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Sample records for growing actin networks

  1. 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.

  2. Curvature and torsion in growing actin networks

    PubMed Central

    Shaevitz, Joshua W; Fletcher, Daniel A

    2011-01-01

    Intracellular pathogens such as Listeria monocytogenes and Rickettsia rickettsii move within a host cell by polymerizing a comet-tail of actin fibers that ultimately pushes the cell forward. This dense network of cross-linked actin polymers typically exhibits a striking curvature that causes bacteria to move in gently looping paths. Theoretically, tail curvature has been linked to details of motility by considering force and torque balances from a finite number of polymerizing filaments. Here we track beads coated with a prokaryotic activator of actin polymerization in three dimensions to directly quantify the curvature and torsion of bead motility paths. We find that bead paths are more likely to have low rather than high curvature at any given time. Furthermore, path curvature changes very slowly in time, with an autocorrelation decay time of 200 s. Paths with a small radius of curvature, therefore, remain so for an extended period resulting in loops when confined to two dimensions. When allowed to explore a three-dimensional (3D) space, path loops are less evident. Finally, we quantify the torsion in the bead paths and show that beads do not exhibit a significant left- or right-handed bias to their motion in 3D. These results suggest that paths of actin-propelled objects may be attributed to slow changes in curvature, possibly associated with filament debranching, rather than a fixed torque. PMID:18560043

  3. Growing actin networks regulated by obstacle size and shape

    NASA Astrophysics Data System (ADS)

    Gong, Bo; Lin, Ji; Qian, Jin

    2017-01-01

    Growing actin networks provide the driving force for the motility of cells and intracellular pathogens. Based on the molecular-level processes of actin polymerization, branching, capping, and depolymerization, we have developed a modeling framework to simulate the stochastic and cooperative behaviors of growing actin networks in propelling obstacles, with an emphasis on the size and shape effects on work capacity and filament orientation in the growing process. Our results show that the characteristic size of obstacles changes the protrusion power per unit length, without influencing the orientation distribution of actin filaments in growing networks. In contrast, the geometry of obstacles has a profound effect on filament patterning, which influences the orientation of filaments differently when the drag coefficient of environment is small, intermediate, or large. We also discuss the role of various parameters, such as the aspect ratio of obstacles, branching rate, and capping rate, in affecting the protrusion power of network growth.

  4. Mesoscopic model for filament orientation in growing actin networks: the role of obstacle geometry

    NASA Astrophysics Data System (ADS)

    Weichsel, Julian; Schwarz, Ulrich S.

    2013-03-01

    Propulsion by growing actin networks is a universal mechanism used in many different biological systems, ranging from the sheet-like lamellipodium of crawling animal cells to the actin comet tails induced by certain bacteria and viruses in order to move within their host cells. Although the core molecular machinery for actin network growth is well preserved in all of these cases, the geometry of the propelled obstacle varies considerably. During recent years, filament orientation distribution has emerged as an important observable characterizing the structure and dynamical state of the growing network. Here we derive several continuum equations for the orientation distribution of filaments growing behind stiff obstacles of various shapes and validate the predicted steady state orientation patterns by stochastic computer simulations based on discrete filaments. We use an ordinary differential equation approach to demonstrate that for flat obstacles of finite size, two fundamentally different orientation patterns peaked at either ±35° or +70°/0°/ - 70° exhibit mutually exclusive stability, in agreement with earlier results for flat obstacles of very large lateral extension. We calculate and validate phase diagrams as a function of model parameters and show how this approach can be extended to obstacles with piecewise straight contours. For curved obstacles, we arrive at a partial differential equation in the continuum limit, which again is in good agreement with the computer simulations. In all cases, we can identify the same two fundamentally different orientation patterns, but only within an appropriate reference frame, which is adjusted to the local orientation of the obstacle contour. Our results suggest that two fundamentally different network architectures compete with each other in growing actin networks, irrespective of obstacle geometry, and clarify how simulated and electron tomography data have to be analyzed for non-flat obstacle geometries.

  5. Fluorescent beads disintegrate actin networks.

    PubMed

    Golde, Tom; Schuldt, Carsten; Schnauß, Jörg; Strehle, Dan; Glaser, Martin; Käs, Josef

    2013-10-01

    We studied the influence of fluorescent polystyrene beads on both entangled and cross-linked actin networks. Thermal bead fluctuations were observed via video particle tracking and analyzed with one-point microrheology. Illumination of fluorescent beads with their appropriate excitation wavelength leads to a drastic softening of actin gels. Other wavelengths and bright field microscopy do not increase thermal bead fluctuations. This effect cannot be significantly reduced by adding common oxygen scavengers. We conclude that the usage of fluorescent beads impairs results when studying the microrheology of actin networks.

  6. 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

  7. Growing an actin gel on spherical surfaces.

    PubMed Central

    Noireaux, V; Golsteyn, R M; Friederich, E; Prost, J; Antony, C; Louvard, D; Sykes, C

    2000-01-01

    Inspired by the motility of the bacteria Listeria monocytogenes, we have experimentally studied the growth of an actin gel around spherical beads grafted with ActA, a protein known to be the promoter of bacteria movement. On ActA-grafted beads F-actin is formed in a spherical manner, whereas on the bacteria a "comet-like" tail of F-actin is produced. We show experimentally that the stationary thickness of the gel depends on the radius of the beads. Moreover, the actin gel is not formed if the ActA surface density is too low. To interpret our results, we propose a theoretical model to explain how the mechanical stress (due to spherical geometry) limits the growth of the actin gel. Our model also takes into account treadmilling of actin. We deduce from our work that the force exerted by the actin gel on the bacteria is of the order of 10 pN. Finally, we estimate from our theoretical model possible conditions for developing actin comet tails. PMID:10692348

  8. 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.

  9. Architecture and Connectivity Govern Actin Network Contractility.

    PubMed

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

    2016-03-07

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

  10. Reversible stress softening of actin networks

    PubMed Central

    Chaudhuri, Ovijit; Parekh, Sapun H.; Fletcher, Daniel A.

    2011-01-01

    The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes1–3. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension4–8. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks. PMID:17230186

  11. Reversible stress softening of actin networks

    NASA Astrophysics Data System (ADS)

    Chaudhuri, Ovijit; Parekh, Sapun H.; Fletcher, Daniel A.

    2007-01-01

    The mechanical properties of cells play an essential role in numerous physiological processes. Organized networks of semiflexible actin filaments determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes. Although numerous actin-binding proteins have been identified that organize networks, the mechanical properties of actin networks with physiological architectures and concentrations have been difficult to measure quantitatively. Studies of mechanical properties in vitro have found that crosslinked networks of actin filaments formed in solution exhibit stress stiffening arising from the entropic elasticity of individual filaments or crosslinkers resisting extension. Here we report reversible stress-softening behaviour in actin networks reconstituted in vitro that suggests a critical role for filaments resisting compression. Using a modified atomic force microscope to probe dendritic actin networks (like those formed in the lamellipodia of motile cells), we observe stress stiffening followed by a regime of reversible stress softening at higher loads. This softening behaviour can be explained by elastic buckling of individual filaments under compression that avoids catastrophic fracture of the network. The observation of both stress stiffening and softening suggests a complex interplay between entropic and enthalpic elasticity in determining the mechanical properties of actin networks.

  12. Actin filaments growing against a barrier with fluctuating shape

    NASA Astrophysics Data System (ADS)

    Sadhu, Raj Kumar; Chatterjee, Sakuntala

    2016-06-01

    We study force generation by a set of parallel actin filaments growing against a nonrigid obstacle, in the presence of an external load. The filaments polymerize by either moving the whole obstacle, with a large energy cost, or by causing local distortion in its shape which costs much less energy. The nonrigid obstacle also has local thermal fluctuations due to which its shape can change with time and we describe this using fluctuations in the height profile of a one-dimensional interface with Kardar-Parisi-Zhang dynamics. We find the shape fluctuations of the barrier strongly affect the force generation mechanism. The qualitative nature of the force-velocity curve is crucially determined by the relative time scale of filament and barrier dynamics. The height profile of the barrier also shows interesting variation with the external load. Our analytical calculations within mean-field theory show reasonable agreement with our simulation results.

  13. Force-Velocity Measurements of a Few Growing Actin Filaments

    PubMed Central

    Brangbour, Coraline; du Roure, Olivia; Helfer, Emmanuèle; Démoulin, Damien; Mazurier, Alexis; Fermigier, Marc; Carlier, Marie-France; Bibette, Jérôme; Baudry, Jean

    2011-01-01

    The polymerization of actin in filaments generates forces that play a pivotal role in many cellular processes. We introduce a novel technique to determine the force-velocity relation when a few independent anchored filaments grow between magnetic colloidal particles. When a magnetic field is applied, the colloidal particles assemble into chains under controlled loading or spacing. As the filaments elongate, the beads separate, allowing the force-velocity curve to be precisely measured. In the widely accepted Brownian ratchet model, the transduced force is associated with the slowing down of the on-rate polymerization. Unexpectedly, in our experiments, filaments are shown to grow at the same rate as when they are free in solution. However, as they elongate, filaments are more confined in the interspace between beads. Higher repulsive forces result from this higher confinement, which is associated with a lower entropy. In this mechanism, the production of force is not controlled by the polymerization rate, but is a consequence of the restriction of filaments' orientational fluctuations at their attachment point. PMID:21541364

  14. Growth of branched actin networks against obstacles.

    PubMed Central

    Carlsson, A E

    2001-01-01

    A method for simulating the growth of branched actin networks against obstacles has been developed. The method is based on simple stochastic events, including addition or removal of monomers at filament ends, capping of filament ends, nucleation of branches from existing filaments, and detachment of branches; the network structure for several different models of the branching process has also been studied. The models differ with regard to their inclusion of effects such as preferred branch orientations, filament uncapping at the obstacle, and preferential branching at filament ends. The actin ultrastructure near the membrane in lamellipodia is reasonably well produced if preferential branching in the direction of the obstacle or barbed-end uncapping effects are included. Uncapping effects cause the structures to have a few very long filaments that are similar to those seen in pathogen-induced "actin tails." The dependence of the growth velocity, branch spacing, and network density on the rate parameters for the various processes is quite different among the branching models. An analytic theory of the growth velocity and branch spacing of the network is described. Experiments are suggested that could distinguish among some of the branching models. PMID:11566765

  15. Growing networks with superjoiners.

    PubMed

    Jabr-Hamdan, Ameerah; Sun, Jie; Ben-Avraham, Daniel

    2014-11-01

    We study the Krapivsky-Redner (KR) network growth model, but where new nodes can connect to any number of existing nodes, m, picked from a power-law distribution p(m)∼m^{-α}. Each of the m new connections is still carried out as in the KR model with probability redirection r (corresponding to degree exponent γ_{KR}=1+1/r in the original KR model). The possibility to connect to any number of nodes resembles a more realistic type of growth in several settings, such as social networks, routers networks, and networks of citations. Here we focus on the in-, out-, and total-degree distributions and on the potential tension between the degree exponent α, characterizing new connections (outgoing links), and the degree exponent γ_{KR}(r) dictated by the redirection mechanism.

  16. Growing networks with superjoiners

    NASA Astrophysics Data System (ADS)

    Jabr-Hamdan, Ameerah; Sun, Jie; ben-Avraham, Daniel

    2014-11-01

    We study the Krapivsky-Redner (KR) network growth model, but where new nodes can connect to any number of existing nodes, m , picked from a power-law distribution p (m ) ˜m-α . Each of the m new connections is still carried out as in the KR model with probability redirection r (corresponding to degree exponent γKR=1 +1 /r in the original KR model). The possibility to connect to any number of nodes resembles a more realistic type of growth in several settings, such as social networks, routers networks, and networks of citations. Here we focus on the in-, out-, and total-degree distributions and on the potential tension between the degree exponent α , characterizing new connections (outgoing links), and the degree exponent γKR(r ) dictated by the redirection mechanism.

  17. Cyclic hardening in bundled actin networks.

    PubMed

    Schmoller, K M; Fernández, P; Arevalo, R C; Blair, D L; Bausch, A R

    2010-01-01

    Nonlinear deformations can irreversibly alter the mechanical properties of materials. Most soft materials, such as rubber and living tissues, display pronounced softening when cyclically deformed. Here we show that, in contrast, reconstituted networks of crosslinked, bundled actin filaments harden when subject to cyclical shear. As a consequence, they exhibit a mechano-memory where a significant stress barrier is generated at the maximum of the cyclic shear strain. This unique response is crucially determined by the network architecture: at lower crosslinker concentrations networks do not harden, but soften showing the classic Mullins effect known from rubber-like materials. By simultaneously performing macrorheology and confocal microscopy, we show that cyclic shearing results in structural reorganization of the network constituents such that the maximum applied strain is encoded into the network architecture.

  18. Tau co-organizes dynamic microtubule and actin networks

    PubMed Central

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

    2015-01-01

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

  19. Actin network architecture can determine myosin motor activity.

    PubMed

    Reymann, Anne-Cécile; Boujemaa-Paterski, Rajaa; Martiel, Jean-Louis; Guérin, Christophe; Cao, Wenxiang; Chin, Harvey F; De La Cruz, Enrique M; Théry, Manuel; Blanchoin, Laurent

    2012-06-08

    The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This "orientation selection" mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.

  20. Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network

    PubMed Central

    Luo, Weiwei; Lieu, Zi Zhao; Manser, Ed; Bershadsky, Alexander D.; Sheetz, Michael P.

    2016-01-01

    A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization. PMID:27760153

  1. Encoding Mechano-Memories in Actin Networks

    NASA Astrophysics Data System (ADS)

    Foucard, Louis; Majumdar, Sayantan; Levine, Alex; Gardel, Margaret

    The ability of cells to sense and adapt to external mechanical stimuli is vital to many of its biological functions. A critical question is therefore to understand how mechanosensory mechanisms arise in living matter, with implications in both cell biology and smart materials design. Experimental work has demonstrated that the mechanical properties of semiflexible actin networks in Eukaryotic cells can be modulated (either transiently or irreversibly) via the application of external forces. Previous work has also shown with a combination of numerical simulations and analytic calculations shows that the broken rotational symmetry of the filament orientational distribution in semiflexible networks leads to dramatic changes in the mechanical response. Here we demonstrate with a combination of numerical and analytic calculations that the observed long-lived mechano-memory in the actin networks arise from changes in the nematic order of the constituent filaments. These stress-induced changes in network topology relax slowly under zero stress and can be observed through changes in the nonlinear mechanics. Our results provide a strategy for designing a novel class of materials and demonstrate a new putative mechanism of mechanical sensing in eukaryotic cells.

  2. Comparative analysis of tools for live cell imaging of actin network architecture

    PubMed Central

    Belin, Brittany J; Goins, Lauren M; Mullins, R Dyche

    2014-01-01

    Abstract Fluorescent derivatives of actin and actin-binding domains are powerful tools for studying actin filament architecture and dynamics in live cells. Growing evidence, however, indicates that these probes are biased, and their cellular distribution does not accurately reflect that of the cytoskeleton. To understand the strengths and weaknesses of commonly used live-cell probes—fluorescent protein fusions of actin, Lifeact, F-tractin, and actin-binding domains from utrophin—we compared their distributions in cells derived from various model organisms. We focused on five actin networks: the peripheral cortex, lamellipodial and lamellar networks, filopodial bundles, and stress fibers. Using phalloidin as a standard, we identified consistent biases in the distribution of each probe. The localization of F-tractin is the most similar to that of phalloidin but induces organism-specific changes in cell morphology. Both Lifeact and GFP-actin concentrate in lamellipodial actin networks but are excluded from lamellar networks and filopodia. In contrast, the full utrophin actin-binding domain (Utr261) binds filaments of the lamellum but only weakly localizes to lamellipodia, while a shorter variant (Utr230) is restricted to the most stable subpopulations of actin filaments: cortical networks and stress fibers. In some cells, Utr230 also detects Golgi-associated filaments, previously detected by immunofluorescence but not visible by phalloidin staining. Consistent with its localization, Utr230 exhibits slow rates of fluorescence recovery after photobleaching (FRAP) compared to F-tractin, Utr261 and Lifeact, suggesting that it may be more useful for FRAP- and photo-activation-based studies of actin network dynamics. PMID:26317264

  3. Comparative analysis of tools for live cell imaging of actin network architecture.

    PubMed

    Belin, Brittany J; Goins, Lauren M; Mullins, R Dyche

    2014-01-01

    Fluorescent derivatives of actin and actin-binding domains are powerful tools for studying actin filament architecture and dynamics in live cells. Growing evidence, however, indicates that these probes are biased, and their cellular distribution does not accurately reflect that of the cytoskeleton. To understand the strengths and weaknesses of commonly used live-cell probes--fluorescent protein fusions of actin, Lifeact, F-tractin, and actin-binding domains from utrophin--we compared their distributions in cells derived from various model organisms. We focused on five actin networks: the peripheral cortex, lamellipodial and lamellar networks, filopodial bundles, and stress fibers. Using phalloidin as a standard, we identified consistent biases in the distribution of each probe. The localization of F-tractin is the most similar to that of phalloidin but induces organism-specific changes in cell morphology. Both Lifeact and GFP-actin concentrate in lamellipodial actin networks but are excluded from lamellar networks and filopodia. In contrast, the full utrophin actin-binding domain (Utr261) binds filaments of the lamellum but only weakly localizes to lamellipodia, while a shorter variant (Utr230) is restricted to the most stable subpopulations of actin filaments: cortical networks and stress fibers. In some cells, Utr230 also detects Golgi-associated filaments, previously detected by immunofluorescence but not visible by phalloidin staining. Consistent with its localization, Utr230 exhibits slow rates of fluorescence recovery after photobleaching (FRAP) compared to F-tractin, Utr261 and Lifeact, suggesting that it may be more useful for FRAP- and photo-activation-based studies of actin network dynamics.

  4. 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

  5. Organization of growing random networks

    SciTech Connect

    Krapivsky, P. L.; Redner, S.

    2001-06-01

    The organizational development of growing random networks is investigated. These growing networks are built by adding nodes successively, and linking each to an earlier node of degree k with an attachment probability A{sub k}. When A{sub k} grows more slowly than linearly with k, the number of nodes with k links, N{sub k}(t), decays faster than a power law in k, while for A{sub k} growing faster than linearly in k, a single node emerges which connects to nearly all other nodes. When A{sub k} is asymptotically linear, N{sub k}(t){similar_to}tk{sup {minus}{nu}}, with {nu} dependent on details of the attachment probability, but in the range 2{lt}{nu}{lt}{infinity}. The combined age and degree distribution of nodes shows that old nodes typically have a large degree. There is also a significant correlation in the degrees of neighboring nodes, so that nodes of similar degree are more likely to be connected. The size distributions of the in and out components of the network with respect to a given node{emdash}namely, its {open_quotes}descendants{close_quotes} and {open_quotes}ancestors{close_quotes}{emdash}are also determined. The in component exhibits a robust s{sup {minus}2} power-law tail, where s is the component size. The out component has a typical size of order lnt, and it provides basic insights into the genealogy of the network.

  6. 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.

  7. 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.

  8. Actin network disassembly powers dissemination of Listeria monocytogenes

    PubMed Central

    Talman, Arthur M.; Chong, Ryan; Chia, Jonathan; Svitkina, Tatyana; Agaisse, Hervé

    2014-01-01

    ABSTRACT Several bacterial pathogens hijack the actin assembly machinery and display intracellular motility in the cytosol of infected cells. At the cell cortex, intracellular motility leads to bacterial dissemination through formation of plasma membrane protrusions that resolve into vacuoles in adjacent cells. Here, we uncover a crucial role for actin network disassembly in dissemination of Listeria monocytogenes. We found that defects in the disassembly machinery decreased the rate of actin tail turnover but did not affect the velocity of the bacteria in the cytosol. By contrast, defects in the disassembly machinery had a dramatic impact on bacterial dissemination. Our results suggest a model of L. monocytogenes dissemination in which the disassembly machinery, through local recycling of the actin network in protrusions, fuels continuous actin assembly at the bacterial pole and concurrently exhausts cytoskeleton components from the network distal to the bacterium, which enables membrane apposition and resolution of protrusions into vacuoles. PMID:24155331

  9. Diffusing wave spectroscopy microrheology of actin filament networks.

    PubMed Central

    Palmer, A; Xu, J; Kuo, S C; Wirtz, D

    1999-01-01

    Filamentous actin (F-actin), one of the constituents of the cytoskeleton, is believed to be the most important participant in the motion and mechanical integrity of eukaryotic cells. Traditionally, the viscoelastic moduli of F-actin networks have been measured by imposing a small mechanical strain and quantifying the resulting stress. The magnitude of the viscoelastic moduli, their concentration dependence and strain dependence, as well as the viscoelastic nature (solid-like or liquid-like) of networks of uncross-linked F-actin, have been the subjects of debate. Although this paper helps to resolve the debate and establishes the extent of the linear regime of F-actin networks' rheology, we report novel measurements of the high-frequency behavior of networks of F-actin, using a noninvasive light-scattering based technique, diffusing wave spectroscopy (DWS). Because no external strain is applied, our optical assay generates measurements of the mechanical properties of F-actin networks that avoid many ambiguities inherent in mechanical measurements. We observe that the elastic modulus has a small magnitude, no strain dependence, and a weak concentration dependence. Therefore, F-actin alone is not sufficient to generate the elastic modulus necessary to sustain the structural rigidity of most cells or support new cellular protrusions. Unlike previous studies, our measurements show that the mechanical properties of F-actin are highly dependent on the frequency content of the deformation. We show that the loss modulus unexpectedly dominates the elastic modulus at high frequencies, which are key for fast transitions. Finally, the measured mean square displacement of the optical probes, which is also generated by DWS measurements, offers new insight into the local bending fluctuations of the individual actin filaments and shows how they generate enhanced dissipation at short time scales. PMID:9916038

  10. Universal properties of growing networks

    NASA Astrophysics Data System (ADS)

    Krapivsky, P. L.; Derrida, B.

    2004-09-01

    Networks growing according to the rule that every new node has a probability pk of being attached to k preexisting nodes, have a universal phase diagram and exhibit power-law decays of the distribution of cluster sizes in the non-percolating phase. The percolation transition is continuous but of infinite order and the size of the giant component is infinitely differentiable at the transition (though of course non-analytic). At the transition the average cluster size (of the finite components) is discontinuous.

  11. Formation of actin networks in microfluidic concentration gradients

    NASA Astrophysics Data System (ADS)

    Strelnikova, Natalja; Herren, Florian; Schoenenberger, Cora-Ann; Pfohl, Thomas

    2016-05-01

    The physical properties of cytoskeletal networks are contributors in a number of mechanical responses of cells including cellular deformation and locomotion, and are crucial for the proper action of living cells. Local chemical gradients modulate cytoskeletal functionality including the interactions of the cytoskeleton with other cellular components. Actin is a major constituent of the cytoskeleton. Introducing a microfluidic-based platform, we explored the impact of concentration gradients on the formation and structural properties of actin networks. Microfluidics-controlled flow-free steady state experimental conditions allow for the generation of chemical gradients of different profiles, such as linear or step-like. We discovered specific features of actin networks emerging in defined gradients. In particular, we analyzed the effects of spatial conditions on network properties, bending rigidities of network links, and the network elasticity.

  12. Global treadmilling coordinates actin turnover and controls the size of actin networks.

    PubMed

    Carlier, Marie-France; Shekhar, Shashank

    2017-03-01

    Various cellular processes (including cell motility) are driven by the regulated, polarized assembly of actin filaments into distinct force-producing arrays of defined size and architecture. Branched, linear, contractile and cytosolic arrays coexist in vivo, and cells intricately control the number, length and assembly rate of filaments in these arrays. Recent in vitro and in vivo studies have revealed novel molecular mechanisms that regulate the number of filament barbed and pointed ends and their respective assembly and disassembly rates, thus defining classes of dynamically different filaments, which coexist in the same cell. We propose that a global treadmilling process, in which a steady-state amount of polymerizable actin monomers is established by the dynamics of each network, is responsible for defining the size and turnover of coexisting actin networks. Furthermore, signal-induced changes in the partitioning of actin to distinct arrays (mediated by RHO GTPases) result in the establishment of various steady-state concentrations of polymerizable monomers, thereby globally influencing the growth rate of actin filaments.

  13. 3D actin network centerline extraction with multiple active contours.

    PubMed

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2014-02-01

    Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and actin cables. While these cytoskeletal structures are often dilute enough to allow imaging of individual filaments or bundles of them, quantitative analysis of these images is challenging. To facilitate quantitative, reproducible and objective analysis of the image data, we propose a semi-automated method to extract actin networks and retrieve their topology in 3D. Our method uses multiple Stretching Open Active Contours (SOACs) that are automatically initialized at image intensity ridges and then evolve along the centerlines of filaments in the network. SOACs can merge, stop at junctions, and reconfigure with others to allow smooth crossing at junctions of filaments. The proposed approach is generally applicable to images of curvilinear networks with low SNR. We demonstrate its potential by extracting the centerlines of synthetic meshwork images, actin networks in 2D Total Internal Reflection Fluorescence Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy. Quantitative evaluation of the method using synthetic images shows that for images with SNR above 5.0, the average vertex error measured by the distance between our result and ground truth is 1 voxel, and the average Hausdorff distance is below 10 voxels.

  14. 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

  15. Self-organization of actin networks by a monomeric myosin

    PubMed Central

    Saczko-Brack, Dario; Warchol, Ewa; Rogez, Benoit; Kröss, Markus; Heissler, Sarah M.; Sellers, James R.; Batters, Christopher; Veigel, Claudia

    2016-01-01

    The organization of actomyosin networks lies at the center of many types of cellular motility, including cell polarization and collective cell migration during development and morphogenesis. Myosin-IXa is critically involved in these processes. Using total internal reflection fluorescence microscopy, we resolved actin bundles assembled by myosin-IXa. Electron microscopic data revealed that the bundles consisted of highly ordered lattices with parallel actin polarity. The myosin-IXa motor domains aligned across the network, forming cross-links at a repeat distance of precisely 36 nm, matching the helical repeat of actin. Single-particle image processing resolved three distinct conformations of myosin-IXa in the absence of nucleotide. Using cross-correlation of a modeled actomyosin crystal structure, we identified sites of additional mass, which can only be accounted for by the large insert in loop 2 exclusively found in the motor domain of class IX myosins. We show that the large insert in loop 2 binds calmodulin and creates two coordinated actin-binding sites that constrain the actomyosin interactions generating the actin lattices. The actin lattices introduce orientated tracks at specific sites in the cell, which might install platforms allowing Rho-GTPase–activating protein (RhoGAP) activity to be focused at a definite locus. In addition, the lattices might introduce a myosin-related, force-sensing mechanism into the cytoskeleton in cell polarization and collective cell migration. PMID:27956608

  16. Microstructure and Mechanical Properties of Composite Actin Networks

    NASA Astrophysics Data System (ADS)

    Gardel, Margaret; Shin, Jennifer; Mahadevan, L.; Matsudaira, Paul; Weitz, D. A.

    2003-03-01

    There exits a family of actin-binding proteins (ABPs) and each protein has a distinct function for bundling, networking, gelating, capping, or simply binding to actin. Whether actin serves as a structural or motile component, its mechanical properties are determined by its degree and kinds of association with different ABPs and these properties are often closely related to its functional needs. For instance, in a cell actin is highly crosslinked with multiple ABPs (fimbrin, alpha-actinin, etc.) to generate thrust and strength for locomotion. In the acrosomal reaction of horseshoe crab sperm, actin exists as a bundle of preassembled filaments crosslinked with scruin to form a rigid structure to penetrate into an egg without yielding. We study the effects three different ABPs (scruin,fimbrin and alpha-actinin) have on the rheology and microstructure of actin networks using multiparticle tracking, imaging, and bulk rheology. From these experiments we can deduce how an evolving microstructure affects the bulk rheological properties and the role different concentrations and kinds of ABPs have in these changes.

  17. The role of actin networks in cellular mechanosensing

    NASA Astrophysics Data System (ADS)

    Azatov, Mikheil

    Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant

  18. Growing Networks with Positive and Negative Links

    NASA Astrophysics Data System (ADS)

    Dech, Corynne; Antwi, Shadrack; Shaw, Leah

    Scale-free networks grown via preferential attachment have been used to model real-world networks such as the Internet, citation networks, and social networks. Here we investigate signed scale-free networks where an edge represents a positive or negative connection. We present analytic results and simulation for a growing signed network model. We compare the signed network to an unsigned scale-free network. We discuss several options for preferential attachment in a signed network that could be further adapted to model the accumulation of links over time in real-world signed networks.

  19. 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

  20. Growing local likelihood network: Emergence of communities

    NASA Astrophysics Data System (ADS)

    Chen, S.; Small, M.

    2015-10-01

    In many real situations, networks grow only via local interactions. New nodes are added to the growing network with information only pertaining to a small subset of existing nodes. Multilevel marketing, social networks, and disease models can all be depicted as growing networks based on local (network path-length) distance information. In these examples, all nodes whose distance from a chosen center is less than d form a subgraph. Hence, we grow networks with information only from these subgraphs. Moreover, we use a likelihood-based method, where at each step we modify the networks by changing their likelihood to be closer to the expected degree distribution. Combining the local information and the likelihood method, we grow networks that exhibit novel features. We discover that the likelihood method, over certain parameter ranges, can generate networks with highly modulated communities, even when global information is not available. Communities and clusters are abundant in real-life networks, and the method proposed here provides a natural mechanism for the emergence of communities in scale-free networks. In addition, the algorithmic implementation of network growth via local information is substantially faster than global methods and allows for the exploration of much larger networks.

  1. Viscoelastic properties of actin networks influence material transport

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Weirich, Kimberly; Gardel, Margaret

    2015-03-01

    Directed flows of cytoplasmic material are important in a variety of biological processes including assembly of a mitotic spindle, retraction of the cell rear during migration, and asymmetric cell division. Networks of cytoskeletal polymers and molecular motors are known to be involved in these events, but how the network mechanical properties are tuned to perform such functions is not understood. Here, we construct networks of either semiflexible actin filaments or rigid bundles with varying connectivity. We find that solutions of rigid rods, where unimpeded sliding of filaments may enhance transport in comparison to unmoving tracks, are the fastest at transporting network components. Entangled solutions of semiflexible actin filaments also transport material, but the entanglements provide resistance. Increasing the elasticity of the actin networks with crosslinking proteins slows network deformation further. However, the length scale of correlated transport in these networks is increased. Our results reveal how the rigidity and connectivity of biopolymers allows material transport to occur over time and length scales required for physiological processes. This work was supported by the U. Chicago MRSEC

  2. Interactions with Actin Monomers, Actin Filaments, and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks*

    PubMed Central

    Helgeson, Luke A.; Prendergast, Julianna G.; Wagner, Andrew R.; Rodnick-Smith, Max; Nolen, Brad J.

    2014-01-01

    Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks. PMID:25160634

  3. Coupled actin-lamin biopolymer networks and protecting DNA

    NASA Astrophysics Data System (ADS)

    Zhang, Tao; Rocklin, D. Zeb; Mao, Xiaoming; Schwarz, J. M.

    The mechanical properties of cells are largely determined by networks of semiflexible biopolymers forming the cytoskeleton. Similarly, the mechanical properties of cell nuclei are also largely determined by networks of semiflexible biopolymers forming the nuclear cytoskeleton. In particular, a network of filamentous lamin sits just inside the inner nuclear membrane to presumably protect the heart of the cell nucleus--the DNA. It has been demonstrated over the past decade that the actin cytoskeletal biopolymer network and the lamin biopolymer network are coupled via a sequence of proteins bridging the outer and inner nuclear membranes, known as the LINC complex. We, therefore, probe the consequences of such a coupling in a model biopolymer network system via numerical simulations to understand the resulting deformations in the lamin network in response to perturbations in the actin cytoskeletal network. We find, for example, that the force transmission across the coupled system can depend sensitively on the concentration of LINC complexes. Such study could have implications for mechanical mechanisms of the regulation of transcription since DNA couples to lamin via lamin-binding domains so that deformations in the lamin network may result in deformations in the DNA.

  4. Branching influences force-velocity curves and length fluctuations in actin networks

    NASA Astrophysics Data System (ADS)

    Hansda, Deepak Kumar; Sen, Shamik; Padinhateeri, Ranjith

    2014-12-01

    We investigate collective dynamics of branched actin networks growing against a rigid movable wall constrained by a resistive force. Computing the force velocity relations, we show that the stall force of such networks depends not only on the average number of filaments touching the wall, but also on the amount of fluctuation of the leading edge of the network. These differences arise due to differences in the network architecture, namely, distance between two adjacent branching points and the initial distance of the starting filament from the wall, with their relative magnitudes influencing the nature of the force velocity curves (convex versus concave). We also show that the introduction of branching results in nonmonotonic diffusion constant, a quantity that measures the growth in length fluctuation of the leading edge of the network, as a function of externally applied force. Together our results demonstrate how the collective dynamics of a branched network differs from that of a parallel filament network.

  5. 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

  6. Mechanics and multiple-particle tracking microheterogeneity of alpha-actinin-cross-linked actin filament networks.

    PubMed Central

    Tseng, Y; Wirtz, D

    2001-01-01

    Cell morphology is controlled by the actin cytoskeleton organization and mechanical properties, which are regulated by the available contents in actin and actin regulatory proteins. Using rheometry and the recently developed multiple-particle tracking method, we compare the mechanical properties and microheterogeneity of actin filament networks containing the F-actin cross-linking protein alpha-actinin. The elasticity of F-actin/alpha-actinin networks increases with actin concentration more rapidly for a fixed molar ratio of actin to alpha-actinin than in the absence of alpha-actinin, for networks of fixed alpha-actinin concentration and of fixed actin concentration, but more slowly than theoretically predicted for a homogeneous cross-linked semiflexible polymer network. These rheological measurements are complemented by multiple-particle tracking of fluorescent microspheres imbedded in the networks. The distribution of the mean squared displacements of these microspheres becomes progressively more asymmetric and wider for increasing concentration in alpha-actinin and, to a lesser extent, for increasing actin concentration, which suggests that F-actin networks become progressively heterogeneous for increasing protein content. This may explain the slower-than-predicted rise in elasticity of F-actin/alpha-actinin networks. Together these in vitro results suggest that actin and alpha-actinin provides the cell with an unsuspected range of regulatory pathways to modulate its cytoskeleton's micromechanics and local organization in vivo. PMID:11509377

  7. Dynamic actin cycling through mitochondrial subpopulations locally regulates the fission-fusion balance within mitochondrial networks.

    PubMed

    Moore, Andrew S; Wong, Yvette C; Simpson, Cory L; Holzbaur, Erika L F

    2016-09-30

    Mitochondria form interconnected networks that dynamically remodel in response to cellular needs. Using live-cell imaging, we investigate the role of the actin cytoskeleton in regulating mitochondrial fission and fusion. We identify cycling of actin filaments onto and off of subsets of cellular mitochondria. The association of actin filaments with mitochondrial subpopulations is transient; actin quickly disassembles, then reassembles around a distinct subpopulation, efficiently cycling through all cellular mitochondria within 14 min. The focal assembly of actin induces local, Drp1-dependent fragmentation of the mitochondrial network. On actin disassembly, fragmented mitochondria undergo rapid fusion, leading to regional recovery of the tubular mitochondrial network. Cycling requires dynamic actin polymerization and is blocked by inhibitors of both Arp2/3 and formins. We propose that cyclic assembly of actin onto mitochondria modulates the fission/fusion balance, promotes network remodelling and content mixing, and thus may serve as an essential mechanism regulating mitochondrial network homeostasis.

  8. Dynamic actin cycling through mitochondrial subpopulations locally regulates the fission–fusion balance within mitochondrial networks

    PubMed Central

    Moore, Andrew S.; Wong, Yvette C.; Simpson, Cory L.; Holzbaur, Erika L. F.

    2016-01-01

    Mitochondria form interconnected networks that dynamically remodel in response to cellular needs. Using live-cell imaging, we investigate the role of the actin cytoskeleton in regulating mitochondrial fission and fusion. We identify cycling of actin filaments onto and off of subsets of cellular mitochondria. The association of actin filaments with mitochondrial subpopulations is transient; actin quickly disassembles, then reassembles around a distinct subpopulation, efficiently cycling through all cellular mitochondria within 14 min. The focal assembly of actin induces local, Drp1-dependent fragmentation of the mitochondrial network. On actin disassembly, fragmented mitochondria undergo rapid fusion, leading to regional recovery of the tubular mitochondrial network. Cycling requires dynamic actin polymerization and is blocked by inhibitors of both Arp2/3 and formins. We propose that cyclic assembly of actin onto mitochondria modulates the fission/fusion balance, promotes network remodelling and content mixing, and thus may serve as an essential mechanism regulating mitochondrial network homeostasis. PMID:27686185

  9. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  10. Microtubules Remodel Actomyosin Networks in Xenopus Egg Extracts via Two Mechanisms of F-Actin Transport

    PubMed Central

    Waterman-Storer, Clare; Duey, Devin Y.; Weber, Kari L.; Keech, John; Cheney, Richard E.; Salmon, E.D.; Bement, William M.

    2000-01-01

    Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in interphase Xenopus egg extracts. In the absence of microtubules, networks of F-actin bundles zippered together or exhibited serpentine gliding along the coverslip. When microtubules were nucleated from Xenopus sperm centrosomes, they were released and translocated away from the aster center. In the presence of microtubules, F-actin exhibited two distinct, microtubule-dependent motilities: rapid (∼250–300 nm/s) jerking and slow (∼50 nm/s), straight gliding. Microtubules remodeled the F-actin network, as F-actin jerking caused centrifugal clearing of F-actin from around aster centers. F-actin jerking occurred when F-actin bound to motile microtubules powered by cytoplasmic dynein. F-actin straight gliding occurred when F-actin bundles translocated along the microtubule lattice. These interactions required Xenopus cytosolic factors. Localization of myosin-II to F-actin suggested it may power F-actin zippering, while localization of myosin-V on microtubules suggested it could mediate interactions between microtubules and F-actin. We examine current models for cytokinesis and cell motility in light of these findings. PMID:10908578

  11. Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue.

    PubMed

    Clark, Abbot F; Brotchie, Daniel; Read, A Thomas; Hellberg, Peggy; English-Wright, Sherry; Pang, Iok-Hou; Ethier, C Ross; Grierson, Ian

    2005-02-01

    Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.

  12. Myosin lever arm directs collective motion on cellular actin network

    PubMed Central

    Hariadi, Rizal F.; Cale, Mario; Sivaramakrishnan, Sivaraj

    2014-01-01

    The molecular motor myosin teams up to drive muscle contraction, membrane traffic, and cell division in biological cells. Myosin function in cells emerges from the interaction of multiple motors tethered to a scaffold, with surrounding actin filaments organized into 3D networks. Despite the importance of myosin function, the influence of intermotor interactions on collective motion remains poorly understood. In this study, we used precisely engineered myosin assemblies to examine emergence in collective myosin movement. We report that tethering multiple myosin VI motors, but not myosin V motors, modifies their movement trajectories on keratocyte actin networks. Single myosin V and VI dimers display similar skewed trajectories, albeit in opposite directions, when traversing the keratocyte actin network. In contrast, tethering myosin VI motors, but not myosin V motors, progressively straightens the trajectories with increasing myosin number. Trajectory shape of multimotor scaffolds positively correlates with the stiffness of the myosin lever arm. Swapping the flexible myosin VI lever arm for the relatively rigid myosin V lever increases trajectory skewness, and vice versa. A simplified model of coupled motor movement demonstrates that the differences in flexural rigidity of the two myosin lever arms is sufficient to account for the differences in observed behavior of groups of myosin V and VI motors. In accordance with this model trajectory, shapes for scaffolds containing both myosin V and VI are dominated by the myosin with a stiffer lever arm. Our findings suggest that structural features unique to each myosin type may confer selective advantages in cellular functions. PMID:24591646

  13. Myosin lever arm directs collective motion on cellular actin network.

    PubMed

    Hariadi, Rizal F; Cale, Mario; Sivaramakrishnan, Sivaraj

    2014-03-18

    The molecular motor myosin teams up to drive muscle contraction, membrane traffic, and cell division in biological cells. Myosin function in cells emerges from the interaction of multiple motors tethered to a scaffold, with surrounding actin filaments organized into 3D networks. Despite the importance of myosin function, the influence of intermotor interactions on collective motion remains poorly understood. In this study, we used precisely engineered myosin assemblies to examine emergence in collective myosin movement. We report that tethering multiple myosin VI motors, but not myosin V motors, modifies their movement trajectories on keratocyte actin networks. Single myosin V and VI dimers display similar skewed trajectories, albeit in opposite directions, when traversing the keratocyte actin network. In contrast, tethering myosin VI motors, but not myosin V motors, progressively straightens the trajectories with increasing myosin number. Trajectory shape of multimotor scaffolds positively correlates with the stiffness of the myosin lever arm. Swapping the flexible myosin VI lever arm for the relatively rigid myosin V lever increases trajectory skewness, and vice versa. A simplified model of coupled motor movement demonstrates that the differences in flexural rigidity of the two myosin lever arms is sufficient to account for the differences in observed behavior of groups of myosin V and VI motors. In accordance with this model trajectory, shapes for scaffolds containing both myosin V and VI are dominated by the myosin with a stiffer lever arm. Our findings suggest that structural features unique to each myosin type may confer selective advantages in cellular functions.

  14. Criticalities in crosslinked actin networks due to myosin activity

    NASA Astrophysics Data System (ADS)

    Sheinman, Michael

    2013-03-01

    Many essential processes in cells and tissues, like motility and morphogenesis, are orchestrated by molecular motors applying internal, active stresses on crosslinked networks of actin filaments. Using scaling analysis, mean-field calculation, numerical modelling and in vitro experiments of such active networks we predict and observe different mechanical regimes exhibiting interesting critical behaviours with non-trivial power-law dependencies. Firstly, we find that the presence of active stresses can dramatically increase the stiffness of a floppy network, as was observed in reconstituted intracellular F-actin networks with myosin motors and extracellular gels with contractile cells. Uniform internal stress results in an anomalous, critical mechanical regime only in the vicinity of the rigidity percolation points of the network. However, taking into account heterogeneity of motors, we demonstrate that the motors, stiffening any floppy network, induce large non-affine fluctuations, giving rise to a critical mechanical regime. Secondly, upon increasing motor concentration, the resulting large internal stress is able to significantly enhance unbinding of the network's crosslinks and, therefore, disconnect the initially well-connected network to isolated clusters. However, during this process, when the network approaches marginal connectivity the internal stresses are expected to drop drastically such that the connectivity stabilizes. This general argument and detailed numerical simulations show that motors should drive a well connected network to a close vicinity of a critical point of marginal connectivity. Experiments clearly confirm this conclusion and demonstrate robust critical connectivity of initially well-connected networks, ruptured by the motor activity for a wide range of parameters. M. Sheinman, C.P. Broedersz and F.C. MacKintosh, Phys. Rev. Lett, in press. J. Alvarado, M. Sheinman, A. Sharma, F.C. MacKintosh and G. Koenderink, in preparation.

  15. Cytoskeletal actin networks in motile cells are critically self-organized systems synchronized by mechanical interactions

    PubMed Central

    Cardamone, Luca; Laio, Alessandro; Torre, Vincent; Shahapure, Rajesh; DeSimone, Antonio

    2011-01-01

    Growing networks of actin fibers are able to organize into compact, stiff two-dimensional structures inside lamellipodia of crawling cells. We put forward the hypothesis that the growing actin network is a critically self-organized system, in which long-range mechanical stresses arising from the interaction with the plasma membrane provide the selective pressure leading to organization. We show that a simple model based only on this principle reproduces the stochastic nature of lamellipodia protrusion (growth periods alternating with fast retractions) and several of the features observed in experiments: a growth velocity initially insensitive to the external force; the capability of the network to organize its orientation; a load-history-dependent growth velocity. Our model predicts that the spectrum of the time series of the height of a growing lamellipodium decays with the inverse of the frequency. This behavior is a well-known signature of self-organized criticality and is confirmed by unique optical tweezer measurements performed in vivo on neuronal growth cones. PMID:21825142

  16. Cytoskeletal actin networks in motile cells are critically self-organized systems synchronized by mechanical interactions.

    PubMed

    Cardamone, Luca; Laio, Alessandro; Torre, Vincent; Shahapure, Rajesh; DeSimone, Antonio

    2011-08-23

    Growing networks of actin fibers are able to organize into compact, stiff two-dimensional structures inside lamellipodia of crawling cells. We put forward the hypothesis that the growing actin network is a critically self-organized system, in which long-range mechanical stresses arising from the interaction with the plasma membrane provide the selective pressure leading to organization. We show that a simple model based only on this principle reproduces the stochastic nature of lamellipodia protrusion (growth periods alternating with fast retractions) and several of the features observed in experiments: a growth velocity initially insensitive to the external force; the capability of the network to organize its orientation; a load-history-dependent growth velocity. Our model predicts that the spectrum of the time series of the height of a growing lamellipodium decays with the inverse of the frequency. This behavior is a well-known signature of self-organized criticality and is confirmed by unique optical tweezer measurements performed in vivo on neuronal growth cones.

  17. Multidimensional neural growing networks and computer intelligence

    SciTech Connect

    Yashchenko, V.A.

    1995-03-01

    This paper examines information-computation processes in time and in space and some aspects of computer intelligence using multidimensional matrix neural growing networks. In particular, issues of object-oriented {open_quotes}thinking{close_quotes} of computers are considered.

  18. Mechanical detection of a long-range actin network emanating from a biomimetic cortex.

    PubMed

    Bussonnier, Matthias; Carvalho, Kevin; Lemière, Joël; Joanny, Jean-François; Sykes, Cécile; Betz, Timo

    2014-08-19

    Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the "actin cloud" and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.

  19. Monitoring the Topology of Growing Dynamical Networks

    NASA Astrophysics Data System (ADS)

    Wu, Zhaoyan; Fu, Xinchu; Chen, Guanrong

    In this paper, topology monitoring of growing networks is studied. When some new nodes are added into a network, the topology of the network is changed, which needs to be monitored in many applications. Some auxiliary systems (network monitors) are designed to achieve this goal. Both linear feedback control and adaptive strategy are applied to designing such network monitors. Based on the Lyapunov function method via constructing a potential or energy function decreasing along any solution of the system, and the LaSalle's invariance principle, which is a generalization of the Lyapunov function method, some sufficient conditions for achieving topology monitoring are obtained. Illustrative examples are provided to demonstrate the effectiveness of the new method.

  20. The Effect of Crosslinking on the Microscale Stress Response and Molecular Deformations in Actin Networks

    NASA Astrophysics Data System (ADS)

    Gurmessa, Bekele; Fitzpatrick, Robert; Valdivia, Jonathon; Anderson, Rae M. R.

