dermatological consultations and it presents a strong association with squamous cell carcinoma. Many substances are used for treatment and prevention, such as retinoids. Nevertheless, many studies on retinoids emphasize their application in treating and preventing non melanoma skin cancers. In this article, we reviewed studies about systemic and topical retinoids used with immunocompetent patients and organ transplant recipients with actinic keratosis, as primary or secondary outcomes. The majority of these papers pointed to a reduction in actinic keratosis count after treatment with retinoids. However, studies need to be better-defined in order to address the lack of a standardized dose, the absence of control groups, the low number of patients and short follow-up periods. Blind, randomized and controlled clinical trials with adequate sample sizes, specifically focused on actinic keratosis, are needed to clarify the real benefit of topical and/or oral retinoids. Comparison of efficacy and safety between oral and topical retinoids in the prevention and treatment of non-melanoma skin cancers and actinic keratosis is an essential pre requisite to establish new strategies to control these conditions. PMID:24068130