    Actin, the most abundant protein in eukaryotic cells, is a semi-flexible biopolymer in the cytoskeleton that plays a crucial structural and mechanical role in cell stability, motion and replication, as well as muscle contraction. Most of these mechanically driven structural changes in cells stem from the complex viscoelastic nature of entangled actin networks and the presence of a myriad of proteins that cross-link actin filaments. Despite their importance, the mechanical response of actin networks is not yet well understood, particularly at the molecular level. Here, we use optical trapping - coupled with fluorescence microscopy - to characterize the microscale stress response and induced filament deformations in entangled and cross-linked actin networks subject to localized mechanical perturbations. In particular, we actively drive a microsphere 10 microns through an entangled or cross- linked actin network at a constant speed and measure the resistive force that the deformed actin filaments exert on the bead during and following strain. We simultaneously visualize and track individual sparsely-labeled actin filaments to directly link force response to molecular deformations, and map the propagation of the initially localized perturbation field throughout the rest of the network (~100 um). By varying the concentration of actin and cross-linkers we directly determine the role of crosslinking and entanglements on the length and time scales of stress propagation, molecular deformation and relaxation mechanisms in actin networks.

  1. Competition between Tropomyosin, Fimbrin, and ADF/Cofilin drives their sorting to distinct actin filament networks.

    PubMed

    Christensen, Jenna R; Hocky, Glen M; Homa, Kaitlin E; Morganthaler, Alisha N; Hitchcock-DeGregori, Sarah E; Voth, Gregory A; Kovar, David R

    2017-03-10

    The fission yeast actin cytoskeleton is an ideal, simplified system to investigate fundamental mechanisms behind cellular self-organization. By focusing on the stabilizing protein tropomyosin Cdc8, bundling protein fimbrin Fim1, and severing protein coffin Adf1, we examined how their pairwise and collective interactions with actin filaments regulate their activity and segregation to functionally diverse F-actin networks. Utilizing multi-color TIRF microscopy of in vitro reconstituted F-actin networks, we observed and characterized two distinct Cdc8 cables loading and spreading cooperatively on individual actin filaments. Furthermore, Cdc8, Fim1, and Adf1 all compete for association with F-actin by different mechanisms, and their cooperative association with actin filaments affects their ability to compete. Finally, competition between Fim1 and Adf1 for F-actin synergizes their activities, promoting rapid displacement of Cdc8 from a dense F-actin network. Our findings reveal that competitive and cooperative interactions between actin binding proteins help define their associations with different F-actin networks.

  2. Features and heterogeneities in growing network models

    NASA Astrophysics Data System (ADS)

    Ferretti, Luca; Cortelezzi, Michele; Yang, Bin; Marmorini, Giacomo; Bianconi, Ginestra

    2012-06-01

    Many complex networks from the World Wide Web to biological networks grow taking into account the heterogeneous features of the nodes. The feature of a node might be a discrete quantity such as a classification of a URL document such as personal page, thematic website, news, blog, search engine, social network, etc., or the classification of a gene in a functional module. Moreover the feature of a node can be a continuous variable such as the position of a node in the embedding space. In order to account for these properties, in this paper we provide a generalization of growing network models with preferential attachment that includes the effect of heterogeneous features of the nodes. The main effect of heterogeneity is the emergence of an “effective fitness” for each class of nodes, determining the rate at which nodes acquire new links. The degree distribution exhibits a multiscaling behavior analogous to the the fitness model. This property is robust with respect to variations in the model, as long as links are assigned through effective preferential attachment. Beyond the degree distribution, in this paper we give a full characterization of the other relevant properties of the model. We evaluate the clustering coefficient and show that it disappears for large network size, a property shared with the Barabási-Albert model. Negative degree correlations are also present in this class of models, along with nontrivial mixing patterns among features. We therefore conclude that both small clustering coefficients and disassortative mixing are outcomes of the preferential attachment mechanism in general growing networks.

  3. Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins.

    PubMed

    Blanchoin, L; Amann, K J; Higgs, H N; Marchand, J B; Kaiser, D A; Pollard, T D

    2000-04-27

    Most nucleated cells crawl about by extending a pseudopod that is driven by the polymerization of actin filaments in the cytoplasm behind the leading edge of the plasma membrane. These actin filaments are linked into a network by Y-branches, with the pointed end of each filament attached to the side of another filament and the rapidly growing barbed end facing forward. Because Arp2/3 complex nucleates actin polymerization and links the pointed end to the side of another filament in vitro, a dendritic nucleation model has been proposed in which Arp2/3 complex initiates filaments from the sides of older filaments. Here we report, by using a light microscopy assay, many new features of the mechanism. Branching occurs during, rather than after, nucleation by Arp2/3 complex activated by the Wiskott-Aldrich syndrome protein (WASP) or Scar protein; capping protein and profilin act synergistically with Arp2/3 complex to favour branched nucleation; phosphate release from aged actin filaments favours dissociation of Arp2/3 complex from the pointed ends of filaments; and branches created by Arp2/3 complex are relatively rigid. These properties result in the automatic assembly of the branched actin network after activation by proteins of the WASP/Scar family and favour the selective disassembly of proximal regions of the network.

  4. Reconstitution of cortical actin networks within water-in-oil emulsions.

    PubMed

    Abu Shah, Enas; Malik-Garbi, Maya; Keren, Kinneret

    2015-01-01

    We describe the reconstitution of dynamic cortical actin networks within cell-like compartments. The approach is based on encapsulation of Xenopus egg extract within water-in-oil emulsions. The growth of cortical actin networks is catalyzed by an amphiphilic actin nucleation-promoting factor that localizes to the water/oil interface. We first describe the preparation of cell-free Xenopus egg extract that provides all the soluble components of the actin machinery. We then describe the preparation of the amphiphilic, fluorescent actin nucleation-promoting factor that directs actin polymerization to the interface. Finally, we describe the steps required for assembly of dynamic actin cortices within water-in-oil emulsions, including the emulsification process and the sample preparation procedures. We provide recommendations for handling sensitive system components and discuss potential uses of this reconstitution approach for cytoskeletal research. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Two Distinct Actin Networks Mediate Traction Oscillations to Confer Focal Adhesion Mechanosensing.

    PubMed

    Wu, Zhanghan; Plotnikov, Sergey V; Moalim, Abdiwahab Y; Waterman, Clare M; Liu, Jian

    2017-02-28

    Focal adhesions (FAs) are integrin-based transmembrane assemblies that connect a cell to its extracellular matrix (ECM). They are mechanosensors through which cells exert actin cytoskeleton-mediated traction forces to sense the ECM stiffness. Interestingly, FAs themselves are dynamic structures that adapt their growth in response to mechanical force. It is unclear how the cell manages the plasticity of the FA structure and the associated traction force to accurately sense ECM stiffness. Strikingly, FA traction forces oscillate in time and space, and govern the cell mechanosensing of ECM stiffness. However, precisely how and why the FA traction oscillates is unknown. We developed a model of FA growth that integrates the contributions of the branched actin network and stress fibers (SFs). Using the model in combination with experimental tests, we show that the retrograde flux of the branched actin network promotes the proximal growth of the FA and contributes to a traction peak near the FA's distal tip. The resulting traction gradient within the growing FA favors SF formation near the FA's proximal end. The SF-mediated actomyosin contractility further stabilizes the FA and generates a second traction peak near the center of the FA. Formin-mediated SF elongation negatively feeds back with actomyosin contractility, resulting in central traction peak oscillation. This underpins the observed FA traction oscillation and, importantly, broadens the ECM stiffness range over which FAs can accurately adapt to traction force generation. Actin cytoskeleton-mediated FA growth and maturation thus culminate with FA traction oscillation to drive efficient FA mechanosensing. Published by Elsevier Inc.

  6. Actin-myosin network is required for proper assembly of influenza virus particles

    SciTech Connect

    Kumakura, Michiko; Kawaguchi, Atsushi Nagata, Kyosuke

    2015-02-15

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.

  7. Spatially embedded growing small-world networks

    PubMed Central

    Zitin, Ari; Gorowara, Alexander; Squires, Shane; Herrera, Mark; Antonsen, Thomas M.; Girvan, Michelle; Ott, Edward

    2014-01-01

    Networks in nature are often formed within a spatial domain in a dynamical manner, gaining links and nodes as they develop over time. Motivated by the growth and development of neuronal networks, we propose a class of spatially-based growing network models and investigate the resulting statistical network properties as a function of the dimension and topology of the space in which the networks are embedded. In particular, we consider two models in which nodes are placed one by one in random locations in space, with each such placement followed by configuration relaxation toward uniform node density, and connection of the new node with spatially nearby nodes. We find that such growth processes naturally result in networks with small-world features, including a short characteristic path length and nonzero clustering. We find no qualitative differences in these properties for two different topologies, and we suggest that results for these properties may not depend strongly on the topology of the embedding space. The results do depend strongly on dimension, and higher-dimensional spaces result in shorter path lengths but less clustering. PMID:25395180

  8. Force feedback controls motor activity and mechanical properties of self-assembling branched actin networks

    PubMed Central

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

    2016-01-01

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

  9. Avalanches, hardening and softening in dense cross-linked actin networks

    NASA Astrophysics Data System (ADS)

    Astrom, Jan; Kumar, Sunil; Vattulainen, Ilpo; Karttunen, Mikko

    2008-03-01

    Actin filament networks enable the cytoskeleton to adjust to internal and external forcing. These active networks can adapt to changes by dynamically adjusting their crosslinks. Here, we study actin filaments as elastic fibers having finite dimensions. We employ a full three-dimensional model to study the elastic properties of actin networks by computer simulations. We model a dense actin network with the crosslinks being approximately 1μm apart. The results show that dense actin networks, without any pre-straining, are characterized by (a) strain hardening without entropic elasticity, (b) 'viscotic' hysteresis in the case of strong crosslinks, (c) avalanches of crosslink slippage leading to strain softening in the case of breakable crosslinks, and (d) spontaneous formation of stress fibers in the case of active crosslink formation and destruction. We will discuss the relation to recent experimental observations.

  10. Effects of filament rigidity in myosin II-induced actin network contractility and dynamics

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Gardel, Margaret

    2014-03-01

    Cells change shape, deforming to move and divide. The dynamic protein scaffold that shapes the cell is the cortex, a disordered, thin network of actin filaments. Random, local stresses generated by myosin II in the network create cellular-scale deformations. Myosin induced buckling and severing of actin filaments has been shown to underlie the contractility of two-dimensional disordered actin networks. This non-linear elastic response of actin filaments is thought to be an essential symmetry breaking mechanism to produce robust contractility in disordered actomyosin networks. To test this idea, we explore the effects of an actin bundling protein fascin, a crosslinker which induces polarity specific bundling of actin filaments, to create a network of F-actin bundles. We investigate myosin-induced stresses in a network of randomly oriented actin filaments, confined to a thin sheet at a supported lipid bilayer surface through a crowding agent. We find fascin-bundled filaments are less prone to filament buckling and show increased filament sliding, causing the myosin activity to induce network reorganization rather than contraction. Thus, changes in the filament bending rigidity in motor-filament systems can drive the system between distinct states with unique dynamic and mechanical signatures.

  11. A variational approach to the growth dynamics of pre-stressed actin filament networks

    NASA Astrophysics Data System (ADS)

    John, Karin; Stöter, Thomas; Misbah, Chaouqi

    2016-09-01

    In order to model the growth dynamics of elastic bodies with residual stresses a thermodynamically consistent approach is needed such that the cross-coupling between growth and mechanics can be correctly described. In the present work we apply a variational principle to the formulation of the interfacial growth dynamics of dendritic actin filament networks growing from biomimetic beads, an experimentally well studied system, where the buildup of residual stresses governs the network growth. We first introduce the material model for the network via a strain energy density for an isotropic weakly nonlinear elastic material and then derive consistently from this model the dynamic equations for the interfaces, i.e. for a polymerizing internal interface in contact with the bead and a depolymerizing external interface directed towards the solvent. We show that (i) this approach automatically preserves thermodynamic symmetry-properties, which is not the case for the often cited ‘rubber-band-model’ (Sekimoto et al 2004 Eur. Phys. J. E 13 247-59, Plastino et al 2004 Eur. Biophys. J. 33 310-20) and (ii) leads to a robust morphological instability of the treadmilling network interfaces. The nature of the instability depends on the interplay of the two dynamic interfaces. Depending on the biochemical conditions the network envelope evolves into a comet-like shape (i.e. the actin envelope thins out at one side and thickens on the opposite side of the bead) via a varicose instability or it breaks the symmetry via higher order zigzag modes. We conclude that morphological instabilities due to mechano-chemical coupling mechanisms and the presences of mechancial pre-stresses can play a major role in locally organizing the cytoskeleton of living cells.

  12. Self-organized DNA/F-actin gels: entangled networks of nematic domains with tunable density

    NASA Astrophysics Data System (ADS)

    Butler, John; Zribi, Olena; Smalyukh, Ivan; Hwee Lai, Ghee; Golestanian, Ramin; Angelini, Thomas; Wong, Gerard

    2008-03-01

    We examine mixtures of DNA and F-actin as a model system of like-charged rigid rods and flexible chains. Confocal microscopy reveals the formation of elongated nematic F-actin domains reticulated via defect-free vertices into a network, all embedded in a mesh of random DNA. Synchrotron x-ray scattering results indicate that the DNA mesh squeezes the F-actin domains into a nematic state via the osmotic pressure of uncondensed counterions, so that the inter-actin spacing within the domains decreases with increasing DNA concentration. These observations are consistent with arguments based on electrostatics and nematic elasticity.

  13. Multi-layered greedy network-growing algorithm: extension of greedy network-growing algorithm to multi-layered networks.

    PubMed

    Kamimura, Ryotaro

    2004-02-01

    In this paper, we extend our greedy network-growing algorithm to multi-layered networks. With multi-layered networks, we can solve many complex problems that single-layered networks fail to solve. In addition, the network-growing algorithm is used in conjunction with teacher-directed learning that produces appropriate outputs without computing errors between targets and outputs. Thus, the present algorithm is a very efficient network-growing algorithm. The new algorithm was applied to three problems: the famous vertical-horizontal lines detection problem, a medical data problem and a road classification problem. In all these cases, experimental results confirmed that the method could solve problems that single-layered networks failed to. In addition, information maximization makes it possible to extract salient features in input patterns.

  14. Cross-linked actin networks (CLANs) in glaucoma.

    PubMed

    Bermudez, Jaclyn Y; Montecchi-Palmer, Michela; Mao, Weiming; Clark, Abbot F

    2017-06-01

    One of the major causes of decreased vision, irreversible vision loss and blindness worldwide is glaucoma. Increased intraocular pressure (IOP) is a major risk factor associated with glaucoma and its molecular mechanisms are not fully understood. The trabecular meshwork (TM) is the primary site of injury in glaucoma, and its dysfunction results in elevated IOP. The glaucomatous TM has increased extracellular matrix deposition as well as cytoskeletal rearrangements referred to as cross-linked actin networks (CLANs) that consist of dome like structures consisting of hubs and spokes. CLANs are thought to play a role in increased aqueous humor outflow resistance and increased IOP by creating stiffer TM cells and tissue. CLANs are inducible by glucocorticoids (GCs) and TGFβ2 in confluent TM cells and TM tissues. The signaling pathways of these induction agents give insight into the possible mechanisms of CLAN formation, but to date, the mechanism of CLANs regulation by these pathways has yet to be determined. Understanding the role CLANs play in IOP elevation and their mechanisms of induction and regulation may lead to novel treatment options to help prevent or intervene in glaucomatous damage to the trabecular meshwork. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Passive and active microrheology for cross-linked F-actin networks in vitro.

    PubMed

    Lee, Hyungsuk; Ferrer, Jorge M; Nakamura, Fumihiko; Lang, Matthew J; Kamm, Roger D

    2010-04-01

    Actin filament (F-actin) is one of the dominant structural constituents in the cytoskeleton. Orchestrated by various actin-binding proteins (ABPs), F-actin is assembled into higher-order structures such as bundles and networks that provide mechanical support for the cell and play important roles in numerous cellular processes. Although mechanical properties of F-actin networks have been extensively studied, the underlying mechanisms for network elasticity are not fully understood, in part because different measurements probe different length and force scales. Here, we developed both passive and active microrheology techniques using optical tweezers to estimate the mechanical properties of F-actin networks at a length scale comparable to cells. For the passive approach we tracked the motion of a thermally fluctuating colloidal sphere to estimate the frequency-dependent complex shear modulus of the network. In the active approach, we used an optical trap to oscillate an embedded microsphere and monitored the response in order to obtain network viscoelasticity over a physiologically relevant force range. While both active and passive measurements exhibit similar results at low strain, the F-actin network subject to high strain exhibits non-linear behavior which is analogous to the strain-hardening observed in macroscale measurements. Using confocal and total internal reflection fluorescent microscopy, we also characterize the microstructure of reconstituted F-actin networks in terms of filament length, mesh size and degree of bundling. Finally, we propose a model of network connectivity by investigating the effect of filament length on the mechanical properties and structure.

  16. RefilinB (FAM101B) targets FilaminA to organize perinuclear actin networks and regulates nuclear shape

    PubMed Central

    Gay, Olivia; Gilquin, Benoît; Nakamura, Fumihiko; Jenkins, Zandra A.; McCartney, Rosannah; Krakow, Deborah; Deshiere, Alexandre; Assard, Nicole; Hartwig, John H.; Robertson, Stephen P.; Baudier, Jacques

    2011-01-01

    The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actin-binding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial–mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actin-based network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations. PMID:21709252

  17. Morphology and viscoelasticity of actin networks formed with the mutually interacting crosslinkers: palladin and alpha-actinin.

    PubMed

    Grooman, Brian; Fujiwara, Ikuko; Otey, Carol; Upadhyaya, Arpita

    2012-01-01

    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 clearly understood. The ABP, palladin, is essential for the maintenance of cell morphology and the regulation of cell movement. Palladin coexists with α-actinin in stress fibers and focal adhesions and binds to both actin and α-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we characterized the micro-structure and mechanics of actin networks crosslinked with palladin and α-actinin. We first showed that palladin crosslinks actin filaments into bundled networks which are viscoelastic in nature. Our studies also showed that composite networks of α-actinin/palladin/actin behave very similar to pure palladin or pure [Formula: see text]-actinin networks. However, we found evidence that palladin and α-actinin synergistically modify network viscoelasticity. To our knowledge, this is the first quantitative characterization of the physical properties of actin networks crosslinked with two mutually interacting crosslinkers.

  18. Symmetric cell division of the mouse zygote requires an actin network.

    PubMed

    Chew, Ting Gang; Lorthongpanich, Chanchao; Ang, Wei Xia; Knowles, Barbara B; Solter, Davor

    2012-12-01

    Positioning of the cleavage plane is regulated to ensure proper animal development. Most animal cells rely on the astral microtubules to position the mitotic spindle, which in turn specifies the cleavage plane. The mouse zygote lacks discernible astral microtubules but still divides symmetrically. Here, we demonstrate a cloud-like accumulation of F-actin surrounds the spindle in zygotes and when this actin network is disassembled, the spindle assumes an off-center position, and the resulting zygote divides asymmetrically into two unequal size blastomeres. Interestingly, when the spindle is micromanipulated to the subcortical region, the zygote without the actin network is unable to reposition the spindle and cleavage plane at the cell center. This study reveals that an actin network maintains the central spindle position in anastral mitosis, and ensures the first embryonic mitosis is symmetrical. © 2012 Wiley Periodicals, Inc. Copyright © 2012 Wiley Periodicals, Inc.

  19. 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

  20. 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.

  1. Mesoscopic Model of Actin-Based Propulsion

    PubMed Central

    Zhu, Jie; Mogilner, Alex

    2012-01-01

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

  2. Dendritic Branching and Homogenization of Actin Networks Mediated by Arp2/3 Complex

    NASA Astrophysics Data System (ADS)

    Tseng, Yiider; Wirtz, Denis

    2004-12-01

    The cytoskeleton of motile cells exploits accessory proteins to locally modulate its organization and micromechanics. Here, we demonstrate that the Arp2/3 complex plays the role, unique among other cytoskeleton proteins, of an actin network “homogenizer,” promoting the extremely rapid formation of homogeneous and stiff networks. Nanotracking of microspheres imbedded in F-actin networks reveals that the Arp2/3 complex promotes the formation of networks that are remarkably more homogeneous than control networks, a distinctive feature that coordinates a dramatic burst of elasticity. These results suggest that the Arp2/3 complex possesses a unique function of stabilizing membrane protrusions through the formation of homogeneous and stiff actin cytoskeleton at the leading edge of crawling cells.

  3. Orientational Order of the Lamellipodial Actin Network as Demonstrated in Living Motile CellsV⃞

    PubMed Central

    Verkhovsky, Alexander B.; Chaga, Oleg Y.; Schaub, Sébastien; Svitkina, Tatyana M.; Meister, Jean-Jacques; Borisy, Gary G.

    2003-01-01

    Lamellipodia of crawling cells represent both the motor for cell advance and the primary building site for the actin cytoskeleton. The organization of actin in the lamellipodium reflects actin dynamics and is of critical importance for the mechanism of cell motility. In previous structural studies, the lamellipodial actin network was analyzed primarily by electron microscopy (EM). An understanding of lamellipodial organization would benefit significantly if the EM data were complemented and put into a kinetic context by establishing correspondence with structural features observable at the light microscopic level in living cells. Here, we use an enhanced phase contrast microscopy technique to visualize an apparent long-range diagonal actin meshwork in the advancing lamellipodia of living cells. Visualization of this meshwork permitted a correlative light and electron microscopic approach that validated the underlying organization of lamellipodia. The linear features in the light microscopic meshwork corresponded to regions of greater actin filament density. Orientation of features was analyzed quantitatively and compared with the orientation of actin filaments at the EM level. We infer that the light microscopic meshwork reflects the orientational order of actin filaments which, in turn, is related to their branching angle. PMID:13679520

  4. In vitro studies of actin filament and network dynamics

    PubMed Central

    Mullins, R Dyche; Hansen, Scott D

    2013-01-01

    Now that many genomes have been sequenced, a central concern of cell biology is to understand how the proteins they encode work together to create living matter. In vitro studies form an essential part of this program because understanding cellular functions of biological molecules often requires isolating them and reconstituting their activities. In particular, many elements of the actin cytoskeleton were first discovered by biochemical methods and their cellular functions deduced from in vitro experiments. We highlight recent advances that have come from in vitro studies, beginning with studies of actin filaments, and ending with multi-component reconstitutions of complex actin-based processes, including force-generation and cell spreading. We describe both scientific results and the technical innovations that made them possible. PMID:23267766

  5. Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts

    PubMed Central

    1993-01-01

    Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilament bundles and the expression of alpha-SM actin, the actin isoform typical of vascular SM cells. Myofibroblasts have been proposed to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. We show here that the subcutaneous administration of transforming growth factor- beta 1 (TGF beta 1) to rats results in the formation of a granulation tissue in which alpha-SM actin expressing myofibroblasts are particularly abundant. Other cytokines and growth factors, such as platelet-derived growth factor and tumor necrosis factor-alpha, despite their profibrotic activity, do not induce alpha-SM actin in myofibroblasts. In situ hybridization with an alpha-SM actin probe shows a high level of alpha-SM actin mRNA expression in myofibroblasts of TGF beta 1-induced granulation tissue. Moreover, TGF beta 1 induces alpha-SM actin protein and mRNA expression in growing and quiescent cultured fibroblasts and preincubation of culture medium containing whole blood serum with neutralizing antibodies to TGF beta 1 results in a decrease of alpha-SM actin expression by fibroblasts in replicative and non-replicative conditions. These results suggest that TGF beta 1 plays an important role in myofibroblast differentiation during wound healing and fibrocontractive diseases by regulating the expression of alpha-SM actin in these cells. PMID:8314838

  6. 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.

  7. Cytoskeletal social networking in the growth cone: how +TIPs mediate microtubule-actin cross-linking to drive axon outgrowth and guidance

    PubMed Central

    Cammarata, Garrett M.; Bearce, Elizabeth A.; Lowery, Laura Anne

    2016-01-01

    The growth cone is a unique structure capable of guiding axons to their proper destinations. Within the growth cone, extracellular guidance cues are interpreted and then transduced into physical changes in the actin filament (F-actin) and microtubule cytoskeletons, providing direction and movement. While both cytoskeletal networks individually possess important growth cone-specific functions, recent data over the past several years point towards a more cooperative role between the two systems. Facilitating this interaction between F-actin and microtubules, microtubule plus-end tracking proteins (+TIPs) have been shown to link the two cytoskeletons together. Evidence suggests that many +TIPs can couple microtubules to F-actin dynamics, supporting both microtubule advance and retraction in the growth cone periphery. In addition, growing in vitro and in vivo data support a secondary role for +TIPs in which they may participate as F-actin nucleators, thus directly influencing F-actin dynamics and organization. This review focuses on how +TIPs may link F-actin and microtubules together in the growth cone, and how these interactions may influence axon guidance. PMID:26783725

  8. Structural and viscoelastic properties of actin networks formed by espin or pathologically relevant espin mutants.

    PubMed

    Lieleg, Oliver; Schmoller, Kurt M; Purdy Drew, Kirstin R; Claessens, Mireille M A E; Semmrich, Christine; Zheng, Lili; Bartles, James R; Bausch, Andreas R

    2009-11-09

    The structural organization of the cytoskeleton determines its viscoelastic response which is crucial for the correct functionality of living cells. Both the mechanical response and microstructure of the cytoskeleton are regulated on a microscopic level by the local activation of different actin binding and/or bundling proteins (ABPs). Misregulations in the expression of these ABPs or mutations in their sequence can entail severe cellular dysfunctions and diseases. Here, we study the structural and viscoelastic properties of reconstituted actin networks cross-linked by the ABP espin and compare the obtained network properties to those of other bundled actin networks. Moreover, we quantify the impact of pathologically relevant espin mutations on the viscoelastic properties of these cytoskeletal networks.

  9. Quantifying the contribution of actin networks to the elastic strength of fibroblasts.

    PubMed

    Ananthakrishnan, Revathi; Guck, Jochen; Wottawah, Falk; Schinkinger, Stefan; Lincoln, Bryan; Romeyke, Maren; Moon, Tess; Käs, Josef

    2006-09-21

    The structural models created to understand the cytoskeletal mechanics of cells in suspension are described here. Suspended cells can be deformed by well-defined surface stresses in an Optical Stretcher [Guck, J., Ananthakrishnan, R., Mahmood, H., Moon, T.J., Cunningham, C.C., Käs, J., 2001. The optical stretcher: a novel laser tool to micromanipulate cells. Biophys. J. 81(2), 767-784], a two-beam optical trap designed for the contact-free deformation of cells. Suspended cells have a well-defined cytoskeleton, displaying a radially symmetric actin cortical network underlying the cell membrane with no actin stress fibers, and microtubules and intermediate filaments in the interior. Based on experimental data using suspended fibroblasts, we create two structural models: a thick shell actin cortex model that describes cell deformation for a localized stress distribution on these cells and a three-layered model that considers the entire cytoskeleton when a broad stress distribution is applied. Applying the models to data, we obtain a (actin) cortical shear moduli G of approximately 220 Pa for normal fibroblasts and approximately 185 Pa for malignantly transformed fibroblasts. Additionally, modeling the cortex as a transiently crosslinked isotropic actin network, we show that actin and its crosslinkers must be co-localized into a tight shell to achieve these cortical strengths. The similar moduli values and cortical actin and crosslinker densities but different deformabilities of the normal and cancerous cells suggest that a cell's structural strength is not solely determined by cytoskeletal composition but equally importantly by (actin) cytoskeletal architecture via differing cortical thicknesses. We also find that although the interior structural elements (microtubules, nucleus) contribute to the deformed cell's exact shape via their loose coupling to the cortex, it is the outer actin cortical shell (and its thickness) that mainly determines the cell's structural

  10. Surface adsorption and hopping cause probe-size-dependent microrheology of actin networks

    NASA Astrophysics Data System (ADS)

    He, Jun; Tang, Jay X.

    2011-04-01

    A network of filaments formed primarily by the abundant cytoskeletal protein actin gives animal cells their shape and elasticity. The rheological properties of reconstituted actin networks have been studied by tracking micron-sized probe beads embedded within the networks. We investigate how microrheology depends on surface properties of probe particles by varying the stickiness of their surface. For this purpose, we chose carboxylate polystyrene (PS) beads, silica beads, bovine serum albumin (BSA) -coated PS beads, and polyethylene glycol (PEG) -grafted PS beads, which show descending stickiness to actin filaments, characterized by confocal imaging and microrheology. Probe size dependence of microrheology is observed for all four types of beads. For the slippery PEG beads, particle-tracking microrheology detects weaker networks using smaller beads, which tend to diffuse through the network by hopping from one confinement “cage” to another. This trend is reversed for the other three types of beads, for which microrheology measures stiffer networks for smaller beads due to physisorption of nearby filaments to the bead surface. We explain the probe size dependence with two simple models. We also evaluate depletion effect near nonadsorption bead surface using quantitative image analysis and discuss the possible impact of depletion on microrheology. Analysis of these effects is necessary in order to accurately define the actin network rheology both in vitro and in vivo.

  11. Formation of regularly spaced networks as a general feature of actin bundle condensation by entropic forces

    NASA Astrophysics Data System (ADS)

    Huber, Florian; Strehle, Dan; Schnauß, Jörg; Käs, Josef

    2015-04-01

    Biopolymer networks contribute mechanical integrity as well as functional organization to living cells. One of their major constituents, the protein actin, is present in a large variety of different network architectures, ranging from extensive networks to densely packed bundles. The shape of the network is directly linked to its mechanical properties and essential physiological functions. However, a profound understanding of architecture-determining mechanisms and their physical constraints remains elusive. We use experimental bottom-up systems to study the formation of confined actin networks by entropic forces. Experiments based on molecular crowding as well as counterion condensation reveal a generic tendency of homogeneous filament solutions to aggregate into regular actin bundle networks connected by aster-like centers. The network architecture is found to critically rely on network formation history. Starting from identical biochemical compositions, we observe drastic changes in network architecture as a consequence of initially biased filament orientation or mixing-induced perturbations. Our experiments suggest that the tendency to form regularly spaced bundle networks is a rather general feature of isotropic, homogeneous filament solutions subject to uniform attractive interactions. Due to the fundamental nature of the considered interactions, we expect that the investigated type of network formation further implies severe physical constraints for cytoskeleton self-organization on the more complex level of living cells.

  12. Auxins and Cytokinins as Antipodal Modulators of Elasticity within the Actin Network of Plant Cells.

    PubMed Central

    Grabski, S.; Schindler, M.

    1996-01-01

    The cytoskeleton of plant and animal cells serves as a transmitter, transducer, and effector of cell signaling mechanisms. In plants, pathways for proliferation, differentiation, intracellular vesicular transport, cell-wall biosynthesis, symbiosis, secretion, and membrane recycling depend on the organization and dynamic properties of actin- and tubulin-based structures that are either associated with the plasma membrane or traverse the cytoplasm. Recently, a new in vivo cytoskeletal assay (cell optical displacement assay) was introduced to measure the tension within subdomains (cortical, transvacuolar, and perinuclear) of the actin network in living plant cells. Cell optical displacement assay measurements within soybean (Glycine max [L.]) root cells previously demonstrated that lipophilic signals, e.g. linoleic acid and arachidonic acid or changes in cytoplasmic pH gradients, could induce significant reductions in the tension within the actin network of transvacuolar strands. In contrast, enhancement of cytoplasmic free Ca2+ resulted in an increase in tension. In the present communication we have used these measurements to show that a similar antipodal pattern of activity exists for auxins and cytokinins (in their ability to modify the tension within the actin network of plant cells). It is suggested that these growth substances exert their effect on the cytoskeleton through the activation of signaling cascades, which result in the production of lipophilic and ionic second messengers, both of which have been demonstrated to directly effect the tension within the actin network of soybean root cells. PMID:12226233

  13. Strain hardening, avalanches, and strain softening in dense cross-linked actin networks

    NASA Astrophysics Data System (ADS)

    Åström, Jan A.; Kumar, P. B. Sunil; Vattulainen, Ilpo; Karttunen, Mikko

    2008-05-01

    Actin filament networks enable the cytoskeleton to adjust to internal and external forcing. These dynamic networks can adapt to changes by dynamically adjusting their cross-links. Here, we model actin filaments as cross-linked elastic fibers of finite dimensions, with the cross-links being approximately 1μm apart, and employ a full three-dimensional model to study their elastic properties by computer simulations. The results show compelling evidence that dense actin networks are characterized by (a) strain hardening without entropic elasticity, (b) avalanches of cross-link slippage leading to strain softening in the case of breakable cross-links, and (c) spontaneous formation of stress fibers in the case of dynamic cross-link formation and destruction.

  14. Mechanics of actin networks crosslinked with mutant human α-actinin-4

    NASA Astrophysics Data System (ADS)

    Volkmer, Sabine; Blair, Daniel; Kasza, Karen; Weitz, David

    2007-03-01

    Globular actin can be polymerized in vitro to form F-actin in the presence of various binding proteins. These networks often exhibit dramatic nonlinear rheological response to imposed strains. We study the rheological properties of F-actin networks crosslinked with human α-actinin-4. A single genetic mutation of the α-actinin-4 protein is associated with focal and segmented glomerulosclerosis (FSGS), a genetic disorder which leads to renal failure. Mechanically, the mutant crosslinker has an increased binding strength compared to the wild type. We will show that human α-actinin-4, displays a unique stiffening response. Moreover, we also demonstrate that a single point mutation dramatically effects the inherent relaxation time of the crosslinked network.

  15. Natural Gas Pipeline Network: Changing and Growing

    EIA Publications

    1996-01-01

    This chapter focuses upon the capabilities of the national natural gas pipeline network, examining how it has expanded during this decade and how it may expand further over the coming years. It also looks at some of the costs of this expansion, including the environmental costs which may be extensive. Changes in the network as a result of recent regional market shifts are also discussed.

  16. How does the antagonism between capping and anti-capping proteins affect actin network dynamics?

    NASA Astrophysics Data System (ADS)

    Hu, Longhua; Papoian, Garegin A.

    2011-09-01

    Actin-based cell motility is essential to many biological processes. We built a simplified, three-dimensional computational model and subsequently performed stochastic simulations to study the growth dynamics of lamellipodia-like branched networks. In this work, we shed light on the antagonism between capping and anti-capping proteins in regulating actin dynamics in the filamentous network. We discuss detailed mechanisms by which capping and anti-capping proteins affect the protrusion speed of the actin network and the rate of nucleation of filaments. We computed a phase diagram showing the regimes of motility enhancement and inhibition by these proteins. Our work shows that the effects of capping and anti-capping proteins are mainly transmitted by modulation of the filamentous network density and local availability of monomeric actin. We discovered that the combination of the capping/anti-capping regulatory network with nucleation-promoting proteins introduces robustness and redundancy in cell motility machinery, allowing the cell to easily achieve maximal protrusion speeds under a broader set of conditions. Finally, we discuss distributions of filament lengths under various conditions and speculate on their potential implication for the emergence of filopodia from the lamellipodial network.

  17. How Do Online Social Networks Grow?

    PubMed Central

    Zhu, Konglin; Li, Wenzhong; Fu, Xiaoming; Nagler, Jan

    2014-01-01

    Online social networks such as Facebook, Twitter and Gowalla allow people to communicate and interact across borders. In past years online social networks have become increasingly important for studying the behavior of individuals, group formation, and the emergence of online societies. Here we focus on the characterization of the average growth of online social networks and try to understand which are possible processes behind seemingly long-range temporal correlated collective behavior. In agreement with recent findings, but in contrast to Gibrat's law of proportionate growth, we find scaling in the average growth rate and its standard deviation. In contrast, Renren and Twitter deviate, however, in certain important aspects significantly from those found in many social and economic systems. Whereas independent methods suggest no significance for temporally long-range correlated behavior for Renren and Twitter, a scaling analysis of the standard deviation does suggest long-range temporal correlated growth in Gowalla. However, we demonstrate that seemingly long-range temporal correlations in the growth of online social networks, such as in Gowalla, can be explained by a decomposition into temporally and spatially independent growth processes with a large variety of entry rates. Our analysis thus suggests that temporally or spatially correlated behavior does not play a major role in the growth of online social networks. PMID:24940744

  18. An epidermal plakin that integrates actin and microtubule networks at cellular junctions.

    PubMed

    Karakesisoglou, I; Yang, Y; Fuchs, E

    2000-04-03

    Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.

  19. Complex growing networks with intrinsic vertex fitness

    SciTech Connect

    Bedogne, C.; Rodgers, G. J.

    2006-10-15

    One of the major questions in complex network research is to identify the range of mechanisms by which a complex network can self organize into a scale-free state. In this paper we investigate the interplay between a fitness linking mechanism and both random and preferential attachment. In our models, each vertex is assigned a fitness x, drawn from a probability distribution {rho}(x). In Model A, at each time step a vertex is added and joined to an existing vertex, selected at random, with probability p and an edge is introduced between vertices with fitnesses x and y, with a rate f(x,y), with probability 1-p. Model B differs from Model A in that, with probability p, edges are added with preferential attachment rather than randomly. The analysis of Model A shows that, for every fixed fitness x, the network's degree distribution decays exponentially. In Model B we recover instead a power-law degree distribution whose exponent depends only on p, and we show how this result can be generalized. The properties of a number of particular networks are examined.

  20. The actin network in the ciliary stalk of photoreceptors functions in the generation of new outer segment discs.

    PubMed

    Hale, I L; Fisher, S K; Matsumoto, B

    1996-12-02

    Cytochalasin D (CD) interferes with the morphogenesis of outer segment disc membrane in photoreceptors. Disruption of either the actin network in the ciliary stalk, where membrane evagination is initiated, or the actin core of the calycal processes, whose position could define the disc perimeter, could be responsible. We have attempted to determine which of these local F-actin populations is involved in membrane morphogenesis and what step in the process is actin-dependent. Biocytin accumulation in nascent discs, detected by fluorescent avidin and laser scanning confocal microscopy (LSCM), provided a means of labeling abnormal discs and a measure of disc membrane addition. F-actin content and distribution were assessed using fluorescent phalloidin and LSCM. First, we examined the effects of a range of CD dosages (0.1, 1.0, or 10.0 microM) on rod photoreceptors in Xenopus laevis eyecup cultures. Ectopic outgrowth of discs, evaluated by LSCM and transmission electron microscopy (TEM), occurred at each concentration. Phalloidin labeling intensified in the ciliary stalk with increasing CD concentration, indicating F-actin aggregation. In contrast, it diminished in the calycal processes, indicating dispersal; TEM showed that calycal process collapse ensued. Disruption was evident at a lower concentration in the ciliary stalk (0.1 microM) than in the calycal processes (1.0 microM). TEM confirmed that the calycal processes remained intact at 0.1 microM. Thus, CD's action on the ciliary stalk network is sufficient to disrupt disc morphogenesis. Second, we examined the effect of CD on temperature-induced acceleration of the rate of disc formation. In the absence of CD, a 10 degrees C temperature shift increased the disc formation rate nearly three-fold. CD (5 microM) caused a 94% inhibition (P < 0.025) of this response; yet, the rate of membrane addition to ectopically growing discs exhibited the expected three-fold increase. Thus, CD's action interferes with the generation

  1. Growing networks with geographical attachment preference: emergence of small worlds.

    PubMed

    Ozik, Jonathan; Hunt, Brian R; Ott, Edward

    2004-02-01

    We introduce a simple mechanism for the evolution of small world networks. Our model is a growing network in which all connections are made locally to geographically nearby sites. Although connections are made purely locally, network growth leads to stretching of old connections and to high clustering. Our results suggest that the abundance of small world networks in geographically constrained systems is a natural consequence of system growth and local interactions.

  2. Growing multiplex networks with arbitrary number of layers

    NASA Astrophysics Data System (ADS)

    Momeni, Naghmeh; Fotouhi, Babak

    2015-12-01

    This paper focuses on the problem of growing multiplex networks. Currently, the results on the joint degree distribution of growing multiplex networks present in the literature pertain to the case of two layers and are confined to the special case of homogeneous growth and are limited to the state state (that is, the limit of infinite size). In the present paper, we first obtain closed-form solutions for the joint degree distribution of heterogeneously growing multiplex networks with arbitrary number of layers in the steady state. Heterogeneous growth means that each incoming node establishes different numbers of links in different layers. We consider both uniform and preferential growth. We then extend the analysis of the uniform growth mechanism to arbitrary times. We obtain a closed-form solution for the time-dependent joint degree distribution of a growing multiplex network with arbitrary initial conditions. Throughout, theoretical findings are corroborated with Monte Carlo simulations. The results shed light on the effects of the initial network on the transient dynamics of growing multiplex networks and takes a step towards characterizing the temporal variations of the connectivity of growing multiplex networks, as well as predicting their future structural properties.

  3. Action of the mechanical disruption of the actin network on the gravisensitivity of the root statocyte

    NASA Astrophysics Data System (ADS)

    Lefranc, A.; Jeune, B.; Driss-Ecole, D.; Perbal, G.

    The effects of the mechanical disruption of the thin actin network of statocytes on gravisensitivity have been studied on lentil roots. Seedling roots were first inverted for 7 min (root tip upward) and then placed in the downward (normal) position for 7 min before gravitropic stimulation in the horizontal position. The period of inversion allowed the amyloplasts to move from the distal part to the proximal part of the statocyte, but did not fully sediment. When the roots were returned to the tip down position, the amyloplasts moved toward the distal part, but also did not completely sediment by the time the roots were placed horizontally. Thus, in these roots the amyloplasts could be still moving toward the distal wall after they had been replaced in the normal position and the actin network should not be fully restored. Gravisensitivity was estimated by the analysis of the dose-response curves of vertical and treated (inverted and returned to downward position) roots. The only effect, which has been observed on treated roots, was a delay of graviresponse for about 1 min. Our interpretation of this result is that in vertical roots the amyloplasts can exert tensions in the actin network that are directly transmitted to mechanoreceptors located in the plasma membrane. In roots with a partially disrupted actin network, a delay of 1 min is necessary for the amyloplasts to activate mechanoreceptors.

  4. Buckling-induced F-actin fragmentation modulates the contraction of active cytoskeletal networks.

    PubMed

    Li, Jing; Biel, Thomas; Lomada, Pranith; Yu, Qilin; Kim, Taeyoon

    2017-04-11

    Actomyosin contractility originating from interactions between F-actin and myosin facilitates various structural reorganizations of the actin cytoskeleton. Cross-linked actomyosin networks show a tendency to contract to single or multiple foci, which has been investigated extensively in numerous studies. Recently, it was suggested that suppression of F-actin buckling via an increase in bending rigidity significantly reduces network contraction. In this study, we demonstrate that networks may show the largest contraction at intermediate bending rigidity, not at the lowest rigidity, if filaments are severed by buckling arising from myosin activity as demonstrated in recent experiments; if filaments are very flexible, frequent severing events can severely deteriorate network connectivity, leading to the formation of multiple small foci and low network contraction. By contrast, if filaments are too stiff, the networks exhibit minimal contraction due to the inhibition of filament buckling. This study reveals that buckling-induced filament severing can modulate the contraction of active cytoskeletal networks, which has been neglected to date.

  5. Region growing with pulse-coupled neural networks: an alternative to seeded region growing.

    PubMed

    Stewart, R D; Fermin, I; Opper, M

    2002-01-01

    The seeded region growing (SRG) algorithm is a fast robust parameter-free method for segmenting intensity images given initial seed locations for each region. The requirement of predetermined seeds means that the model cannot operate fully autonomously. In this paper, we demonstrate a novel region growing variant of the pulse-coupled neural network (PCNN), which offers comparable performance to the SRG and is able to generate seed locations internally, opening the way to fully autonomous operation.

  6. A novel p21-activated kinase binds the actin and microtubule networks and induces microtubule stabilization

    PubMed Central

    Cau, Julien; Faure, Sandrine; Comps, Michel; Delsert, Claude; Morin, Nathalie

    2001-01-01

    Coordination of the different cytoskeleton networks in the cell is of central importance for morphogenesis, organelle transport, and motility. The Rho family proteins are well characterized for their effects on the actin cytoskeleton, but increasing evidence indicates that they may also control microtubule (MT) dynamics. Here, we demonstrate that a novel Cdc42/Rac effector, X-p21-activated kinase (PAK)5, colocalizes and binds to both the actin and MT networks and that its subcellular localization is regulated during cell cycle progression. In transfected cells, X-PAK5 promotes the formation of stabilized MTs that are associated in bundles and interferes with MTs dynamics, slowing both the elongation and shrinkage rates and inducing long paused periods. X-PAK5 subcellular localization is regulated tightly, since coexpression with active Rac or Cdc42 induces its shuttling to actin-rich structures. Thus, X-PAK5 is a novel MT-associated protein that may communicate between the actin and MT networks during cellular responses to environmental conditions. PMID:11733543

  7. An Epidermal Plakin That Integrates Actin and Microtubule Networks at Cellular Junctions

    PubMed Central

    Karakesisoglou, Iakowos; Yang, Yanmin; Fuchs, Elaine

    2000-01-01

    Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal–muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (Kd = 0.35 μM) and one microtubule (Kd ∼6 μM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell–cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin–microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies. PMID:10747097

  8. Local F-actin network links synapse formation and axon branching.

    PubMed

    Chia, Poh Hui; Chen, Baoyu; Li, Pengpeng; Rosen, Michael K; Shen, Kang

    2014-01-16

    Axonal branching and synapse formation are tightly linked developmental events during the establishment of synaptic circuits. Newly formed synapses promote branch initiation and stability. However, little is known about molecular mechanisms that link these two processes. Here, we show that local assembly of an F-actin cytoskeleton at nascent presynaptic sites initiates both synapse formation and axon branching. We further find that assembly of the F-actin network requires a direct interaction between the synaptic cell adhesion molecule SYG-1 and a key regulator of actin cytoskeleton, the WVE-1/WAVE regulatory complex (WRC). SYG-1 cytoplasmic tail binds to the WRC using a consensus WRC interacting receptor sequence (WIRS). WRC mutants or mutating the SYG-1 WIRS motif leads to loss of local F-actin, synaptic material, and axonal branches. Together, these data suggest that synaptic adhesion molecules, which serve as a necessary component for both synaptogenesis and axonal branch formation, directly regulate subcellular actin cytoskeletal organization. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2013-01-01

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

  10. Actin and microtubule networks contribute differently to cell response for small and large strains

    NASA Astrophysics Data System (ADS)

    Kubitschke, H.; Schnauss, J.; Nnetu, K. D.; Warmt, E.; Stange, R.; Kaes, J.

    2017-09-01

    Cytoskeletal filaments provide cells with mechanical stability and organization. The main key players are actin filaments and microtubules governing a cell’s response to mechanical stimuli. We investigated the specific influences of these crucial components by deforming MCF-7 epithelial cells at small (≤5% deformation) and large strains (>5% deformation). To understand specific contributions of actin filaments and microtubules, we systematically studied cellular responses after treatment with cytoskeleton influencing drugs. Quantification with the microfluidic optical stretcher allowed capturing the relative deformation and relaxation of cells under different conditions. We separated distinctive deformational and relaxational contributions to cell mechanics for actin and microtubule networks for two orders of magnitude of drug dosages. Disrupting actin filaments via latrunculin A, for instance, revealed a strain-independent softening. Stabilizing these filaments by treatment with jasplakinolide yielded cell softening for small strains but showed no significant change at large strains. In contrast, cells treated with nocodazole to disrupt microtubules displayed a softening at large strains but remained unchanged at small strains. Stabilizing microtubules within the cells via paclitaxel revealed no significant changes for deformations at small strains, but concentration-dependent impact at large strains. This suggests that for suspended cells, the actin cortex is probed at small strains, while at larger strains; the whole cell is probed with a significant contribution from the microtubules.

  11. Prestressed F-actin networks cross-linked by hinged filamins replicate mechanical properties of cells

    NASA Astrophysics Data System (ADS)

    Gardel, M. L.; Nakamura, F.; Hartwig, J. H.; Crocker, J. C.; Stossel, T. P.; Weitz, D. A.

    2006-02-01

    We show that actin filaments, shortened to physiological lengths by gelsolin and cross-linked with recombinant human filamins (FLNs), exhibit dynamic elastic properties similar to those reported for live cells. To achieve elasticity values of comparable magnitude to those of cells, the in vitro network must be subjected to external prestress, which directly controls network elasticity. A molecular requirement for the strain-related behavior at physiological conditionsis a flexible hinge found in FLNa and some FLNb molecules. Basic physical properties of the in vitro filamin-F-actin network replicate the essential mechanical properties of living cells. This physical behavior could accommodate passive deformation and internal organelle trafficking at low strains yet resist externally or internally generated high shear forces. cytoskeleton | cell mechanics | nonlinear rheology

  12. The Eps8/IRSp53/VASP Network Differentially Controls Actin Capping and Bundling in Filopodia Formation

    PubMed Central

    Milanesi, Francesca; Di Fiore, Pier Paolo; Menna, Elisabetta; Matteoli, Michela; Gov, Nir S.; Scita, Giorgio; Ciliberto, Andrea

    2011-01-01

    There is a body of literature that describes the geometry and the physics of filopodia using either stochastic models or partial differential equations and elasticity and coarse-grained theory. Comparatively, there is a paucity of models focusing on the regulation of the network of proteins that control the formation of different actin structures. Using a combination of in-vivo and in-vitro experiments together with a system of ordinary differential equations, we focused on a small number of well-characterized, interacting molecules involved in actin-dependent filopodia formation: the actin remodeler Eps8, whose capping and bundling activities are a function of its ligands, Abi-1 and IRSp53, respectively; VASP and Capping Protein (CP), which exert antagonistic functions in controlling filament elongation. The model emphasizes the essential role of complexes that contain the membrane deforming protein IRSp53, in the process of filopodia initiation. This model accurately accounted for all observations, including a seemingly paradoxical result whereby genetic removal of Eps8 reduced filopodia in HeLa, but increased them in hippocampal neurons, and generated quantitative predictions, which were experimentally verified. The model further permitted us to explain how filopodia are generated in different cellular contexts, depending on the dynamic interaction established by Eps8, IRSp53 and VASP with actin filaments, thus revealing an unexpected plasticity of the signaling network that governs the multifunctional activities of its components in the formation of filopodia. PMID:21814501

  13. Synergistic action of dendritic mitochondria and creatine kinase maintains ATP homeostasis and actin dynamics in growing neuronal dendrites.

    PubMed

    Fukumitsu, Kansai; Fujishima, Kazuto; Yoshimura, Azumi; Wu, You Kure; Heuser, John; Kengaku, Mineko

    2015-04-08

    The distribution of mitochondria within mature, differentiated neurons is clearly adapted to their regional physiological needs and can be perturbed under various pathological conditions, but the function of mitochondria in developing neurons has been less well studied. We have studied mitochondrial distribution within developing mouse cerebellar Purkinje cells and have found that active delivery of mitochondria into their dendrites is a prerequisite for proper dendritic outgrowth. Even when mitochondria in the Purkinje cell bodies are functioning normally, interrupting the transport of mitochondria into their dendrites severely disturbs dendritic growth. Additionally, we find that the growth of atrophic dendrites lacking mitochondria can be rescued by activating ATP-phosphocreatine exchange mediated by creatine kinase (CK). Conversely, inhibiting cytosolic CKs decreases dendritic ATP levels and also disrupts dendrite development. Mechanistically, this energy depletion appears to perturb normal actin dynamics and enhance the aggregation of cofilin within growing dendrites, reminiscent of what occurs in neurons overexpressing the dephosphorylated form of cofilin. These results suggest that local ATP synthesis by dendritic mitochondria and ATP-phosphocreatine exchange act synergistically to sustain the cytoskeletal dynamics necessary for dendritic development.

  14. Dynamics of epidemic diseases on a growing adaptive network

    NASA Astrophysics Data System (ADS)

    Demirel, Güven; Barter, Edmund; Gross, Thilo

    2017-02-01

    The study of epidemics on static networks has revealed important effects on disease prevalence of network topological features such as the variance of the degree distribution, i.e. the distribution of the number of neighbors of nodes, and the maximum degree. Here, we analyze an adaptive network where the degree distribution is not independent of epidemics but is shaped through disease-induced dynamics and mortality in a complex interplay. We study the dynamics of a network that grows according to a preferential attachment rule, while nodes are simultaneously removed from the network due to disease-induced mortality. We investigate the prevalence of the disease using individual-based simulations and a heterogeneous node approximation. Our results suggest that in this system in the thermodynamic limit no epidemic thresholds exist, while the interplay between network growth and epidemic spreading leads to exponential networks for any finite rate of infectiousness when the disease persists.

  15. Dynamics of epidemic diseases on a growing adaptive network

    PubMed Central

    Demirel, Güven; Barter, Edmund; Gross, Thilo

    2017-01-01

    The study of epidemics on static networks has revealed important effects on disease prevalence of network topological features such as the variance of the degree distribution, i.e. the distribution of the number of neighbors of nodes, and the maximum degree. Here, we analyze an adaptive network where the degree distribution is not independent of epidemics but is shaped through disease-induced dynamics and mortality in a complex interplay. We study the dynamics of a network that grows according to a preferential attachment rule, while nodes are simultaneously removed from the network due to disease-induced mortality. We investigate the prevalence of the disease using individual-based simulations and a heterogeneous node approximation. Our results suggest that in this system in the thermodynamic limit no epidemic thresholds exist, while the interplay between network growth and epidemic spreading leads to exponential networks for any finite rate of infectiousness when the disease persists. PMID:28186146

  16. Temporal dynamics of connectivity and epidemic properties of growing networks

    NASA Astrophysics Data System (ADS)

    Fotouhi, Babak; Shirkoohi, Mehrdad Khani

    2016-01-01

    Traditional mathematical models of epidemic disease had for decades conventionally considered static structure for contacts. Recently, an upsurge of theoretical inquiry has strived towards rendering the models more realistic by incorporating the temporal aspects of networks of contacts, societal and online, that are of interest in the study of epidemics (and other similar diffusion processes). However, temporal dynamics have predominantly focused on link fluctuations and nodal activities, and less attention has been paid to the growth of the underlying network. Many real networks grow: Online networks are evidently in constant growth, and societal networks can grow due to migration flux and reproduction. The effect of network growth on the epidemic properties of networks is hitherto unknown, mainly due to the predominant focus of the network growth literature on the so-called steady state. This paper takes a step towards alleviating this gap. We analytically study the degree dynamics of a given arbitrary network that is subject to growth. We use the theoretical findings to predict the epidemic properties of the network as a function of time. We observe that the introduction of new individuals into the network can enhance or diminish its resilience against endemic outbreaks and investigate how this regime shift depends upon the connectivity of newcomers and on how they establish connections to existing nodes. Throughout, theoretical findings are corroborated with Monte Carlo simulations over synthetic and real networks. The results shed light on the effects of network growth on the future epidemic properties of networks and offers insights for devising a priori immunization strategies.

  17. MicroFilament Analyzer identifies actin network organizations in epidermal cells of Arabidopsis thaliana roots.

    PubMed

    Jacques, Eveline; Lewandowski, Michal; Buytaert, Jan; Fierens, Yves; Verbelen, Jean-Pierre; Vissenberg, Kris

    2013-07-01

    The plant cytoskeleton plays a crucial role in the cells' growth and development during different developmental stages and it undergoes many rearrangements. In order to describe the arrangements of the F-actin cytoskeleton in root epidermal cells of Arabidopsis thaliana, the recently developed software MicroFilament Analyzer (MFA) was exploited. This software enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way. Using confocal microscopy and transgenic GFP-FABD2-GFP plants the actin cytoskeleton was visualized in the root epidermis. MFA analysis revealed that during the early stages of cell development F-actin is organized in a mainly random pattern. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. In the evolution from young to old cells, an approximately even distribution of transverse, oblique or combined orientations is always present besides the switch from random to a longitudinal oriented actin cytoskeleton.

  18. MicroFilament Analyzer identifies actin network organizations in epidermal cells of Arabidopsis thaliana roots

    PubMed Central

    Jacques, Eveline; Lewandowski, Michal; Buytaert, Jan; Fierens, Yves; Verbelen, Jean-Pierre; Vissenberg, Kris

    2013-01-01

    The plant cytoskeleton plays a crucial role in the cells’ growth and development during different developmental stages and it undergoes many rearrangements. In order to describe the arrangements of the F-actin cytoskeleton in root epidermal cells of Arabidopsis thaliana, the recently developed software MicroFilament Analyzer (MFA) was exploited. This software enables high-throughput identification and quantification of the orientation of filamentous structures on digital images in a highly standardized and fast way. Using confocal microscopy and transgenic GFP-FABD2-GFP plants the actin cytoskeleton was visualized in the root epidermis. MFA analysis revealed that during the early stages of cell development F-actin is organized in a mainly random pattern. As the cells grow, they preferentially adopt a longitudinal organization, a pattern that is also preserved in the largest cells. In the evolution from young to old cells, an approximately even distribution of transverse, oblique or combined orientations is always present besides the switch from random to a longitudinal oriented actin cytoskeleton. PMID:23656865

  19. Nonequilibrium dynamics of probe filaments in actin-myosin networks

    NASA Astrophysics Data System (ADS)

    Gladrow, J.; Broedersz, C. P.; Schmidt, C. F.

    2017-08-01

    Active dynamic processes of cells are largely driven by the cytoskeleton, a complex and adaptable semiflexible polymer network, motorized by mechanoenzymes. Small dimensions, confined geometries, and hierarchical structures make it challenging to probe dynamics and mechanical response of such networks. Embedded semiflexible probe polymers can serve as nonperturbing multiscale probes to detect force distributions in active polymer networks. We show here that motor-induced forces transmitted to the probe polymers are reflected in nonequilibrium bending dynamics, which we analyze in terms of spatial eigenmodes of an elastic beam under steady-state conditions. We demonstrate how these active forces induce correlations among the mode amplitudes, which furthermore break time-reversal symmetry. This leads to a breaking of detailed balance in this mode space. We derive analytical predictions for the magnitude of resulting probability currents in mode space in the white-noise limit of motor activity. We relate the structure of these currents to the spatial profile of motor-induced forces along the probe polymers and provide a general relation for observable currents on two-dimensional hyperplanes.

  20. Nonequilibrium dynamics of probe filaments in actin-myosin networks.

    PubMed

    Gladrow, J; Broedersz, C P; Schmidt, C F

    2017-08-01

    Active dynamic processes of cells are largely driven by the cytoskeleton, a complex and adaptable semiflexible polymer network, motorized by mechanoenzymes. Small dimensions, confined geometries, and hierarchical structures make it challenging to probe dynamics and mechanical response of such networks. Embedded semiflexible probe polymers can serve as nonperturbing multiscale probes to detect force distributions in active polymer networks. We show here that motor-induced forces transmitted to the probe polymers are reflected in nonequilibrium bending dynamics, which we analyze in terms of spatial eigenmodes of an elastic beam under steady-state conditions. We demonstrate how these active forces induce correlations among the mode amplitudes, which furthermore break time-reversal symmetry. This leads to a breaking of detailed balance in this mode space. We derive analytical predictions for the magnitude of resulting probability currents in mode space in the white-noise limit of motor activity. We relate the structure of these currents to the spatial profile of motor-induced forces along the probe polymers and provide a general relation for observable currents on two-dimensional hyperplanes.

  1. Crosslinked actin networks show liquid crystal elastomer behaviour, including soft-mode elasticity

    NASA Astrophysics Data System (ADS)

    Dalhaimer, Paul; Discher, Dennis E.; Lubensky, Tom C.

    2007-05-01

    Actin filament networks with protein crosslinks of distinct length and flexibility resemble liquid crystal elastomers. We simulate actin filament systems with flexible crosslinkers of varying length and connectivity to understand general phase behaviour and elasticity. Simulated networks with very short filaments and long crosslinkers resemble the cytoskeleton of the red blood cell and remain isotropic in compression and shear, seeming well-suited to blood flow. In contrast, networks with longer filaments as found in many cell types show three regimes of nematic phase behaviour dependent on crosslinker length: (1) `loose' networks are isotropic at zero stress but align under compression or shear; (2) `semi-loose' networks are nematic at low stress but become isotropic under dilation and (3) `tight' networks possess a locked-in nematic order as represented by the cytoskeleton of the outer hair cell in the ear, for which anisotropic compliance directs sound propagation. Furthermore, for a subset of loose networks with `periodic' connections among filaments, extremely soft stress-strain behaviour is found, as predicted for liquid crystal elastomers.

  2. The spatial response of nonlinear strain propagation in response to actively driven microspheres through entangled actin networks

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    The semiflexible biopolymer actin, a ubiquitous component of nearly all biological organisms, plays an important role in many mechanically-driven processes such as muscle contraction, cancer invasion and cell motility. As such, entangled actin networks, which possess unique and complex viscoelastic properties, have been the subject of much theoretical and experimental work. However, due to this viscoelastic complexity, much is still unknown regarding the correlation of the applied stress on actin networks to the induced filament strain at the molecular and micro scale. Here, we use simultaneous optical trapping and fluorescence microscopy to characterize the link between applied microscopic forces and strain propagation as a function of strain rate and concentration. Specifically, we track fiduciary markers on entangled actin filaments before, during and after actively driving embedded microspheres through the network. These measurements provide much needed insight into the molecular-level dynamics connecting stress and strain in semiflexible polymer networks.

  3. AKAP220 manages apical actin networks that coordinate aquaporin-2 location and renal water reabsorption

    PubMed Central

    Whiting, Jennifer L.; Ogier, Leah; Forbush, Katherine A.; Bucko, Paula; Gopalan, Janani; Seternes, Ole-Morten; Langeberg, Lorene K.; Scott, John D.

    2016-01-01

    Filtration through the kidney eliminates toxins, manages electrolyte balance, and controls water homeostasis. Reabsorption of water from the luminal fluid of the nephron occurs through aquaporin-2 (AQP2) water pores in principal cells that line the kidney-collecting duct. This vital process is impeded by formation of an “actin barrier” that obstructs the passive transit of AQP2 to the plasma membrane. Bidirectional control of AQP2 trafficking is managed by hormones and signaling enzymes. We have discovered that vasopressin-independent facets of this homeostatic mechanism are under the control of A-Kinase Anchoring Protein 220 (AKAP220; product of the Akap11 gene). CRISPR/Cas9 gene editing and imaging approaches show that loss of AKAP220 disrupts apical actin networks in organoid cultures. Similar defects are evident in tissue sections from AKAP220-KO mice. Biochemical analysis of AKAP220-null kidney extracts detected reduced levels of active RhoA GTPase, a well-known modulator of the actin cytoskeleton. Fluorescent imaging of kidney sections from these genetically modified mice revealed that RhoA and AQP2 accumulate at the apical surface of the collecting duct. Consequently, these animals are unable to appropriately dilute urine in response to overhydration. We propose that membrane-proximal signaling complexes constrained by AKAP220 impact the actin barrier dynamics and AQP2 trafficking to ensure water homeostasis. PMID:27402760

  4. Mixing properties of growing networks and Simpson's paradox.

    PubMed

    Capocci, Andrea; Colaiori, Francesca

    2006-08-01

    The mixing properties of networks are usually inferred by comparing the degree of a node with the average degree of its neighbors. This kind of analysis often leads to incorrect conclusions: Assortative patterns may appear reversed by a mechanism known as Simpson's paradox. We prove this fact by analytical calculations and simulations on three classes of growing networks based on preferential attachment and fitness, where the disassortative behavior observed is a spurious effect. Our results give a crucial contribution to the debate about the origin of disassortative mixing, since networks previously classified as disassortative reveal instead assortative behavior to a careful analysis.

  5. Competition for actin between two distinct F-actin networks defines a bistable switch for cell polarization.

    PubMed

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

    2015-11-01

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

  6. Two-population dynamics in a growing network model

    NASA Astrophysics Data System (ADS)

    Ivanova, Kristinka; Iordanov, Ivan

    2012-02-01

    We introduce a growing network evolution model with nodal attributes. The model describes the interactions between potentially violent V and non-violent N agents who have different affinities in establishing connections within their own population versus between the populations. The model is able to generate all stable triads observed in real social systems. In the framework of rate equations theory, we employ the mean-field approximation to derive analytical expressions of the degree distribution and the local clustering coefficient for each type of nodes. Analytical derivations agree well with numerical simulation results. The assortativity of the potentially violent network qualitatively resembles the connectivity pattern in terrorist networks that was recently reported. The assortativity of the network driven by aggression shows clearly different behavior than the assortativity of the networks with connections of non-aggressive nature in agreement with recent empirical results of an online social system.

  7. Mechanical output of myosin II motors is regulated by myosin filament size and actin network mechanics

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Alberts, Jonathan; Gardel, Margaret; Munro, Edwin

    2013-03-01

    The interactions of bipolar myosin II filaments with actin arrays are a predominate means of generating forces in numerous physiological processes including muscle contraction and cell migration. However, how the spatiotemporal regulation of these forces depends on motor mechanochemistry, bipolar filament size, and local actin mechanics is unknown. Here, we simulate myosin II motors with an agent-based model in which the motors have been benchmarked against experimental measurements. Force generation occurs in two distinct regimes characterized either by stable tension maintenance or by stochastic buildup and release; transitions between these regimes occur by changes to duty ratio and myosin filament size. The time required for building force to stall scales inversely with the stiffness of a network and the actin gliding speed of a motor. Finally, myosin motors are predicted to contract a network toward stiffer regions, which is consistent with experimental observations. Our representation of myosin motors can be used to understand how their mechanical and biochemical properties influence their observed behavior in a variety of in vitro and in vivo contexts.

  8. Actin-myosin network influences morphological response of neuronal cells to altered osmolarity.

    PubMed

    Bober, Brian G; Love, James M; Horton, Steven M; Sitnova, Mariya; Shahamatdar, Sina; Kannan, Ajay; Shah, Sameer B

    2015-04-01

    Acute osmotic fluctuations in the brain occur during a number of clinical conditions and can result in a variety of adverse neurological symptoms. Osmotic perturbation can cause changes in the volumes of intra- and extracellular fluid and, due to the rigidity of the skull, can alter intracranial pressure thus making it difficult to analyze purely osmotic effects in vivo. The present study aims to determine the effects of changes in osmolarity on SH-SY5Y human neuroblastoma cells in vitro, and the role of the actin-myosin network in regulating this response. Cells were exposed to hyper- or hypoosmotic media and morphological and cytoskeletal responses were recorded. Hyperosmotic shock resulted in a drop in cell body volume and planar area, a persisting shape deformation, and increases in cellular translocation. Hypoosmotic shock did not significantly alter planar area, but caused a transient increase in cell body volume and an increase in cellular translocation via the development of small protrusions rich in actin. Disruption of the actin-myosin network with latrunculin and blebbistatin resulted in changes to volume and shape regulation, and a decrease in cellular translocation. In both osmotic perturbations, no apparent disruptions to cytoskeletal integrity were observed by light microscopy. Overall, because osmotically induced changes persisted even after volume regulation occurred, it is possible that osmotic stress may play a larger role in neurological dysfunction than currently believed.

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

    PubMed Central

    Ingerman, Elena; Hsiao, Jennifer Ying

    2013-01-01

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

  10. Elasticity, adhesion and actin based propulsion

    NASA Astrophysics Data System (ADS)

    Gopinathan, Ajay

    2006-03-01

    When a cells crawls, its shape re-organizes via polymerization and depolymerization of actin filaments. The growing ends of the filaments are oriented towards the outside of the cell, and their polymerization pushes the cell membrane forwards. The same mechanism comes into play when the bacterial pathogen Listeria monocytogenes infects a cell. The bacterium hijacks the host cell's actin machinery to create an actin network (the actin comet tail) that propels the bacterium through cells and into neighboring cells. We propose a mechanism for how polymerization gives rise to motility that incorporates the effects of inhomogeneous polymerization. We treat the actin comet tail as an elastic continuum tethered to the rear of the bacterium. The interplay of polymerization and tethering gives rise to inhomogeneous stresses calculated with a finite element analysis. We quantitatively reproduce many distinctive features of actin propulsion that have been observed experimentally, including stepped motion, hopping, tail shape and the propulsion of flat surfaces.

  11. Mechanical Stress Induces Remodeling of Vascular Networks in Growing Leaves.

    PubMed

    Bar-Sinai, Yohai; Julien, Jean-Daniel; Sharon, Eran; Armon, Shahaf; Nakayama, Naomi; Adda-Bedia, Mokhtar; Boudaoud, Arezki

    2016-04-01

    Differentiation into well-defined patterns and tissue growth are recognized as key processes in organismal development. However, it is unclear whether patterns are passively, homogeneously dilated by growth or whether they remodel during tissue expansion. Leaf vascular networks are well-fitted to investigate this issue, since leaves are approximately two-dimensional and grow manyfold in size. Here we study experimentally and computationally how vein patterns affect growth. We first model the growing vasculature as a network of viscoelastic rods and consider its response to external mechanical stress. We use the so-called texture tensor to quantify the local network geometry and reveal that growth is heterogeneous, resembling non-affine deformations in composite materials. We then apply mechanical forces to growing leaves after veins have differentiated, which respond by anisotropic growth and reorientation of the network in the direction of external stress. External mechanical stress appears to make growth more homogeneous, in contrast with the model with viscoelastic rods. However, we reconcile the model with experimental data by incorporating randomness in rod thickness and a threshold in the rod growth law, making the rods viscoelastoplastic. Altogether, we show that the higher stiffness of veins leads to their reorientation along external forces, along with a reduction in growth heterogeneity. This process may lead to the reinforcement of leaves against mechanical stress. More generally, our work contributes to a framework whereby growth and patterns are coordinated through the differences in mechanical properties between cell types.

  12. Mechanical Stress Induces Remodeling of Vascular Networks in Growing Leaves

    PubMed Central

    Bar-Sinai, Yohai; Julien, Jean-Daniel; Sharon, Eran; Armon, Shahaf; Nakayama, Naomi; Adda-Bedia, Mokhtar; Boudaoud, Arezki

    2016-01-01

    Differentiation into well-defined patterns and tissue growth are recognized as key processes in organismal development. However, it is unclear whether patterns are passively, homogeneously dilated by growth or whether they remodel during tissue expansion. Leaf vascular networks are well-fitted to investigate this issue, since leaves are approximately two-dimensional and grow manyfold in size. Here we study experimentally and computationally how vein patterns affect growth. We first model the growing vasculature as a network of viscoelastic rods and consider its response to external mechanical stress. We use the so-called texture tensor to quantify the local network geometry and reveal that growth is heterogeneous, resembling non-affine deformations in composite materials. We then apply mechanical forces to growing leaves after veins have differentiated, which respond by anisotropic growth and reorientation of the network in the direction of external stress. External mechanical stress appears to make growth more homogeneous, in contrast with the model with viscoelastic rods. However, we reconcile the model with experimental data by incorporating randomness in rod thickness and a threshold in the rod growth law, making the rods viscoelastoplastic. Altogether, we show that the higher stiffness of veins leads to their reorientation along external forces, along with a reduction in growth heterogeneity. This process may lead to the reinforcement of leaves against mechanical stress. More generally, our work contributes to a framework whereby growth and patterns are coordinated through the differences in mechanical properties between cell types. PMID:27074136

  13. Self-organized gels in DNA/F-actin mixtures without crosslinkers: networks of induced nematic domains with tunable density.

    PubMed

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

    2008-11-21

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

  14. A growing social network model in geographical space

    NASA Astrophysics Data System (ADS)

    Antonioni, Alberto; Tomassini, Marco

    2017-09-01

    In this work we propose a new model for the generation of social networks that includes their often ignored spatial aspects. The model is a growing one and links are created either taking space into account, or disregarding space and only considering the degree of target nodes. These two effects can be mixed linearly in arbitrary proportions through a parameter. We numerically show that for a given range of the combination parameter, and for given mean degree, the generated network class shares many important statistical features with those observed in actual social networks, including the spatial dependence of connections. Moreover, we show that the model provides a good qualitative fit to some measured social networks.

  15. Microstructural model for cyclic hardening in F-actin networks crosslinked by α-actinin

    NASA Astrophysics Data System (ADS)

    López-Menéndez, Horacio; Rodríguez, José Félix

    2016-06-01

    The rheology of F-actin networks has attracted a great attention during the last years. In order to gain a complete understanding of the rheological properties of these novel materials, it is necessary the study in a large deformations regime to alter their internal structure. In this sense, Schmoller et al. (2010) showed that the reconstituted networks of F-actin crosslinked with α-actinin unexpectedly harden when they are subjected to a cyclical shear. This observation contradicts the expected Mullins effect observed in most soft materials, such as rubber and living tissues, where a pronounced softening is observed when they are cyclically deformed. We think that the key to understand this stunning effect is the gelation process. To define it, the most relevant constituents are the chemical crosslinks - α-actinin -, the physical crosslinks - introduced by the entanglement of the semiflexible network - and the interaction between them. As a consequence of this interaction, a pre-stressed network emerges and introduces a feedback effect, where the pre-stress also regulates the adhesion energy of the α-actinin, setting the structure in a metastable reference configuration. Therefore, the external loads and the evolvement of the trapped stress drive the microstructural changes during the cyclic loading protocol. In this work, we propose a micromechanical model into the framework of nonlinear continuum mechanics. The mechanics of the F-actin filaments is modelled using the wormlike chain model for semiflexible filaments and the gelation process is modelled as mesoscale dynamics for the α-actinin and physical crosslink. The model has been validated with reported experimental results.

  16. F-actin cross-linking enhances the stability of force generation in disordered actomyosin networks

    NASA Astrophysics Data System (ADS)

    Jung, Wonyeong; Murrell, Michael P.; Kim, Taeyoon

    2015-12-01

    Myosin molecular motors and actin cross-linking proteins (ACPs) are known to mediate the generation and transmission of mechanical forces within the cortical F-actin cytoskeleton that drive major cellular processes such as cell division and migration. However, how motors and ACPs interact collectively over diverse timescales to modulate the time-dependent mechanical properties of the cytoskeleton remains unclear. In this study, we present a three-dimensional agent-based computational model of the cortical actomyosin network to quantitatively determine the effects of motor activity and the density and kinetics of ACPs on the accumulation and maintenance of mechanical tension within a disordered actomyosin network. We found that motors accumulate large stress quickly by behaving as temporary cross-linkers although this stress is relaxed over time unless there are sufficient passive ACPs to stabilize the network. Stabilization by ACPs helps motors to generate forces up to their maximum potential, leading to significant enhancement of the efficiency and stability of stress generation. Thus, we demonstrated that the force-dependent kinetics of ACP dissociation plays a critical role for the accumulation and sustainment of stress and the structural remodeling of networks.

  17. Detection of systolic ejection click using time growing neural network.

    PubMed

    Gharehbaghi, Arash; Dutoit, Thierry; Ask, Per; Sörnmo, Leif

    2014-04-01

    In this paper, we present a novel neural network for classification of short-duration heart sounds: the time growing neural network (TGNN). The input to the network is the spectral power in adjacent frequency bands as computed in time windows of growing length. Children with heart systolic ejection click (SEC) and normal children are the two groups subjected to analysis. The performance of the TGNN is compared to that of a time delay neural network (TDNN) and a multi-layer perceptron (MLP), using training and test datasets of similar sizes with a total of 614 normal and abnormal cardiac cycles. From the test dataset, the classification rate/sensitivity is found to be 97.0%/98.1% for the TGNN, 85.1%/76.4% for the TDNN, and 92.7%/85.7% for the MLP. The results show that the TGNN performs better than do TDNN and MLP when frequency band power is used as classifier input. The performance of TGNN is also found to exhibit better immunity to noise.

  18. Statistical validation of high-dimensional models of growing networks

    NASA Astrophysics Data System (ADS)

    Medo, Matúš

    2014-03-01

    The abundance of models of complex networks and the current insufficient validation standards make it difficult to judge which models are strongly supported by data and which are not. We focus here on likelihood maximization methods for models of growing networks with many parameters and compare their performance on artificial and real datasets. While high dimensionality of the parameter space harms the performance of direct likelihood maximization on artificial data, this can be improved by introducing a suitable penalization term. Likelihood maximization on real data shows that the presented approach is able to discriminate among available network models. To make large-scale datasets accessible to this kind of analysis, we propose a subset sampling technique and show that it yields substantial model evidence in a fraction of time necessary for the analysis of the complete data.

  19. Topology of growing networks accelerated by intermediary process

    NASA Astrophysics Data System (ADS)

    Ikeda, Nobutoshi

    2017-10-01

    We examined the effect of accelerated growth (increase in the mean vertex degree) caused by internal links between pre-existing nodes on the topology of growing networks and showed that a kind of local rule, edge additions stimulated by randomly chosen intermediaries (intermediary process), not only guarantees a highly clustered structure but also induces the scale-free property on growing networks without an explicit preferential rule with the aid of accelerating growth. We relate the power-law exponent γ describing the degree distribution to the rate of acceleration and an internal factor which expresses the contribution of internal links to the rate of increase in degrees. The behavior of the internal factor generates different forms of time dependence of degrees according to whether γ > 2 or γ < 2. Accelerating growth plays a dominant role in the structural formation of networks based on local rules, in contrast to cases when non-local rules are applied, because similar studies showed that a simple preferential linking rule (a non-local rule) easily induces power laws without accelerating growth.

  20. STAR syndrome-associated CDK10/Cyclin M regulates actin network architecture and ciliogenesis.

    PubMed

    Guen, Vincent J; Gamble, Carly; Perez, Dahlia E; Bourassa, Sylvie; Zappel, Hildegard; Gärtner, Jutta; Lees, Jacqueline A; Colas, Pierre

    2016-01-01

    CDK10/CycM is a protein kinase deficient in STAR (toe Syndactyly, Telecanthus and Anogenital and Renal malformations) syndrome, which results from mutations in the X-linked FAM58A gene encoding Cyclin M. The biological functions of CDK10/CycM and etiology of STAR syndrome are poorly understood. Here, we report that deficiency of CDK10/Cyclin M promotes assembly and elongation of primary cilia. We establish that this reflects a key role for CDK10/Cyclin M in regulation of actin network organization, which is known to govern ciliogenesis. In an unbiased screen, we identified the RhoA-associated kinase PKN2 as a CDK10/CycM phosphorylation substrate. We establish that PKN2 is a bone fide regulator of ciliogenesis, acting in a similar manner to CDK10/CycM. We discovered that CDK10/Cyclin M binds and phosphorylates PKN2 on threonines 121 and 124, within PKN2's core RhoA-binding domain. Furthermore, we demonstrate that deficiencies in CDK10/CycM or PKN2, or expression of a non-phosphorylatable version of PKN2, destabilize both the RhoA protein and the actin network architecture. Importantly, we established that ectopic expression of RhoA is sufficient to override the induction of ciliogenesis resulting from CDK10/CycM knockdown, indicating that RhoA regulation is critical for CDK10/CycM's negative effect on ciliogenesis. Finally, we show that kidney sections from a STAR patient display dilated renal tubules and abnormal, elongated cilia. Altogether, these results reveal CDK10/CycM as a key regulator of actin dynamics and a suppressor of ciliogenesis through phosphorylation of PKN2 and promotion of RhoA signaling. Moreover, they suggest that STAR syndrome is a ciliopathy.

  1. Growing network model for community with group structure

    NASA Astrophysics Data System (ADS)

    Noh, Jae Dong; Jeong, Hyeong-Chai; Ahn, Yong-Yeol; Jeong, Hawoong

    2005-03-01

    We propose a growing network model for a community with a group structure. The community consists of individual members and groups, gatherings of members. The community grows as a new member is introduced by an existing member at each time step. The new member then creates a new group or joins one of the groups of the introducer. We investigate the emerging community structure analytically and numerically. The group size distribution shows a power-law distribution for a variety of growth rules, while the activity distribution follows an exponential or a power law depending on the details of the growth rule. We also present an analysis of empirical data from online communities the “Groups” in http://www.yahoo.com and the “Cafe” in http://www.daum.net, which show a power-law distribution for a wide range of group sizes.

  2. Elastic Coupling of Nascent apCAM Adhesions to Flowing Actin Networks

    PubMed Central

    Mejean, Cecile O.; Schaefer, Andrew W.; Buck, Kenneth B.; Kress, Holger; Shundrovsky, Alla; Merrill, Jason W.; Dufresne, Eric R.; Forscher, Paul

    2013-01-01

    Adhesions are multi-molecular complexes that transmit forces generated by a cell’s acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions’ mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement. PMID:24039928

  3. Two distinct actin networks mediate traction oscillations to confer mechanosensitivity of focal adhesions

    NASA Astrophysics Data System (ADS)

    Wu, Zhanghan; Plotnikov, Sergey; Waterman, Clare; Liu, Jian

    Cells sense the mechanical stiffness of their extracellular matrix (ECM) by exerting traction force through focal adhesions (FAs), which are integrin-based protein assemblies. Strikingly, FA-mediated traction forces oscillate in time and space and govern durotaxis - the tendency of most cell types to migrate toward stiffer ECM. The underlying mechanism of this intriguing oscillation of FA traction force is unknown. Combing theory and experiment, we develop a model of FA growth, which integrates coordinated contributions of a branched actin network and stress fibers in the process. We show that retrograde flux of branched actin network contributes to a traction peak near the FA distal tip and that stress fiber-mediated actomyosin Contractility generates a second traction peak near the FA center. Formin-mediated stress fiber elongation negatively feeds back with actomyosin Contractility, resulting in the central traction peak oscillation. This underpins observed spatio-temporal patterns of the FA traction, and broadens the ECM stiffness range, over which FAs could accurately adapt with traction force generation. Our findings shed light on the fundamental mechanism of FA mechanosensing and hence durotaxis.

  4. Elastic coupling of nascent apCAM adhesions to flowing actin networks.

    PubMed

    Mejean, Cecile O; Schaefer, Andrew W; Buck, Kenneth B; Kress, Holger; Shundrovsky, Alla; Merrill, Jason W; Dufresne, Eric R; Forscher, Paul

    2013-01-01

    Adhesions are multi-molecular complexes that transmit forces generated by a cell's acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions' mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.

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

    NASA Astrophysics Data System (ADS)

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

    2008-11-01

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

  6. Constitutive modeling of the stress-strain behavior of F-actin filament networks.

    PubMed

    Palmer, Jeffrey S; Boyce, Mary C

    2008-05-01

    The central role of the cytoskeleton in both healthy and diseased cellular functions makes it a compelling subject for detailed three-dimensional (3D) micromechanical modeling. Microstructural features of the cytoskeleton govern the cell's mechanical behavior in many of the regulating cellular functions including cell division, adhesion, spreading, migration, contraction, and other mechanotransductive effects which influence biochemical processes. Actin microfilaments (AF) combine to form one of the predominant cytoskeletal networks important to these biological processes. Here, the AF cytoskeletal microstructure and stress-strain behavior is modeled via a microstructurally-informed continuum mechanics approach. The force-extension behavior of the individual filaments is captured using the MacKintosh derivation of the worm-like chain (WLC) constitutive relationship for short chains where a new and direct analytical expression for the filament force as a function of filament extension is developed in this paper. The filament force-extension behavior is then used in conjunction with the Arruda-Boyce eight-chain network model to capture the 3D multiaxial stress-strain behavior of the network. The resulting 3D cytoskeletal network constitutive model provides the ability to track microstructural stretch and orientation states during 3D macroscopic stretching conditions. The non-affine nature of the network model effectively accommodates the imposed macroscopic shear strain through filament rotation and a relatively small amount of filament stretch. These characteristics enable the network model, using physically realistic material properties, to capture the initial stiffness of the AF network as well as the nonlinear strain stiffening observed at large stresses. The network model predictions compare favorably with published microrheological data of in vitro AF networks.

  7. Convergence and extension at gastrulation require a myosin IIB dependent cortical actin network

    PubMed Central

    Skoglund, Paul; Rolo, Ana; Chen, Xuejun; Gumbiner, Barry M.; Keller, Ray

    2009-01-01

    Summary Force-producing convergence (narrowing) and extension (lengthening) of tissues by active intercalation of cells along the axis of convergence play a major role in axial morphogenesis during development of both vertebrate and invertebrate embryos, and failure of these processes in human embryos leads to embryonic defects including spina bifida and anencephaly. Here we use Xenopus laevis, a system in which the polarized cell motility that drives this active cell intercalation has been related to development of forces that close the blastopore and elongate the body axis, to examine the role of myosin IIB in convergence and extension. We find that myosin IIB is localized in the cortex of intercalating cells, and that morpholino knockdown of this myosin isoform shows that it is essential for maintenance of a stereotypical, cortical actin cytoskeleton that we visualize with time-lapse fluorescent confocal microscopy. We show that this actin network consists of foci or nodes connected by cables and is polarized relative to the embryonic axis, preferentially cyclically shortening and lengthening parallel to the axis of cell polarization, elongation, and intercalation, and also parallel to the axis of convergence forces during gastrulation. MHC-B-depletion results in disruption of this polarized cytoskeleton, loss of the polarized protrusive activity characteristic of intercalating cells, eventual loss of cell-cell and cell matrix adhesion, and dose-dependent failure of blastopore closure, arguably because of failure to develop convergence forces parallel to the myosin IIB-dependent dynamics of the actin cytoskeleton. These findings bridge the gap between a molecular-scale motor protein and tissue-scale embryonic morphogenesis. PMID:18550716

  8. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium

    PubMed Central

    1994-01-01

    Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria. First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin- minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine- coated coverslips. The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development. PMID:8089176

  9. Growing Brazilian demand to spur gas network in South America

    SciTech Connect

    Deffarges, E.H. ); Maurer, L.I.A. )

    1993-01-18

    A recent combination in South America of economic and geopolitical factors is prompting development of a new integrated gas-pipeline network in the continent's Southern Cone. The crucial factors include privatization, regional integration, economic growth, and environmental concerns. The area, Latin America's largest regional entity, includes Brazil (population 150 million and a 1990 GNP of about $375 billion, 9th largest in the world), Argentina (population 32 million and the third largest Latin American economy after Brazil and Mexico), Bolivia, Chile, Paraguay, and Uruguay. Argentina, Brazil, Paraguay, and Uruguay are members of the MercoSur economic bloc whose objective is to develop free trade in the region. There are very few integrated pipeline networks in the world. Besides the giant North American system, with hundreds of producers and pipelines, there is only one other large integrated network. It connects continental European countries to their outside suppliers such as Norway, the C.I.S., and Algeria. The emergence of a new pipeline system is therefore important for the natural-gas industry worldwide and even more so if it occurs in a region now growing rapidly after a decade of economic difficulties.

  10. Growing networks of overlapping communities with internal structure

    NASA Astrophysics Data System (ADS)

    Young, Jean-Gabriel; Hébert-Dufresne, Laurent; Allard, Antoine; Dubé, Louis J.

    2016-08-01

    We introduce an intuitive model that describes both the emergence of community structure and the evolution of the internal structure of communities in growing social networks. The model comprises two complementary mechanisms: One mechanism accounts for the evolution of the internal link structure of a single community, and the second mechanism coordinates the growth of multiple overlapping communities. The first mechanism is based on the assumption that each node establishes links with its neighbors and introduces new nodes to the community at different rates. We demonstrate that this simple mechanism gives rise to an effective maximal degree within communities. This observation is related to the anthropological theory known as Dunbar's number, i.e., the empirical observation of a maximal number of ties which an average individual can sustain within its social groups. The second mechanism is based on a recently proposed generalization of preferential attachment to community structure, appropriately called structural preferential attachment (SPA). The combination of these two mechanisms into a single model (SPA+) allows us to reproduce a number of the global statistics of real networks: The distribution of community sizes, of node memberships, and of degrees. The SPA+ model also predicts (a) three qualitative regimes for the degree distribution within overlapping communities and (b) strong correlations between the number of communities to which a node belongs and its number of connections within each community. We present empirical evidence that support our findings in real complex networks.

  11. Ranking nodes in growing networks: When PageRank fails

    PubMed Central

    Mariani, Manuel Sebastian; Medo, Matúš; Zhang, Yi-Cheng

    2015-01-01

    PageRank is arguably the most popular ranking algorithm which is being applied in real systems ranging from information to biological and infrastructure networks. Despite its outstanding popularity and broad use in different areas of science, the relation between the algorithm’s efficacy and properties of the network on which it acts has not yet been fully understood. We study here PageRank’s performance on a network model supported by real data, and show that realistic temporal effects make PageRank fail in individuating the most valuable nodes for a broad range of model parameters. Results on real data are in qualitative agreement with our model-based findings. This failure of PageRank reveals that the static approach to information filtering is inappropriate for a broad class of growing systems, and suggest that time-dependent algorithms that are based on the temporal linking patterns of these systems are needed to better rank the nodes. PMID:26553630

  12. Regional frequency analysis using Growing Neural Gas network

    NASA Astrophysics Data System (ADS)

    Abdi, Amin; Hassanzadeh, Yousef; Ouarda, Taha B. M. J.

    2017-07-01

    The delineation of hydrologically homogeneous regions is an important issue in regional hydrological frequency analysis. In the present study, an application of the Growing Neural Gas (GNG) network for hydrological data clustering is presented. The GNG is an incremental and unsupervised neural network, which is able to adapt its structure during the training procedure without using a prior knowledge of the size and shape of the network. In the GNG algorithm, the Minimum Description Length (MDL) measure as the cluster validity index is utilized for determining the optimal number of clusters (sub-regions). The capability of the proposed algorithm is illustrated by regionalizing drought severities for 40 synoptic weather stations in Iran. To fulfill this aim, first a clustering method is applied to form the sub-regions and then a heterogeneity measure is used to test the degree of heterogeneity of the delineated sub-regions. According to the MDL measure and considering two different indices namely CS and Davies-Bouldin (DB) in the GNG network, the entire study area is subdivided in two sub-regions located in the eastern and western sides of Iran. In order to evaluate the performance of the GNG algorithm, a number of other commonly used clustering methods, like K-means, fuzzy C-means, self-organizing map and Ward method are utilized in this study. The results of the heterogeneity measure based on the L-moments approach reveal that only the GNG algorithm successfully yields homogeneous sub-regions in comparison to the other methods.

  13. Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells

    NASA Astrophysics Data System (ADS)

    Ory, Eleanor C.; Bhandary, Lekhana; E Boggs, Amanda; Chakrabarti, Kristi R.; Parker, Joshua; Losert, Wolfgang; Martin, Stuart S.

    2017-04-01

    The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems—growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; however, the impacts on microtubule dynamics remain incompletely understood. Using human MCF-7 breast tumor cells expressing GFP-tagged microtubule end-binding-protein-1 (EB1) and coexpression of cytoplasmic fluorescent protein mCherry, we map the trajectories of growing microtubule ends and cytoplasmic boundary respectively. Based on EB1 tracks and cytoplasmic boundary outlines, we calculate the speed, distance from cytoplasmic boundary, and straightness of microtubule growth. Actin depolymerization with Latrunculin-A reduces EB1 growth speed as well as allows the trajectories to extend beyond the cytoplasmic boundary. Blebbistatin, a direct myosin-II inhibitor, reduced EB1 speed and yielded less straight EB1 trajectories. Inhibiting signaling upstream of myosin-II contractility via the Rho-kinase inhibitor, Y-27632, altered EB1 dynamics differently from Blebbistatin. These results indicate that reduced actin cortex integrity can induce distinct alterations in microtubule dynamics. Given recent findings that tumor stem cell characteristics are increased by drugs which reduce actin contractility or stabilize microtubules, it remains important to clearly define how cytoskeletal drugs alter the interactions between these two filament systems in tumor cells.

  14. Analysis of microtubule growth dynamics arising from altered actin network structure and contractility in breast tumor cells.

    PubMed

    Ory, Eleanor; Bhandary, Lekhana; Boggs, Amanda; Chakrabarti, Kristi; Parker, Joshua; Losert, Wolfgang; Martin, Stuart S

    2017-01-16

    The periphery of epithelial cells is shaped by opposing cytoskeletal physical forces generated predominately by two dynamic force generating systems - growing microtubule ends push against the boundary from the cell center, and the actin cortex contracts the attached plasma membrane. Here we investigate how changes to the structure and dynamics of the actin cortex alter the dynamics of microtubules. Current drugs target actin polymerization and contraction to reduce cell division and invasiveness; however, the impacts on microtubule dynamics remain incompletely understood. Using human MCF-7 breast tumor cells expressing GFP-tagged microtubule end-binding-protein-1 (EB1) and coexpression of cytoplasmic fluorescent protein mCherry, we map the trajectories of growing microtubule ends and cytoplasmic boundary respectively. Based on EB1 tracks and cytoplasmic boundary outlines, we calculate the speed, distance from cytoplasmic boundary, and straightness of microtubule growth. Actin depolymerization with Latrunculin-A reduces EB1 growth speed as well as allows the trajectories to extend beyond the cytoplasmic boundary. Blebbistatin, a direct myosin-II inhibitor, reduced EB1 speed and yielded less straight EB1 trajectories. Inhibiting signaling upstream of myosin-II contractility via the Rho-kinase inhibitor, Y-27632, altered EB1 dynamics differently from Blebbistatin. These results indicate that reduced actin cortex integrity can induce distinct alterations in microtubule dynamics. Given recent findings that tumor stem cell characteristics are increased by drugs which reduce actin contractility or stabilize microtubules, it remains important to clearly define how cytoskeletal drugs alter the interactions between these two filament systems in tumor cells.

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

    PubMed

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

    2005-05-01

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

  16. A Gβγ effector, ElmoE, transduces GPCR signaling to the actin network during chemotaxis.

    PubMed

    Yan, Jianshe; Mihaylov, Vassil; Xu, Xuehua; Brzostowski, Joseph A; Li, Hongyan; Liu, Lunhua; Veenstra, Timothy D; Parent, Carole A; Jin, Tian

    2012-01-17

    Activation of G protein-coupled receptors (GPCRs) leads to the dissociation of heterotrimeric G-proteins into Gα and Gβγ subunits, which go on to regulate various effectors involved in a panoply of cellular responses. During chemotaxis, Gβγ subunits regulate actin assembly and migration, but the protein(s) linking Gβγ to the actin cytoskeleton remains unknown. Here, we identified a Gβγ effector, ElmoE in Dictyostelium, and demonstrated that it is required for GPCR-mediated chemotaxis. Remarkably, ElmoE associates with Gβγ and Dock-like proteins to activate the small GTPase Rac, in a GPCR-dependent manner, and also associates with Arp2/3 complex and F-actin. Thus, ElmoE serves as a link between chemoattractant GPCRs, G-proteins and the actin cytoskeleton. The pathway, consisting of GPCR, Gβγ, Elmo/Dock, Rac, and Arp2/3, spatially guides the growth of dendritic actin networks in pseudopods of eukaryotic cells during chemotaxis.

  17. Actin-Regulator Feedback Interactions during Endocytosis

    PubMed Central

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

    2016-01-01

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

  18. Nuclei migrate through constricted spaces using microtubule motors and actin networks in C. elegans hypodermal cells.

    PubMed

    Bone, Courtney R; Chang, Yu-Tai; Cain, Natalie E; Murphy, Shaun P; Starr, Daniel A

    2016-11-15

    Cellular migrations through constricted spaces are a crucial aspect of many developmental and disease processes including hematopoiesis, inflammation and metastasis. A limiting factor in these events is nuclear deformation. Here, we establish an in vivo model in which nuclei can be visualized while moving through constrictions and use it to elucidate mechanisms for nuclear migration. C. elegans hypodermal P-cell larval nuclei traverse a narrow space that is about 5% their width. This constriction is blocked by fibrous organelles, structures that pass through P cells to connect the muscles to cuticle. Fibrous organelles are removed just prior to nuclear migration, when nuclei and lamins undergo extreme morphological changes to squeeze through the space. Both actin and microtubule networks are organized to mediate nuclear migration. The LINC complex, consisting of the SUN protein UNC-84 and the KASH protein UNC-83, recruits dynein and kinesin-1 to the nuclear surface. Both motors function in P-cell nuclear migration, but dynein, functioning through UNC-83, plays a more central role as nuclei migrate towards minus ends of polarized microtubule networks. Thus, the nucleoskeleton and cytoskeleton are coordinated to move nuclei through constricted spaces.

  19. Directed actin assembly and motility.

    PubMed

    Boujemaa-Paterski, Rajaa; Galland, Rémi; Suarez, Cristian; Guérin, Christophe; Théry, Manuel; Blanchoin, Laurent

    2014-01-01

    The actin cytoskeleton is a key component of the cellular architecture. However, understanding actin organization and dynamics in vivo is a complex challenge. Reconstitution of actin structures in vitro, in simplified media, allows one to pinpoint the cellular biochemical components and their molecular interactions underlying the architecture and dynamics of the actin network. Previously, little was known about the extent to which geometrical constraints influence the dynamic ultrastructure of these networks. Therefore, in order to study the balance between biochemical and geometrical control of complex actin organization, we used the innovative methodologies of UV and laser patterning to design a wide repertoire of nucleation geometries from which we assembled branched actin networks. Using these methods, we were able to reconstitute complex actin network organizations, closely related to cellular architecture, to precisely direct and control their 3D connections. This methodology mimics the actin networks encountered in cells and can serve in the fabrication of innovative bioinspired systems.

  20. Networks Models of Actin Dynamics during Spermatozoa Postejaculatory Life: A Comparison among Human-Made and Text Mining-Based Models.

    PubMed

    Bernabò, Nicola; Ordinelli, Alessandra; Ramal Sanchez, Marina; Mattioli, Mauro; Barboni, Barbara

    2016-01-01

    Here we realized a networks-based model representing the process of actin remodelling that occurs during the acquisition of fertilizing ability of human spermatozoa (HumanMade_ActinSpermNetwork, HM_ASN). Then, we compared it with the networks provided by two different text mining tools: Agilent Literature Search (ALS) and PESCADOR. As a reference, we used the data from the online repository Kyoto Encyclopaedia of Genes and Genomes (KEGG), referred to the actin dynamics in a more general biological context. We found that HM_ALS and the networks from KEGG data shared the same scale-free topology following the Barabasi-Albert model, thus suggesting that the information is spread within the network quickly and efficiently. On the contrary, the networks obtained by ALS and PESCADOR have a scale-free hierarchical architecture, which implies a different pattern of information transmission. Also, the hubs identified within the networks are different: HM_ALS and KEGG networks contain as hubs several molecules known to be involved in actin signalling; ALS was unable to find other hubs than "actin," whereas PESCADOR gave some nonspecific result. This seems to suggest that the human-made information retrieval in the case of a specific event, such as actin dynamics in human spermatozoa, could be a reliable strategy.

  1. Networks Models of Actin Dynamics during Spermatozoa Postejaculatory Life: A Comparison among Human-Made and Text Mining-Based Models

    PubMed Central

    Ordinelli, Alessandra; Ramal Sanchez, Marina; Mattioli, Mauro; Barboni, Barbara

    2016-01-01

    Here we realized a networks-based model representing the process of actin remodelling that occurs during the acquisition of fertilizing ability of human spermatozoa (HumanMade_ActinSpermNetwork, HM_ASN). Then, we compared it with the networks provided by two different text mining tools: Agilent Literature Search (ALS) and PESCADOR. As a reference, we used the data from the online repository Kyoto Encyclopaedia of Genes and Genomes (KEGG), referred to the actin dynamics in a more general biological context. We found that HM_ALS and the networks from KEGG data shared the same scale-free topology following the Barabasi-Albert model, thus suggesting that the information is spread within the network quickly and efficiently. On the contrary, the networks obtained by ALS and PESCADOR have a scale-free hierarchical architecture, which implies a different pattern of information transmission. Also, the hubs identified within the networks are different: HM_ALS and KEGG networks contain as hubs several molecules known to be involved in actin signalling; ALS was unable to find other hubs than “actin,” whereas PESCADOR gave some nonspecific result. This seems to suggest that the human-made information retrieval in the case of a specific event, such as actin dynamics in human spermatozoa, could be a reliable strategy. PMID:27642606

  2. Sequential interaction of actin-related proteins 2 and 3 (Arp2/3) complex with neural Wiscott-Aldrich syndrome protein (N-WASP) and cortactin during branched actin filament network formation.

    PubMed

    Uruno, Takehito; Liu, Jiali; Li, Yansong; Smith, Nicole; Zhan, Xi

    2003-07-11

    The WASP and cortactin families constitute two distinct classes of Arp2/3 modulators in mammalian cells. Physical and functional interactions among the Arp2/3 complex, VCA (a functional domain of N-WASP), and cortactin were examined under conditions that were with or without actin polymerization. In the absence of actin, cortactin binds significantly weaker to the Arp2/3 complex than VCA. At concentrations of VCA 20-fold lower than cortactin, the association of cortactin with the Arp2/3 complex was nearly abolished. Analysis of the cells infected with Shigella demonstrated that N-WASP located at the tip of the bacterium, whereas cortactin accumulated in the comet tail. Interestingly, cortactin promotes Arp2/3 complex-mediated actin polymerization and actin branching in the presence of VCA at a saturating concentration, and cortactin acquired 20 nm affinity for the Arp2/3 complex during actin polymerization. The interaction of VCA with the Arp2/3 complex was reduced in the presence of both cortactin and actin. Moreover, VCA reduced its affinity for Arp2/3 complex at branching sites that were stabilized by phalloidin. These data imply a novel mechanism for the de novo assembly of a branched actin network that involves a coordinated sequential interaction of N-WASP and cortactin with the Arp2/3 complex.

  3. Reversible mechano-memory in sheared cross-linked actin networks

    NASA Astrophysics Data System (ADS)

    Majumdar, Sayantan; Gardel, Margaret L.

    2015-03-01

    Is it possible to control the shear modulus of a material mechanically? We reconstitute a network of actin filaments cross-linked with Filamin A and show that the system has remarkable property to respond under shear in a deformation history dependent manner. When a large shear stress pulse is applied to the system, the system remembers the direction of deformation long after the stress pulse is removed. For the next loading cycle, shear response of the system becomes anisotropic; if the applied pulse direction is same as the previous one, the system behaves like a viscoelastic solid but a transient liquefaction is observed if the pulse direction is reversed. Imaging and normal force measurements under shear suggest that this anisotropic response comes from stretching and bending dominated deformation directions induced by the large shear deformation giving rise to a direction dependent mechano-memory. The long time scale over which the memory effect persists has relevance in various deformations in cellular and multicellular systems. S.M. acknowledges support from a Kadanoff-Rice Post Doctoral fellowship from MRSEC, University of Chicago.

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

    PubMed

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

    2014-09-08

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

  5. Why is Actin Patchy?

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2009-03-01

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

  6. The Role of Temporal Trends in Growing Networks

    PubMed Central

    Ruppin, Eytan; Shavitt, Yuval

    2016-01-01

    The rich get richer principle, manifested by the Preferential attachment (PA) mechanism, is widely considered one of the major factors in the growth of real-world networks. PA stipulates that popular nodes are bound to be more attractive than less popular nodes; for example, highly cited papers are more likely to garner further citations. However, it overlooks the transient nature of popularity, which is often governed by trends. Here, we show that in a wide range of real-world networks the recent popularity of a node, i.e., the extent by which it accumulated links recently, significantly influences its attractiveness and ability to accumulate further links. We proceed to model this observation with a natural extension to PA, named Trending Preferential Attachment (TPA), in which edges become less influential as they age. TPA quantitatively parametrizes a fundamental network property, namely the network’s tendency to trends. Through TPA, we find that real-world networks tend to be moderately to highly trendy. Networks are characterized by different susceptibilities to trends, which determine their structure to a large extent. Trendy networks display complex structural traits, such as modular community structure and degree-assortativity, occurring regularly in real-world networks. In summary, this work addresses an inherent trait of complex networks, which greatly affects their growth and structure, and develops a unified model to address its interaction with preferential attachment. PMID:27486847

  7. Growing Homophilic Networks Are Natural Navigable Small Worlds

    PubMed Central

    Malkov, Yury A.; Ponomarenko, Alexander

    2016-01-01

    Navigability, an ability to find a logarithmically short path between elements using only local information, is one of the most fascinating properties of real-life networks. However, the exact mechanism responsible for the formation of navigation properties remained unknown. We show that navigability can be achieved by using only two ingredients present in the majority of networks: network growth and local homophily, giving a persuasive answer how the navigation appears in real-life networks. A very simple algorithm produces hierarchical self-similar optimally wired navigable small world networks with exponential degree distribution by using only local information. Adding preferential attachment produces a scale-free network which has shorter greedy paths, but worse (power law) scaling of the information extraction locality (algorithmic complexity of a search). Introducing saturation of the preferential attachment leads to truncated scale-free degree distribution that offers a good tradeoff between these parameters and can be useful for practical applications. Several features of the model are observed in real-life networks, in particular in the brain neural networks, supporting the earlier suggestions that they are navigable. PMID:27348120

  8. Transcriptional Network Growing Models Using Motif-Based Preferential Attachment

    PubMed Central

    Abdelzaher, Ahmed F.; Al-Musawi, Ahmad F.; Ghosh, Preetam; Mayo, Michael L.; Perkins, Edward J.

    2015-01-01

    Understanding relationships between architectural properties of gene-regulatory networks (GRNs) has been one of the major goals in systems biology and bioinformatics, as it can provide insights into, e.g., disease dynamics and drug development. Such GRNs are characterized by their scale-free degree distributions and existence of network motifs – i.e., small-node subgraphs that occur more abundantly in GRNs than expected from chance alone. Because these transcriptional modules represent “building blocks” of complex networks and exhibit a wide range of functional and dynamical properties, they may contribute to the remarkable robustness and dynamical stability associated with the whole of GRNs. Here, we developed network-construction models to better understand this relationship, which produce randomized GRNs by using transcriptional motifs as the fundamental growth unit in contrast to other methods that construct similar networks on a node-by-node basis. Because this model produces networks with a prescribed lower bound on the number of choice transcriptional motifs (e.g., downlinks, feed-forward loops), its fidelity to the motif distributions observed in model organisms represents an improvement over existing methods, which we validated by contrasting their resultant motif and degree distributions against existing network-growth models and data from the model organism of the bacterium Escherichia coli. These models may therefore serve as novel testbeds for further elucidating relationships between the topology of transcriptional motifs and network-wide dynamical properties. PMID:26528473

  9. Transcriptional Network Growing Models Using Motif-Based Preferential Attachment.

    PubMed

    Abdelzaher, Ahmed F; Al-Musawi, Ahmad F; Ghosh, Preetam; Mayo, Michael L; Perkins, Edward J

    2015-01-01

    Understanding relationships between architectural properties of gene-regulatory networks (GRNs) has been one of the major goals in systems biology and bioinformatics, as it can provide insights into, e.g., disease dynamics and drug development. Such GRNs are characterized by their scale-free degree distributions and existence of network motifs - i.e., small-node subgraphs that occur more abundantly in GRNs than expected from chance alone. Because these transcriptional modules represent "building blocks" of complex networks and exhibit a wide range of functional and dynamical properties, they may contribute to the remarkable robustness and dynamical stability associated with the whole of GRNs. Here, we developed network-construction models to better understand this relationship, which produce randomized GRNs by using transcriptional motifs as the fundamental growth unit in contrast to other methods that construct similar networks on a node-by-node basis. Because this model produces networks with a prescribed lower bound on the number of choice transcriptional motifs (e.g., downlinks, feed-forward loops), its fidelity to the motif distributions observed in model organisms represents an improvement over existing methods, which we validated by contrasting their resultant motif and degree distributions against existing network-growth models and data from the model organism of the bacterium Escherichia coli. These models may therefore serve as novel testbeds for further elucidating relationships between the topology of transcriptional motifs and network-wide dynamical properties.

  10. Growing optimal scale-free networks via likelihood.

    PubMed

    Small, Michael; Li, Yingying; Stemler, Thomas; Judd, Kevin

    2015-04-01

    Preferential attachment, by which new nodes attach to existing nodes with probability proportional to the existing nodes' degree, has become the standard growth model for scale-free networks, where the asymptotic probability of a node having degree k is proportional to k^{-γ}. However, the motivation for this model is entirely ad hoc. We use exact likelihood arguments and show that the optimal way to build a scale-free network is to attach most new links to nodes of low degree. Curiously, this leads to a scale-free network with a single dominant hub: a starlike structure we call a superstar network. Asymptotically, the optimal strategy is to attach each new node to one of the nodes of degree k with probability proportional to 1/N+ζ(γ)(k+1)(γ) (in a N node network): a stronger bias toward high degree nodes than exhibited by standard preferential attachment. Our algorithm generates optimally scale-free networks (the superstar networks) as well as randomly sampling the space of all scale-free networks with a given degree exponent γ. We generate viable realization with finite N for 1≪γ<2 as well as γ>2. We observe an apparently discontinuous transition at γ≈2 between so-called superstar networks and more treelike realizations. Gradually increasing γ further leads to reemergence of a superstar hub. To quantify these structural features, we derive a new analytic expression for the expected degree exponent of a pure preferential attachment process and introduce alternative measures of network entropy. Our approach is generic and can also be applied to an arbitrary degree distribution.

  11. Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges.

    PubMed

    Fujiwara, Ikuko; Remmert, Kirsten; Piszczek, Grzegorz; Hammer, John A

    2014-05-13

    Although capping protein (CP) terminates actin filament elongation, it promotes Arp2/3-dependent actin network assembly and accelerates actin-based motility both in vitro and in vivo. In vitro, capping protein Arp2/3 myosin I linker (CARMIL) antagonizes CP by reducing its affinity for the barbed end and by uncapping CP-capped filaments, whereas the protein V-1/myotrophin sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP:V-1 complex, thereby converting inactive CP into a version with moderate affinity for the barbed end. Here we further clarify the mechanism of this exchange reaction, and we demonstrate that the CP:CARMIL complex created by complex exchange slows the rate of barbed-end elongation by rapidly associating with, and dissociating from, the barbed end. Importantly, the cellular concentrations of V-1 and CP determined here argue that most CP is sequestered by V-1 at steady state in vivo. Finally, we show that CARMIL is recruited to the plasma membrane and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of freely diffusing, inactive CP (CP:V-1) feeds, via CARMIL-driven complex exchange, the formation of weak-capping complexes (CP:CARMIL) at the plasma membrane of protruding edges. In vivo, therefore, CARMIL should promote Arp2/3-dependent actin network assembly at the leading edge by promoting barbed-end capping there.

  12. Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges

    PubMed Central

    Fujiwara, Ikuko; Remmert, Kirsten; Piszczek, Grzegorz; Hammer, John A.

    2014-01-01

    Although capping protein (CP) terminates actin filament elongation, it promotes Arp2/3-dependent actin network assembly and accelerates actin-based motility both in vitro and in vivo. In vitro, capping protein Arp2/3 myosin I linker (CARMIL) antagonizes CP by reducing its affinity for the barbed end and by uncapping CP-capped filaments, whereas the protein V-1/myotrophin sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP:V-1 complex, thereby converting inactive CP into a version with moderate affinity for the barbed end. Here we further clarify the mechanism of this exchange reaction, and we demonstrate that the CP:CARMIL complex created by complex exchange slows the rate of barbed-end elongation by rapidly associating with, and dissociating from, the barbed end. Importantly, the cellular concentrations of V-1 and CP determined here argue that most CP is sequestered by V-1 at steady state in vivo. Finally, we show that CARMIL is recruited to the plasma membrane and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of freely diffusing, inactive CP (CP:V-1) feeds, via CARMIL-driven complex exchange, the formation of weak-capping complexes (CP:CARMIL) at the plasma membrane of protruding edges. In vivo, therefore, CARMIL should promote Arp2/3-dependent actin network assembly at the leading edge by promoting barbed-end capping there. PMID:24778263

  13. Microtubules Modulate F-actin Dynamics during Neuronal Polarization.

    PubMed

    Zhao, Bing; Meka, Durga Praveen; Scharrenberg, Robin; König, Theresa; Schwanke, Birgit; Kobler, Oliver; Windhorst, Sabine; Kreutz, Michael R; Mikhaylova, Marina; Calderon de Anda, Froylan

    2017-08-29

    Neuronal polarization is reflected by different dynamics of microtubule and filamentous actin (F-actin). Axonal microtubules are more stable than those in the remaining neurites, while dynamics of F-actin in axonal growth cones clearly exceed those in their dendritic counterparts. However, whether a functional interplay exists between the microtubule network and F-actin dynamics in growing axons and whether this interplay is instrumental for breaking cellular symmetry is currently unknown. Here, we show that an increment on microtubule stability or number of microtubules is associated with increased F-actin dynamics. Moreover, we show that Drebrin E, an F-actin and microtubule plus-end binding protein, mediates this cross talk. Drebrin E segregates preferentially to growth cones with a higher F-actin treadmilling rate, where more microtubule plus-ends are found. Interruption of the interaction of Drebrin E with microtubules decreases F-actin dynamics and arrests neuronal polarization. Collectively the data show that microtubules modulate F-actin dynamics for initial axon extension during neuronal development.

  14. Growing Fully Distributed Robust Topologies in a Sensor Network

    NASA Astrophysics Data System (ADS)

    Gasparri, Andrea; Meloni, Sandro; Panzieri, Stefano

    Wireless Sensor Networks (WSN) are at the forefront of emerging technologies due to the recent advances in Micro-Electro-Mechanical Systems (MEMS) technology. WSN are considered to be unattended systems with applications ranging from environmental sensing, structural monitoring, and industrial process control to emergency response and mobile target tracking.The distributed nature and the limited hardware capabilities of WSN challenge the development of effective applications. The strength of a sensor network, which turns out to be also its weakness, is the capability to perform inter-node processing while sharing data across the network. However, the limited reliability of a node, due to the low-cost nature of the hardware components, drastically constrains this aspect. For this reason, the availability of a mechanism to build distributed robust connectivity topologies, where robustness is meant against random failures of nodes and intentional attacks of nodes, is crucial. The complex network theory along with the percolation theory provides a suitable framework to achieve that. Indeed, topologies such as multi-modal and scale free ones, show interesting properties which might be embedded into a sensor network to significantly increase its robustness. In this work, a mechanisms to build robust topologies in a distributed fashion is proposed, its effectiveness is analytically investigated and results are validated through simulations.

  15. 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.

  16. Swelling-induced surface instabilities in growing poroelastic polymer networks

    NASA Astrophysics Data System (ADS)

    Hennessy, Matthew G.; Vitale, Alessandra; Cabral, Joao T.; Matar, Omar K.

    2016-11-01

    The swelling that occurs when a deformable polymer network absorbs solvent can generate large compressive stresses which, in turn, can lead to a rich variety of surface instabilities. In this talk, we will discuss recent experiments by our group which suggest that the growth of a polymer network by photopolymerisation and the onset of swelling-induced surface instabilities can simultaneously occur and drive the self-assembly of complex three-dimensional structures. In addition, we will present a theoretical model of photopolymersation that captures the growth, swelling, and mechanical response of the polymer network. The model is based on an Eulerian formulation of nonlinear poroelasticity. The transport of monomer is described by a generalisation of Darcy's law that accounts for flow due to gradients in the pressure and composition. A combination of asymptotic analysis and finite-element simulations is used to explore the coupling between growth and instability as well as the resulting surface morphologies.

  17. Nucleocapsid of Tomato spotted wilt tospovirus forms mobile particles that traffic on an actin/endoplasmic reticulum network driven by myosin XI-K.

    PubMed

    Feng, Zhike; Chen, Xiaojiao; Bao, Yiqun; Dong, Jiahong; Zhang, Zhongkai; Tao, Xiaorong

    2013-12-01

    A number of viral proteins from plant viruses, other than movement proteins, have been shown to traffic intracellularly along actin filaments and to be involved in viral infection. However, there has been no report that a viral capsid protein may traffic within a cell by utilizing the actin/endoplasmic reticulum (ER) network. We used Tomato spotted wilt tospovirus (TSWV) as a model virus to study the cell biological properties of a nucleocapsid (N) protein. We found that TSWV N protein was capable of forming highly motile cytoplasmic inclusions that moved along the ER and actin network. The disruption of actin filaments by latrunculin B, an actin-depolymerizing agent, almost stopped the intracellular movement of N inclusions, whereas treatment with a microtubule-depolymerizing reagent, oryzalin, did not. The over-expression of a myosin XI-K tail, functioning in a dominant-negative manner, completely halted the movement of N inclusions. Latrunculin B treatment strongly inhibited the formation of TSWV local lesions in Nicotiana tabacum cv Samsun NN and delayed systemic infection in N. benthamiana. Collectively, our findings provide the first evidence that the capsid protein of a plant virus has the novel property of intracellular trafficking. The findings add capsid protein as a new class of viral protein that traffics on the actin/ER system.

  18. Combinatorial genetic analysis of a network of actin disassembly‐promoting factors

    PubMed Central

    Ydenberg, Casey A.; Johnston, Adam; Weinstein, Jaclyn; Bellavance, Danielle; Jansen, Silvia

    2015-01-01

    The patterning of actin cytoskeleton structures in vivo is a product of spatially and temporally regulated polymer assembly balanced by polymer disassembly. While in recent years our understanding of actin assembly mechanisms has grown immensely, our knowledge of actin disassembly machinery and mechanisms has remained comparatively sparse. Saccharomyces cerevisiae is an ideal system to tackle this problem, both because of its amenabilities to genetic manipulation and live‐cell imaging and because only a single gene encodes each of the core disassembly factors: cofilin (COF1), Srv2/CAP (SRV2), Aip1 (AIP1), GMF (GMF1/AIM7), coronin (CRN1), and twinfilin (TWF1). Among these six factors, only the functions of cofilin are essential and have been well defined. Here, we investigated the functions of the nonessential actin disassembly factors by performing genetic and live‐cell imaging analyses on a combinatorial set of isogenic single, double, triple, and quadruple mutants in S. cerevisiae. Our results show that each disassembly factor makes an important contribution to cell viability, actin organization, and endocytosis. Further, our data reveal new relationships among these factors, providing insights into how they work together to orchestrate actin turnover. Finally, we observe specific combinations of mutations that are lethal, e.g., srv2Δ aip1Δ and srv2Δ crn1Δ twf1Δ, demonstrating that while cofilin is essential, it is not sufficient in vivo, and that combinations of the other disassembly factors perform vital functions. © 2015 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc. PMID:26147656

  19. 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

  20. Memo-RhoA-mDia1 signaling controls microtubules, the actin network, and adhesion site formation in migrating cells.

    PubMed

    Zaoui, Kossay; Honoré, Stéphane; Isnardon, Daniel; Braguer, Diane; Badache, Ali

    2008-11-03

    Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form lamellipodia, they show defective localization of lamellipodial markers such as alpha-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo-RhoA-mDia1 signaling coordinates the organization of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.

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

    PubMed Central

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

    2015-01-01

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

  2. Amplification of actin polymerization forces

    PubMed Central

    Dmitrieff, Serge; Nédélec, François

    2016-01-01

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

  3. Amplification of actin polymerization forces.

    PubMed

    Dmitrieff, Serge; Nédélec, François

    2016-03-28

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

  4. Growing up with Social Networks and Online Communities

    ERIC Educational Resources Information Center

    Strom, Paris; Strom, Robert

    2012-01-01

    This presentation examines child and adolescent social networking with an emphasis on how this unprecedented form of communication can be used to contribute to healthy growth and development. Most literature about child and adolescent relationships reflects yesterday's world, a time when face-to-face encounters were the only concern. Students saw…

  5. Growing up with Social Networks and Online Communities

    ERIC Educational Resources Information Center

    Strom, Paris; Strom, Robert

    2012-01-01

    This presentation examines child and adolescent social networking with an emphasis on how this unprecedented form of communication can be used to contribute to healthy growth and development. Most literature about child and adolescent relationships reflects yesterday's world, a time when face-to-face encounters were the only concern. Students saw…

  6. Emergence of disassortative mixing from pruning nodes in growing scale-free networks

    PubMed Central

    Wang, Sheng-Jun; Wang, Zhen; Jin, Tao; Boccaletti, Stefano

    2014-01-01

    Disassortative mixing is ubiquitously found in technological and biological networks, while the corresponding interpretation of its origin remains almost virgin. We here give evidence that pruning the largest-degree nodes of a growing scale-free network has the effect of decreasing the degree correlation coefficient in a controllable and tunable way, while keeping both the trait of a power-law degree distribution and the main properties of network's resilience and robustness under failures or attacks. The essence of these observations can be attributed to the fact the deletion of large-degree nodes affects the delicate balance of positive and negative contributions to degree correlation in growing scale-free networks, eventually leading to the emergence of disassortativity. Moreover, these theoretical prediction will get further validation in the empirical networks. We support our claims via numerical results and mathematical analysis, and we propose a generative model for disassortative growing scale-free networks. PMID:25520244

  7. Emergence of disassortative mixing from pruning nodes in growing scale-free networks.

    PubMed

    Wang, Sheng-Jun; Wang, Zhen; Jin, Tao; Boccaletti, Stefano

    2014-12-18

    Disassortative mixing is ubiquitously found in technological and biological networks, while the corresponding interpretation of its origin remains almost virgin. We here give evidence that pruning the largest-degree nodes of a growing scale-free network has the effect of decreasing the degree correlation coefficient in a controllable and tunable way, while keeping both the trait of a power-law degree distribution and the main properties of network's resilience and robustness under failures or attacks. The essence of these observations can be attributed to the fact the deletion of large-degree nodes affects the delicate balance of positive and negative contributions to degree correlation in growing scale-free networks, eventually leading to the emergence of disassortativity. Moreover, these theoretical prediction will get further validation in the empirical networks. We support our claims via numerical results and mathematical analysis, and we propose a generative model for disassortative growing scale-free networks.

  8. Analytical results for stochastically growing networks: Connection to the zero-range process

    NASA Astrophysics Data System (ADS)

    Mohanty, P. K.; Jalan, Sarika

    2008-04-01

    We introduce a stochastic model of growing networks where both the number of new nodes which join the network and the number of connections vary stochastically. We provide an exact mapping between this model and the zero-range process, and calculate analytically the degree distribution for any given evolution rule. We argue that this mapping can be used to infer a possible evolution rule for any given network. This is being demonstrated for a protein-protein interaction network of Saccharomyces cerevisiae.

  9. Modulation of cargo release from dense core granules by size and actin network.

    PubMed

    Felmy, Felix

    2007-08-01

    During regulated fusion of secretory granules with the plasma membrane, a fusion pore first opens and then dilates. The dilating pore allows cargo proteins from the dense core to be released into the extracellular space. Using real-time evanescent field fluorescence microscopy of live PC12 cells, it was determined how rapidly proteins of different sizes escape from single granules after fusion. Tissue plasminogen activator (tPA)-Venus is released 40-fold slower than the three times smaller neuropeptide Y [NPY-monomeric GFP (mGFP)]. An NPY bearing two mGFPs in tandem [NPY-(mGFP)(2)] as an intermediate-sized fusion probe is released most slowly. Although, the time-course of release varies substantially for a given probe. Coexpression of beta-actin, actin-related protein 3 or mAbp1 slowed the release of the two larger cargo molecules but did not affect release of NPY-mGFP or of the granule-membrane-bound probe Vamp-pHluorin. Additionally, high concentrations of cytochalasin D slowed release of the tPA-Venus. Together these results suggest that fusion pore dilation is not the only determinate of release time-course and that actin rearrangements similar to those mediating actin-mediated motility influences the time-course of release without directly interfering with the granule membrane to cell membrane connection.

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

    PubMed Central

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

    2016-01-01

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

  11. Growing up wired: social networking sites and adolescent psychosocial development.

    PubMed

    Spies Shapiro, Lauren A; Margolin, Gayla

    2014-03-01

    Since the advent of social networking site (SNS) technologies, adolescents' use of these technologies has expanded and is now a primary way of communicating with and acquiring information about others in their social network. Overall, adolescents and young adults' stated motivations for using SNSs are quite similar to more traditional forms of communication-to stay in touch with friends, make plans, get to know people better, and present oneself to others. We begin with a summary of theories that describe the role of SNSs in adolescents' interpersonal relationships, as well as common methodologies used in this field of research thus far. Then, with the social changes that occur throughout adolescence as a backdrop, we address the ways in which SNSs intersect with key tasks of adolescent psychosocial development, specifically peer affiliation and friendship quality, as well as identity development. Evidence suggests that SNSs differentially relate to adolescents' social connectivity and identity development, with sociability, self-esteem, and nature of SNS feedback as important potential moderators. We synthesize current findings, highlight unanswered questions, and recommend both methodological and theoretical directions for future research.

  12. Kv3.3 Channels Bind Hax-1 and Arp2/3 to Assemble a Stable Local Actin Network that Regulates Channel Gating.

    PubMed

    Zhang, Yalan; Zhang, Xiao-Feng; Fleming, Matthew R; Amiri, Anahita; El-Hassar, Lynda; Surguchev, Alexei A; Hyland, Callen; Jenkins, David P; Desai, Rooma; Brown, Maile R; Gazula, Valeswara-Rao; Waters, Michael F; Large, Charles H; Horvath, Tamas L; Navaratnam, Dhasakumar; Vaccarino, Flora M; Forscher, Paul; Kaczmarek, Leonard K

    2016-04-07

    Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.

  13. The 5' cap of tobacco mosaic virus (TMV) is required for virion attachment to the actin/endoplasmic reticulum network during early infection.

    PubMed

    Christensen, Nynne; Tilsner, Jens; Bell, Karen; Hammann, Philippe; Parton, Richard; Lacomme, Christophe; Oparka, Karl

    2009-05-01

    Almost nothing is known of the earliest stages of plant virus infections. To address this, we microinjected Cy3 (UTP)-labelled tobacco mosaic virus (TMV) into living tobacco trichome cells. The Cy3-virions were infectious, and the viral genome trafficked from cell to cell. However, neither the fluorescent vRNA pool nor the co-injected green fluorescent protein (GFP) left the injected trichome, indicating that the synthesis of (unlabelled) progeny viral (v)RNA is required to initiate cell-to-cell movement, and that virus movement is not accompanied by passive plasmodesmatal gating. Cy3-vRNA formed granules that became anchored to the motile cortical actin/endoplasmic reticulum (ER) network within minutes of injection. Granule movement on actin/ER was arrested by actin inhibitors indicating actin-dependent RNA movement. The 5' methylguanosine cap was shown to be required for vRNA anchoring to the actin/ER. TMV vRNA lacking the 5' cap failed to form granules and was degraded in the cytoplasm. Removal of the 3' untranslated region or replicase both inhibited replication but did not prevent granule formation and movement. Dual-labelled TMV virions in which the vRNA and the coat protein were highlighted with different fluorophores showed that both fluorescent signals were initially located on the same ER-bound granules, indicating that TMV virions may become attached to the ER prior to uncoating of the viral genome.

  14. Responses to cell loss become restricted as the supporting cells in mammalian vestibular organs grow thick junctional actin bands that develop high stability.

    PubMed

    Burns, Joseph C; Corwin, Jeffrey T

    2014-01-29

    Sensory hair cell (HC) loss is a major cause of permanent hearing and balance impairments for humans and other mammals. Yet, fish, amphibians, reptiles, and birds readily replace HCs and recover from such sensory deficits. It is unknown what prevents replacement in mammals, but cell replacement capacity declines contemporaneously with massive postnatal thickening of F-actin bands at the junctions between vestibular supporting cells (SCs). In non-mammals, SCs can give rise to regenerated HCs, and the bands remain thin even in adults. Here we investigated the stability of the F-actin bands between SCs in ears from chickens and mice and Madin-Darby canine kidney cells. Pharmacological experiments and fluorescence recovery after photobleaching (FRAP) of SC junctions in utricles from mice that express a γ-actin-GFP fusion protein showed that the thickening F-actin bands develop increased resistance to depolymerization and exceptional stability that parallels a sharp decline in the cell replacement capacity of the maturing mammalian ear. The FRAP recovery rate and the mobile fraction of γ-actin-GFP both decreased as the bands thickened with age and became highly stabilized. In utricles from neonatal mice, time-lapse recordings in the vicinity of dying HCs showed that numerous SCs change shape and organize multicellular actin purse strings that reseal the epithelium. In contrast, adult SCs appeared resistant to deformation, with resealing responses limited to just a few neighboring SCs that did not form purse strings. The exceptional stability of the uniquely thick F-actin bands at the junctions of mature SCs may play an important role in restricting dynamic repair responses in mammalian vestibular epithelia.

  15. Growing Up Wired: Social Networking Sites and Adolescent Psychosocial Development

    PubMed Central

    Shapiro, Lauren A. Spies; Margolin, Gayla

    2013-01-01

    Since the advent of SNS technologies, adolescents' use of these technologies has expanded and is now a primary way of communicating with and acquiring information about others in their social network. Overall, adolescents and young adults’ stated motivations for using SNSs are quite similar to more traditional forms of communication—to stay in touch with friends, make plans, get to know people better, and present oneself to others. We begin with a summary of theories that describe the role of SNSs in adolescents’ interpersonal relationships, as well as common methodologies used in this field of research thus far. Then, with the social changes that occur throughout adolescence as a backdrop, we address the ways in which SNSs intersect with key tasks of adolescent psychosocial development, specifically peer affiliation and friendship quality, as well as identity development. Evidence suggests that SNSs differentially relate to adolescents’ social connectivity and identity development, with sociability, self-esteem, and nature of SNS feedback as important potential moderators. We synthesize current findings, highlight unanswered questions, and recommend both methodological and theoretical directions for future research. PMID:23645343

  16. The Interaction of Arp2/3 Complex with Actin: Nucleation, High Affinity Pointed End Capping, and Formation of Branching Networks of Filaments

    NASA Astrophysics Data System (ADS)

    Dyche Mullins, R.; Heuser, John A.; Pollard, Thomas D.

    1998-05-01

    The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μ M. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7 degrees. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

  17. Actinic keratosis

    MedlinePlus

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

  18. Semi-retentive cytoskeletal fractionation (SERCYF): A novel method for the biochemical analysis of the organization of microtubule and actin cytoskeleton networks.

    PubMed

    Sato, Yuta; Murakami, Yota; Takahashi, Masayuki

    2017-07-08

    A variety of biochemical fractionation methods are available for the quantification of cytoskeletal components. However, each method is designed to target only one cytoskeletal network, either the microtubule (MT) or actin cytoskeleton, and non-targeted cytoskeletal networks are ignored. Considering the importance of MT-actin crosstalk, the organization of both the targeted and non-targeted cytoskeletal networks must be retained intact during fractionation for the accurate analysis of cytoskeletal organization. In this study, we reveal that existing fractionation methods, represented by the MT-sedimentation-method for MTs and the Triton X-100 solubility assay-method for actin cytoskeletons, disrupt the organizations of the non-targeted cytoskeletons. We demonstrate a novel fractionation method for the accurate analysis of the cytoskeletal organizations using a taxol-containing PEM-based permeabilization buffer, which we name "semi-retentive cytoskeletal fractionation (SERCYF)-method". The organizations of both MTs and actin cytoskeletons were retained intact even after permeabilization with this buffer. By using the SERCYF-method, we analyzed the effects of nocodazole on the cytoskeletal organizations biochemically and showed promotion of the actin cytoskeletal organization by MT depolymerization. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Accelerated actin filament polymerization from microtubule plus-ends

    PubMed Central

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

    2016-01-01

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

  20. 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.

  1. Actin Mechanics and Fragmentation*

    PubMed Central

    De La Cruz, Enrique M.; Gardel, Margaret L.

    2015-01-01

    Cell physiological processes require the regulation and coordination of both mechanical and dynamical properties of the actin cytoskeleton. Here we review recent advances in understanding the mechanical properties and stability of actin filaments and how these properties are manifested at larger (network) length scales. We discuss how forces can influence local biochemical interactions, resulting in the formation of mechanically sensitive dynamic steady states. Understanding the regulation of such force-activated chemistries and dynamic steady states reflects an important challenge for future work that will provide valuable insights as to how the actin cytoskeleton engenders mechanoresponsiveness of living cells. PMID:25957404

  2. Chromosome translocation may lead to PRK1-dependent anticancer drug resistance in yeast via endocytic actin network deregulation.

    PubMed

    Nikitin, Dmitri V; Bruschi, Carlo V; Sims, Jason; Breitenbach, Michael; Rinnerthaler, Mark; Tosato, Valentina

    2014-04-01

    Chromosome translocations are often observed in cancer cells, being in some cases the cause of neoplastic transformation while in others the results of it. In previous works, we reproduced this major genomic rearrangement by bridge-induced chromosome translocation (BIT) technology in the model eukaryote Saccharomyces cerevisiae and reported that it affects DNA replication, cell cycle, karyogamy, and cytokinesis while it produces genetic instability. In the present work, we further discovered that this event can lead to increased resistance to anticancer chemicals like Doxorubicin and Latrunculin A via an endocytic actin network deregulation triggered by over-expression of the PRK1 serine/threonine protein kinase gene. This effect is further enhanced by the overexpression of PDR1 and PDR3 transcriptional regulators of pleiotropic drug resistance factors. However, when the actin depolymerizing drug Latrunculin A is forcefully allowed to penetrate through their altered cell wall and membrane barriers, it can kill translocants more efficiently than wild type cells. These observations provide an example of an acquired anticancer drug resistance mechanism and could serve as a lead to how it might be overcome, as any treatment inhibiting genome rearrangements could increase the positive outcome of anticancer therapy by lowering cellular drug resistance. Copyright © 2014 Elsevier GmbH. All rights reserved.

  3. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

    PubMed Central

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. DOI: http://dx.doi.org/10.7554/eLife.06585.001 PMID:26295568

  4. 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

  5. Responses to Cell Loss Become Restricted as the Supporting Cells in Mammalian Vestibular Organs Grow Thick Junctional Actin Bands That Develop High Stability

    PubMed Central

    Burns, Joseph C.

    2014-01-01

    Sensory hair cell (HC) loss is a major cause of permanent hearing and balance impairments for humans and other mammals. Yet, fish, amphibians, reptiles, and birds readily replace HCs and recover from such sensory deficits. It is unknown what prevents replacement in mammals, but cell replacement capacity declines contemporaneously with massive postnatal thickening of F-actin bands at the junctions between vestibular supporting cells (SCs). In non-mammals, SCs can give rise to regenerated HCs, and the bands remain thin even in adults. Here we investigated the stability of the F-actin bands between SCs in ears from chickens and mice and Madin-Darby canine kidney cells. Pharmacological experiments and fluorescence recovery after photobleaching (FRAP) of SC junctions in utricles from mice that express a γ-actin–GFP fusion protein showed that the thickening F-actin bands develop increased resistance to depolymerization and exceptional stability that parallels a sharp decline in the cell replacement capacity of the maturing mammalian ear. The FRAP recovery rate and the mobile fraction of γ-actin–GFP both decreased as the bands thickened with age and became highly stabilized. In utricles from neonatal mice, time-lapse recordings in the vicinity of dying HCs showed that numerous SCs change shape and organize multicellular actin purse strings that reseal the epithelium. In contrast, adult SCs appeared resistant to deformation, with resealing responses limited to just a few neighboring SCs that did not form purse strings. The exceptional stability of the uniquely thick F-actin bands at the junctions of mature SCs may play an important role in restricting dynamic repair responses in mammalian vestibular epithelia. PMID:24478379

  6. Arbuscular Mycorrhizal Fungal Networks Vary throughout the Growing Season and between Successional Stages

    PubMed Central

    Bennett, Alison Elizabeth; Daniell, Tim John; Öpik, Maarja; Davison, John; Moora, Mari; Zobel, Martin; Selosse, Marc-André; Evans, Darren

    2013-01-01

    To date, few analyses of mutualistic networks have investigated successional or seasonal dynamics. Combining interaction data from multiple time points likely creates an inaccurate picture of the structure of networks (because these networks are aggregated across time), which may negatively influence their application in ecosystem assessments and conservation. Using a replicated bipartite mutualistic network of arbuscular mycorrhizal (AM) fungal-plant associations, detected using large sample numbers of plants and AM fungi identified through molecular techniques, we test whether the properties of the network are temporally dynamic either between different successional stages or within the growing season. These questions have never been directly tested in the AM fungal-plant mutualism or the vast majority of other mutualisms. We demonstrate the following results: First, our examination of two different successional stages (young and old forest) demonstrated that succession increases the proportion of specialists within the community and decreases the number of interactions. Second, AM fungal-plant mutualism structure changed throughout the growing season as the number of links between partners increased. Third, we observed shifts in associations between AM fungal and plant species throughout the growing season, potentially reflecting changes in biotic and abiotic conditions. Thus, this analysis opens up two entirely new areas of research: 1) identifying what influences changes in plant-AM fungal associations in these networks, and 2) what aspects of temporal variation and succession are of general importance in structuring bipartite networks and plant-AM fungal communities. PMID:24358265

  7. Arbuscular mycorrhizal fungal networks vary throughout the growing season and between successional stages.

    PubMed

    Bennett, Alison Elizabeth; Daniell, Tim John; Öpik, Maarja; Davison, John; Moora, Mari; Zobel, Martin; Selosse, Marc-André; Evans, Darren

    2013-01-01

    To date, few analyses of mutualistic networks have investigated successional or seasonal dynamics. Combining interaction data from multiple time points likely creates an inaccurate picture of the structure of networks (because these networks are aggregated across time), which may negatively influence their application in ecosystem assessments and conservation. Using a replicated bipartite mutualistic network of arbuscular mycorrhizal (AM) fungal-plant associations, detected using large sample numbers of plants and AM fungi identified through molecular techniques, we test whether the properties of the network are temporally dynamic either between different successional stages or within the growing season. These questions have never been directly tested in the AM fungal-plant mutualism or the vast majority of other mutualisms. We demonstrate the following results: First, our examination of two different successional stages (young and old forest) demonstrated that succession increases the proportion of specialists within the community and decreases the number of interactions. Second, AM fungal-plant mutualism structure changed throughout the growing season as the number of links between partners increased. Third, we observed shifts in associations between AM fungal and plant species throughout the growing season, potentially reflecting changes in biotic and abiotic conditions. Thus, this analysis opens up two entirely new areas of research: 1) identifying what influences changes in plant-AM fungal associations in these networks, and 2) what aspects of temporal variation and succession are of general importance in structuring bipartite networks and plant-AM fungal communities.

  8. An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus

    PubMed Central

    1987-01-01

    We have studied the F-actin network in cycling suspension culture cells of carrot (Daucus carota L.) using rhodaminyl lysine phallotoxin (RLP). In addition to conventional fixation with formaldehyde, we have used two different nonfixation methods before adding RLP: extracting cells in a stabilizing buffer; inducing transient pores in the plasma membrane with pulses of direct current (electroporation). These alternative methods for introducing RLP revealed additional features of the actin network not seen in aldehyde-fixed cells. The three- dimensional organization of this network in nonflattened cells was demonstrated by projecting stereopairs derived from through-focal series of computer-enhanced images. F-actin is present in interphase cells in four interconnected configurations: a meshwork surrounding the nucleus; thick cables in transvacuolar strands and deep in the cytoplasm; a finer network of bundles within the cortical cytoplasm; even finer filaments that run in ordered transverse array around the cell periphery. The actin network is organized differently during division but it does not disappear as do the cortical microtubules. RLP stains a central filamentous cortical band as the chromatin begins to condense (preprophase); it stains the mitotic spindle (as recently shown by Seagull et al. [Seagull, R. W., M. Falconer, and C. A. Weerdenburg, 1987, J. Cell Biol., 104:995-1004] for aldehyde fixed suspension cells) and the cytokinetic apparatus (as shown by Clayton, L., and C. W. Lloyd, 1985, Exp. Cell Res., 156:231-238). However, it is now shown that an additional network of F-actin persists in the cytoplasm throughout division associating in turn with the preprophase band, the mitotic spindle, and the cytokinetic phragmoplast. PMID:2440896

  9. Cortactin Branches Out: Roles in Regulating Protrusive Actin Dynamics

    PubMed Central

    Ammer, Amanda Gatesman; Weed, Scott A.

    2008-01-01

    Since its discovery in the early 1990’s, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures. PMID:18615630

  10. Self-growing neural network architecture using crisp and fuzzy entropy

    NASA Technical Reports Server (NTRS)

    Cios, Krzysztof J.

    1992-01-01

    The paper briefly describes the self-growing neural network algorithm, CID3, which makes decision trees equivalent to hidden layers of a neural network. The algorithm generates a feedforward architecture using crisp and fuzzy entropy measures. The results for a real-life recognition problem of distinguishing defects in a glass ribbon, and for a benchmark problen of telling two spirals apart are shown and discussed.

  11. Self-growing neural network architecture using crisp and fuzzy entropy

    NASA Technical Reports Server (NTRS)

    Cios, Krzysztof J.

    1992-01-01

    The paper briefly describes the self-growing neural network algorithm, CID3, which makes decision trees equivalent to hidden layers of a neural network. The algorithm generates a feedforward architecture using crisp and fuzzy entropy measures. The results for a real-life recognition problem of distinguishing defects in a glass ribbon, and for a benchmark problen of telling two spirals apart are shown and discussed.

  12. Self-growing neural network architecture using crisp and fuzzy entropy

    NASA Technical Reports Server (NTRS)

    Cios, Krzysztof J.

    1992-01-01

    The paper briefly describes the self-growing neural network algorithm, CID2, which makes decision trees equivalent to hidden layers of a neural network. The algorithm generates a feedforward architecture using crisp and fuzzy entropy measures. The results of a real-life recognition problem of distinguishing defects in a glass ribbon and of a benchmark problem of differentiating two spirals are shown and discussed.

  13. Growing scale-free networks with tunable distributions of triad motifs

    NASA Astrophysics Data System (ADS)

    Li, Shuguang; Yuan, Jianping; Shi, Yong; Zagal, Juan Cristóbal

    2015-06-01

    Network motifs are local structural patterns and elementary functional units of complex networks in real world, which can have significant impacts on the global behavior of these systems. Many models are able to reproduce complex networks mimicking a series of global features of real systems, however the local features such as motifs in real networks have not been well represented. We propose a model to grow scale-free networks with tunable motif distributions through a combined operation of preferential attachment and triad motif seeding steps. Numerical experiments show that the constructed networks have adjustable distributions of the local triad motifs, meanwhile preserving the global features of power-law distributions of node degree, short average path lengths of nodes, and highly clustered structures.

  14. Actin Dynamics in Growth Cone Motility and Navigation

    PubMed Central

    Gomez, Timothy M.; Letourneau, Paul C.

    2014-01-01

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

  15. A growing and pruning sequential learning algorithm of hyper basis function neural network for function approximation.

    PubMed

    Vuković, Najdan; Miljković, Zoran

    2013-10-01

    Radial basis function (RBF) neural network is constructed of certain number of RBF neurons, and these networks are among the most used neural networks for modeling of various nonlinear problems in engineering. Conventional RBF neuron is usually based on Gaussian type of activation function with single width for each activation function. This feature restricts neuron performance for modeling the complex nonlinear problems. To accommodate limitation of a single scale, this paper presents neural network with similar but yet different activation function-hyper basis function (HBF). The HBF allows different scaling of input dimensions to provide better generalization property when dealing with complex nonlinear problems in engineering practice. The HBF is based on generalization of Gaussian type of neuron that applies Mahalanobis-like distance as a distance metrics between input training sample and prototype vector. Compared to the RBF, the HBF neuron has more parameters to optimize, but HBF neural network needs less number of HBF neurons to memorize relationship between input and output sets in order to achieve good generalization property. However, recent research results of HBF neural network performance have shown that optimal way of constructing this type of neural network is needed; this paper addresses this issue and modifies sequential learning algorithm for HBF neural network that exploits the concept of neuron's significance and allows growing and pruning of HBF neuron during learning process. Extensive experimental study shows that HBF neural network, trained with developed learning algorithm, achieves lower prediction error and more compact neural network.

  16. Contribution of space experiments to the study of the actin network structure of the lentil root statocyte

    NASA Astrophysics Data System (ADS)

    Driss-Ecole, D.; Perbal, G.

    Two space experiments performed in the frame of the S/MM03 and S/MM05 Missions of Spacehab have permitted to study the structure of the actin network in the lentil root statocyte. In the first experiment, lentil seedlings were grown on a 1g centrifuge (with a root-tip-directed 1g acceleration) and then were placed in microgravity for increasing periods of time (13, 29, 46 or 122min). In the second experiments lentil seedlings were either grown in microgravity or on a 1g centrifuge and then were subjected to various centrifugal accelerations (from 0.39 to 0.93g for 9 or 22 min) perpendicular to the longitudinal axis the roots. At the end of the stimulation period, gravitropic curvature of roots was followed by time-lapse photography for 3 h in microgravity. In both experiments, some seedlings were chemically fixed in space in order to analyse the distribution of the amyloplasts within the statocytes in the different samples. The S/MM03 experiment has demonstrated that, in microgravity, the amyloplasts move from the distal pole to the proximal pole of the statocytes. This autonomous and directed movement is due to an actomyosin system and lead to a stable position of the statoliths near the nucleus after about 2h. A treatment with cytochalasin D did not stop this movement but slowed down the velocity of their displacement. The S/MM05 experiment showed that after a stimulation of 22 min at 0.93g the sedimentation of statoliths along the longitudinal wall is similar in statocytes of roots grown first in microgravity or on the 1g centrifuge although their initial position was not the same. When these stimulated seedlings were kept for 3 h in microgravity the amyloplasts in both cases move towards the nucleus. This overall oblique displacement could be due to the oblique orientation of the actin filaments with their + ends directed toward the nucleus. This hypothesis is reinforced by the results obtained with the S/MM03 experiment: the value of the force responsible for

  17. Action selection in growing state spaces: control of network structure growth

    NASA Astrophysics Data System (ADS)

    Thalmeier, Dominik; Gómez, Vicenç; Kappen, Hilbert J.

    2017-01-01

    The dynamical processes taking place on a network depend on its topology. Influencing the growth process of a network therefore has important implications on such dynamical processes. We formulate the problem of influencing the growth of a network as a stochastic optimal control problem in which a structural cost function penalizes undesired topologies. We approximate this control problem with a restricted class of control problems that can be solved using probabilistic inference methods. To deal with the increasing problem dimensionality, we introduce an adaptive importance sampling method for approximating the optimal control. We illustrate this methodology in the context of formation of information cascades, considering the task of influencing the structure of a growing conversation thread, as in Internet forums. Using a realistic model of growing trees, we show that our approach can yield conversation threads with better structural properties than the ones observed without control.

  18. Reply to ``Comment on `Linking population-level models with growing networks: A class of epidemic models' ''

    NASA Astrophysics Data System (ADS)

    Breban, Romulus; Vardavas, Raffaele; Blower, Sally

    2006-07-01

    We disagree with Bootsma and Diekmann that our formula for the basic reproduction ratio (R0) has an underlying conceptual mistake. In Phys. Rev. E 72, 046110 (2005), we propose a large class of growing networks (which we call Kinetic Monte Carlo (KMC) growing networks) as individual-level models for the transmission of infectious diseases. KMC growing networks are conceptually different from the well-established Crump-Mode-Jagers continuous-time branching processes. Thus, the branching process definition of R0 is not valid for KMC growing networks, and a different implementation of the biological definition of R0 is necessary.

  19. Bacterial nucleators: actin' on actin

    PubMed Central

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

    2015-01-01

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

  20. Bistability in the Rac1, PAK, and RhoA Signaling Network Drives Actin Cytoskeleton Dynamics and Cell Motility Switches

    PubMed Central

    Byrne, Kate M.; Monsefi, Naser; Dawson, John C.; Degasperi, Andrea; Bukowski-Wills, Jimi-Carlo; Volinsky, Natalia; Dobrzyński, Maciej; Birtwistle, Marc R.; Tsyganov, Mikhail A.; Kiyatkin, Anatoly; Kida, Katarzyna; Finch, Andrew J.; Carragher, Neil O.; Kolch, Walter; Nguyen, Lan K.; von Kriegsheim, Alex; Kholodenko, Boris N.

    2016-01-01

    Summary Dynamic interactions between RhoA and Rac1, members of the Rho small GTPase family, play a vital role in the control of cell migration. Using predictive mathematical modeling, mass spectrometry-based quantitation of network components, and experimental validation in MDA-MB-231 mesenchymal breast cancer cells, we show that a network containing Rac1, RhoA, and PAK family kinases can produce bistable, switch-like responses to a graded PAK inhibition. Using a small chemical inhibitor of PAK, we demonstrate that cellular RhoA and Rac1 activation levels respond in a history-dependent, bistable manner to PAK inhibition. Consequently, we show that downstream signaling, actin dynamics, and cell migration also behave in a bistable fashion, displaying switches and hysteresis in response to PAK inhibition. Our results demonstrate that PAK is a critical component in the Rac1-RhoA inhibitory crosstalk that governs bistable GTPase activity, cell morphology, and cell migration switches. PMID:27136688

  1. [Actinic keratoses].

    PubMed

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

    2003-06-01

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

  2. A study of the temporal robustness of the growing global container-shipping network

    PubMed Central

    Wang, Nuo; Wu, Nuan; Dong, Ling-ling; Yan, Hua-kun; Wu, Di

    2016-01-01

    Whether they thrive as they grow must be determined for all constantly expanding networks. However, few studies have focused on this important network feature or the development of quantitative analytical methods. Given the formation and growth of the global container-shipping network, we proposed the concept of network temporal robustness and quantitative method. As an example, we collected container liner companies’ data at two time points (2004 and 2014) and built a shipping network with ports as nodes and routes as links. We thus obtained a quantitative value of the temporal robustness. The temporal robustness is a significant network property because, for the first time, we can clearly recognize that the shipping network has become more vulnerable to damage over the last decade: When the node failure scale reached 50% of the entire network, the temporal robustness was approximately −0.51% for random errors and −12.63% for intentional attacks. The proposed concept and analytical method described in this paper are significant for other network studies. PMID:27713549

  3. A study of the temporal robustness of the growing global container-shipping network

    NASA Astrophysics Data System (ADS)

    Wang, Nuo; Wu, Nuan; Dong, Ling-Ling; Yan, Hua-Kun; Wu, Di

    2016-10-01

    Whether they thrive as they grow must be determined for all constantly expanding networks. However, few studies have focused on this important network feature or the development of quantitative analytical methods. Given the formation and growth of the global container-shipping network, we proposed the concept of network temporal robustness and quantitative method. As an example, we collected container liner companies’ data at two time points (2004 and 2014) and built a shipping network with ports as nodes and routes as links. We thus obtained a quantitative value of the temporal robustness. The temporal robustness is a significant network property because, for the first time, we can clearly recognize that the shipping network has become more vulnerable to damage over the last decade: When the node failure scale reached 50% of the entire network, the temporal robustness was approximately ‑0.51% for random errors and ‑12.63% for intentional attacks. The proposed concept and analytical method described in this paper are significant for other network studies.

  4. A study of the temporal robustness of the growing global container-shipping network.

    PubMed

    Wang, Nuo; Wu, Nuan; Dong, Ling-Ling; Yan, Hua-Kun; Wu, Di

    2016-10-07

    Whether they thrive as they grow must be determined for all constantly expanding networks. However, few studies have focused on this important network feature or the development of quantitative analytical methods. Given the formation and growth of the global container-shipping network, we proposed the concept of network temporal robustness and quantitative method. As an example, we collected container liner companies' data at two time points (2004 and 2014) and built a shipping network with ports as nodes and routes as links. We thus obtained a quantitative value of the temporal robustness. The temporal robustness is a significant network property because, for the first time, we can clearly recognize that the shipping network has become more vulnerable to damage over the last decade: When the node failure scale reached 50% of the entire network, the temporal robustness was approximately -0.51% for random errors and -12.63% for intentional attacks. The proposed concept and analytical method described in this paper are significant for other network studies.

  5. Densification and structural transitions in networks that grow by node copying

    NASA Astrophysics Data System (ADS)

    Bhat, U.; Krapivsky, P. L.; Lambiotte, R.; Redner, S.

    2016-12-01

    We introduce a growing network model, the copying model, in which a new node attaches to a randomly selected target node and, in addition, independently to each of the neighbors of the target with copying probability p . When p <1/2 , this algorithm generates sparse networks, in which the average node degree is finite. A power-law degree distribution also arises, with a nonuniversal exponent whose value is determined by a transcendental equation in p . In the sparse regime, the network is "normal," e.g., the relative fluctuations in the number of links are asymptotically negligible. For p ≥1/2 , the emergent networks are dense (the average degree increases with the number of nodes N ), and they exhibit intriguing structural behaviors. In particular, the N dependence of the number of m cliques (complete subgraphs of m nodes) undergoes m -1 transitions from normal to progressively more anomalous behavior at an m -dependent critical values of p . Different realizations of the network, which start from the same initial state, exhibit macroscopic fluctuations in the thermodynamic limit: absence of self-averaging. When linking to second neighbors of the target node can occur, the number of links asymptotically grows as N2 as N →∞ , so that the network is effectively complete as N →∞ .

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

    PubMed Central

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

    2013-01-01

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

  7. 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

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

    PubMed Central

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

    2016-01-01

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

  9. GhCFE1A, a dynamic linker between the ER network and actin cytoskeleton, plays an important role in cotton fibre cell initiation and elongation.

    PubMed

    Lv, Fenni; Wang, Haihai; Wang, Xinyu; Han, Libo; Ma, Yinping; Wang, Sen; Feng, Zhidi; Niu, Xiaowei; Cai, Caiping; Kong, Zhaosheng; Zhang, Tianzhen; Guo, Wangzhen

    2015-04-01

    Fibre cell initiation and elongation is critical for cotton fibre development. However, little is known about the regulation of initiation and elongation during fibre cell development. Here, the regulatory role of a novel protein GhCFE1A was uncovered. GhCFE1A is preferentially expressed at initiation and rapid elongation stages during fibre development; in addition, much higher expression of GhCFE1A was detected at the fibre initiation stage in fibreless cotton mutants than in the fibre-bearing TM-1 wild-type. Importantly, overexpression of GhCFE1A in cotton not only delayed fibre cell elongation but also significantly reduced the density of lint and fuzz fibre initials and stem trichomes. Yeast two-hybrid assay showed that GhCFE1A interacted with several actin proteins, and the interaction was further confirmed by co-sedimentation assay. Interestingly, a subcellular localization assay showed that GhCFE1A resided on the cortical endoplasmic reticulum (ER) network and co-localized with actin cables. Moreover, the density of F-actin filaments was shown to be reduced in GhCFE1A-overexpressing fibres at the rapid elongation stage compared with the wild-type control. Taken together, the results demonstrate that GhCFE1A probably functions as a dynamic linker between the actin cytoskeleton and the ER network, and plays an important role in fibre cell initiation and elongation during cotton fibre development.

  10. Chemotaxis and Actin Oscillations

    NASA Astrophysics Data System (ADS)

    Bodenschatz, Eberhard; Hsu, Hsin-Fang; Negrete, Jose; Beta, Carsten; Pumir, Alain; Gholami, Azam; Tarantola, Marco; Westendorf, Christian; Zykov, Vladimir

    Recently, self-oscillations of the cytoskeletal actin have been observed in Dictyostelium, a model system for studying chemotaxis. Here we report experimental results on the self-oscillation mechanism and the role of regulatory proteins and myosin II. We stimulate cells rapidly and periodically by using photo un-caging of the chemoattractant in a micro-fluidic device and measured the cellular responses. We found that the response amplitude grows with stimulation strength only in a very narrow region of stimulation, after which the response amplitude reaches a plateau. Moreover, the frequency-response is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and de-polymerization time in the single cell level. Despite of the large cell-to-cell variability, we found that the polymerization time is independent of external stimuli and the de-polymerization time is prolonged as the stimulation strength increases. Our conclusions will be summarized and the role of noise in the signaling network will be discussed. German Science Foundation CRC 937.

  11. Rictor/mTORC2 regulates blood-testis barrier dynamics via its effects on gap junction communications and actin filament network.

    PubMed

    Mok, Ka-Wai; Mruk, Dolores D; Lee, Will M; Cheng, C Yan

    2013-03-01

    In the mammalian testis, coexisting tight junctions (TJs), basal ectoplasmic specializations, and gap junctions (GJs), together with desmosomes near the basement membrane, constitute the blood-testis barrier (BTB). The most notable feature of the BTB, however, is the extensive network of actin filament bundles, which makes it one of the tightest blood-tissue barriers. The BTB undergoes restructuring to facilitate the transit of preleptotene spermatocytes at stage VIII-IX of the epithelial cycle. Thus, the F-actin network at the BTB undergoes cyclic reorganization via a yet-to-be explored mechanism. Rictor, the key component of mTORC2 that is known to regulate actin cytoskeleton, was shown to express stage-specifically at the BTB in the seminiferous epithelium. Its expression was down-regulated at the BTB in stage VIII-IX tubules, coinciding with BTB restructuring at these stages. Using an in vivo model, a down-regulation of rictor at the BTB was also detected during adjudin-induced BTB disruption, illustrating rictor expression is positively correlated with the status of the BTB integrity. Indeed, the knockdown of rictor by RNAi was found to perturb the Sertoli cell TJ-barrier function in vitro and the BTB integrity in vivo. This loss of barrier function was accompanied by changes in F-actin organization at the Sertoli cell BTB in vitro and in vivo, associated with a loss of interaction between actin and α-catenin or ZO-1. Rictor knockdown by RNAi was also found to impede Sertoli cell-cell GJ communication, disrupting protein distribution (e.g., occludin, ZO-1) at the BTB, illustrating that rictor is a crucial BTB regulator.

  12. Direct actin binding to A- and B-type lamin tails and actin filament bundling by the lamin A tail

    PubMed Central

    Simon, Dan N; Zastrow, Michael S

    2010-01-01

    Nuclear intermediate filament networks formed by A- and B-type lamins are major components of the nucleoskeleton. Lamins have growing links to human physiology and disease including Emery-Dreifuss muscular dystrophy (EDMD), lipodystrophy, cardiomyopathy, neuropathy, cerebellar disorders and segmental accelerated ‘aging’ syndromes. How lamins interact with other nucleoskeletal components, and even the identities of these other components, are open questions. Previous studies suggested lamins might bind actin. We report that the recombinant C-terminal tail domain of human A- and B-type lamins binds directly to purified actin in high-speed pelleting assays. This interaction maps to a conserved Actin Binding site (AB-1) comprising lamin A residues 461–536 in the Ig-fold domain, which are 54% identical in lamin B1. Two EDMD-causing missense mutations (R527P and L530P) in lamin A that are predicted to disrupt the Ig-fold, each reduced F-actin binding by ∼66%, whereas the surface-exposed lipodystrophy-causing R482Q mutation had no significant effect. The lamin A tail was unique among lamins in having a second actin-binding site (AB-2). This second site was mapped to lamin A tail residues 564–608, based on actin-binding results for the lamin C tail and internal deletions in the lamin A tail that cause Hutchinson-Gilford Progeria Syndrome (Δ35, Δ50) or restrictive dermopathy (Δ90). Supporting the presence of two actin-binding sites, recombinant precursor (unmodified) and mature lamin A tails (not C or B1 tails) each bundled F-actin in vitro: furthermore F-actin bundling was reduced 25–40% by the R527P, L530P, Δ35 and Δ50 mutations, and was abolished by Δ90. Unexpectedly, the mature lamin A tail bound F-actin significantly more efficiently than did the prelamin A tail; this suggested unmodified residues 647–664, unique to prelamin A, might auto-inhibit binding to actin (and potentially other partners). These biochemical results suggest direct mechanisms

  13. Asymptotic behavior of connecting-nearest-neighbor models for growing networks

    NASA Astrophysics Data System (ADS)

    Juher, David; Saldaña, Joan; Soler, Jaume

    2006-02-01

    This paper deals with the mathematical description of the asymptotic behavior of the solutions of a couple of models for the dynamics of growing networks based on connecting, with a higher probability, nodes that have a neighbor in common. The first model, proposed by A. Vázquez, is nonlinear and, in general, the long-time behavior of the solutions differs from the one predicted by the linear reduction proposed in its original treatment. A second model is specifically derived from the rules defining an in silico model also proposed by Vázquez to simulate the growth of a network under the mechanism of connecting nearest neighbors. The two analytical models lead to very different predictions for the configuration of the network that are tested using the simulations of the in silico model.

  14. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein

    PubMed Central

    1983-01-01

    Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three- dimensional network resembling the peripheral cytoskeleton of motile cells. PMID:6682423

  15. Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

    PubMed

    Niederman, R; Amrein, P C; Hartwig, J

    1983-05-01

    Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

  16. Temperature-induced sol-gel transition and microgel formation in α-actinin cross-linked actin networks: A rheological study

    NASA Astrophysics Data System (ADS)

    Tempel, M.; Isenberg, G.; Sackmann, E.

    1996-08-01

    We have studied the sol-gel transition, the viscoelastic and the structural properties of networks constituted of semiflexible actin filaments cross-linked by α-actinin. Cross-linking was regulated in a reversible way by varying the temperature through the association-dissociation equilibrium of the actin-α-actinin system. Viscoelastic parameters [shear storage modulus G'(ω), phase shift tan(Φ)(ω), creep compliance J(t)] were measured as a function of temperature and actin-to-cross-linker ratio by a magnetically driven rotating disc rheometer. G'(ω) and tan(Φ)(ω) were studied at a frequency ω corresponding to the elastic plateau regime of the G'(ω) versus ω spectrum of the purely entangled solution. The microstructure of the networks was viewed by negative staining electron microscopy (EM). The phase shift tan(Φ) (or equivalently the viscosity η) diverges and reaches a maximum when approaching the apparent gel point from lower and higher temperatures, and the maximum defines the gel point (temperature Tg). The elastic plateau modulus G'N diverges at temperatures beyond this gel point TTg. The cross-linking transition (corresponding to a sol-gel transition at zero frequency) is interpreted in terms of a percolation model and the divergence of G'N at TTg), (2) that microscopic segregation takes place at T<=Tg leading to local formation of clusters (a state termed microgel), and (3) that at low actin-α-actinin ratios (rAα<=10) and low temperatures (T<=10 °C) macroscopic segregation into bundles of cross-linked actin filaments and a diluted solution of actin filaments is observed. The three regimes of network structure are represented by an

  17. Fabrication of three-dimensional electrical connections by means of directed actin self-organization.

    PubMed

    Galland, Rémi; Leduc, Patrick; Guérin, Christophe; Peyrade, David; Blanchoin, Laurent; Théry, Manuel

    2013-05-01

    A promising approach to improve the performance of microelectronic devices is to build three-dimensional (3D) chips made of stacked circuits. However, a major hurdle lies in the fabrication of dense arrays of electrical interconnections between these layers, where accessibility is limited. Here we show that the directed growth and self-organization of actin filaments can offer a solution to this problem. We defined the shape and orientation of 3D actin networks through both micropatterning of actin nucleation factors and biochemical control of actin filament polymerization. Networks growing from two opposing layers were able to interpenetrate and form mechanically stable connections, which were then coated with gold using a selective metallization process. The electrical conductivity, robustness and modularity of the metallized self-organized connections make this approach potentially attractive for 3D chip manufacturing.

  18. A geometric graph model for citation networks of exponentially growing scientific papers

    NASA Astrophysics Data System (ADS)

    Xie, Zheng; Ouyang, Zhenzheng; Liu, Qi; Li, Jianping

    2016-08-01

    In citation networks, the content relativity of papers is a precondition of engendering citations, which is hard to model by a topological graph. A geometric graph is proposed to predict some features of the citation networks with exponentially growing papers, which addresses the precondition by using coordinates of nodes to model the research contents of papers, and geometric distances between nodes to diversities of research contents between papers. Citations between modeled papers are drawn according to a geometric rule, which addresses the precondition as well as some other factors engendering citations, namely academic influences of papers, aging of those influences, and incomplete copying of references. Instead of cumulative advantage of degree, the model illustrates that the scale-free property of modeled networks arises from the inhomogeneous academic influences of modeled papers. The model can also reproduce some other statistical features of citation networks, e.g. in- and out-assortativities, which show the model provides a suitable tool to understand some aspects of citation networks by geometry.

  19. Application of growing hierarchical SOM for visualisation of network forensics traffic data.

    PubMed

    Palomo, E J; North, J; Elizondo, D; Luque, R M; Watson, T

    2012-08-01

    Digital investigation methods are becoming more and more important due to the proliferation of digital crimes and crimes involving digital evidence. Network forensics is a research area that gathers evidence by collecting and analysing network traffic data logs. This analysis can be a difficult process, especially because of the high variability of these attacks and large amount of data. Therefore, software tools that can help with these digital investigations are in great demand. In this paper, a novel approach to analysing and visualising network traffic data based on growing hierarchical self-organising maps (GHSOM) is presented. The self-organising map (SOM) has been shown to be successful for the analysis of highly-dimensional input data in data mining applications as well as for data visualisation in a more intuitive and understandable manner. However, the SOM has some problems related to its static topology and its inability to represent hierarchical relationships in the input data. The GHSOM tries to overcome these limitations by generating a hierarchical architecture that is automatically determined according to the input data and reflects the inherent hierarchical relationships among them. Moreover, the proposed GHSOM has been modified to correctly treat the qualitative features that are present in the traffic data in addition to the quantitative features. Experimental results show that this approach can be very useful for a better understanding of network traffic data, making it easier to search for evidence of attacks or anomalous behaviour in a network environment.

  20. Actinic reticuloid

    SciTech Connect

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

    1982-09-01

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

  1. A finite-element mesh generator based on growing neural networks.

    PubMed

    Triantafyllidis, D G; Labridis, D P

    2002-01-01

    A mesh generator for the production of high-quality finite-element meshes is being proposed. The mesh generator uses an artificial neural network, which grows during the training process in order to adapt itself to a prespecified probability distribution. The initial mesh is a constrained Delaunay triangulation of the domain to be triangulated. Two new algorithms to accelerate the location of the best matching unit are introduced. The mesh generator has been found able to produce meshes of high quality in a number of classic cases examined and is highly suited for problems where the mesh density vector can be calculated in advance.

  2. Intelligent Phonocardiography for Screening Ventricular Septal Defect Using Time Growing Neural Network.

    PubMed

    Gharehbaghi, Arash; Sepehri, Amir A; Lindén, Maria; Babic, Ankica

    2017-01-01

    This paper presents results of a study on the applicability of the intelligent phonocardiography in discriminating between Ventricular Spetal Defect (VSD) and regurgitation of the atrioventricular valves. An original machine learning method, based on the Time Growing Neural Network (TGNN), is employed for classifying the phonocardiographic recordings collected from the pediatric referrals to a children hospital. 90 individuals, 30 VSD, 30 with the valvular regurgitation, and 30 healthy subjects, participated in the study after obtaining the informed consents. The accuracy and sensitivity of the approach is estimated to be 86.7% and 83.3%, respectively, showing a good performance to be used as a decision support system.

  3. Expansion of cooperatively growing populations: Optimal migration rates and habitat network structures

    NASA Astrophysics Data System (ADS)

    Yang, Kai-Cheng; Wu, Zhi-Xi; Holme, Petter; Nonaka, Etsuko

    2017-01-01

    Range expansion of species is driven by the interactions among individual- and population-level processes and the spatial pattern of habitats. In this work we study how cooperatively growing populations spread on networks representing the skeleton of complex landscapes. By separating the slow and fast variables of the expansion process, we are able to give analytical predictions for the critical conditions that divide the dynamic behaviors into different phases (extinction, localized survival, and global expansion). We observe a resonance phenomenon in how the critical condition depends on the expansion rate, indicating the existence of an optimal strategy for global expansion. We derive the conditions for such optimal migration in locally treelike graphs and numerically study other structured networks. Our results highlight the importance of both the underlying interaction pattern and migration rate of the expanding populations for range expansion. We also discuss potential applications of the results to biological control and conservation.

  4. Structural phase transition in a growing network model with tunable member intimacy

    NASA Astrophysics Data System (ADS)

    Kim, Kibum; Jo, Woo Seong; Kim, Beom Jun

    2017-05-01

    Users of online communities become more intimate in time by writing posts and exchanging comments to each other. Although a certain level of intimacy among a group of members can be beneficial for the activity of the whole community, too strong intimacy among existing members can make newcomers feel alienated, driving them to leave the community. In this letter, we introduce a growing network model in which we systematically study the effect of member intimacy on the formation of connected component of the network. We introduce a parameter called clinginess and control how the member intimacy affects the communication activity. We observe that cumulative number of users who leave the community exhibits a transition-like behavior, similarly to the discontinuous transition in statistical mechanics models. Implication of our result in constructing a sustainable online community is also discussed.

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

    PubMed

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

    2015-12-07

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

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

    PubMed Central

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

    2015-01-01

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

  7. Scaling in complex systems: a link between the dynamics of networks and growing interfaces

    PubMed Central

    Brú, A.; Alós, E.; Nuño, J. C.; de Dios, M. Fernández

    2014-01-01

    We consider growing interfaces as dynamical networks whose nodes are the discrete points of the interface and the edges the physical interactions among them. We map the points of the interface formed at each time into a graph by means of a visibility algorithm. As the corresponding interfaces grow, their visibility graphs change over time. We show that the visibility graphs are all scale free for each time. We use the variance of the node degrees as a measure of the dynamical properties of these graphs. This magnitude reveals an unexpected scaling behaviour of these graphs in both the number of nodes and time. This enables to define three robust exponents that characterize any type of dynamics with more detail than the classical scaling analysis applied directly to the physical interfaces. To check the feasibility of this approach we study and classify six different dynamical processes and estimate their critical exponents. We conclude that the dynamics of physical systems far from equilibrium can be determined by its corresponding visibility network. Indeed, this methodology is able to discern among dynamical processes that hitherto have been classified in the same universality class according to the scaling analysis of their interfaces. PMID:25518729

  8. Bacterial Actins.

    PubMed

    Izoré, Thierry; van den Ent, Fusinita

    2017-01-01

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

  9. IQGAP and mitotic exit network (MEN) proteins are required for cytokinesis and re-polarization of the actin cytoskeleton in the budding yeast, Saccharomyces cerevisiae.

    PubMed

    Corbett, Mark; Xiong, Yulan; Boyne, James R; Wright, Daniel J; Munro, Ewen; Price, Clive

    2006-11-01

    In budding yeast the final stages of the cell division cycle, cytokinesis and cell separation, are distinct events that require to be coupled, both together and with mitotic exit. Here we demonstrate that mutations in genes of the mitotic exit network (MEN) prevent cell separation and are synthetically lethal in combination with both cytokinesis and septation defective mutations. Analysis of the synthetic lethal phenotypes reveals that Iqg1p functions in combination with the MEN components, Tem1p, Cdc15p Dbf20p and Dbf2p to govern the re-polarization of the actin cytoskeleton to either side of the bud neck. In addition phosphorylation of the conserved PCH protein, Hof1p, is dependent upon these activities and requires actin ring assembly. Recruitment of Dbf2p to the bud neck is dependent upon actin ring assembly and correlates with Hof1p phosphorylation. Failure to phosphorylate Hof1p results in the increased stability of the protein and its persistence at the bud neck. These data establish a mechanistic dependency of cell separation upon an intermediate step requiring actomyosin ring assembly.

  10. Growing hierarchical tree SOM: an unsupervised neural network with dynamic topology.

    PubMed

    Forti, Alberto; Foresti, Gian Luca

    2006-12-01

    In this paper we introduce a tree structured self-organizing network, called the Growing Hierarchical Tree SOM (GHTSOM), that combines unsupervised learning with a dynamic topology for hierarchical classification of unlabelled data sets. The main feature of the proposed model is a SOM-like self-organizing process that allows the network to adapt the topology of each layer of the hierarchy to the characteristics of the training set. In particular the self-organization is obtained in two steps: the first one concerns the learning phase and is finalized with the creation of a tree of SOMs, while the second one is in regard to the clustering phase and provides the formation of classes for each level of the tree (hence self-organization not only for training but also for the creation of topological connections). As a result the network works without the need for user-defined parameters. Experimental results are proposed on both synthetic and real data sets.

  11. Actin cortex architecture regulates cell surface tension.

    PubMed

    Chugh, Priyamvada; Clark, Andrew G; Smith, Matthew B; Cassani, Davide A D; Dierkes, Kai; Ragab, Anan; Roux, Philippe P; Charras, Guillaume; Salbreux, Guillaume; Paluch, Ewa K

    2017-06-01

    Animal cell shape is largely determined by the cortex, a thin actin network underlying the plasma membrane in which myosin-driven stresses generate contractile tension. Tension gradients result in local contractions and drive cell deformations. Previous cortical tension regulation studies have focused on myosin motors. Here, we show that cortical actin network architecture is equally important. First, we observe that actin cortex thickness and tension are inversely correlated during cell-cycle progression. We then show that the actin filament length regulators CFL1, CAPZB and DIAPH1 regulate mitotic cortex thickness and find that both increasing and decreasing thickness decreases tension in mitosis. This suggests that the mitotic cortex is poised close to a tension maximum. Finally, using a computational model, we identify a physical mechanism by which maximum tension is achieved at intermediate actin filament lengths. Our results indicate that actin network architecture, alongside myosin activity, is key to cell surface tension regulation.

  12. Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells.

    PubMed

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

    2013-09-30

    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.

  13. 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

  14. Recognition of handwritten similar Chinese characters by self-growing probabilistic decision-based neural network.

    PubMed

    Fu, H C; Xu, Y Y; Chang, H Y

    1999-12-01

    Recognition of similar (confusion) characters is a difficult problem in optical character recognition (OCR). In this paper, we introduce a neural network solution that is capable of modeling minor differences among similar characters, and is robust to various personal handwriting styles. The Self-growing Probabilistic Decision-based Neural Network (SPDNN) is a probabilistic type neural network, which adopts a hierarchical network structure with nonlinear basis functions and a competitive credit-assignment scheme. Based on the SPDNN model, we have constructed a three-stage recognition system. First, a coarse classifier determines a character to be input to one of the pre-defined subclasses partitioned from a large character set, such as Chinese mixed with alphanumerics. Then a character recognizer determines the input image which best matches the reference character in the subclass. Lastly, the third module is a similar character recognizer, which can further enhance the recognition accuracy among similar or confusing characters. The prototype system has demonstrated a successful application of SPDNN to similar handwritten Chinese recognition for the public database CCL/HCCR1 (5401 characters x200 samples). Regarding performance, experiments on the CCL/HCCR1 database produced 90.12% recognition accuracy with no rejection, and 94.11% accuracy with 6.7% rejection, respectively. This recognition accuracy represents about 4% improvement on the previously announced performance. As to processing speed, processing before recognition (including image preprocessing, segmentation, and feature extraction) requires about one second for an A4 size character image, and recognition consumes approximately 0.27 second per character on a Pentium-100 based personal computer, without use of any hardware accelerator or co-processor.

  15. [The reorganization of actin cytoskeleton and microtubule system of human endothelial vein in the intercellular contacts formation].

    PubMed

    Shahov, A S; Dugina, V B; Alieva, I B

    2015-01-01

    Endothelial cells are tightly fitted to each other and lining the interior surface of all vessels of living organism to provide vascular permeability regulation and interchange between the blood circulating in vessels and tissue fluids of those organs in which these vessels are located. In vitro endothelial monolayer conserve it's basic barrier function which is native for vessels endothelium. Based on this fact we used endothelial cells growing in vitro as a model system in experimental studies of cytoskeletal and adhesion cell components interaction. In current paper, cultured human vein endothelial cells monolayer was used to quantify cytoskeleton alterations in the of endothelial cells from spreading and formation of the first cell-cell contacts to confluent monolayer formation. The system of actin filaments formed two different cytoskeletal structures in the cells of venous endothelium: 1) cortical actin network; 2) actin stress fibers (bundles) arranged parallel to the substrate. Two actin isoforms, β- and γ-cytoplasmic (non-muscle) actins, are expressed in endothelial cells. The bundles of actin stress fibers were detected by immunofluorescent staining with antibody against β-actin, whereas antibodies against γ-actin identified cortical and lamellar networks. For assessment of the actin cytoskeleton organization it's fluorescence intensity on the area of 10 μM2 located (1) near the free edge, and (2) in the zone of cell-cell contacts were analyzed. Fluorescence intensity of β-actin structures was higher in the areas of cell-cell contact. The fluorescence of γ-actin structures was more intensive at the leading edges of the lamellae, and was the lowest on the stable edges of the cells with formed cell-cell contacts. The endothelial monolayer formation was accompanied by microtubule system alteration: the number of microtubules increased at the cell edge, and besides the microtubules quantity in the area of already formed cell-cell contact was always

  16. 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

  17. Actin-binding proteins take the reins in growth cones.

    PubMed

    Pak, Chi W; Flynn, Kevin C; Bamburg, James R

    2008-02-01

    Higher-order actin-based networks (actin superstructures) are important for growth-cone motility and guidance. Principles for generating, organizing and remodelling actin superstructures have emerged from recent findings in cell-free systems, non-neuronal cells and growth cones. This Review examines how actin superstructures are initiated de novo at the leading-edge membrane and how the spontaneous organization of actin superstructures is driven by ensembles of actin-binding proteins. How the regulation of actin-binding proteins can affect growth-cone turning and axonal regeneration is also discussed.

  18. Empirical test of the origin of Zipf’s law in growing social networks

    NASA Astrophysics Data System (ADS)

    Zhang, Qunzhi; Sornette, Didier

    2011-11-01

    Zipf’s power law is a general empirical regularity found in many systems. We report a detailed analysis of a burgeoning network of social groups, in which all ingredients needed for Zipf’s law to apply are verifiable and verified. A recently developed theory predicts that Zipf’s law corresponds to systems that are growing according to a maximally sustainable path in the presence of random proportional growth, stochastic birth and death processes. We estimate empirically the average growth r and its standard deviation σ as well as the death rate h and predict without adjustable parameters the exponent μ of the power law distribution P(s) of the group sizes s. Using numerical simulations of the underlying growth model, we demonstrate that the empirical stability of Zipf’s law over the whole lifetime of the social network can be attributed to the interplay between a finite lifetime effect and a large σ value. Our analysis and the corresponding results demonstrate that Zipf’s law can be observed with a good precision even when the balanced growth condition is not realized, if the random proportional growth has a strong stochastic component and is acting on young systems under development.

  19. Nuclear Actin in Development and Transcriptional Reprogramming.

    PubMed

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

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

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

    PubMed

    Marsick, Bonnie M; Letourneau, Paul C

    2011-03-17

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

  1. The yeast gene, MDM20, is necessary for mitochondrial inheritance and organization of the actin cytoskeleton.

    PubMed

    Hermann, G J; King, E J; Shaw, J M

    1997-04-07

    In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament-binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20 delta cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.

  2. Functional synergy of actin filament cross-linking proteins.

    PubMed

    Tseng, Yiider; Schafer, Benjamin W; Almo, Steven C; Wirtz, Denis

    2002-07-12

    The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.

  3. 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.

  4. TGFβ2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways

    PubMed Central

    Montecchi-Palmer, Michela; Bermudez, Jaclyn Y.; Webber, Hannah C.; Patel, Gaurang C.; Clark, Abbot F.; Mao, Weiming

    2017-01-01

    Purpose Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor β2 (TGFβ2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFβ2 signaling pathways in CLAN formation. Methods Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFβ receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4′,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test. Results TGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFβ receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFβ receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. Conclusions TGFβ2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers. PMID:28241317

  5. Live Cell Imaging of Actin Dynamics in the Filamentous Fungus Aspergillus nidulans.

    PubMed

    Schultzhaus, Zachary; Quintanilla, Laura; Hilton, Angelyn; Shaw, Brian D

    2016-04-01

    Hyphal cells of filamentous fungi grow at their tips in a method analogous to pollen tube and root hair elongation. This process, generally referred to as tip growth, requires precise regulation of the actin cytoskeleton, and characterizing the various actin structures in these cell types is currently an active area of research. Here, the actin marker Lifeact was used to document actin dynamics in the filamentous fungus Aspergillus nidulans. Contractile double rings were observed at septa, and annular clusters of puncta were seen subtending growing hyphal tips, corresponding to the well-characterized subapical endocytic collar. However, Lifeact also revealed two additional structures. One, an apical array, was dynamic on the face opposite the tip, while a subapical web was dynamic on the apical face and was located several microns behind the growth site. Each was observed turning into the other over time, implying that they could represent different localizations of the same structure, although hyphae with a subapical web grew faster than those exhibiting an apical array. The subapical web has not been documented in any filamentous fungus to date, and is separate from the networks of F-actin seen in other tip-growing organisms surrounding septa or stationary along the plasmalemma.

  6. Mechanical Response of Cytoskeletal Networks

    PubMed Central

    Gardel, Margaret L.; Kasza, Karen E.; Brangwynne, Clifford P.; Liu, Jiayu; Weitz, David A.

    2015-01-01

    The cellular cytoskeleton is a dynamic network of filamentous proteins, consisting of filamentous actin (F-actin), microtubules, and intermediate filaments. However, these networks are not simple linear, elastic solids; they can exhibit highly nonlinear elasticity and athermal dynamics driven by ATP-dependent processes. To build quantitative mechanical models describing complex cellular behaviors, it is necessary to understand the underlying physical principles that regulate force transmission and dynamics within these networks. In this chapter, we review our current understanding of the physics of networks of cytoskeletal proteins formed in vitro. We introduce rheology, the technique used to measure mechanical response. We discuss our current understanding of the mechanical response of F-actin networks, and how the biophysical properties of F-actin and actin cross-linking proteins can dramatically impact the network mechanical response. We discuss how incorporating dynamic and rigid microtubules into F-actin networks can affect the contours of growing microtubules and composite network rigidity. Finally, we discuss the mechanical behaviors of intermediate filaments. PMID:19118688

  7. Nuclear Actin in Development and Transcriptional Reprogramming

    PubMed Central

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

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

  8. Monitoring of Solar Radiation Intensity using Wireless Sensor Network for Plant Growing

    NASA Astrophysics Data System (ADS)

    Siregar, B.; Fadli, F.; Andayani, U.; Harahap, LA; Fahmi, F.

    2017-01-01

    Abstract— Plant growth is highly depending on the sunlight, if the consumption of sunlight is enough, it will grow well. The plant will be green because of its chlorophyll and it can perform photosynthesis at maximum; but if the plants get less sunlight, it will make the plants be yellowing. Radiation is electromagnetic waves that are good for plants, so-called visible light. In the electromagnetic wave spectrum the best wavelength range from 400-700 nm for the plant. A monitoring of sun intensity is needed in order to obtain sufficient solar radiation consumption and provide notification if there is a high radiation. In this study, several sensors and devices were combined such as photosynthetic solar radiation sensors, GSM / GPRS and waspmote as a main board or a microcontroller. The test was carried out on at least three occasions; the system has a stable radiation in the morning with an average of 505.51 micrometers. IN this study, we have successfully developed a monitoring tools for solar radiation intensity applied on plant growth by using wireless sensor network.

  9. Actinic Prurigo.

    PubMed

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

    2015-01-01

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

  10. The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network.

    PubMed

    Bladt, Friedhelm; Aippersbach, Elke; Gelkop, Sigal; Strasser, Geraldine A; Nash, Piers; Tafuri, Anna; Gertler, Frank B; Pawson, Tony

    2003-07-01

    Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated beta-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1(-/-) Nck2(-/-) embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization.

  11. The Murine Nck SH2/SH3 Adaptors Are Important for the Development of Mesoderm-Derived Embryonic Structures and for Regulating the Cellular Actin Network

    PubMed Central

    Bladt, Friedhelm; Aippersbach, Elke; Gelkop, Sigal; Strasser, Geraldine A.; Nash, Piers; Tafuri, Anna; Gertler, Frank B.; Pawson, Tony

    2003-01-01

    Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated β-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1−/− Nck2−/− embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization. PMID:12808099

  12. Logical gates in actin monomer.

    PubMed

    Adamatzky, Andrew

    2017-09-18

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

  13. Modeling actin waves in dictyostelium cells

    NASA Astrophysics Data System (ADS)

    Wasnik, Vaibhav; Mukhopadhyay, Ranjan

    2011-03-01

    Actin networks in living cells demonstrate a high capacity for self-organization and are responsible for the formation of a variety of structures such as lamellopodia, phagocytic cups, and cleavage furrows. Recent experiments have studied actin waves formed on the surface of dictyostelium cells that have been treated with a depolymerizing agent. These waves are believed to be physiologically important, for example, for the formation of phagocytic cups. We propose and study a minimal model, based on the dendritic nucleation of actin polymers, to explain the formation of these waves. This model can be extended to study the dynamics of the coupled actin-membrane system.

  14. The effect of the neural activity on topological properties of growing neural networks.

    PubMed

    Gafarov, F M; Gafarova, V R

    2016-09-01

    The connectivity structure in cortical networks defines how information is transmitted and processed, and it is a source of the complex spatiotemporal patterns of network's development, and the process of creation and deletion of connections is continuous in the whole life of the organism. In this paper, we study how neural activity influences the growth process in neural networks. By using a two-dimensional activity-dependent growth model we demonstrated the neural network growth process from disconnected neurons to fully connected networks. For making quantitative investigation of the network's activity influence on its topological properties we compared it with the random growth network not depending on network's activity. By using the random graphs theory methods for the analysis of the network's connections structure it is shown that the growth in neural networks results in the formation of a well-known "small-world" network.

  15. Grow Your Personal Learning Network: New Technologies Can Keep You Connected and Help You Manage Information Overload

    ERIC Educational Resources Information Center

    Warlick, David

    2009-01-01

    Personal learning networks (PLNs) are not new. People have long relied on their families, friends, colleagues, and acquaintances to supplement their knowledge about the world. But the times are changing. Information and communication technologies (ICT), including an ever-growing repertoire of open source applications, have freed content from the…

  16. The Interaction of Vinculin with Actin

    PubMed Central

    Golji, Javad; Mofrad, Mohammad R. K.

    2013-01-01

    Vinculin can interact with F-actin both in recruitment of actin filaments to the growing focal adhesions and also in capping of actin filaments to regulate actin dynamics. Using molecular dynamics, both interactions are simulated using different vinculin conformations. Vinculin is simulated either with only its vinculin tail domain (Vt), with all residues in its closed conformation, with all residues in an open I conformation, and with all residues in an open II conformation. The open I conformation results from movement of domain 1 away from Vt; the open II conformation results from complete dissociation of Vt from the vinculin head domains. Simulation of vinculin binding along the actin filament showed that Vt alone can bind along the actin filaments, that vinculin in its closed conformation cannot bind along the actin filaments, and that vinculin in its open I conformation can bind along the actin filaments. The simulations confirm that movement of domain 1 away from Vt in formation of vinculin 1 is sufficient for allowing Vt to bind along the actin filament. Simulation of Vt capping actin filaments probe six possible bound structures and suggest that vinculin would cap actin filaments by interacting with both S1 and S3 of the barbed-end, using the surface of Vt normally occluded by D4 and nearby vinculin head domain residues. Simulation of D4 separation from Vt after D1 separation formed the open II conformation. Binding of open II vinculin to the barbed-end suggests this conformation allows for vinculin capping. Three binding sites on F-actin are suggested as regions that could link to vinculin. Vinculin is suggested to function as a variable switch at the focal adhesions. The conformation of vinculin and the precise F-actin binding conformation is dependent on the level of mechanical load on the focal adhesion. PMID:23633939

  17. ENA/VASP proteins regulate exocytosis by mediating myosin VI-dependent recruitment of secretory granules to the cortical actin network.

    PubMed

    Tomatis, Vanesa M; Josh, Peter; Papadopulos, Andreas; Gormal, Rachel S; Lanoue, Vanessa; Martin, Sally; Meunier, Frédéric A

    2017-08-04

    In neurosecretory cells, myosin VI associated with secretory granules (SGs) mediates their activity-dependent recruitment to the cortical actin network and is necessary to sustain exocytosis. The mechanism by which myosin VI interacts with SGs is unknown. Using a myosin VI pull-down assay and mass spectrometry we identified Mena, a member of the ENA/VASP family, as a myosin VI binding partner in PC12 cells, and confirmed that Mena colocalized with myosin VI on SGs. Using a knock-sideways approach to inactivate the ENA/VASP family members by mitochondrial relocation, we revealed a concomitant redistribution of myosin VI. This was ensued by a reduction in the association of myosin VI with SGs, a decreased SG mobility and density in proximity to the plasma membrane as well as decreased evoked exocytosis. These data demonstrate that ENA/VASP proteins regulate SG exocytosis through modulating the activity of myosin VI. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Guiding Principles for Designing and Growing a Campus Network for the Future.

    ERIC Educational Resources Information Center

    Long, Philip E.

    2000-01-01

    Offers guidelines to help colleges and universities ensure a course of telecommunications network growth and renewal, provide continuous network upgrades, and maintain a position of flexibility. Discusses focuses on principles that guide network planning and design; how to apply principles to standard building blocks and network design; voice,…

  19. 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

  20. Actin and Actin-Binding Proteins.

    PubMed

    Pollard, Thomas D

    2016-08-01

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

  1. Energy efficient flexible hybrid wavelength division multiplexing-time division multiplexing passive optical network with pay as you grow deployment

    NASA Astrophysics Data System (ADS)

    Garg, Amit Kumar; Madavi, Amresh Ashok; Janyani, Vijay

    2017-02-01

    A flexible hybrid wavelength division multiplexing-time division multiplexing passive optical network architecture that allows dual rate signals to be sent at 1 and 10 Gbps to each optical networking unit depending upon the traffic load is proposed. The proposed design allows dynamic wavelength allocation with pay-as-you-grow deployment capability. This architecture is capable of providing up to 40 Gbps of equal data rates to all optical distribution networks (ODNs) and up to 70 Gbps of a asymmetrical data rate to the specific ODN. The proposed design handles broadcasting capability with simultaneous point-to-point transmission, which further reduces energy consumption. In this architecture, each module sends a wavelength to each ODN, thus making the architecture fully flexible; this flexibility allows network providers to use only required OLT components and switch off others. The design is also reliable to any module or TRx failure and provides services without any service disruption. Dynamic wavelength allocation and pay-as-you-grow deployment support network extensibility and bandwidth scalability to handle future generation access networks.

  2. Myosin 1b promotes the formation of post-Golgi carriers by regulating actin assembly and membrane remodelling at the trans-Golgi network.

    PubMed

    Almeida, Claudia G; Yamada, Ayako; Tenza, Danièle; Louvard, Daniel; Raposo, Graça; Coudrier, Evelyne

    2011-06-12

    The function of organelles is intimately associated with rapid changes in membrane shape. By exerting force on membranes, the cytoskeleton and its associated motors have an important role in membrane remodelling. Actin and myosin 1 have been implicated in the invagination of the plasma membrane during endocytosis. However, whether myosin 1 and actin contribute to the membrane deformation that gives rise to the formation of post-Golgi carriers is unknown. Here we report that myosin 1b regulates the actin-dependent post-Golgi traffic of cargo, generates force that controls the assembly of F-actin foci and, together with the actin cytoskeleton, promotes the formation of tubules at the TGN. Our results provide evidence that actin and myosin 1 regulate organelle shape and uncover an important function for myosin 1b in the initiation of post-Golgi carrier formation by regulating actin assembly and remodelling TGN membranes.

  3. The role of substrate curvature in actin-based pushing forces.

    PubMed

    Schwartz, Ian M; Ehrenberg, Morton; Bindschadler, Michael; McGrath, James L

    2004-06-22

    The extension of the plasma membrane during cell crawling or spreading is known to require actin polymerization; however, the question of how pushing forces derive from actin polymerization remains open. A leading theory (herein referred to as elastic propulsion) illustrates how elastic stresses in networks growing on curved surfaces can result in forces that push particles. To date all examples of reconstituted motility have used curved surfaces, raising the possibility that such squeezing forces are essential for actin-based pushing. By contrast, other theories, such as molecular ratchets, neither require nor consider surface curvature to explain pushing forces. Here, we critically test the requirement of substrate curvature by reconstituting actin-based motility on polystyrene disks. We find that disks move through extracts in a manner that indicates pushing forces on their flat surfaces and that disks typically move faster than the spheres they are manufactured from. For a subset of actin tails that form on the perimeter of disks, we find no correlation between local surface curvature and tail position. Collectively the data indicate that curvature-dependent mechanisms are not required for actin-based pushing.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-04-01

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

  6. GPCRs and actin-cytoskeleton dynamics.

    PubMed

    Vázquez-Victorio, Genaro; González-Espinosa, Claudia; Espinosa-Riquer, Zyanya P; Macías-Silva, Marina

    2016-01-01

    A multitude of physiological processes regulated by G protein-coupled receptors (GPCRs) signaling are accomplished by the participation of active rearrangements of the cytoskeleton. In general, it is common that a cross talk occurs among networks of microfilaments, microtubules, and intermediate filaments in order to reach specific cell responses. In particular, actin-cytoskeleton dynamics regulate processes such as cell shape, cell division, cell motility, and cell polarization, among others. This chapter describes the current knowledge about the regulation of actin-cytoskeleton dynamic by diverse GPCR signaling pathways, and also includes some protocols combining immunofluorescence and confocal microscopy for the visualization of the different rearrangements of the actin-cytoskeleton. We report how both the S1P-GPCR/G12/13/Rho/ROCK and glucagon-GPCR/Gs/cAMP axes induce differential actin-cytoskeleton rearrangements in epithelial cells. We also show that specific actin-binding molecules, like phalloidin and LifeAct, are very useful to analyze F-actin reorganization by confocal microscopy, and also that both molecules show similar results in fixed cells, whereas the anti-actin antibody is useful to detect both the G- and F-actin, as well as their compartmentalization. Thus, it is highly recommended to utilize different approaches to investigate the regulation of actin dynamics by GPCR signaling, with the aim to get a better picture of the phenomenon under study.

  7. Boolean gates on actin filaments

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

  8. Teacher and Principal Responses to the Grow Network: A Preliminary Evaluation of Use in Chicago Public Elementary Schools. Research Data Brief

    ERIC Educational Resources Information Center

    Easton, John Q.; Luppescu, Stuart

    2004-01-01

    During the 2001-02 school year, Chicago Public Schools (CPS) officials contracted with the Grow Network[TM] to provide supplemental reporting of student test results to parents, teachers, and school administrators. Under terms of its contract with CPS, the Grow Network provides a variety of resources, including individualized, printed score…

  9. Actin filament curvature biases branching direction

    PubMed Central

    Risca, Viviana I.; Wang, Evan B.; Chaudhuri, Ovijit; Chia, Jia Jun; Geissler, Phillip L.; Fletcher, Daniel A.

    2012-01-01

    Mechanical cues affect many important biological processes in metazoan cells, such as migration, proliferation, and differentiation. Such cues are thought to be detected by specialized mechanosensing molecules linked to the cytoskeleton, an intracellular network of protein filaments that provide mechanical rigidity to the cell and drive cellular shape change. The most abundant such filament, actin, forms branched networks nucleated by the actin-related protein (Arp) 2/3 complex that support or induce membrane protrusions and display adaptive behavior in response to compressive forces. Here we show that filamentous actin serves in a mechanosensitive capacity itself, by biasing the location of actin branch nucleation in response to filament bending. Using an in vitro assay to measure branching from curved sections of immobilized actin filaments, we observed preferential branch formation by the Arp2/3 complex on the convex face of the curved filament. To explain this behavior, we propose a fluctuation gating model in which filament binding or branch nucleation by Arp2/3 occur only when a sufficiently large, transient, local curvature fluctuation causes a favorable conformational change in the filament, and we show with Monte Carlo simulations that this model can quantitatively account for our experimental data. We also show how the branching bias can reinforce actin networks in response to compressive forces. These results demonstrate how filament curvature can alter the interaction of cytoskeletal filaments with regulatory proteins, suggesting that direct mechanotransduction by actin may serve as a general mechanism for organizing the cytoskeleton in response to force. PMID:22308368

  10. Orthogonal (transverse) arrangements of actin in endothelia and fibroblasts

    PubMed Central

    Curtis, Adam; Aitchison, Gregor; Tsapikouni, Theodora

    2006-01-01

    Though actin filaments running across the cell (transverse actin) have been occasionally reported for epithelial cells in groups and for cells growing on fibres, there has been no report heretofore of transverse actin in cells grown on planar substrata. This paper describes evidence in support of this possibility derived from actin staining, polarization microscopy and force measurements. The paper introduces two new methods for detecting the orientation and activity of contractile elements in cells. The orthogonal actin is most obvious in cells grown on groove ridge structures, but can be detected in cells grown on flat surfaces. PMID:17015307

  11. Dynamic reorganization of the actin cytoskeleton

    PubMed Central

    Gressin, Laurène; Théry, Manuel; Blanchoin, Laurent

    2015-01-01

    Cellular processes, including morphogenesis, polarization, and motility, rely on a variety of actin-based structures. Although the biochemical composition and filament organization of these structures are different, they often emerge from a common origin. This is possible because the actin structures are highly dynamic. Indeed, they assemble, grow, and disassemble in a time scale of a second to a minute. Therefore, the reorganization of a given actin structure can promote the formation of another. Here, we discuss such transitions and illustrate them with computer simulations. PMID:26989473

  12. Dynamic reorganization of the actin cytoskeleton.

    PubMed

    Letort, Gaëlle; Ennomani, Hajer; Gressin, Laurène; Théry, Manuel; Blanchoin, Laurent

    2015-01-01

    Cellular processes, including morphogenesis, polarization, and motility, rely on a variety of actin-based structures. Although the biochemical composition and filament organization of these structures are different, they often emerge from a common origin. This is possible because the actin structures are highly dynamic. Indeed, they assemble, grow, and disassemble in a time scale of a second to a minute. Therefore, the reorganization of a given actin structure can promote the formation of another. Here, we discuss such transitions and illustrate them with computer simulations.

  13. Dendritic Actin Nucleation Causes Traveling Waves and Patches

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

    2010-03-01

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

  14. Bootstrapping disaster: The challenge of growing and maintaining a cross-sector collaborative network.

    PubMed

    Wachhaus, Aaron

    This article examines the interaction of nonprofit and private actors with the traditional bureaucratic structures of government in central Pennsylvania&s recovery from hurricane Irene and tropical storm Lee. That effort relied heavily on private and nonprofit organizations as drivers of the response and recovery. The author maps the organizations involved in the recovery effort and explores the impact of the recovery effort on those organizations. A social network analysis was conducted and complemented with follow-up interviews with key actors. The network analysis reveals weak communication between sectors and a reliance on nonprofits to deliver services; interviews uncover the challenges of intersectoral collaboration. The author addresses the successes and limitations of the means by which a network of nonprofit efforts were coordinated with federal and state relief efforts and draw lessons for improving future practices. The author finds that this case deviates from theory in several ways that complicated community response and recovery. In particular, the challenges of developing and maintaining a recovery network while simultaneously delivering services placed great strain on several organizations, as well as on the fledgling network as a whole.

  15. Stochastic Dynamical Model of a Growing Citation Network Based on a Self-Exciting Point Process

    NASA Astrophysics Data System (ADS)

    Golosovsky, Michael; Solomon, Sorin

    2012-08-01

    We put under experimental scrutiny the preferential attachment model that is commonly accepted as a generating mechanism of the scale-free complex networks. To this end we chose a citation network of physics papers and traced the citation history of 40 195 papers published in one year. Contrary to common belief, we find that the citation dynamics of the individual papers follows the superlinear preferential attachment, with the exponent α=1.25-1.3. Moreover, we show that the citation process cannot be described as a memoryless Markov chain since there is a substantial correlation between the present and recent citation rates of a paper. Based on our findings we construct a stochastic growth model of the citation network, perform numerical simulations based on this model and achieve an excellent agreement with the measured citation distributions.

  16. Actin cytoskeleton demonstration in Trichomonas vaginalis and in other trichomonads.

    PubMed

    Brugerolle, G; Bricheux, G; Coffe, G

    1996-01-01

    The flagellate form of Trichomonas vaginalis (T v) transforms to amoeboid cells upon adherence to converslips. They grow and their nuclei divide without undergoing cytokinesis, yielding giant cells and a monolayer of T v F-actin was demonstrated in Trichomonas vaginalis by fluorescence microscopy using phalloidin and an anti-actin mAb which labelled the cytoplasm of both the flagellate and amoeboid forms. Comparative electrophoresis and immunoblotting established that the actin band has the same 42 kDa as muscle actin, but 2-D electrophoresis resolved the actin band into four spots; the two major spots observed were superimposable with major muscle actin isoforms. Electron microscopy demonstrated an ectoplasmic microfibrillar layer along the adhesion zone of amoeboid T v adhering to coverslips. Immunogold staining, using anti-actin monoclonal antibodies demonstrated that this layer was mainly composed of actin microfilaments. A comparative immunoblotting study comprising seven trichomonad species showed that all trichomonads studied expressed actin. The mAb Sigma A-4700 specific for an epitope on the actin C-terminal sequence labelled only actin of Trichomonas vaginalis, Tetratrichomonas gallinarum. Trichomitus batrachorum and Hypotrichomonas acosta, but not the actin of Tritrichomonas foetus, Tritrichomonas augusta and Monocercomonas sp. This discrimination between a 'trichomonas branch' and a 'tritrichomonas branch' is congruent with inferred sequence phylogeny from SSu rRNA and with classical phylogeny of trichomonads.

  17. The City in the Country: Growing Alternative Food Networks in Metropolitan Areas

    ERIC Educational Resources Information Center

    Jarosz, Lucy

    2008-01-01

    Alternative food networks (AFNs) are commonly defined by attributes such as the spatial proximity between farmers and consumers, the existence of retail venues such as farmers markets, community supported agriculture (CSA) and a commitment to sustainable food production and consumption. Focusing upon processes rather than attributes, this paper…

  18. Growing complex network of citations of scientific papers: Modeling and measurements.

    PubMed

    Golosovsky, Michael; Solomon, Sorin

    2017-01-01

    We consider the network of citations of scientific papers and use a combination of the theoretical and experimental tools to uncover microscopic details of this network growth. Namely, we develop a stochastic model of citation dynamics based on the copying-redirection-triadic closure mechanism. In a complementary and coherent way, the model accounts both for statistics of references of scientific papers and for their citation dynamics. Originating in empirical measurements, the model is cast in such a way that it can be verified quantitatively in every aspect. Such validation is performed by measuring citation dynamics of physics papers. The measurements revealed nonlinear citation dynamics, the nonlinearity being intricately related to network topology. The nonlinearity has far-reaching consequences including nonstationary citation distributions, diverging citation trajectories of similar papers, runaways or "immortal papers" with infinite citation lifetime, etc. Thus nonlinearity in complex network growth is our most important finding. In a more specific context, our results can be a basis for quantitative probabilistic prediction of citation dynamics of individual papers and of the journal impact factor.

  19. Growing complex network of citations of scientific papers: Modeling and measurements

    NASA Astrophysics Data System (ADS)

    Golosovsky, Michael; Solomon, Sorin

    2017-01-01

    We consider the network of citations of scientific papers and use a combination of the theoretical and experimental tools to uncover microscopic details of this network growth. Namely, we develop a stochastic model of citation dynamics based on the copying-redirection-triadic closure mechanism. In a complementary and coherent way, the model accounts both for statistics of references of scientific papers and for their citation dynamics. Originating in empirical measurements, the model is cast in such a way that it can be verified quantitatively in every aspect. Such validation is performed by measuring citation dynamics of physics papers. The measurements revealed nonlinear citation dynamics, the nonlinearity being intricately related to network topology. The nonlinearity has far-reaching consequences including nonstationary citation distributions, diverging citation trajectories of similar papers, runaways or "immortal papers" with infinite citation lifetime, etc. Thus nonlinearity in complex network growth is our most important finding. In a more specific context, our results can be a basis for quantitative probabilistic prediction of citation dynamics of individual papers and of the journal impact factor.

  20. Going to Scale: As KIPP Network Grows, Positive Impacts Are Sustained. In Focus Brief

    ERIC Educational Resources Information Center

    Tuttle, Christina Clark; Gleason, Philip; Knechtel, Virginia; Nichols-Barrer, Ira; Booker, Kevin; Chojnacki, Gregory; Coen, Thomas; Goble, Lisbeth

    2015-01-01

    KIPP (Knowledge is Power Program) is a national network of public charter schools whose stated mission is to help underserved students enroll in and graduate from college. Prior studies (see Tuttle et al. 2013) have consistently found that attending a KIPP middle school positively affects student achievement, but few have addressed longer-term…

  1. Joint estimation of preferential attachment and node fitness in growing complex networks

    NASA Astrophysics Data System (ADS)

    Pham, Thong; Sheridan, Paul; Shimodaira, Hidetoshi

    2016-09-01

    Complex network growth across diverse fields of science is hypothesized to be driven in the main by a combination of preferential attachment and node fitness processes. For measuring the respective influences of these processes, previous approaches make strong and untested assumptions on the functional forms of either the preferential attachment function or fitness function or both. We introduce a Bayesian statistical method called PAFit to estimate preferential attachment and node fitness without imposing such functional constraints that works by maximizing a log-likelihood function with suitably added regularization terms. We use PAFit to investigate the interplay between preferential attachment and node fitness processes in a Facebook wall-post network. While we uncover evidence for both preferential attachment and node fitness, thus validating the hypothesis that these processes together drive complex network evolution, we also find that node fitness plays the bigger role in determining the degree of a node. This is the first validation of its kind on real-world network data. But surprisingly the rate of preferential attachment is found to deviate from the conventional log-linear form when node fitness is taken into account. The proposed method is implemented in the R package PAFit.

  2. Joint estimation of preferential attachment and node fitness in growing complex networks

    PubMed Central

    Pham, Thong; Sheridan, Paul; Shimodaira, Hidetoshi

    2016-01-01

    Complex network growth across diverse fields of science is hypothesized to be driven in the main by a combination of preferential attachment and node fitness processes. For measuring the respective influences of these processes, previous approaches make strong and untested assumptions on the functional forms of either the preferential attachment function or fitness function or both. We introduce a Bayesian statistical method called PAFit to estimate preferential attachment and node fitness without imposing such functional constraints that works by maximizing a log-likelihood function with suitably added regularization terms. We use PAFit to investigate the interplay between preferential attachment and node fitness processes in a Facebook wall-post network. While we uncover evidence for both preferential attachment and node fitness, thus validating the hypothesis that these processes together drive complex network evolution, we also find that node fitness plays the bigger role in determining the degree of a node. This is the first validation of its kind on real-world network data. But surprisingly the rate of preferential attachment is found to deviate from the conventional log-linear form when node fitness is taken into account. The proposed method is implemented in the R package PAFit. PMID:27601314

  3. The City in the Country: Growing Alternative Food Networks in Metropolitan Areas

    ERIC Educational Resources Information Center

    Jarosz, Lucy

    2008-01-01

    Alternative food networks (AFNs) are commonly defined by attributes such as the spatial proximity between farmers and consumers, the existence of retail venues such as farmers markets, community supported agriculture (CSA) and a commitment to sustainable food production and consumption. Focusing upon processes rather than attributes, this paper…

  4. A statistically inferred microRNA network identifies breast cancer target miR-940 as an actin cytoskeleton regulator

    NASA Astrophysics Data System (ADS)

    Bhajun, Ricky; Guyon, Laurent; Pitaval, Amandine; Sulpice, Eric; Combe, Stéphanie; Obeid, Patricia; Haguet, Vincent; Ghorbel, Itebeddine; Lajaunie, Christian; Gidrol, Xavier

    2015-02-01

    MiRNAs are key regulators of gene expression. By binding to many genes, they create a complex network of gene co-regulation. Here, using a network-based approach, we identified miRNA hub groups by their close connections and common targets. In one cluster containing three miRNAs, miR-612, miR-661 and miR-940, the annotated functions of the co-regulated genes suggested a role in small GTPase signalling. Although the three members of this cluster targeted the same subset of predicted genes, we showed that their overexpression impacted cell fates differently. miR-661 demonstrated enhanced phosphorylation of myosin II and an increase in cell invasion, indicating a possible oncogenic miRNA. On the contrary, miR-612 and miR-940 inhibit phosphorylation of myosin II and cell invasion. Finally, expression profiling in human breast tissues showed that miR-940 was consistently downregulated in breast cancer tissues

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

    PubMed

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

    1998-06-01

    IB at the leading edge of E. histolytica. ABP-120 organizes F-actin in a network and myosin IB participates in the pseudopod formation. Similar approaches using T. vaginalis resulted in the discovery of an actin-binding protein that participate in the F-actin reorganization during adhesion of parasites to target cells. This protein is homologous to alpha-actinin from other eukaryotic cells. Finally, by using cell biology approaches, F-actin was observed in the cytoplasm as well as in the nucleus of Dinoflagellates. The recent developments in the molecular genetics of protozoa will provide new insights to understand the roles of actin-binding proteins during cytoskeleton activities.

  6. Temporal evolution of the macropore network and saturated hydraulic conductivity in an arable, clayey topsoil during one growing season

    NASA Astrophysics Data System (ADS)

    Sandin, Maria; Köstel, Johannes; Jarvis, Nicholas; Larsbo, Mats

    2015-04-01

    Soil macropore networks and thus hydraulic properties at and close to saturation vary considerably with time, as a result of the dynamic nature of a diverse range of interacting soil structure-forming and degrading factors such as tillage and traffic events, faunal and plant root activity, swell/shrink arising from wetting and drying cycles, freeze-thaw etc. These properties are nevertheless treated as constants in most hydrological modelling studies. This is mostly justified by a lack of understanding of the processes driving these changes. Temporal variations of saturated and near-saturated hydraulic conductivity have been studied in the field (e.g. by tension disc infiltrometer), but these measurements only indirectly reflect the characteristics of the macropore network. In this study, we used non-destructive X-ray tomography to investigate the temporal changes in the macropore network characteristics occurring in the harrowed layer of a conventionally-tilled agricultural field over one growing season. Undisturbed soil cores (60-70 mm height, 68 mm diameter) were sampled on five different occasions. Changes in the geometric and topological properties of the X-ray imaged macropore system (voxel resolution = 120 µm) were compared with variations in saturated hydraulic conductivity measured on the same samples. Image analysis showed that total porosity, specific surface area, average pore diameter and the connectivity of the pore system in the uppermost 60-70 mm of ploughed and harrowed soil decreased from the first sampling occasion shortly after seedbed preparation and sowing until the middle of the growing season after which it slightly increased again. Separate analysis of the total porosity of the top 5 mm showed a marked decrease between the first two sampling occasions, followed by a gradual increase. Despite these structural changes in the macropore system, saturated hydraulic conductivity was only weakly correlated with macropore network characteristics.

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

    PubMed

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

    2013-01-08

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

  8. A growing global network's role in outbreak response: AFHSC-GEIS 2008-2009.

    PubMed

    Johns, Matthew C; Burke, Ronald L; Vest, Kelly G; Fukuda, Mark; Pavlin, Julie A; Shrestha, Sanjaya K; Schnabel, David C; Tobias, Steven; Tjaden, Jeffrey A; Montgomery, Joel M; Faix, Dennis J; Duffy, Mark R; Cooper, Michael J; Sanchez, Jose L; Blazes, David L; Wangchuk, Sonam; Dorji, Tandin; Gibbons, Robert; Iamsirithaworn, Sopon; Richardson, Jason; Buathong, Rome; Jarman, Richard; Yoon, In-Kyu; Shakya, Geeta; Ofula, Victor; Coldren, Rodney; Bulimo, Wallace; Sang, Rosemary; Omariba, Duke; Obura, Beryl; Mwala, Dennis; Kasper, Matthew; Brice, Gary; Williams, Maya; Yasuda, Chad; Barthel, Robert V; Pimentel, Guillermo; Meyers, Chris; Kammerer, Peter; Baynes, Darcie E; Metzgar, David; Hawksworth, Anthony; Blair, Patrick; Ellorin, Melody; Coon, Robert; Macintosh, Victor; Burwell, Kristen; Macias, Elizabeth; Palys, Thomas; Jerke, Kurt

    2011-03-04

    A cornerstone of effective disease surveillance programs comprises the early identification of infectious threats and the subsequent rapid response to prevent further spread. Effectively identifying, tracking and responding to these threats is often difficult and requires international cooperation due to the rapidity with which diseases cross national borders and spread throughout the global community as a result of travel and migration by humans and animals. From Oct.1, 2008 to Sept. 30, 2009, the United States Department of Defense's (DoD) Armed Forces Health Surveillance Center Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) identified 76 outbreaks in 53 countries. Emerging infectious disease outbreaks were identified by the global network and included a wide spectrum of support activities in collaboration with host country partners, several of which were in direct support of the World Health Organization's (WHO) International Health Regulations (IHR) (2005). The network also supported military forces around the world affected by the novel influenza A/H1N1 pandemic of 2009. With IHR (2005) as the guiding framework for action, the AFHSC-GEIS network of international partners and overseas research laboratories continues to develop into a far-reaching system for identifying, analyzing and responding to emerging disease threats.

  9. Pharmacological characterization of actin-binding (-)-doliculide.

    PubMed

    Foerster, Florian; Braig, Simone; Chen, Tao; Altmann, Karl-Heinz; Vollmar, Angelika M

    2014-09-15

    Natural compounds offer a broad spectrum of potential drug candidates against human malignancies. Several cytostatic drugs, which are in clinical use for decades, derive directly from natural sources or are synthetically optimized derivatives of natural lead structures. An eukaryote target molecule to which many natural derived anti-cancer drugs bind to is the microtubule network. Of similar importance for the cell is the actin cytoskeleton, responsible for cell movements, migration of cells and cytokinesis. Nature provides also a broad range of compounds directed against actin as intracellular target, but none of these actin-targeting compounds has ever been brought to clinical trials. One reason why actin-binding compounds have not yet been considered for further clinical investigations is that little is known about their pharmacological properties in cancer cells. Herein, we focused on the closer characterization of doliculide, an actin binding natural compound of marine origin in the breast cancer cell lines MCF7 and MDA-MB-231. We used fluorescence-recovery-after-photobleaching (FRAP) analysis to determine doliculide's early effects on the actin cytoskeleton and rhodamin-phalloidin staining for long-term effects on the actin CSK. After validating the disruption of the actin network, we further investigated the functional effects of doliculide. Doliculide treatment leads to inhibition of proliferation and impairs the migratory potential. Finally, we could also show that doliculide leads to the induction of apoptosis in both cell lines. Our data for the first time provide a closer characterization of doliculide in breast cancer cells and propagate doliculide for further investigations as lead structure and potential therapeutic option as actin-targeting compound.

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

    PubMed Central

    Shinohara, Satoko; Takaoka, Shunpei; Miyake, Jun

    2017-01-01

    Filamentous actin (F-actin) forms many types of structures and dynamically regulates cell morphology and movement, and plays a mechanosensory role for extracellular stimuli. In this study, we determined that the smooth muscle-related transcription factor, cysteine-rich protein 2 (CRP2), regulates the supramolecular networks of F-actin. The structures of CRP2 and F-actin in solution were analyzed by small-angle X-ray solution scattering (SAXS). The general shape of CRP2 was partially unfolded and relatively ellipsoidal in structure, and the apparent cross sectional radius of gyration (Rc) was about 15.8 Å. The predicted shape, derived by ab initio modeling, consisted of roughly four tandem clusters: LIM domains were likely at both ends with the middle clusters being an unfolded linker region. From the SAXS analysis, the Rc of F-actin was about 26.7 Å, and it was independent of CRP2 addition. On the other hand, in the low angle region of the CRP2-bound F-actin scattering, the intensities showed upward curvature with the addition of CRP2, which indicates increasing branching of F-actin following CRP2 binding. From biochemical analysis, the actin filaments were augmented and clustered by the addition of CRP2. This F-actin clustering activity of CRP2 was cooperative with α-actinin. Thus, binding of CRP2 to F-actin accelerates actin polymerization and F-actin cluster formation. PMID:28813482

  11. The Yeast Gene, MDM20, Is Necessary for Mitochondrial Inheritance and Organization of the Actin Cytoskeleton

    PubMed Central

    Hermann, Greg J.; King, Edward J.; Shaw, Janet M.

    1997-01-01

    In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton. PMID:9105043

  12. Pattern formation in actin gels: A study in the mechanics of gels formed by the important cytoskeletal protein actin, especially as applied to cellular motility

    NASA Astrophysics Data System (ADS)

    Balter, Ariel

    We have studied pattern formation in actin gels to better understand how they function in biological systems, especially in the motility mechanism used by some pathogenic bacteria such as Listeria. By coating themselves with certain enzymes, these bacteria appropriate actin (a protein) from the surrounding host cell's cytoplasm and cause a network or "gel" of actin filaments to grow on their outer surface. As the resulting "comet tail" shaped protrusion grows, it pushes the bacterium away. In experiments, polystyrene beads coated with the same enzymes will also generate comet tails and swim in a very similar manner. However, these bead experiments have also generated anomalous results such as the formation of many comet tails. In some experiments, when two comet tails formed they systematically grew into regular, oppositely handed helices. The formation of any comet tails on a bead poses a physical conundrum. The bacterial enzyme coating is asymmetrical so the comet tail forms in a particular place. But the beads are symmetrical, so comet tails formation constitutes symmetry breaking and spontaneous pattern formation. We have modeled this process as a competition between elastic energy (which favors many tails) and chemical energy (which favors few tails). Our analytical model explains the factors that experimentally determine the number of tails, and numerical simulations confirm these predictions. To understand the helical tails, we did extensive data analysis involving image processing, statistical analysis and mathematical modeling of images of the helical tails. We identified some important features of how the twin tails form. For instance, the tail growth rate is independent of drag force, and bead rotation must accompany helical tail formation. We also created a physical model for helical growth. Numerical simulations of our model show that at very low Reynolds number, a cylindrical object growing under the conditions of an actin comet tail can spontaneously

  13. Confinement induces actin flow in a meiotic cytoplasm

    PubMed Central

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

    2012-01-01

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

  14. Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile, long-lived plaques.

    PubMed

    Meijer, Harold J G; Hua, Chenlei; Kots, Kiki; Ketelaar, Tijs; Govers, Francine

    2014-06-01

    The actin cytoskeleton is a dynamic but well-organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact-eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub-apical region whereas in the extreme apex in growing hyphae, waves of fine F-actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ∼30 s while disassembly was accomplished in ∼10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.

  15. Time-resolved studies of actin organization by multivalent ions and actin-binding proteins

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Purdy, Kirstin; Bartles, James R.; Chee Lai Wong, Gerard

    2007-03-01

    Actin is one of the principal components in the eukaryotic cytoskeleton, the architecture of which is highly regulated for a wide range of biological functions. In the presence of multivalent salts or actin-binding proteins, it is known that F-actin can organize into bundles or networks. In this work, we use time-resolved confocal microscopy to study the dynamics of actin bundle growth induced by multivalent ions and by espin, a prototypical actin binding protein that is known to induce bundles. For divalent ion induced bundles, we observe a rapid lateral saturation followed by longitudinal growth of bundles, in sharp contrast to the bundling mechanism of espin, which favors finite length bundles.

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

    NASA Astrophysics Data System (ADS)

    Wang, Xinxin

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

  17. Self-organizing tree-growing network for the classification of protein sequences.

    PubMed Central

    Wang, H. C.; Dopazo, J.; de la Fraga, L. G.; Zhu, Y. P.; Carazo, J. M.

    1998-01-01

    The self-organizing tree algorithm (SOTA) was recently introduced to construct phylogenetic trees from biological sequences, based on the principles of Kohonen's self-organizing maps and on Fritzke's growing cell structures. SOTA is designed in such a way that the generation of new nodes can be stopped when the sequences assigned to a node are already above a certain similarity threshold. In this way a phylogenetic tree resolved at a high taxonomic level can be obtained. This capability is especially useful to classify sets of diversified sequences. SOTA was originally designed to analyze pre-aligned sequences. It is now adapted to be able to analyze patterns associated to the frequency of residues along a sequence, such as protein dipeptide composition and other n-gram compositions. In this work we show that the algorithm applied to these data is able to not only successfully construct phylogenetic trees of protein families, such as cytochrome c, triosephophate isomerase, and hemoglobin alpha chains, but also classify very diversified sequence data sets, such as a mixture of interleukins and their receptors. PMID:9865956

  18. Growing organizational capacity through a systems approach: one health network's experience.

    PubMed

    MacKenzie, Richard; Capuano, Terry; Durishin, Linda Drexinger; Stern, Glen; Burke, James B

    2008-02-01

    Hospitals are reporting unexpected surges in demand for services. Lehigh Valley Hospital challenged its clinical and administrative staff to increase capacity by at least 4% per year using an interdepartmental, systemwide initiative, Growing Organizational Capacity (GOC). Following a systemwide leadership retreat that yielded more than 1,000 ideas, the initiative's principal sponsor convened a cross-functional improvement team. During a two-year period, 17 projects were implemented. Using a complex systems approach, improvement ideas "emerged" from microsystems at the points of care. Through rigorous reporting and testing of process adaptations, need, data, and people drove innovation. Hundreds of multilevel clinical and administrative staff redesigned processes and roles to increase organizational capacity. Admissions rose by 6.1%, 5.5 %, 8.7%, 5.0%, and 3.8% in fiscal years 2003 through 2007, respectively. Process enhancements cost approximately $1 million, while increased revenues attributable to increased capacity totaled $2.5 million. Multiple, coordinated, and concurrent projects created a greater impact than that possible with a single project. GOC and its success, best explained in the context of complex adaptive systems and microsystem theories, are transferrable to throughput issues that challenge efficiency and effectiveness in other health care systems.

  19. 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.

  20. 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.

  1. International network on endocrine complications in thalassaemia (I-CET): an opportunity to grow.

    PubMed

    De Sanctis, V; Soliman, A T; Angastiniotis, M; Eleftheriou, A; Kattamis, Ch; Karimi, M; El Kholy, M; Elsedfy, H; Yassin, Mohd Abdel Daem Mohd; El Awwa, A; Stoeva, I; Skordis, N; Raiola, G; Fiscina, B

    2012-04-01

    Most of the endocrine complications in thalassaemia are attributable to iron overload which may be the result of economic circumstances (expense of the chelation therapy), late onset of chelation therapy or poor compliance with the iron chelation therapy. The major difficulties reported by hematologists or pediatric endocrinologists experienced in thalassaemias or thalassaemia syndromes in following growth disorders and endocrine complications were: lack of familiarity with medical treatment of endocrine complications (40%), interpretation of endocrine tests (30%), costs (65%), absence of paediatric endocrinologist for consultation on growth disorders and endocrine complications (27%), facilities (27%), other (e.g. lack of collaboration and on-time consultation between thalassaemic Centers supervised by hematologists and endocrinologists) (17%). Because any progress we make in research into growth disorders and endocrine complications in thalassaemia should be passed on to all those suffering from it, guaranteeing them the same therapeutic benefits and the same quality of life, on the 8th of May, 2009 in Ferrara (Italy), the International Network on Endocrine Complications in Thalassemia (I-CET) was founded. The I-CET group is planning to conduct, in Ferrara in May 2012, a workshop, "MRI and Endocrine Complications in Thalassaemia", and in Doha (Qatar) in September 2012, a 3-day intensive course entitled, "Growth disorders and Endocrine Complications in Thalassaemia", to provide interested pediatricians, physicians and hematologists from all over the world with an in-depth approach to the diagnosis and management of growth and endocrine disorders in thalassaemic patients.

  2. Actin machinery and mechanosensitivity in invadopodia, podosomes and focal adhesions

    PubMed Central

    Albiges-Rizo, Corinne; Destaing, Olivier; Fourcade, Bertrand; Planus, Emmanuelle; Block, Marc R.

    2009-01-01

    Summary The invasiveness of cells is correlated with the presence of dynamic actin-rich membrane structures called invadopodia, which are membrane protrusions that are associated with localized polymerization of sub-membrane actin filaments. Similar to focal adhesions and podosomes, invadopodia are cell matrix adhesion sites. Indeed, invadopodia share several features with podosomes, but whether they are distinct structures is still a matter of debate. Invadopodia are built upon an N-WASP-dependent branched actin network, and the Rho GTPase Cdc42 is involved in inducing invadopodial-membrane protrusion, which is mediated by actin filaments that are organized in bundles to form an actin core. Actin-core formation is thought to be an early step in invadopodium assembly, and the actin core is perpendicular to the extracellular matrix and the plasma membrane; this contrasts with the tangential orientation of actin stress fibers anchored to focal adhesions. In this Commentary, we attempt to summarize recent insights into the actin dynamics of invadopodia and podosomes, and the forces that are transmitted through these invasive structures. Although the mechanisms underlying force-dependent regulation of invadopodia and podosomes are largely unknown compared with those of focal adhesions, these structures do exhibit mechanosensitivity. Actin dynamics and associated forces might be key elements in discriminating between invadopodia, podosomes and focal adhesions. Targeting actin regulatory molecules that specifically promote invadopodium formation is an attractive strategy against cancer-cell invasion. PMID:19692590

  3. Geometrical and mechanical properties control actin filament organization.

    PubMed

    Letort, Gaëlle; Politi, Antonio Z; Ennomani, Hajer; Théry, Manuel; Nedelec, Francois; Blanchoin, Laurent

    2015-05-01

    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.

  4. 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

  5. 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

  6. Cyclase-associated protein (CAP) acts directly on F-actin to accelerate cofilin-mediated actin severing across the range of physiological pH.

    PubMed

    Normoyle, Kieran P M; Brieher, William M

    2012-10-12

    Fast actin depolymerization is necessary for cells to rapidly reorganize actin filament networks. Utilizing a Listeria fluorescent actin comet tail assay to monitor actin disassembly rates, we observed that although a mixture of actin disassembly factors (cofilin, coronin, and actin-interacting protein 1 is sufficient to disassemble actin comet tails in the presence of physiological G-actin concentrations this mixture was insufficient to disassemble actin comet tails in the presence of physiological F-actin concentrations. Using biochemical complementation, we purified cyclase-associated protein (CAP) from thymus extracts as a factor that protects against the inhibition of excess F-actin. CAP has been shown to participate in actin dynamics but has been thought to act by liberating cofilin from ADP·G-actin monomers to restore cofilin activity. However, we found that CAP augments cofilin-mediated disassembly by accelerating the rate of cofilin-mediated severing. We also demonstrated that CAP acts directly on F-actin and severs actin filaments at acidic, but not neutral, pH. At the neutral pH characteristic of cytosol in most mammalian cells, we demonstrated that neither CAP nor cofilin are capable of severing actin filaments. However, the combination of CAP and cofilin rapidly severed actin at all pH values across the physiological range. Therefore, our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization.

  7. Cyclase-associated Protein (CAP) Acts Directly on F-actin to Accelerate Cofilin-mediated Actin Severing across the Range of Physiological pH*

    PubMed Central

    Normoyle, Kieran P. M.; Brieher, William M.

    2012-01-01

    Fast actin depolymerization is necessary for cells to rapidly reorganize actin filament networks. Utilizing a Listeria fluorescent actin comet tail assay to monitor actin disassembly rates, we observed that although a mixture of actin disassembly factors (cofilin, coronin, and actin-interacting protein 1 is sufficient to disassemble actin comet tails in the presence of physiological G-actin concentrations this mixture was insufficient to disassemble actin comet tails in the presence of physiological F-actin concentrations. Using biochemical complementation, we purified cyclase-associated protein (CAP) from thymus extracts as a factor that protects against the inhibition of excess F-actin. CAP has been shown to participate in actin dynamics but has been thought to act by liberating cofilin from ADP·G-actin monomers to restore cofilin activity. However, we found that CAP augments cofilin-mediated disassembly by accelerating the rate of cofilin-mediated severing. We also demonstrated that CAP acts directly on F-actin and severs actin filaments at acidic, but not neutral, pH. At the neutral pH characteristic of cytosol in most mammalian cells, we demonstrated that neither CAP nor cofilin are capable of severing actin filaments. However, the combination of CAP and cofilin rapidly severed actin at all pH values across the physiological range. Therefore, our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization. PMID:22904322

  8. Diclofenac Topical (actinic keratosis)

    MedlinePlus

    Solaraze® Gel ... Diclofenac topical gel (Solaraze) is used to treat actinic keratosis (flat, scaly growths on the skin caused by too much sun ... nonsteroidal anti-inflammatory drugs (NSAIDs). The way diclofenac gel works to treat actinic keratosis is not known. ...

  9. Tropomyosin - master regulator of actin filament function in the cytoskeleton.

    PubMed

    Gunning, Peter W; Hardeman, Edna C; Lappalainen, Pekka; Mulvihill, Daniel P

    2015-08-15

    Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.

  10. A network meta-analysis of the relative efficacy of treatments for actinic keratosis of the face or scalp in Europe.

    PubMed

    Vegter, Stefan; Tolley, Keith

    2014-01-01

    Several treatments are available for actinic keratosis (AK) on the face and scalp. Most treatment modalities were compared to placebo and therefore little is known on their relative efficacy. To compare the different treatments for mild to moderate AK on the face and scalp available in clinical practice in Europe. A network meta-analysis (NMA) was performed on the outcome "complete patient clearance". Ten treatment modalities were included: two 5-aminolaevulinic acid photodynamic therapies (ALA-PDT), applied as gel (BF-200 ALA) or patch; methyl-aminolevulinate photodynamic therapy (MAL-PDT); three modalities with imiquimod (IMI), applied as a 4-week or 16-week course with 5% imiquimod, or a 2-3 week course with 3.75% imiquimod; cryotherapy; diclofenac 3% in 2.5% hyaluronic acid; 0.5% 5-fluorouracil (5-FU); and ingenol mebutate (IMB). The only data available for 5% 5-FU was from one small study and was determined to be too limited to be reliably included in the analysis. For BF-200 ALA and MAL-PDT, data from illumination with narrow-band lights were selected as these are typically used in clinical practice. The NMA was performed with a random-effects Bayesian model. 25 trials on 5,562 patients were included in the NMA. All active treatments were significantly better than placebo. BF-200 ALA showed the highest efficacy compared to placebo to achieve total patient clearance. BF-200 ALA had the highest probability to be the best treatment and the highest SUCRA score (64.8% and 92.1%), followed by IMI 5% 4 weeks (10.1% and 74.2%) and 5-FU 0.5% (7.2% and 66.8%). This NMA showed that BF-200 ALA, using narrow-band lights, was the most efficacious treatment for mild to moderate AK on the face and scalp. This analysis is relevant for clinical decision making and health technology assessment, assisting the improved management of AK.

  11. Spontaneous actin dynamics in contractile rings

    NASA Astrophysics Data System (ADS)

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

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

  12. The yeast actin cytoskeleton: from cellular function to biochemical mechanism.

    PubMed

    Moseley, James B; Goode, Bruce L

    2006-09-01

    All cells undergo rapid remodeling of their actin networks to regulate such critical processes as endocytosis, cytokinesis, cell polarity, and cell morphogenesis. These events are driven by the coordinated activities of a set of 20 to 30 highly conserved actin-associated proteins, in addition to many cell-specific actin-associated proteins and numerous upstream signaling molecules. The combined activities of these factors control with exquisite precision the spatial and temporal assembly of actin structures and ensure dynamic turnover of actin structures such that cells can rapidly alter their cytoskeletons in response to internal and external cues. One of the most exciting principles to emerge from the last decade of research on actin is that the assembly of architecturally diverse actin structures is governed by highly conserved machinery and mechanisms. With this realization, it has become apparent that pioneering efforts in budding yeast have contributed substantially to defining the universal mechanisms regulating actin dynamics in eukaryotes. In this review, we first describe the filamentous actin structures found in Saccharomyces cerevisiae (patches, cables, and rings) and their physiological functions, and then we discuss in detail the specific roles of actin-associated proteins and their biochemical mechanisms of action.

  13. Carbon nanotubes reorganize actin structures in cells and ex vivo.

    PubMed

    Holt, Brian D; Short, Philip A; Rape, Andrew D; Wang, Yu-li; Islam, Mohammad F; Dahl, Kris Noel

    2010-08-24

    The ability of globular actin to form filaments and higher-order network structures of the cytoskeleton is essential for cells to maintain their shape and perform essential functions such as force generation, motility, and division. Alterations of actin structures can dramatically change a cell's ability to function. We found that purified and dispersed single wall carbon nanotubes (SWCNTs) can induce actin bundling in cells and in purified model actin systems. SWCNTs do not induce acute cell death, but cell proliferation is greatly reduced in SWCNT-treated cells with an increase in actin-related division defects. Actin, normally present in basal stress fibers in control cells, is located in heterogeneous structures throughout the SWCNT-treated cell. These SWCNT-induced changes in actin structures are seen functionally in multinucleated cells and with reduced force generation. Ex vivo, purified actin filaments cross-linked with alpha-actinin and formed isotropic networks, whereas SWCNTs caused purified actin filaments to assemble into bundles. While purified, isolated SWCNTs do not appear acutely toxic, this subcellular reorganization may cause chronic changes to cellular functions.

  14. What we talk about when we talk about nuclear actin

    PubMed Central

    Belin, Brittany J; Mullins, R Dyche

    2013-01-01

    In the cytoplasm, actin filaments form crosslinked networks that enable eukaryotic cells to transport cargo, change shape, and move. Actin is also present in the nucleus but, in this compartment, its functions are more cryptic and controversial. If we distill the substantial literature on nuclear actin down to its essentials, we find four, recurring, and more-or-less independent, claims: (1) crosslinked networks of conventional actin filaments span the nucleus and help maintain its structure and organize its contents; (2) assembly or contraction of filaments regulates specific nuclear events; (3) actin monomers moonlight as subunits of chromatin remodeling complexes, independent of their ability to form filaments; and (4) modified actin monomers or oligomers, structurally distinct from canonical, cytoskeletal filaments, mediate nuclear events by unknown mechanisms. We discuss the evidence underlying these claims and as well as their strengths and weaknesses. Next, we describe our recent work, in which we built probes specific for nuclear actin and used them to describe the form and distribution of actin in somatic cell nuclei. Finally, we discuss how different forms of nuclear actin may play different roles in different cell types and physiological contexts. PMID:23934079

  15. Actin protofilament orientation in deformation of the erythrocyte membrane skeleton.

    PubMed Central

    Picart, C; Dalhaimer, P; Discher, D E

    2000-01-01

    The red cell's spectrin-actin network is known to sustain local states of shear, dilation, and condensation, and yet the short actin filaments are found to maintain membrane-tangent and near-random azimuthal orientations. When calibrated with polarization results for single actin filaments, imaging of micropipette-deformed red cell ghosts has allowed an assessment of actin orientations and possible reorientations in the network. At the hemispherical cap of the aspirated projection, where the network can be dilated severalfold, filaments have the same membrane-tangent orientation as on a relatively unstrained portion of membrane. Likewise, over the length of the network projection pulled into the micropipette, where the network is strongly sheared in axial extension and circumferential contraction, actin maintains its tangent orientation and is only very weakly aligned with network extension. Similar results are found for the integral membrane protein Band 3. Allowing for thermal fluctuations, we deduce a bound for the effective coupling constant, alpha, between network shear and azimuthal orientation of the protofilament. The finding that alpha must be about an order of magnitude or more below its tight-coupling value illustrates how nanostructural kinematics can decouple from more macroscopic responses. Monte Carlo simulations of spectrin-actin networks at approximately 10-nm resolution further support this conclusion and substantiate an image of protofilaments as elements of a high-temperature spin glass. PMID:11106606

  16. 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

  17. 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

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

    PubMed Central

    Case, Lindsay B.; Waterman, Clare M.

    2011-01-01

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

  19. Documenting Uncertainty and Error in Gridded Growing Degree Day and Spring Onset Maps Generated by the USA National Phenology Network

    NASA Astrophysics Data System (ADS)

    Crimmins, T. M.; Switzer, J.; Rosemartin, A.; Marsh, L.; Gerst, K.; Crimmins, M.; Weltzin, J. F.

    2016-12-01

    Since 2016 the USA National Phenology Network (USA-NPN; www.usanpn.org) has produced and delivered daily maps and short-term forecasts of accumulated growing degree days and spring onset dates at fine spatial scale for the conterminous United States. Because accumulated temperature is a strong driver of phenological transitions in plants and animals, including leaf-out, flowering, fruit ripening, and migration, these data products have utility for a wide range of natural resource planning and management applications, including scheduling invasive species and pest detection and control activities, determining planting dates, anticipating allergy outbreaks and planning agricultural harvest dates. The USA-NPN is a national-scale program that supports scientific advancement and decision-making by collecting, storing, and sharing phenology data and information. We will be expanding the suite of gridded map products offered by the USA-NPN to include predictive species-specific maps of phenological transitions in plants and animals at fine spatial and temporal resolution in the future. Data products, such as the gridded maps currently produced by the USA-NPN, inherently contain uncertainty and error arising from multiple sources, including error propagated forward from underlying climate data and from the models implemented. As providing high-quality, vetted data in a transparent way is central to the USA-NPN, we aim to identify and report the sources and magnitude of uncertainty and error in gridded maps and forecast products. At present, we compare our real-time gridded products to independent, trustworthy data sources, such as the Climate Reference Network, on a daily basis and report Mean Absolute Error and bias through an interactive online dashboard.

  20. Regulation of myosin II activity by actin architecture

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly; Stam, Samantha; McCall, Patrick; Munro, Edwin; Gardel, Margaret

    2015-03-01

    Networks of actin filaments containing myosin II motors generate forces and motions that promote biological processes such as cell division, motility, and cargo transport. In cells, actin filaments are arranged in various structures from disordered meshworks to tight bundles. Clusters of myosin II motors, known as myosin filaments, crosslink and generate force on neighboring actin filaments. We hypothesized that the local actin architecture controls the magnitude and duration of force generated by myosin II motors. We used fluorescence imaging to directly measure the mobility of myosin II filaments on actin networks and bundles with varying actin filament polarity, orientation, spacing, and length. On unipolar bundles, myosin exhibits fast, unidirectional motion consistent with their unloaded gliding speed. On mixed polarity bundles, myosin speed is reduced by one order of magnitude and marked by direction switching and trapping. Increasing filament spacing and bundle flexibility reduces the duration of trapping and enhances the mobility of motors. Simulations indicate that stable trapping is a signature of large generated forces while increased mobility indicates force release. Our data underscore that the efficiency of force generation by myosin motors in an actin network depends sensitively on its architecture and suggests actin crosslinking proteins are tuned to optimize actomyosin contractility.

  1. Axonal actin in action: Imaging actin dynamics in neurons.

    PubMed

    Ladt, Kelsey; Ganguly, Archan; Roy, Subhojit

    2016-01-01

    Actin is a highly conserved, key cytoskeletal protein involved in numerous structural and functional roles. In neurons, actin has been intensively investigated in axon terminals-growth cones-and dendritic spines, but details about actin structure and dynamics in axon shafts have remained obscure for decades. A major barrier in the field has been imaging actin. Actin exists as soluble monomers (G-actin) as well as actin filaments (F-actin), and labeling actin with conventional fluorescent probes like GFP/RFP typically leads to a diffuse haze that makes it difficult to discern kinetic behaviors. In a recent publication, we used F-actin selective probes to visualize actin dynamics in axons, resolving striking actin behaviors that have not been described before. However, using these probes to visualize actin dynamics is challenging as they can cause bundling of actin filaments; thus, experimental parameters need to be strictly optimized. Here we describe some practical methodological details related to using these probes for visualizing F-actin dynamics in axons.

  2. Actin is an essential component of plant gravitropic signaling pathways

    NASA Astrophysics Data System (ADS)

    Braun, Markus; Hauslage, Jens; Limbach, Christoph

    2003-08-01

    A role of the actin cytoskeleton in the different phases of gravitropism in higher plant organs seems obvious, but experimental evidence is still inconclusive and contradictory. In gravitropically tip-growing rhizoids and protonemata, however, it is well documented that actin is an essential component of the tip-growth machinery and is involved either in the cellular mechanisms that lead to gravity sensing and in the processes of the graviresponses that result in the reorientation of the growth direction. All these processes depend on a complexly organized and highly dynamic organization of actin filaments whose diverse functions are coordinated by numerous associated proteins. Actin filaments and myosins mediate the transport of secretory vehicles to the growing tip and precisely control the delivery of cell wall material. In addition, both cell types use a very efficient actomyosin-based system to control and correct the position of their statoliths and to direct sedimenting statoliths to confined graviperception sites at the plasma membrane. The studies presented in this paper provide evidence for the essential role of actin in plant gravity sensing and the gravitropic responses. A unique actin-organizing center exists in the tip of characean rhizoids and protonemata which is associated with and dynamically regulated by a specific set of actin-dynamizing proteins. It is concluded that this highly dynamic apical actin array is an essential prerequisite for gravity sensing and gravity-oriented tip growth.

  3. Actin-filament disassembly: it takes two to shrink them fast.

    PubMed

    Winterhoff, Moritz; Faix, Jan

    2015-06-01

    Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level.

  4. Actin-binding proteins implicated in the formation of the punctate actin foci stimulated by the self-incompatibility response in Papaver.

    PubMed

    Poulter, Natalie S; Staiger, Christopher J; Rappoport, Joshua Z; Franklin-Tong, Vernonica E

    2010-03-01

    The actin cytoskeleton is a key target for signaling networks and plays a central role in translating signals into cellular responses in eukaryotic cells. Self-incompatibility (SI) is an important mechanism responsible for preventing self-fertilization. The SI system of Papaver rhoeas pollen involves a Ca(2+)-dependent signaling network, including massive actin depolymerization as one of the earliest cellular responses, followed by the formation of large actin foci. However, no analysis of these structures, which appear to be aggregates of filamentous (F-)actin based on phalloidin staining, has been carried out to date. Here, we characterize and quantify the formation of F-actin foci in incompatible Papaver pollen tubes over time. The F-actin foci increase in size over time, and we provide evidence that their formation requires actin polymerization. Once formed, these SI-induced structures are unusually stable, being resistant to treatments with latrunculin B. Furthermore, their formation is associated with changes in the intracellular localization of two actin-binding proteins, cyclase-associated protein and actin-depolymerizing factor. Two other regulators of actin dynamics, profilin and fimbrin, do not associate with the F-actin foci. This study provides, to our knowledge, the first insights into the actin-binding proteins and mechanisms involved in the formation of these intriguing structures, which appear to be actively formed during the SI response.

  5. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane – a minimally invasive investigation by STED-FCS

    PubMed Central

    Andrade, Débora M.; Clausen, Mathias P.; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E.; Hell, Stefan W.; Lagerholm, B. Christoffer; Eggeling, Christian

    2015-01-01

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes. PMID:26118385

  6. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane - a minimally invasive investigation by STED-FCS

    NASA Astrophysics Data System (ADS)

    Andrade, Débora M.; Clausen, Mathias P.; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E.; Hell, Stefan W.; Lagerholm, B. Christoffer; Eggeling, Christian

    2015-06-01

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.

  7. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane--a minimally invasive investigation by STED-FCS.

    PubMed

    Andrade, Débora M; Clausen, Mathias P; Keller, Jan; Mueller, Veronika; Wu, Congying; Bear, James E; Hell, Stefan W; Lagerholm, B Christoffer; Eggeling, Christian

    2015-06-29

    Important discoveries in the last decades have changed our view of the plasma membrane organisation. Specifically, the cortical cytoskeleton has emerged as a key modulator of the lateral diffusion of membrane proteins. Cytoskeleton-dependent compartmentalised lipid diffusion has been proposed, but this concept remains controversial because this phenomenon has thus far only been observed with artefact-prone probes in combination with a single technique: single particle tracking. In this paper, we report the first direct observation of compartmentalised phospholipid diffusion in the plasma membrane of living cells using a minimally invasive, fluorescent dye labelled lipid analogue. These observations were made using optical STED nanoscopy in combination with fluorescence correlation spectroscopy (STED-FCS), a technique which allows the study of membrane dynamics on a sub-millisecond time-scale and with a spatial resolution of down to 40 nm. Specifically, we find that compartmentalised phospholipid diffusion depends on the cortical actin cytoskeleton, and that this constrained diffusion is directly dependent on the F-actin branching nucleator Arp2/3. These findings provide solid evidence that the Arp2/3-dependent cortical actin cytoskeleton plays a pivotal role in the dynamic organisation of the plasma membrane, potentially regulating fundamental cellular processes.

  8. Inside view of cell locomotion through single-molecule: fast F-/G-actin cycle and G-actin regulation of polymer restoration

    PubMed Central

    Watanabe, Naoki

    2010-01-01

    The actin cytoskeleton drives cell locomotion and tissue remodeling. The invention of live-cell fluorescence single-molecule imaging opened a window for direct viewing of the actin remodeling processes in the cell. Since then, a number of unanticipated molecular functions have been revealed. One is the mechanism of F-actin network breakdown. In lamellipodia, one third of newly polymerized F-actin disassembles within 10 seconds. This fast F-actin turnover is facilitated by the filament severing/disrupting activity involving cofilin and AIP1. Astoundingly fast dissociation kinetics of the barbed end interactors including capping protein suggests that F-actin turnover might proceed through repetitive disruption/reassembly of the filament near the barbed end. The picture of actin polymerization is also being revealed. At the leading edge of the cell, Arp2/3 complex is highly activated in a narrow edge region. In contrast, mDia1 and its related Formin homology proteins display a long-distance directional molecular movement using their processive actin capping ability. Recently, these two independently-developed projects converged into a discovery of the spatiotemporal coupling between mDia1-mediated filament nucleation and actin disassembly. Presumably, the local concentration fluctuation of G-actin regulates the actin nucleation efficiency of specific actin nucleators including mDia1. Pharmacological perturbation and quantitative molecular behavior analysis synergize to reveal hidden molecular linkages in the actin turnover cycle and cell signaling. PMID:20075609

  9. The dynamics of filament assembly define cytoskeletal network morphology

    PubMed Central

    Foffano, Giulia; Levernier, Nicolas; Lenz, Martin

    2016-01-01

    The actin cytoskeleton is a key component in the machinery of eukaryotic cells, and it self-assembles out of equilibrium into a wide variety of biologically crucial structures. Although the molecular mechanisms involved are well characterized, the physical principles governing the spatial arrangement of actin filaments are not understood. Here we propose that the dynamics of actin network assembly from growing filaments results from a competition between diffusion, bundling and steric hindrance, and is responsible for the range of observed morphologies. Our model and simulations thus predict an abrupt dynamical transition between homogeneous and strongly bundled networks as a function of the actin polymerization rate. This suggests that cells may effect dramatic changes to their internal architecture through minute modifications of their nonequilibrium dynamics. Our results are consistent with available experimental data. PMID:28000681

  10. The dynamics of filament assembly define cytoskeletal network morphology

    NASA Astrophysics Data System (ADS)

    Foffano, Giulia; Levernier, Nicolas; Lenz, Martin

    2016-12-01

    The actin cytoskeleton is a key component in the machinery of eukaryotic cells, and it self-assembles out of equilibrium into a wide variety of biologically crucial structures. Although the molecular mechanisms involved are well characterized, the physical principles governing the spatial arrangement of actin filaments are not understood. Here we propose that the dynamics of actin network assembly from growing filaments results from a competition between diffusion, bundling and steric hindrance, and is responsible for the range of observed morphologies. Our model and simulations thus predict an abrupt dynamical transition between homogeneous and strongly bundled networks as a function of the actin polymerization rate. This suggests that cells may effect dramatic changes to their internal architecture through minute modifications of their nonequilibrium dynamics. Our results are consistent with available experimental data.

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

    PubMed

    Blotnick, Edna; Sol, Asaf; Muhlrad, Andras

    2017-01-01

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

  12. 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.

  13. Biomimetic systems for studying actin-based motility.

    PubMed

    Upadhyaya, Arpita; van Oudenaarden, Alexander

    2003-09-16

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

  14. Actin cytoskeleton in Arabidopsis thaliana under blue and red light.

    PubMed

    Krzeszowiec, Weronika; Rajwa, Bartek; Dobrucki, Jurek; Gabryś, Halina

    2007-05-01

    Actin cytoskeleton is the basis of chloroplast-orientation movements. These movements are activated by blue light in the leaves of terrestrial angiosperms. Red light has been shown to affect the spatial reorganization of F-actin in water plants, where chloroplast movements are closely connected with cytoplasmic streaming. The aim of the present study was to determine whether blue light, which triggers characteristic responses of chloroplasts, i.e. avoidance and accumulation, also influences F-actin organization in the mesophyll cells of Arabidopsis thaliana. Actin filaments in fixed mesophyll tissue were labelled with Alexa Fluor 488-conjugated phalloidin. The configuration of actin filaments, expressed as a form factor (4 pi x area/perimeter(2)), was determined for all actin formations which were measured in fluorescence confocal images. In the present study, we compare form-factor distributions and the median form factors for strong and weak, blue- and red-irradiated tissues. Spatial organization of the F-actin network did not undergo any changes which could be attributed specifically to blue light. Actin patterns were similar in blue-irradiated wild-type plants and phot2 (phototropin 2) mutants which lack the avoidance response of chloroplasts. However, significant differences in the shape and distribution of F-actin formations were observed between mesophyll cells of phot2 mutants irradiated with strong and weak red light. These differences were absent in wild-type leaves. Actin does not appear to be the main target for the blue-light chloroplast-orientation signal. The modes of actin involvement in chloroplast translocations are different in water and terrestrial angiosperms. The results suggest that co-operation occurs between blue- and red-light photoreceptors in the control of the actin cytoskeleton architecture in Arabidopsis.

  15. Force Generation, Polymerization Dynamics and Nucleation of Actin Filaments

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe

    We study force generation and actin filament dynamics using stochastic and deterministic methods. First, we treat force generation of bundled actin filaments by polymerization via molecular-level stochastic simulations. In the widely-used Brownian Ratchet model, actin filaments grow freely whenever the tip-obstacle gap created by thermal fluctuation exceeds the monomer size. We name this model the Perfect Brownian Ratchet (PBR) model. In the PBR model, actin monomer diffusion is treated implicitly. We perform a series of simulations based on the PBR, in which obstacle motion is treated explicitly; in most previous studies, obstacle motion has been treated implicitly. We find that the cooperativity of filaments is generally weak in the PBR model, meaning that more filaments would grow more slowly given the same force per filament. Closed-form formulas are also developed, which match the simulation results. These portable and accurate formulas provide guidance for experiments and upper and lower bounds for theoretical analyses. We also studied a variation of the PBR, called the Diffusing Brownian Ratchet (DBR) model, in which both actin monomer and obstacle diffusion are treated explicitly. We find that the growth rate of multiple filaments is even lower, compared with that in PBR. This finding challenges the widely-accepted PBR assumption and suggests that pushing the study of actin dynamics down to the sub-nanometer level yields new insights. We subsequently used a rate equation approach to model the effect of local depletion of actin monomers on the nucleation of actin filaments on biomimetic beads, and how the effect is regulated by capping protein (CP). We find that near the bead surface, a higher CP concentration increases local actin concentration, which leads to an enhanced activities of actin filaments' nucleation. Our model analysis matches the experimental results and lends support to an important but undervalued hypothesis proposed by Carlier and

  16. Quantitative Fluorescent Speckle Microscopy (QFSM) to Measure Actin Dynamics

    PubMed Central

    Mendoza, Michelle C.; Besson, Sebastien; Danuser, Gaudenz

    2012-01-01

    Quantitative Fluorescent Speckle Microscopy (QFSM) is a live cell imaging method to analyze the dynamics of macromolecular assemblies with high spatial and temporal resolution. Its greatest successes were in the analysis of actin filament and adhesion dynamics in the context of cell migration and microtubule dynamics in interphase and the meotic/mitotic spindle. Here, we focus on the former application to illustrate the procedures of FSM imaging and the computational image processing that extracts quantitative information from these experiments. QFSM is advantageous over other methods because it measures the movement and turnover kinetics of the actin filament (F-actin) network in living cells across the entire field of view. Experiments begin with microinjection of fluorophore-labeled actin into cells, which generate a low ratio of fluorescently-labeled:endogenous unlabeled actin monomers. Spinning disk confocal or wide-field imaging then visualizes fluorophore clusters (2–8 actin monomers) within the assembled F-actin network as speckles. QFSM software identifies and computationally tracks and utilizes the location, appearance, and disappearance of speckles to derive network flows and maps of the rate of filament assembly and disassembly. PMID:23042526

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

    NASA Astrophysics Data System (ADS)

    John, Karin; Peyla, Philippe; Misbah, Chaouqi

    2007-03-01

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

  18. Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells

    PubMed Central

    Loo, Chin-San; Chen, Cheng-Wei; Wang, Po-Jen; Chen, Pei-Yu; Lin, Shu-Yu; Khoo, Kay-Hooi; Fenton, Robert A.; Knepper, Mark A.; Yu, Ming-Jiun

    2013-01-01

    In kidney collecting duct cells, filamentous actin (F-actin) depolymerization is a critical step in vasopressin-induced trafficking of aquaporin-2 to the apical plasma membrane. However, the molecular components of this response are largely unknown. Using stable isotope-based quantitative protein mass spectrometry and surface biotinylation, we identified 100 proteins that showed significant abundance changes in the apical plasma membrane of mouse cortical collecting duct cells in response to vasopressin. Fourteen of these proteins are involved in actin cytoskeleton regulation, including actin itself, 10 actin-associated proteins, and 3 regulatory proteins. Identified were two integral membrane proteins (Clmn, Nckap1) and one actin-binding protein (Mpp5) that link F-actin to the plasma membrane, five F-actin end-binding proteins (Arpc2, Arpc4, Gsn, Scin, and Capzb) involved in F-actin reorganization, and two actin adaptor proteins (Dbn1, Lasp1) that regulate actin cytoskeleton organization. There were also protease (Capn1), protein kinase (Cdc42bpb), and Rho guanine nucleotide exchange factor 2 (Arhgef2) that mediate signal-induced F-actin changes. Based on these findings, we devised a live-cell imaging method to observe vasopressin-induced F-actin dynamics in polarized mouse cortical collecting duct cells. In response to vasopressin, F-actin gradually disappeared near the center of the apical plasma membrane while consolidating laterally near the tight junction. This F-actin peripheralization was blocked by calcium ion chelation. Vasopressin-induced apical aquaporin-2 trafficking and forskolin-induced water permeability increase were blocked by F-actin disruption. In conclusion, we identified a vasopressin-regulated actin network potentially responsible for vasopressin-induced apical F-actin dynamics that could explain regulation of apical aquaporin-2 trafficking and water permeability increase. PMID:24085853

  19. Viscoelastic properties of actin-coated membranes

    NASA Astrophysics Data System (ADS)

    Helfer, E.; Harlepp, S.; Bourdieu, L.; Robert, J.; Mackintosh, F. C.; Chatenay, D.

    2001-02-01

    In living cells, cytoskeletal filaments interact with the plasma membrane to form structures that play a key role in cell shape and mechanical properties. To study the interaction between these basic components, we designed an in vitro self-assembled network of actin filaments attached to the outer surface of giant unilamellar vesicles. Optical tweezers and single-particle tracking experiments are used to study the rich dynamics of these actin-coated membranes (ACM). We show that microrheology studies can be carried out on such an individual microscopic object. The principle of the experiment consists in measuring the thermally excited position fluctuations of a probe bead attached biochemically to the membrane. We propose a model that relates the power spectrum of these thermal fluctuations to the viscoelastic properties of the membrane. The presence of the actin network modifies strongly the membrane dynamics with respect to a fluid, lipid bilayer one. It induces first a finite (ω=0) two-dimensional (2D) shear modulus G02D~0.5 to 5 μN/m in the membrane plane. Moreover, the frequency dependence at high frequency of the shear modulus [G'2D(f )~f0.85+/-0.07] and of the bending modulus (κACM(f)~f0.55+/-0.21) demonstrate the viscoelastic behavior of the composite membrane. These results are consistent with a common exponent of 0.75 for both moduli as expected from our model and from prior measurements on actin solutions.

  20. A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells.

    PubMed

    Sheahan, Michael B; Staiger, Chris J; Rose, Ray J; McCurdy, David W

    2004-12-01

    The actin cytoskeleton coordinates numerous cellular processes required for plant development. The functions of this network are intricately linked to its dynamic arrangement, and thus progress in understanding how actin orchestrates cellular processes relies on critical evaluation of actin organization and turnover. To investigate the dynamic nature of the actin cytoskeleton, we used a fusion protein between green fluorescent protein (GFP) and the second actin-binding domain (fABD2) of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. The GFP-fABD2 fusion protein labeled highly dynamic and dense actin networks in diverse species and cell types, revealing structural detail not seen with alternative labeling methods, such as the commonly used mouse talin GFP fusion (GFP-mTalin). Further, we show that expression of the GFP-fABD2 fusion protein in Arabidopsis, unlike GFP-mTalin, has no detectable adverse effects on plant morphology or development. Time-lapse confocal microscopy and fluorescence recovery after photobleaching analyses of the actin cytoskeleton labeled with GFP-fABD2 revealed that lateral-filament migration and sliding of individual actin filaments or bundles are processes that contribute to the dynamic and continually reorganizing nature of the actin scaffold. These new observations of the dynamic actin cytoskeleton in plant cells using GFP-fABD2 reveal the value of this probe for future investigations of how actin filaments coordinate cellular processes required for plant development.

  1. Weighted growing simplicial complexes

    NASA Astrophysics Data System (ADS)

    Courtney, Owen T.; Bianconi, Ginestra

    2017-06-01

    Simplicial complexes describe collaboration networks, protein interaction networks, and brain networks and in general network structures in which the interactions can include more than two nodes. In real applications, often simplicial complexes are weighted. Here we propose a nonequilibrium model for weighted growing simplicial complexes. The proposed dynamics is able to generate weighted simplicial complexes with a rich interplay between weights and topology emerging not just at the level of nodes and links, but also at the level of faces of higher dimension.

  2. Verification of Satellite Radar Remote Sensing Based Estimates of Boreal and Subalpine Growing Seasons Using an Ecosystem Process Model and Surface Biophysical Measurement Network Information

    NASA Astrophysics Data System (ADS)

    Kimball, J. S.; McDonald, K. C.; Running, S. W.; Zimmermann, R.

    2002-12-01

    We employ daily surface Radar backscatter data from the SeaWinds Ku-band Scatterometer onboard Quikscat to estimate landscape freeze-thaw state and associated length of the seasonal non-frozen period as a surrogate for determining the annual growing season across boreal and subalpine regions of North America for 2000 and 2001. We compare these results with estimates of growing season length derived from a network of surface stations, utilizing BIOME-BGC stand-level ecosystem process model simulations, site sap flow and tower eddy flux net CO2 exchange measurements for a network of mature evergreen coniferous forest stands. Remote sensing based estimates of spatial patterns in the timing of seasonal freeze-thaw vary by more than 8 weeks, while associated estimates of growing season length span more than 14 weeks across the region. Inter-annual variability between 2000 and 2001 is found to be on the order of 1-4 weeks. Remote sensing estimates of growing season initiation and length are found to be well correlated with both site measurements and model simulations. Remote sensing measurements of the end of the seasonal non-frozen period are also found to be consistent with site based temperature measurements, but not with site based estimates of growing season termination. These findings are attributed to a relatively strong dependence of the onset of the growing season to snowmelt and associated soil thaw in spring and the relative importance of additional factors such as light availability and day length in controlling growing season termination. This work was performed at the University of Montana, and at the Jet Propulsion Laboratory, California Institute of Technology, under contract with the National Aeronautics and Space Administration.

  3. Feisty filaments: actin dynamics in the red blood cell membrane skeleton

    PubMed Central

    Gokhin, David S.; Fowler, Velia M.

    2016-01-01

    STRUCTURED ABSTRACT Purpose of review The purpose of this review is to discuss recent advances and unsolved questions in our understanding of actin filament organization and dynamics in the red blood cell (RBC) membrane skeleton, a two-dimensional quasi-hexagonal network consisting of (α1β1)2-spectrin tetramers interconnecting short actin filament-based junctional complexes. Recent findings In contrast to the long-held view that RBC actin filaments are static structures that do not exchange subunits with the cytosol, RBC actin filaments are dynamic structures that undergo subunit exchange and turnover, as evidenced by monomer incorporation experiments with rhodamine-actin and filament disruption experiments with actin-targeting drugs. The malaria-causing parasite, Plasmodium falciparum, co-opts RBC actin dynamics to construct aberrantly branched actin filament networks. Even though RBC actin filaments are dynamic, RBC actin filament lengths are highly uniform (~37 nm). RBC actin filament lengths are thought to be stabilized by the capping proteins, tropomodulin-1 and αβ-adducin, as well as the side-binding protein tropomyosin, present in an equimolar combination of two isoforms, TM5b (Tpm1.9) and TM5NM1 (Tpm3.1). Summary New evidence indicates that RBC actin filaments are not simply passive cytolinkers, but rather dynamic structures whose assembly and disassembly play important roles in RBC membrane function. PMID:27055045

  4. Aluminum modifies the viscosity of filamentous actin solutions as measured by optical displacement microviscometry.

    PubMed

    Arnoys, E J; Schindler, M

    2000-01-01

    A microtechnique has been developed that is capable of measuring the viscosity of filamentous actin (F-actin) solutions. This method, called optical displacement microviscometry (ODM), was utilized to determine the changes in viscosity of solutions of rabbit muscle, human platelet, and maize pollen actin when measured in the absence and presence of aluminum. Measurements demonstrated that the viscosity of the different actin solutions decreased with aluminum concentration. In contrast, increases in viscosity were observed when aluminum was added to F-actin solutions containing filamin (chicken gizzard), a protein that bundles actin filaments. Confocal fluorescence imaging of pure actin solutions in the presence of aluminum showed a disrupted actin network composed of fragmented actin filaments in the form of small aggregates. In contrast, in the presence of filamin, aluminum promoted the formation of thicker actin filaments. These measurements demonstrate that aluminum can affect actin filaments differentially depending on the presence of an actin-binding protein. In addition, a strong correlation is observed between the changes in viscosity as measured by ODM and the thickness and assembled state of bundles of actin filaments.

  5. Actinic keratosis. Current treatment options.

    PubMed

    Jeffes, E W; Tang, E H

    2000-01-01

    Actinic keratoses are hyperkeratotic skin lesions that represent focal abnormal proliferation of epidermal keratinocytes. Some actinic keratoses evolve into squamous cell carcinoma of the skin, while others resolve spontaneously. The conversion rate of actinic keratosis to squamous cell carcinoma is not accurately known, but appears to be in the range of 0.25 to 1% per year. Although there is a low rate of conversion of actinic keratoses to squamous cell carcinoma, 60% of squamous cell carcinomas of the skin probably arise from actinic keratoses. The main cause of actinic keratoses in otherwise healthy Caucasians appears to be the sun. Therapy for actinic keratoses begins with prevention which starts with sun avoidance and physical protection. Sunprotection with sunscreens actually slows the return of actinic keratoses in patients already getting actinic keratoses. Interestingly, a few studies are available that demonstrate that a high fat diet is associated with the production of more actinic keratoses than is a low fat diet. One of the mainstays of therapy has been local destruction of the actinic keratoses with cryotherapy, and curettage and electrodesiccation. A new addition to this group of therapies to treat individual actinic keratoses is photodynamic therapy with topical aminolevulinic acid and light. In patients who have numerous actinic keratoses in an area of severely sun damaged skin, therapies which are applied to the whole actinic keratosis area are used. The goal of treating such an area of skin is to treat all of the early as well as the numerous clinically evident actinic keratoses at the same time. The classical approaches for treating areas of photodamaged skin without treating actinic keratoses individually include: the use of topically applied fluorouracil cream, dermabrasion, and cutaneous peels with various agents like trichloroacetic acid. Both topically as well as orally administered retinoids have been used to treat actinic keratoses but

  6. Simulation of the effect of confinement in actin ring formation

    NASA Astrophysics Data System (ADS)

    Adeli Koudehi, Maral; Vavylonis, Dimitrios; Haosu Tang Team; Dimitrios Vavylonis Team

    Actin filaments are vital for different network structures in living cells. During cytokinesis, they form a contractile ring containing myosin motor proteins and actin filament cross-linkers to separate one cell into two cells. Recent experimental studies have quantified the bundle, ring, and network structures that form when actin filaments polymerize in confined environments in vitro, in the presence of varying concentrations of cross-linkers. In this study, we performed numerical simulations to investigate the effect of actin spherical confinement and cross-linking in ring formation. We used a spring-bead model and Brownian dynamics to simulate semiflexible actin filaments that polymerize in a confining sphere with a rate proportional to the monomer concentration. Applying the model for different size of the confining spheres shows that the probability of ring formation decreases by increasing the radius (at fixed initial monomer concentration), in agreement with prior experimental data. We describe the effect of persistence length, orientation-dependent cross-linking, and initial actin monomer concentration. Simulations show that equilibrium configurations can be reached through zipping and unzipping of actin filaments in bundles and transient ring formation.

  7. The centrosome is an actin-organizing center

    PubMed Central

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

    2016-01-01

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

  8. The centrosome is an actin-organizing centre.

    PubMed

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

    2016-01-01

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

  9. Lamellipodial actin mechanically links myosin activity with adhesion site formation

    PubMed Central

    Giannone, Gregory; Dubin-Thaler, Benjamin; Rossier, Olivier; Cai, Yunfei; Chaga, Oleg; Jiang, Guoying; Beaver, William; Döbereiner, Hans-Günther; Freund, Yoav; Borisy, Gary; Sheetz, Michael P.

    2013-01-01

    Summary Cell motility proceeds by cycles of edge protrusion, adhesion and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process. PMID:17289574

  10. Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia.

    PubMed

    Cohan, C S; Welnhofer, E A; Zhao, L; Matsumura, F; Yamashiro, S

    2001-02-01

    Growth cones at the distal tips of growing nerve axons contain bundles of actin filaments distributed throughout the lamellipodium and that project into filopodia. The regulation of actin bundling by specific actin binding proteins is likely to play an important role in many growth cone behaviors. Although the actin binding protein, fascin, has been localized in growth cones, little information is available on its functional significance. We used the large growth cones of the snail Helisoma to determine whether fascin was involved in temporal changes in actin filaments during growth cone morphogenesis. Fascin localized to radially oriented actin bundles in lamellipodia (ribs) and filopodia. Using a fascin antibody and a GFP fascin construct, we found that fascin incorporated into actin bundles from the beginning of growth cone formation at the cut end of axons. Fascin associated with most of the actin bundle except the proximal 6--12% adjacent to the central domain, which is the region associated with actin disassembly. Later, during growth cone morphogenesis when actin ribs shortened, the proximal fascin-free zone of bundles increased, but fascin was retained in the distal, filopodial portion of bundles. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which phosphorylates fascin and decreases its affinity for actin, resulted in loss of all actin bundles from growth cones. Our findings suggest that fascin may be particularly important for the linear structure and dynamics of filopodia and for lamellipodial rib dynamics by regulating filament organization in bundles.

  11. Interaction of Phalloidin with Actin

    PubMed Central

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

    1974-01-01

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

  12. IFT88 influences chondrocyte actin organization and biomechanics

    PubMed Central

    Wang, Z.; Wann, A.K.T.; Thompson, C.L.; Hassen, A.; Wang, W.; Knight, M.M.

    2016-01-01

    Summary Objectives 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. Methods The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88orpk). 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. Results IFT88orpk 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 IFT88orpk cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88orpk cells. Following membrane blebbing, IFT88orpk cells exhibited slower reformation of the actin cortex. IFT88orpk 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. Conclusions 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. PMID:26493329

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

    PubMed

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

    2011-02-14

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

  14. 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

  15. Symmetry breaking in reconstituted actin cortices

    PubMed Central

    Abu Shah, Enas; Keren, Kinneret

    2014-01-01

    The actin cortex plays a pivotal role in cell division, in generating and maintaining cell polarity and in motility. In all these contexts, the cortical network has to break symmetry to generate polar cytoskeletal dynamics. Despite extensive research, the mechanisms responsible for regulating cortical dynamics in vivo and inducing symmetry breaking are still unclear. Here we introduce a reconstituted system that self-organizes into dynamic actin cortices at the inner interface of water-in-oil emulsions. This artificial system undergoes spontaneous symmetry breaking, driven by myosin-induced cortical actin flows, which appears remarkably similar to the initial polarization of the embryo in many species. Our in vitro model system recapitulates the rich dynamics of actin cortices in vivo, revealing the basic biophysical and biochemical requirements for cortex formation and symmetry breaking. Moreover, this synthetic system paves the way for further exploration of artificial cells towards the realization of minimal model systems that can move and divide. DOI: http://dx.doi.org/10.7554/eLife.01433.001 PMID:24843007

  16. Disseminated superficial actinic porokeratosis.

    PubMed

    Rouhani, Panta; Fischer, Max; Meehan, Shane; Pomeranz, Miriam Keltz

    2012-12-15

    Disseminated superficial actinic porokeratosis, which was described in 1966, is characterized by small, atrophic patches with distinctive keratin rims that occur on sun-exposed areas of the extremities, shoulders, and back. The diagnosis is based on the histopathologic finding of a cornoid lamella, absence of a granular layer, and often a thin epidermis. It is associated with exposure to ultraviolet radiation. Gene studies suggest a pathway defect in which several mutations in keratinocyte proliferation and differentiation lead to development of porokeratosis.

  17. Single-molecule studies of actin assembly and disassembly factors.

    PubMed

    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.

  18. Moesin and cortactin control actin-dependent multivesicular endosome biogenesis

    PubMed Central

    Muriel, Olivia; Tomas, Alejandra; Scott, Cameron C.; Gruenberg, Jean

    2016-01-01

    We used in vivo and in vitro strategies to study the mechanisms of multivesicular endosome biogenesis. We found that, whereas annexinA2 and ARP2/3 mediate F-actin nucleation and branching, respectively, the ERM protein moesin supports the formation of F-actin networks on early endosomes. We also found that moesin plays no role during endocytosis and recycling to the plasma membrane but is absolutely required, much like actin, for early-to-late-endosome transport and multivesicular endosome formation. Both actin network formation in vitro and early-to-late endosome transport in vivo also depend on the F-actin–binding protein cortactin. Our data thus show that moesin and cortactin are necessary for formation of F-actin networks that mediate endosome biogenesis or maturation and transport through the degradative pathway. We propose that the primary function of endosomal F-actin is to control the membrane remodeling that accompanies endosome biogenesis. We also speculate that this mechanism helps segregate tubular and multivesicular membranes along the recycling and degradation pathways, respectively. PMID:27605702

  19. Auxin deprivation induces a developmental switch in maize somatic embryogenesis involving redistribution of microtubules and actin filaments from endoplasmic to cortical cytoskeletal arrays.

    PubMed

    Samaj, J; Baluska, F; Pretová, A; Volkmann, D

    2003-06-01

    A developmental switch from non-polar pre-embryogenic units to polarized transition units in maize embryogenic callus is caused by auxin deprivation from the culture medium. This switch is accompanied by cytoskeletal rearrangements in embryogenic cells. An immunofluorescence study revealed prominent endoplasmic microtubules and actin filament meshworks radiating from the nuclear surfaces in pre-embryogenic cells growing on medium supplemented with auxin. On the other hand, parallel-organized cortical microtubules and cortical actin filament networks are inherently associated with polarized embryogenic cells of transition units growing on medium without auxin. These results indicate that fine-tuning of the dynamic equilibrium between endoplasmic and cortical cytoskeletal arrays is important for progress in somatic embryogenesis.

  20. 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

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

    SciTech Connect

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

    2003-06-11

    Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin during nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.

  2. A novel form of actin in Leishmania: molecular characterisation, subcellular localisation and association with subpellicular microtubules.

    PubMed

    Sahasrabuddhe, Amogh A; Bajpai, Virendra K; Gupta, Chhitar M

    2004-03-01

    To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 10(6) copies per cell of this protein (M(r) 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network. These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed.

  3. Measuring actin dynamics during phagocytosis using photo-switchable fluorescence

    NASA Astrophysics Data System (ADS)

    Kovari, Daniel T.; Curtis, Jennifer E.

    2013-03-01

    Phagocytosis has traditionally been investigated in terms of the relevant biochemical signaling pathways. However, a growing number of studies investigating the physical aspects of phagocytosis have demonstrated that several distinct forces are exerted throughout particle ingestion. We use variations on FRAP (Fluorescence Recovery After Photobleaching) in combination with photo-switchable fluorescent protein to investigate actin dynamics as a phagocyte attempts to engulf its prey. The goal of our actin studies are to determine the recruitment and polymerization rate of actin in the forming phagosome and whether an organized contractile actin ring is present and responsible for phagosome closure, as proposed in the literature. These experiments are ongoing and contribute to our long term effort of developing a physics based model of phagocytosis.

  4. IKKε inhibits PKC to promote Fascin-dependent actin bundling

    PubMed Central

    Ogura, Yosuke; Misaki, Kazuyo; Maeda, Takuya; Kimpara, Akiyo; Yonemura, Shigenobu; Hayashi, Shigeo

    2016-01-01

    Signaling molecules have pleiotropic functions and are activated by various extracellular stimuli. Protein kinase C (PKC) is activated by diverse receptors, and its dysregulation is associated with diseases including cancer. However, how the undesired activation of PKC is prevented during development remains poorly understood. We have previously shown that a protein kinase, IKKε, is active at the growing bristle tip and regulates actin bundle organization during Drosophila bristle morphogenesis. Here, we demonstrate that IKKε regulates the actin bundle localization of a dynamic actin cross-linker, Fascin. IKKε inhibits PKC, thereby protecting Fascin from inhibitory phosphorylation. Excess PKC activation is responsible for the actin bundle defects in IKKε-deficient bristles, whereas PKC is dispensable for bristle morphogenesis in wild-type bristles, indicating that PKC is repressed by IKKε in wild-type bristle cells. These results suggest that IKKε prevents excess activation of PKC during bristle morphogenesis. PMID:27578797

  5. Early Signaling in Primary T Cells Activated by Antigen Presenting Cells Is Associated with a Deep and Transient Lamellal Actin Network

    PubMed Central

    Roybal, Kole T.; Mace, Emily M.; Mantell, Judith M.; Verkade, Paul; Orange, Jordan S.; Wülfing, Christoph

    2015-01-01

    Cellular signaling transduction critically depends on molecular interactions that are in turn governed by dynamic subcellular distributions of the signaling system components. Comprehensive insight into signal transduction requires an understanding of such distributions and cellular structures driving them. To investigate the activation of primary murine T cells by antigen presenting cells (APC) we have imaged more than 60 signaling intermediates during T cell stimulation with microscopy across resolution limits. A substantial number of signaling intermediates associated with a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell, as characterized in detail here. By mapping the more than 60 signaling intermediates onto the spatiotemporal features of cell biological structures, the lamellum and other ones previously described, we also define distinct spatial and temporal characteristics of T cell signal initiation, amplification, and core signaling in the activation of primary T cells by APCs. These characteristics differ substantially from ones seen when T cells are activated using common reductionist approaches. PMID:26237050

  6. Emergent properties of composite semiflexible biopolymer networks

    PubMed Central

    Jensen, Mikkel H.; Morris, Eliza J.; Goldman, Robert D.; Weitz, David A.

    2014-01-01

    The semiflexible polymers filamentous actin (F-actin) and intermediate filaments (IF) both form complex networks within the cell, and together are key determinants of cellular stiffness. While the mechanics of F-actin networks together with stiff microtubules have been characterized, the interplay between F-actin and IF networks is largely unknown, necessitating the study of composite networks using mixtures of semiflexible biopolymers. We employ bulk rheology in a simplified in vitro system to uncover the fundamental mechanical interactions between networks of the 2 semiflexible polymers, F-actin and vimentin IF. Surprisingly, co-polymerization of actin and vimentin can produce composite networks either stronger or weaker than pure F-actin networks. We show that this effect occurs through steric constraints imposed by IF on F-actin during network formation and filament crosslinking, highlighting novel emergent behavior in composite semiflexible networks. PMID:25759912

  7. Imaging mass spectrometry and MS/MS molecular networking reveals chemical interactions among cuticular bacteria and pathogenic fungi associated with fungus-growing ants.

    PubMed

    Boya P, Cristopher A; Fernández-Marín, Hermógenes; Mejía, Luis C; Spadafora, Carmenza; Dorrestein, Pieter C; Gutiérrez, Marcelino

    2017-07-17

    The fungus-growing ant-microbe symbiosis is an ideal system to study chemistry-based microbial interactions due to the wealth of microbial interactions described, and the lack of information on the molecules involved therein. In this study, we employed a combination of MALDI imaging mass spectrometry (MALDI-IMS) and MS/MS molecular networking to study chemistry-based microbial interactions in this system. MALDI IMS was used to visualize the distribution of antimicrobials at the inhibition zone between bacteria associated to the ant Acromyrmex echinatior and the fungal pathogen Escovopsis sp. MS/MS molecular networking was used for the dereplication of compounds found at the inhibition zones. We identified the antibiotics actinomycins D, X2 and X0β, produced by the bacterium Streptomyces CBR38; and the macrolides elaiophylin, efomycin A and efomycin G, produced by the bacterium Streptomyces CBR53.These metabolites were found at the inhibition zones using MALDI IMS and were identified using MS/MS molecular networking. Additionally, three shearinines D, F, and J produced by the fungal pathogen Escovopsis TZ49 were detected. This is the first report of elaiophylins, actinomycin X0β and shearinines in the fungus-growing ant symbiotic system. These results suggest a secondary prophylactic use of these antibiotics by A. echinatior because of their permanent production by the bacteria.

  8. Actin Skeletons at the Membrane as Liquid Crystal Elastomers

    NASA Astrophysics Data System (ADS)

    Discher, Dennis; Dalhaimer, Paul; Levine, Alex; Lubensky, Tom

    2002-03-01

    Actin filaments crosslinked by proteins such as spectrin form plasma membrane networks in a number of cell-types, including the red blood cell and the outer hair cell of the inner ear. Actin filaments are stiff compared to spectrin and can be considered hard rods. We statistically simulate network phase behavior at finite temperature by Monte Carlo methods, and explore the effects of spectrin and actin length as well as isotropic and shear stresses. Relative lengths required for a zero pressure nematic phase are determined, for exmaple, and indicate structural requirements for obtaining a 2D anisotropic elastomer. Emerging studies of network elasticity examine the anisotropic state and begin to probe the relevance of hyper-soft modes to hearing.

  9. Actin-mediated motion of meiotic chromosomes

    PubMed Central

    Koszul, R.; Kim, K. P.; Prentiss, M.; Kleckner, N.; Kameoka, S.

    2008-01-01

    Summary Chromosome movement is prominent during meiosis. Here, using a combination of in vitro and in vivo approaches, we elucidate the basis for dynamic mid-prophase chromosome movement in budding yeast. Diverse finding reveal a process in which, at the pachytene stage, individual telomere/nuclear envelope (NE) ensembles attach passively to, and then move in concert with, nucleus-hugging actin cables that are continuous with the global cytoskeletal actin network. Other chromosomes move in concert with lead chromosome(s). The same process, in modulated form, explains the zygotene "bouquet" configuration in which, immediately preceding pachytene, chromosome ends colocalize dynamically in a restricted region of the NE. Mechanical properties of the system and biological roles of mid-prophase movement for meiosis, including recombination, are discussed. PMID:18585353

  10. Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP.

    PubMed

    Frieden, C; Patane, K

    1985-07-16

    The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.

  11. CRMP-5 interacts with actin to regulate neurite outgrowth

    PubMed Central

    GONG, XIAOBING; TAN, MINGHUI; GAO, YUAN; CHEN, KEEN; GUO, GUOQING

    2016-01-01

    CRMP family proteins (CRMPs) are abundantly expressed in the developing nervous system mediating growth cone guidance, neuronal polarity and axon elongation. CRMP-5 has been indicated to serve a critical role in neurite outgrowth. However, the detailed mechanisms of how CRMP-5 regulates neurite outgrowth remain unclear. In the current study, co-immunoprecipitation was used to identify the fact that CRMP-5 interacted with the actin and tubulin cytoskeleton networks in the growth cones of developing hippocampal neurons. CRMP-5 exhibited increased affinity towards actin when compared with microtubules. Immunocytochemistry was used to identify the fact that CRMP-5 colocalized with actin predominantly in the C-domain and T-zone in growth cones. In addition, genetic inhibition of CRMP-5 by siRNA suppressed the expression of actin, growth cone development and neurite outgrowth. Overexpression of CRMP-5 promoted the interaction with actin, growth cone development and hippocampal neurite outgrowth. Taken together, these data suggest that CRMP-5 is able to interact with the actin cytoskeleton network in the growth cone and affect growth cone development and neurite outgrowth via this interaction in developing hippocampal neurons. PMID:26677106

  12. Viscoelastic properties of actin-coated membranes.

    PubMed

    Helfer, E; Harlepp, S; Bourdieu, L; Robert, J; MacKintosh, F C; Chatenay, D

    2001-02-01

    In living cells, cytoskeletal filaments interact with the plasma membrane to form structures that play a key role in cell shape and mechanical properties. To study the interaction between these basic components, we designed an in vitro self-assembled network of actin filaments attached to the outer surface of giant unilamellar vesicles. Optical tweezers and single-particle tracking experiments are used to study the rich dynamics of these actin-coated membranes (ACM). We show that microrheology studies can be carried out on such an individual microscopic object. The principle of the experiment consists in measuring the thermally excited position fluctuations of a probe bead attached biochemically to the membrane. We propose a model that relates the power spectrum of these thermal fluctuations to the viscoelastic properties of the membrane. The presence of the actin network modifies strongly the membrane dynamics with respect to a fluid, lipid bilayer one. It induces first a finite (omega=0) two-dimensional (2D) shear modulus G(0)(2D) approximately 0.5 to 5 microN/m in the membrane plane. Moreover, the frequency dependence at high frequency of the shear modulus [G(')(2D)(f ) approximately f(0.85+/-0.07)] and of the bending modulus (kappa(ACM)(f) approximately f(0.55+/-0.21)) demonstrate the viscoelastic behavior of the composite membrane. These results are consistent with a common exponent of 0.75 for both moduli as expected from our model and from prior measurements on actin solutions.

  13. A simple approach to controllably grow network-like branched single-crystalline Si 3N 4 nanostructures

    NASA Astrophysics Data System (ADS)

    Zhu, Na; Peng, Zhijian; Fu, Xiuli; Wang, Chengbiao; Fu, Zhiqiang; Qi, Longhao; Miao, Hezhuo

    2010-07-01

    We reported a simple, large-scale, and controllable growth method for network-like branched single-crystalline Si 3N 4 nanostructures by catalyst-assisted pyrolysis of a polysilazane. The templates were a silicon wafer deposited with a 5 nm Fe film. The processes simply involved in thermal cross-linking of the polymer precursor, crushing of the solidified preceramic polymer chunks into fine powder, and thermal pyrolysis of the powder under the protection of ultra-high purity nitrogen. The collected white network-like branched nanostructures were formed through "metal-absorption on the surface of nanostructures" model by vapor-liquid-solid mechanism. Microstructure characterizations indicate that the nanostructures are single-crystalline hexagonal α-Si 3N 4. The reaction mechanism of Si 3N 4 nanonetworks was also proposed.

  14. Overlapping and distinct functions for cofilin, coronin and Aip1 in actin dynamics in vivo

    PubMed Central

    Lin, Meng-Chi; Galletta, Brian J.; Sept, David; Cooper, John A.

    2010-01-01

    Actin-filament disassembly is crucial for actin-based motility, to control filament network architecture and to regenerate subunits for assembly. Here, we examined the roles of three actin cytoskeletal proteins, coronin, cofilin and Aip1, which have been suggested to combine in various ways to control actin dynamics by promoting or regulating disassembly. We studied their functions during the endocytosis process in budding yeast, where actin-filament dynamics at the cortical actin ‘patch’ contribute to the formation and movement of endocytic vesicles. We found that all three proteins were recruited during the late phase of the life of the actin patch. They all arrived at the same time, when actin and other actin-associated proteins were leaving the patch. Cofilin point mutations influenced the localization of coronin and Aip1, but the complete loss of coronin had no effect on localization of cofilin or Aip1. Using quantitative patch motion analysis and comparing mutant alleles, the phenotypes for mutations of the three genes showed some commonalities, but also some striking differences. Cofilin was clearly the most important; it displayed the most severe mutant phenotypes affecting actin-patch assembly and movement. Together, the results suggest that all three proteins work together to promote actin disassembly, but not in a simple way, and not with equal importance. PMID:20332110

  15. Physical Model for Self-Organization of Actin Cytoskeleton and Adhesion Complexes at the Cell Front

    PubMed Central

    Shemesh, Tom; Bershadsky, Alexander D.; Kozlov, Michael M.

    2012-01-01

    Cell motion is driven by interplay between the actin cytoskeleton and the cell adhesions in the front part of the cell. The actin network segregates into lamellipodium and lamellum, whereas the adhesion complexes are characteristically distributed underneath the actin system. Here, we suggest a computational model for this characteristic organization of the actin-adhesion system. The model is based on the ability of the adhesion complexes to sense mechanical forces, the stick-slip character of the interaction between the adhesions and the moving actin network, and a hypothetical propensity of the actin network to disintegrate upon sufficiently strong stretching stresses. We identify numerically three possible types of system organization, all observed in living cells: two states in which the actin network exhibits segregation into lamellipodium and lamellum, whereas the cell edge either remains stationary or moves, and a state where the actin network does not undergo segregation. The model recovers the asynchronous fluctuations and outward bulging of the cell edge, and the dependence of the edge protrusion velocity on the rate of the nascent adhesion generation, the membrane tension, and the substrate rigidity. PMID:22768930

  16. The Cortical Localization of the Microtubule Orientation Protein, Kar9p, Is Dependent upon Actin and Proteins Required for Polarization

    PubMed Central

    Miller, Rita K.; Matheos, Dina; Rose, Mark D.

    1999-01-01

    In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)– Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Δ, pea2Δ, bud6Δ, and bni1Δ, required for normal polarization and actin cytoskeleton functions and, of these, bni1Δ affected Kar9p localization most severely. Like kar9Δ, bni1Δ mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Δ, the bni1Δ mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Δ bni1Δ double mutants suggested that Kar9p retained some function in bni1Δ mitotic cells. Unlike the polarization mutants, kar9Δ shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Δ. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks. PMID:10085294

  17. Cilia assembly: a role for F-actin in IFT recruitment.

    PubMed

    Quarmby, Lynne

    2014-09-08

    Ciliary growth rates are limited by the availability of precursors at the growing tip. A new paper reveals that the early rapid growth of nascent cilia is supported by F-actin-facilitated delivery of IFT proteins to basal bodies.

  18. Structural organization of the actin cytoskeleton at sites of clathrin-mediated endocytosis.

    PubMed

    Collins, Agnieszka; Warrington, Anthony; Taylor, Kenneth A; Svitkina, Tatyana

    2011-07-26

    The dynamic actin cytoskeleton plays an important role in clathrin-mediated endocytosis (CME). However, its exact functions remain uncertain as a result of a lack of high-resolution structural information regarding actin architecture at endocytic sites. Using platinum replica electron microscopy in combination with electron tomography, we found that actin patches associated with clathrin-coated structures (CCSs) in cultured mouse cells consist of a densely branched actin network, in which actin filament barbed ends are oriented toward the CCS. The shape of the actin network varied from a small lateral patch at the periphery of shallow CCSs, to a collar-like arrangement around partly invaginated CCSs with actin filament barbed ends abutting the CCS neck, to a polarized comet tail in association with highly constricted or fully endocytosed CCSs. Our data suggest that the primary role of the actin cytoskeleton in CME is to constrict and elongate the bud neck and drive the endocytosed vesicles from the plasma membrane. Moreover, in these processes, barbed ends directly push onto the load, as in a conventional propulsion mechanism. Based on our findings, we propose a model for initiation, evolution, and function of the dendritic actin network at CCSs. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. F-actin reorganization upon de- and rehydration in the aeroterrestrial green alga Klebsormidium crenulatum.

    PubMed

    Blaas, Kathrin; Holzinger, Andreas

    2017-03-21

    Filamentous actin (F-actin) is a dynamic network involved in many cellular processes like cell division and cytoplasmic streaming. While many studies have addressed the involvement of F-actin in different cellular processes in cultured cells, little is known on the reactions to environmental stress scenarios, where this system might have essential regulatory functions. We investigated here the de- and rehydration kinetics of breakdown and reassembly of F-actin in the streptophyte green alga Klebsormidium crenulatum. Measurements of the chlorophyll fluorescence (effective quantum yield of photosystem II [ΔF/Fm']) via pulse amplitude modulation were performed as a measure for dehydration induced shut down of physiological activity, which ceased after 141±15min at ∼84% RH. We hypothesized that there is a link between this physiological parameter and the status of the F-actin system. Indeed, 20min of dehydration (ΔF/Fm'=0) leads to a breakdown of the fine cortical F-actin network as visualized by Atto 488 phalloidin staining, and dot-like structures remained. Already 10min after rehydration a beginning reassembly of F-actin is observed, after 25min the F-actin network appeared similar to untreated controls, indicating a full recovery. These results demonstrate the fast kinetics of F-actin dis- and reassembly likely contributing to cellular reorganization upon rehydration.

  20. Formins: Actin nucleators that regulate cytoskeletal dynamics during spermatogenesis

    PubMed Central

    Li, Nan; Mruk, Dolores D; Tang, Elizabeth I; Wong, Chris KC; Lee, Will M; Silvestrini, Bruno; Cheng, C Yan

    2015-01-01

    Formins are a growing class of actin nucleation proteins that promote the polymerization of actin microfilaments, forming long stretches of actin microfilaments to confer actin filament bundling in mammalian cells. As such, microfilament bundles can be formed in specific cellular domains, in particular in motile mammalian cells, such as filopodia. Since ectoplasmic specialization (ES), a testis-specific adherens junction (AJ), at the Sertoli cell-cell and Sertoli-spermatid interface is constituted by arrays of actin microfilament bundles, it is likely that formins are playing a significant physiological role on the homeostasis of ES during the epithelial cycle of spermatogenesis. In this Commentary, we provide a timely discussion on formin 1 which was recently shown to be a crucial regulator of actin microfilaments at the ES in the rat testis (Li N et al. Endocrinology, 2015, in press; DOI: 10.1210/en.2015-1161, PMID:25901598). We also highlight research that is needed to unravel the functional significance of formins in spermatogenesis. PMID:26413414

  1. Formins: Actin nucleators that regulate cytoskeletal dynamics during spermatogenesis.

    PubMed

    Li, Nan; Mruk, Dolores D; Tang, Elizabeth I; Wong, Chris Kc; Lee, Will M; Silvestrini, Bruno; Cheng, C Yan

    2015-01-01

    Formins are a growing class of actin nucleation proteins that promote the polymerization of actin microfilaments, forming long stretches of actin microfilaments to confer actin filament bundling in mammalian cells. As such, microfilament bundles can be formed in specific cellular domains, in particular in motile mammalian cells, such as filopodia. Since ectoplasmic specialization (ES), a testis-specific adherens junction (AJ), at the Sertoli cell-cell and Sertoli-spermatid interface is constituted by arrays of actin microfilament bundles, it is likely that formins are playing a significant physiological role on the homeostasis of ES during the epithelial cycle of spermatogenesis. In this Commentary, we provide a timely discussion on formin 1 which was recently shown to be a crucial regulator of actin microfilaments at the ES in the rat testis (Li N et al. Endocrinology, 2015, in press; DOI: 10.1210/en.2015-1161, PMID:25901598). We also highlight research that is needed to unravel the functional significance of formins in spermatogenesis.

  2. Suppressors of Yeast Actin Mutations

    PubMed Central

    Novick, P.; Osmond, B. C.; Botstein, D.

    1989-01-01

    Suppressors of a temperature-sensitive mutation (act1-1) in the single actin gene of Saccharomyces cerevisiae were selected that had simultaneously acquired a cold-sensitive growth phenotype. Five genes, called SAC (suppressor of actin) were defined by complementation tests; both suppression and cold-sensitive phenotypes were recessive. Three of the genes (SAC1, SAC2 and SAC3) were subjected to extensive genetic and phenotypic analysis, including molecular cloning. Suppression was found to be allele-specific with respect to actin alleles. The sac mutants, even in ACT1(+) genetic backgrounds, displayed phenotypes similar to those of actin mutants, notably aberrant organization of intracellular actin and deposition of chitin at the cell surface. These results are interpreted as being consistent with the idea that the SAC genes encode proteins that interact with actin, presumably as components or controllers of the assembly or stability of the yeast actin cytoskeleton. Two unexpected properties of the SAC1 gene were noted. Disruptions of the gene indicated that its function is essential only at temperatures below about 17° and all sac1 alleles are inviable when combined with act1-2. These properties are interpreted in the context of the evolution of the actin cytoskeleton of yeast. PMID:2656401

  3. Ring closure in actin polymers

    NASA Astrophysics Data System (ADS)

    Sinha, Supurna; Chattopadhyay, Sebanti

    2017-03-01

    We present an analysis for the ring closure probability of semiflexible polymers within the pure bend Worm Like Chain (WLC) model. The ring closure probability predicted from our analysis can be tested against fluorescent actin cyclization experiments. We also discuss the effect of ring closure on bend angle fluctuations in actin polymers.

  4. Nuclear actin depolymerization in transcriptionally active avian and amphibian oocytes leads to collapse of intranuclear structures

    PubMed Central

    Maslova, Antonina; Krasikova, Alla

    2012-01-01

    Actin, which is normally depleted in the nuclei of somatic cells, accumulates in high amounts in giant nuclei of amphibian oocytes. The supramolecular organization and functions of this nuclear pool of actin in growing vertebrate oocyte are controversial. Here, we investigated the role of nuclear actin in the maintenance of the spatial architecture of intranuclear structures in avian and amphibian growing oocytes. A meshwork of filamentous actin was not detected in freshly isolated or fixed oocyte nuclei of Xenopus, chicken or quail. We found that the actin meshwork inside the oocyte nucleus could be induced by phalloidin treatment. Actin polymerization is demonstrated to be required to stabilize the specific spatial organization of nuclear structures in avian and amphibian growing oocytes. In experiments with the actin depolymerizing drugs cytochalasin D and latrunculin A, we showed that disassembly of nuclear actin polymers led to chromosome condensation and their transportation to a limited space within the oocyte nucleus. Experimentally induced “collapsing” of chromosomes and nuclear bodies, together with global inhibition of transcription, strongly resembled the process of karyosphere formation during oocyte growth. PMID:22572951

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

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders

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

  6. Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast.

    PubMed

    Skau, Colleen T; Courson, David S; Bestul, Andrew J; Winkelman, Jonathan D; Rock, Ronald S; Sirotkin, Vladimir; Kovar, David R

    2011-07-29

    Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.

  7. Quantitative Analysis of F-Actin Redistribution in Astrocytoma Cells Treated with Candidate Pharmaceuticals

    PubMed Central

    Lockett, Stephen; Verma, Chrissie; Brafman, Alla; Gudla, Prabhakar; Nandy, Kaustav; Mimaki, Yoshihiro; Fuchs, Philip L.; Jaja, Joseph; Reilly, Karlyne M.; Beutler, John; Turbyville, Thomas J.

    2015-01-01

    Actin fibers (F-actin) control the shape and internal organization of cells, and generate force. It has been long appreciated that these functions are tightly coupled, and in some cases drive cell behavior and cell fate. The distribution and dynamics of F-actin is different in cancer versus normal cells and in response to small molecules, including actin-targeting natural products and anticancer drugs. Therefore, quantifying actin structural changes from high resolution fluorescence micrographs is necessary for further understanding actin cytoskeleton dynamics and phenotypic consequences of drug interactions on cells. We applied an artificial neural network algorithm, which used image intensity and anisotropy measurements, to quantitatively classify F-actin subcellular features into actin along the edges of cells, actin at the protrusions of cells, internal fibers and punctate signals. The algorithm measured significant increase in F-actin at cell edges with concomitant decrease in internal punctate actin in astrocytoma cells lacking functional neurofibromin and p53 when treated with three structurally-distinct anticancer small molecules: OSW1, Schweinfurthin A (SA) and a synthetic marine compound 23′-dehydroxycephalostatin 1. Distinctly different changes were measured in cells treated with the actin inhibitor cytochalasin B. These measurements support published reports that SA acts on F-actin in NF1−/− neurofibromin deficient cancer cells through changes in Rho signaling. Quantitative pattern analysis of cells has wide applications for understanding mechanisms of small molecules, because many anticancer drugs directly or indirectly target cytoskeletal proteins. Furthermore, quantitative information about the actin cytoskeleton may make it possible to further understand cell fate decisions using mathematically testable models. Published 2014 Wiley Periodicals Inc.† PMID:24515854

  8. Quantitative analysis of F-actin redistribution in astrocytoma cells treated with candidate pharmaceuticals.

    PubMed

    Lockett, Stephen; Verma, Chrissie; Brafman, Alla; Gudla, Prabhakar; Nandy, Kaustav; Mimaki, Yoshihiro; Fuchs, Philip L; Jaja, Joseph; Reilly, Karlyne M; Beutler, John; Turbyville, Thomas J

    2014-06-01

    Actin fibers (F-actin) control the shape and internal organization of cells, and generate force. It has been long appreciated that these functions are tightly coupled, and in some cases drive cell behavior and cell fate. The distribution and dynamics of F-actin is different in cancer versus normal cells and in response to small molecules, including actin-targeting natural products and anticancer drugs. Therefore, quantifying actin structural changes from high resolution fluorescence micrographs is necessary for further understanding actin cytoskeleton dynamics and phenotypic consequences of drug interactions on cells. We applied an artificial neural network algorithm, which used image intensity and anisotropy measurements, to quantitatively classify F-actin subcellular features into actin along the edges of cells, actin at the protrusions of cells, internal fibers and punctate signals. The algorithm measured significant increase in F-actin at cell edges with concomitant decrease in internal punctate actin in astrocytoma cells lacking functional neurofibromin and p53 when treated with three structurally-distinct anticancer small molecules: OSW1, Schweinfurthin A (SA) and a synthetic marine compound 23'-dehydroxycephalostatin 1. Distinctly different changes were measured in cells treated with the actin inhibitor cytochalasin B. These measurements support published reports that SA acts on F-actin in NF1(-/-) neurofibromin deficient cancer cells through changes in Rho signaling. Quantitative pattern analysis of cells has wide applications for understanding mechanisms of small molecules, because many anti-cancer drugs directly or indirectly target cytoskeletal proteins. Furthermore, quantitative information about the actin cytoskeleton may make it possible to further understand cell fate decisions using mathematically testable models. Published 2014 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of

  9. Actin binding proteins and spermiogenesis

    PubMed Central

    Mruk, Dolores D

    2011-01-01

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

  10. ER sheet persistence is coupled to myosin 1c-regulated dynamic actin filament arrays.

    PubMed

    Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M; Lowe, Martin; Vartiainen, Maria K; Jokitalo, Eija

    2014-04-01

    The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.

  11. Transport along freely suspended actin cortex models in a controlled microfluidic environment

    NASA Astrophysics Data System (ADS)

    Schulz, Simon; Haraszti, Tamas; Roos, Wouter; Schmitz, Christian; Ulmer, Jens; Graeter, Stefan; Spatz, Joachim P.

    2006-03-01

    Arrays of microfabricated pillars are constructed to serve as a template for mimicking the actin cortex of cells. The three-dimensional template surface prevents interaction of the actin filaments hanging between pillars. A special flow-cell design enables applying flow around a network of actin freely suspended between polydimethylsiloxane pillars. This opens new possibilities to study the mechanics of two-dimensional actin networks as a function of actin-crosslinkers, to observe the active diffusion of molecular motors operating on pending networks and to investigate the alternations in the transport of microscopic particles, coated by different proteins and molecular motors, along these actin cortex models under the drag of flow. The stiffness of the F-actin can be tuned by bundling through various cross-linkers. Additionally, actin filaments act as tracks for guiding passive and active transport of cargo such as organelles or microspheres by molecular motors like myosin-V. These transport problems are biomimetic studies of tracks and external driving force on a statistical process of two-dimensional networks isolated from the complicated and undetermined cellular environment.

  12. ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays

    PubMed Central

    Joensuu, Merja; Belevich, Ilya; Rämö, Olli; Nevzorov, Ilya; Vihinen, Helena; Puhka, Maija; Witkos, Tomasz M.; Lowe, Martin; Vartiainen, Maria K.; Jokitalo, Eija

    2014-01-01

    The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network. PMID:24523293

  13. F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex

    PubMed Central

    Murrell, Michael P.; Gardel, Margaret L.

    2012-01-01

    Here we develop a minimal model of the cell actomyosin cortex by forming a quasi-2D cross-linked filamentous actin (F-actin) network adhered to a model cell membrane and contracted by myosin thick filaments. Myosin motors generate both compressive and tensile stresses on F-actin and consequently induce large bending fluctuations, which reduces their effective persistence length to <1 μm. Over a large range of conditions, we show the extent of network contraction corresponds exactly to the extent of individual F-actin shortening via buckling. This demonstrates an essential role of buckling in breaking the symmetry between tensile and compressive stresses to facilitate mesoscale network contraction of up to 80% strain. Portions of buckled F-actin with a radius of curvature ∼300 nm are prone to severing and thus compressive stresses mechanically coordinate contractility with F-actin severing, the initial step of F-actin turnover. Finally, the F-actin curvature acquired by myosin-induced stresses can be further constrained by adhesion of the network to a membrane, accelerating filament severing but inhibiting the long-range transmission of the stresses necessary for network contractility. Thus, the extent of membrane adhesion can regulate the coupling between network contraction and F-actin severing. These data demonstrate the essential role of the nonlinear response of F-actin to compressive stresses in potentiating both myosin-mediated contractility and filament severing. This may serve as a general mechanism to mechanically coordinate contractility and cortical dynamics across diverse actomyosin assemblies in smooth muscle and nonmuscle cells. PMID:23213249

  14. Bacterial Actins and Their Interactors.

    PubMed

    Gayathri, Pananghat

    2017-01-01

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

  15. Novel actin depolymerizing macrolide aplyronine A.

    PubMed

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

    1996-09-01

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

  16. 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.

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

    PubMed Central

    Appaduray, Mark A.; Masedunskas, Andrius; Lucas, Christine A.; Warren, Sean C.; Timpson, Paul; Stear, Jeffrey H.

    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. PMID:27977753

  18. Cortactin Localization to Sites of Actin Assembly in Lamellipodia Requires Interactions with F-Actin and the Arp2/3 Complex

    PubMed Central

    Weed, Scott A.; Karginov, Andrei V.; Schafer, Dorothy A.; Weaver, Alissa M.; Kinley, Andrew W.; Cooper, John A.; Parsons, J. Thomas

    2000-01-01

    Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA failed to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region were inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain–containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes. PMID:11018051

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

    PubMed

    Stevenson, V A; Theurkauf, W E

    2000-10-05

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

  20. Growing a professional network to over 3000 members in less than 4 years: evaluation of InspireNet, British Columbia's virtual nursing health services research network.

    PubMed

    Frisch, Noreen; Atherton, Pat; Borycki, Elizabeth; Mickelson, Grace; Cordeiro, Jennifer; Novak Lauscher, Helen; Black, Agnes

    2014-02-21

    Use of Web 2.0 and social media technologies has become a new area of research among health professionals. Much of this work has focused on the use of technologies for health self-management and the ways technologies support communication between care providers and consumers. This paper addresses a new use of technology in providing a platform for health professionals to support professional development, increase knowledge utilization, and promote formal/informal professional communication. Specifically, we report on factors necessary to attract and sustain health professionals' use of a network designed to increase nurses' interest in and use of health services research and to support knowledge utilization activities in British Columbia, Canada. "InspireNet", a virtual professional network for health professionals, is a living laboratory permitting documentation of when and how professionals take up Web 2.0 and social media. Ongoing evaluation documents our experiences in establishing, operating, and evaluating this network. Overall evaluation methods included (1) tracking website use, (2) conducting two member surveys, and (3) soliciting member feedback through focus groups and interviews with those who participated in electronic communities of practice (eCoPs) and other stakeholders. These data have been used to learn about the types of support that seem relevant to network growth. Network growth exceeded all expectations. Members engaged with varying aspects of the network's virtual technologies, such as teams of professionals sharing a common interest, research teams conducting their work, and instructional webinars open to network members. Members used wikis, blogs, and discussion groups to support professional work, as well as a members' database with contact information and areas of interest. The database is accessed approximately 10 times per day. InspireNet public blog posts are accessed roughly 500 times each. At the time of writing, 21 research teams

  1. 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.

  2. Formaldehyde fixation is detrimental to actin cables in glucose-depleted S. cerevisiae cells

    PubMed Central

    Vasicova, Pavla; Rinnerthaler, Mark; Haskova, Danusa; Novakova, Lenka; Malcova, Ivana; Breitenbach, Michael; Hasek, Jiri

    2016-01-01

    Actin filaments form cortical patches and emanating cables in fermenting cells of Saccharomyces cerevisiae. This pattern has been shown to be depolarized in glucose-depleted cells after formaldehyde fixation and staining with rhodamine-tagged phalloidin. Loss of actin cables in mother cells was remarkable. Here we extend our knowledge on actin in live glucose-depleted cells co-expressing the marker of actin patches (Abp1-RFP) with the marker of actin cables (Abp140-GFP). Glucose depletion resulted in appearance of actin patches also in mother cells. However, even after 80 min of glucose deprivation these cells showed a clear network of actin cables labeled with Abp140-GFP in contrast to previously published data. In live cells with a mitochondrial dysfunction (rho0 cells), glucose depletion resulted in almost immediate appearance of Abp140-GFP foci partially overlapping with Abp1-RFP patches in mother cells. Residual actin cables were clustered in patch-associated bundles. A similar overlapping “patchy” pattern of both actin markers was observed upon treatment of glucose-deprived rho+ cells with FCCP (the inhibitor of oxidative phosphorylation) and upon treatment with formaldehyde. While the formaldehyde-targeted process stays unknown, our results indicate that published data on yeast actin cytoskeleton obtained from glucose-depleted cells after fixation should be considered with caution.

  3. 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

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

    PubMed Central

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

    2017-01-01

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

  5. Actin dynamics and cofilin-actin rods in Alzheimer disease

    PubMed Central

    Bamburg, James R.; Bernstein, Barbara W.

    2017-01-01

    Cytoskeletal abnormalities and synaptic loss, typical of both familial and sporadic Alzheimer disease (AD), are induced by diverse stresses such as neuroinflammation, oxidative stress, and energetic stress, each of which may be initiated or enhanced by proinflammatory cytokines or amyloid-β (Aβ) peptides. Extracellular Aβ-containing plaques and intracellular phospho-tau-containing neurofibrillary tangles are postmortem pathologies required to confirm AD and have been the focus of most studies. However, AD brain, but not normal brain, also have increased levels of cytoplasmic rod-shaped bundles of filaments composed of ADF/cofilin-actin in a 1:1 complex (rods). Cofilin, the major ADF/cofilin isoform in mammalian neurons, severs actin filaments at low cofilin/actin ratios and stabilizes filaments at high cofilin/actin ratios. It binds cooperatively to ADP-actin subunits in F-actin. Cofilin is activated by dephosphorylation and may be oxidized in stressed neurons to form disulfide-linked dimers, required for bundling cofilin-actin filaments into stable rods. Rods form within neurites causing synaptic dysfunction by sequestering cofilin, disrupting normal actin dynamics, blocking transport, and exacerbating mitochondrial membrane potential loss. Aβ and proinflammatory cytokines induce rods through a cellular prion protein-dependent activation of NADPH oxidase and production of reactive oxygen species. Here we review recent advances in our understanding of cofilin biochemistry, rod formation, and the development of cognitive deficits. We will then discuss rod formation as a molecular pathway for synapse loss that may be common between all three prominent current AD hypotheses, thus making rods an attractive therapeutic target. PMID:26873625

  6. Reactive Oxygen Species Regulate Protrusion Efficiency by Controlling Actin Dynamics

    PubMed Central

    Taulet, Nicolas; Delorme-Walker, Violaine D.; DerMardirossian, Céline

    2012-01-01

    Productive protrusions allowing motile cells to sense and migrate toward a chemotactic gradient of reactive oxygen species (ROS) require a tight control of the actin cytoskeleton. However, the mechanisms of how ROS affect cell protrusion and actin dynamics are not well elucidated yet. We show here that ROS induce the formation of a persistent protrusion. In migrating epithelial cells, protrusion of the leading edge requires the precise regulation of the lamellipodium and lamella F-actin networks. Using fluorescent speckle microscopy, we showed that, upon ROS stimulation, the F-actin retrograde flow is enhanced in the lamellipodium. This event coincides with an increase of cofilin activity, free barbed ends formation, Arp2/3 recruitment, and ERK activity at the cell edge. In addition, we observed an acceleration of the F-actin flow in the lamella of ROS-stimulated cells, which correlates with an enhancement of the cell contractility. Thus, this study demonstrates that ROS modulate both the lamellipodium and the lamella networks to control protrusion efficiency. PMID:22876286

  7. Direct interaction of microtubule- and actin-based transport motors

    NASA Technical Reports Server (NTRS)

    Huang, J. D.; Brady, S. T.; Richards, B. W.; Stenolen, D.; Resau, J. H.; Copeland, N. G.; Jenkins, N. A.

    1999-01-01

    The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.

  8. Direct interaction of microtubule- and actin-based transport motors

    NASA Technical Reports Server (NTRS)

    Huang, J. D.; Brady, S. T.; Richards, B. W.; Stenolen, D.; Resau, J. H.; Copeland, N. G.; Jenkins, N. A.

    1999-01-01

    The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood. For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport. In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination. Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU. As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell. These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules.

  9. Growing a Professional Network to Over 3000 Members in Less Than 4 Years: Evaluation of InspireNet, British Columbia’s Virtual Nursing Health Services Research Network

    PubMed Central

    Atherton, Pat; Borycki, Elizabeth; Mickelson, Grace; Cordeiro, Jennifer; Novak Lauscher, Helen; Black, Agnes

    2014-01-01

    Background Use of Web 2.0 and social media technologies has become a new area of research among health professionals. Much of this work has focused on the use of technologies for health self-management and the ways technologies support communication between care providers and consumers. This paper addresses a new use of technology in providing a platform for health professionals to support professional development, increase knowledge utilization, and promote formal/informal professional communication. Specifically, we report on factors necessary to attract and sustain health professionals’ use of a network designed to increase nurses’ interest in and use of health services research and to support knowledge utilization activities in British Columbia, Canada. Objective “InspireNet”, a virtual professional network for health professionals, is a living laboratory permitting documentation of when and how professionals take up Web 2.0 and social media. Ongoing evaluation documents our experiences in establishing, operating, and evaluating this network. Methods Overall evaluation methods included (1) tracking website use, (2) conducting two member surveys, and (3) soliciting member feedback through focus groups and interviews with those who participated in electronic communities of practice (eCoPs) and other stakeholders. These data have been used to learn about the types of support that seem relevant to network growth. Results Network growth exceeded all expectations. Members engaged with varying aspects of the network’s virtual technologies, such as teams of professionals sharing a common interest, research teams conducting their work, and instructional webinars open to network members. Members used wikis, blogs, and discussion groups to support professional work, as well as a members’ database with contact information and areas of interest. The database is accessed approximately 10 times per day. InspireNet public blog posts are accessed roughly 500 times

  10. Dual pools of actin at presynaptic terminals.

    PubMed

    Bleckert, Adam; Photowala, Huzefa; Alford, Simon

    2012-06-01

    We investigated actin's function in vesicle recycling and exocytosis at lamprey synapses and show that FM1-43 puncta and phalloidin-labeled filamentous actin (F-actin) structures are colocalized, yet recycling vesicles are not contained within F-actin clusters. Additionally, phalloidin also labels a plasma membrane-associated cortical actin. Injection of fluorescent G-actin revealed activity-independent dynamic actin incorporation into presynaptic synaptic vesicle clusters but not into cortical actin. Latrunculin-A, which sequesters G-actin, dispersed vesicle-associated actin structures and prevented subsequent labeled G-actin and phalloidin accumulation at presynaptic puncta, yet cortical phalloidin labeling persisted. Dispersal of presynaptic F-actin structures by latrunculin-A did not disrupt vesicle clustering or recycling or alter the amplitude or kinetics of excitatory postsynaptic currents (EPSCs). However, it slightly enhanced release during repetitive stimulation. While dispersal of presynaptic actin puncta with latrunculin-A failed to disperse synaptic vesicles or inhibit synaptic transmission, presynaptic phalloidin injection blocked exocytosis and reduced endocytosis measured by action potential-evoked FM1-43 staining. Furthermore, phalloidin stabilization of only cortical actin following pretreatment with latrunculin-A was sufficient to inhibit synaptic transmission. Conversely, treatment of axons with jasplakinolide, which induces F-actin accumulation but disrupts F-actin structures in vivo, resulted in increased synaptic transmission accompanied by a loss of phalloidin labeling of cortical actin but no loss of actin labeling within vesicle clusters. Marked synaptic deficits seen with phalloidin stabilization of cortical F-actin, in contrast to the minimal effects of disruption of a synaptic vesicle-associated F-actin, led us to conclude that two structurally and functionally distinct pools of actin exist at presynaptic sites.

  11. [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.

  12. Thermal unfolding and aggregation of actin.

    PubMed

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

    2008-09-01

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

  13. Feeling for Filaments: Quantification of the Cortical Actin Web in Live Vascular Endothelium

    PubMed Central

    Kronlage, Cornelius; Schäfer-Herte, Marco; Böning, Daniel; Oberleithner, Hans; Fels, Johannes

    2015-01-01

    Contact-mode atomic force microscopy (AFM) has been shown to reveal cortical actin structures. Using live endothelial cells, we visualized cortical actin dynamics simultaneously by AFM and confocal fluorescence microscopy. We present a method that quantifies dynamic changes in the mechanical ultrastructure of the cortical actin web. We argue that the commonly used, so-called error signal imaging in AFM allows a qualitative, but not quantitative, analysis of cortical actin dynamics. The approach we used comprises fast force-curve-based topography imaging and subsequent image processing that enhances local height differences. Dynamic changes in the organization of the cytoskeleton network can be observed and quantified by surface roughness calculations and automated morphometrics. Upon treatment with low concentrations of the actin-destabilizing agent cytochalasin D, the cortical cytoskeleton network is thinned out and the average mesh size increases. In contrast, jasplakinolide, a drug that enhances actin polymerization, consolidates the cytoskeleton network and reduces the average mesh area. In conclusion, cortical actin dynamics can be quantified in live cells. To our knowledge, this opens a new pathway for conducting quantitative structure-function analyses of the endothelial actin web just beneath the apical plasma membrane. PMID:26287621

  14. Distribution of actin of the human erythrocyte membrane cytoskeleton after interaction with radiographic contrast media.

    PubMed

    Franke, R P; Scharnweber, T; Fuhrmann, R; Krüger, A; Wenzel, F; Mrowietz, C; Jung, F

    2013-01-01

    A type-dependent chemotoxic effect of radiographic contrast media on erythrocytes and endothelial cells was reported several times. While mechanisms of toxicity are still unclear the cellular reactions e.g. echinocyte formation in erythrocytes and the buckling of endothelial cells coincided with deterioration of capillary perfusion (in patients with coronary artery disease) and tissue oxygen tension (in the myocardium of pigs). Whether the shape changes in erythrocytes coincide with changes in the arrangement of actin, the core of the actin-spectrin cytoskeletal network and possible actor in membrane stresses and deformation is not known until now. To get specific informations actin was stained using two different staining methods (antibodies to β-actin staining oligomeric G-actin and polymeric F-actin and Phalloidin-Rhodamin staining polymeric F-actin only). In addition, an advanced version of confocal laser scanning microscopes was used enabling the display of the actin arrangement near substrate surfaces. Blood smears were produced after erythrocyte suspension in autologous plasma or in two different plasma/RCM mixtures. In this study an even homogenous distribution of fine grained globular actin in the normal human erythrocyte could be demonstrated. After suspension of erythrocytes in a plasma/Iodixanol mixture an increased number of membrane protrusions appeared densely filled with intensely stained actin similar to cells suspended in autologous plasma, however, there in less numbers. Suspension in Iopromide, in contrast, induced a complete reorganization of the cytoskeletal actin: the fine grained globular actin distribution disappeared and only few, long and thick actin filaments bundled and possibly polymerized appeared, instead, shown here for the first time.

  15. Characterization of Ring-Like F-Actin Structure as a Mechanical Partner for Spindle Positioning in Mitosis

    PubMed Central

    Jiang, Hao; Zhu, Tongge; Xia, Peng; Seffens, William; Aikhionbare, Felix; Wang, Dongmei; Dou, Zhen; Yao, Xuebiao

    2014-01-01

    Proper spindle positioning and orientation are essential for accurate mitosis which requires dynamic interactions between microtubule and actin filament (F-actin). Although mounting evidence demonstrates the role of F-actin in cortical cytoskeleton dynamics, it remains elusive as to the structure and function of F-actin-based networks in spindle geometry. Here we showed a ring-like F-actin structure surrounding the mitotic spindle which forms since metaphase and maintains in MG132-arrested metaphase HeLa cells. This cytoplasmic F-actin structure is relatively isotropic and less dynamic. Our computational modeling of spindle position process suggests a possible mechanism by which the ring-like F-actin structure can regulate astral microtubule dynamics and thus mitotic spindle orientation. We further demonstrated that inhibiting Plk1, Mps1 or Myosin, and disruption of microtubules or F-actin polymerization perturbs the formation of the ring-like F-actin structure and alters spindle position and symmetric division. These findings reveal a previously unrecognized but important link between mitotic spindle and ring-like F-actin network in accurate mitosis and enables the development of a method to theoretically illustrate the relationship between mitotic spindle and cytoplasmic F-actin. PMID:25299690

  16. Characterization of ring-like F-actin structure as a mechanical partner for spindle positioning in mitosis.

    PubMed

    Lu, Huan; Zhao, Qun; Jiang, Hao; Zhu, Tongge; Xia, Peng; Seffens, William; Aikhionbare, Felix; Wang, Dongmei; Dou, Zhen; Yao, Xuebiao

    2014-01-01

    Proper spindle positioning and orientation are essential for accurate mitosis which requires dynamic interactions between microtubule and actin filament (F-actin). Although mounting evidence demonstrates the role of F-actin in cortical cytoskeleton dynamics, it remains elusive as to the structure and function of F-actin-based networks in spindle geometry. Here we showed a ring-like F-actin structure surrounding the mitotic spindle which forms since metaphase and maintains in MG132-arrested metaphase HeLa cells. This cytoplasmic F-actin structure is relatively isotropic and less dynamic. Our computational modeling of spindle position process suggests a possible mechanism by which the ring-like F-actin structure can regulate astral microtubule dynamics and thus mitotic spindle orientation. We further demonstrated that inhibiting Plk1, Mps1 or Myosin, and disruption of microtubules or F-actin polymerization perturbs the formation of the ring-like F-actin structure and alters spindle position and symmetric division. These findings reveal a previously unrecognized but important link between mitotic spindle and ring-like F-actin network in accurate mitosis and enables the development of a method to theoretically illustrate the relationship between mitotic spindle and cytoplasmic F-actin.

  17. New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales.

    PubMed

    Yamashiro, Sawako; Mizuno, Hiroaki; Smith, Matthew B; Ryan, Gillian L; Kiuchi, Tai; Vavylonis, Dimitrios; Watanabe, Naoki

    2014-04-01

    Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein-actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8-8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network.

  18. New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales

    PubMed Central

    Yamashiro, Sawako; Mizuno, Hiroaki; Smith, Matthew B.; Ryan, Gillian L.; Kiuchi, Tai; Vavylonis, Dimitrios; Watanabe, Naoki

    2014-01-01

    Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein–actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8–8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network. PMID:24501425

  19. Dynamic network morphology and tension buildup in a 3D model of cytokinetic ring assembly.

    PubMed

    Bidone, Tamara C; Tang, Haosu; Vavylonis, Dimitrios

    2014-12-02

    During fission yeast cytokinesis, actin filaments nucleated by cortical formin Cdc12 are captured by myosin motors bound to a band of cortical nodes and bundled by cross-linking proteins. The myosin motors exert forces on the actin filaments, resulting in a net pulling of the nodes into a contractile ring, while cross-linking interactions help align actin filaments and nodes into a single bundle. We used these mechanisms in a three-dimensional computational model of contractile ring assembly, with semiflexible actin filaments growing from formins at cortical nodes, capturing of filaments by neighboring nodes, and cross-linking among filaments through attractive interactions. The model was used to predict profiles of actin filament density at the cell cortex, morphologies of condensing node-filament networks, and regimes of cortical tension by varying the node pulling force and strength of cross-linking among actin filaments. Results show that cross-linking interactions can lead to confinement of actin filaments at the simulated cortical boundary. We show that the ring-formation region in parameter space lies close to regions leading to clumps, meshworks or double rings, and stars/cables. Since boundaries between regions are not sharp, transient structures that resemble clumps, stars, and meshworks can appear in the process of ring assembly. These results are consistent with prior experiments with mutations in actin-filament turnover regulators, myosin motor activity, and changes in the concentration of cross-linkers that alter the morphology of the condensing network. Transient star shapes appear in some simulations, and these morphologies offer an explanation for star structures observed in prior experimental images. Finally, we quantify tension along actin filaments and forces on nodes during ring assembly and show that the mechanisms describing ring assembly can also drive ring constriction once the ring is formed. Copyright © 2014 Biophysical Society

  20. Computational Tension Mapping of Adherent Cells Based on Actin Imaging

    PubMed Central

    Manifacier, Ian; Milan, Jean-Louis; Jeanneau, Charlotte; Chmilewsky, Fanny; Chabrand, Patrick; About, Imad

    2016-01-01

    Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension. PMID:26812601

  1. Computational Tension Mapping of Adherent Cells Based on Actin Imaging.

    PubMed

    Manifacier, Ian; Milan, Jean-Louis; Jeanneau, Charlotte; Chmilewsky, Fanny; Chabrand, Patrick; About, Imad

    2016-01-01

    Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension.

  2. Soft viscoelastic properties of nuclear actin age oocytes due to gravitational creep.

    PubMed

    Feric, Marina; Broedersz, Chase P; Brangwynne, Clifford P

    2015-11-18

    The actin cytoskeleton helps maintain structural organization within living cells. In large X. laevis oocytes, gravity becomes a dominant force and is countered by a nuclear actin network that prevents liquid-like nuclear bodies from immediate sedimentation and coalescence. However, nuclear actin's mechanical properties, and how they facilitate the stabilization of nuclear bodies, remain unknown. Using active microrheology, we find that nuclear actin forms a weak viscoelastic network, with a modulus of roughly 0.1 Pa. Embedded probe particles subjected to a constant force exhibit continuous displacement, due to viscoelastic creep. Gravitational forces also cause creep displacement of nuclear bodies, resulting in their asymmetric nuclear distribution. Thus, nuclear actin does not indefinitely support the emulsion of nuclear bodies, but only kinetically stabilizes them by slowing down gravitational creep to ~2 months. This is similar to the viability time of large oocytes, suggesting gravitational creep ages oocytes, with fatal consequences on long timescales.

  3. Rho GTPases, phosphoinositides, and actin

    PubMed Central

    Croisé, Pauline; Estay-Ahumada, Catherine; Gasman, Stéphane; Ory, Stéphane

    2014-01-01

    Rho GTPases are well known regulators of the actin cytoskeleton that act by binding and activating actin nucleators. They are therefore involved in many actin-based processes, including cell migration, cell polarity, and membrane trafficking. With the identification of phosphoinositide kinases and phosphatases as potential binding partners or effectors, Rho GTPases also appear to participate in the regulation of phosphoinositide metabolism. Since both actin dynamics and phosphoinositide turnover affect the efficiency and the fidelity of vesicle transport between cell compartments, Rho GTPases have emerged as critical players in membrane trafficking. Rho GTPase activity, actin remodeling, and phosphoinositide metabolism need to be coordinated in both space and time to ensure the progression of vesicles along membrane trafficking pathways. Although most molecular pathways are still unclear, in this review, we will highlight recent advances made in our understanding of how Rho-dependent signaling pathways organize actin dynamics and phosphoinositides and how phosphoinositides potentially provide negative feedback to Rho GTPases during endocytosis, exocytosis and membrane exchange between intracellular compartments. PMID:24914539

  4. Clamped-filament elongation model for actin-based motors.

    PubMed Central

    Dickinson, Richard B; Purich, Daniel L

    2002-01-01

    Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility. PMID:11806905

  5. Crystal growing

    NASA Technical Reports Server (NTRS)

    Neville, J. P.

    1990-01-01

    One objective is to demonstrate the way crystals grow and how they affect the behavior of material. Another objective is to compare the growth of crystals in metals and nonmetals. The procedures, which involve a supersaturated solution of a salt that will separate into crystals on cooling and the pouring off of an eutectic solution to expose the crystals formed by a solid solution when an alloy of two metals forms a solid and eutectic solution on cooling, are described.

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

    PubMed

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

    2015-11-05

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

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

    PubMed Central

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

    2015-01-01

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

  8. Formation of filopodia-like bundles in vitro from a dendritic network

    PubMed Central

    Vignjevic, Danijela; Yarar, Defne; Welch, Matthew D.; Peloquin, John; Svitkina, Tatyana; Borisy, Gary G.

    2003-01-01

    We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)–activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)–coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin. PMID:12642617

  9. 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

  10. Tracer diffusion through F-actin: effect of filament length and cross-linking.

    PubMed Central

    Jones, J D; Luby-Phelps, K

    1996-01-01

    We have determined diffusion coefficients for small (50- to 70-nm diameter) fluorescein-thiocarbamoyl-labeled Ficoll tracers through F-actin as a function of filament length and cross-linking. fx45 was used to regulate filament length and avidin/biotinylated actin or ABP-280 was used to prepare cross-linked actin gels. We found that tracer diffusion was generally independent of filament length in agreement with theoretical predictions for diffusion through solutions of rods. However, in some experiments diffusion was slower through short (< or = 1.0 micron) filaments, although this result was not consistently reproducible. Measured diffusion coefficients through unregulated F-actin and filaments of lengths > 1.0 micron were more rapid than predicted by theory for tracer diffusion through rigid, random networks, which was consistent with some degree of actin bundling. Avidin-induced cross-linking of biotinylated F-actin did not affect diffusion through unregulated F-actin, but in cases where diffusion was slower through short filaments this cross-linking method resulted in enhanced tracer diffusion rates indistinguishable from unregulated F-actin. This finding, in conjunction with increased turbidity of 1.0-micron filaments upon avidin cross-linking, indicated that this cross-linking method induces F-actin bundling. By contrast, ABP-280 cross-linking retarded diffusion through unregulated F-actin and decreased turbidity. Tracer diffusion under these conditions was well approximated by the diffusion theory. Both cross-linking procedures resulted in gel formation as determined by falling ball viscometry. These results demonstrate that network microscopic geometry is dependent on the cross-linking method, although both methods markedly increase F-actin macroscopic viscosity. PMID:8913611

  11. Unconventional myosin traffic in cells reveals a selective actin cytoskeleton

    PubMed Central

    Brawley, Crista M.; Rock, Ronald S.

    2009-01-01

    Eukaryotic cells have a self-organizing cytoskeleton where motors transport cargoes along cytoskeletal tracks. To understand the sorting process, we developed a system to observe single-molecule motility in a cellular context. We followed myosin classes V, VI, and X on triton-extracted actin cytoskeletons from Drosophila S2, mammalian COS-7, and mammalian U2OS cells. We find that these cells vary considerably in their global traffic patterns. The S2 and U2OS cells have regions of actin that either enhance or inhibit specific myosin classes. U2OS cells allow for 1 motor class, myosin VI, to move along stress fiber bundles, while motility of myosin V and X are suppressed. Myosin X motors are recruited to filopodia and the lamellar edge in S2 cells, whereas myosin VI motility is excluded from the same regions. Furthermore, we also see different velocities of myosin V motors in central regions of S2 cells, suggesting regional control of motor motility by the actin cytoskeleton. We also find unexpected features of the actin cytoskeletal network, including a population of reversed filaments with the barbed-end toward the cell center. This myosin motor regulation demonstrates that native actin cytoskeletons are more than just a collection of filaments. PMID:19478066

  12. Slow down of actin depolymerization by cross-linking molecules.

    PubMed

    Schmoller, Kurt M; Semmrich, Christine; Bausch, Andreas R

    2011-02-01

    The ability to control the assembly and disassembly dynamics of actin filaments is an essential property of the cellular cytoskeleton. While many different proteins are known which accelerate the polymerization of monomers into filaments or promote their disintegration, much less is known on mechanisms which guarantee the kinetic stability of the cytoskeletal filaments. Previous studies indicate that cross-linking molecules might fulfill these stabilizing tasks, which in addition facilitates their ability to regulate the organization of cytoskeletal structures in vivo. The effect of depolymerization factors on such structures or the mechanism which leads finally to their disintegration remain unknown. Here, we use multiple depolymerization methods in order to directly demonstrate that cross-linking and bundling proteins effectively suppress the actin depolymerization in a concentration dependent manner. Even the actin depolymerizing factor cofilin is not sufficient to facilitate a fast disintegration of highly cross-linked actin networks unless molecular motors are used simultaneously. The drastic modification of actin kinetics by cross-linking molecules can be expected to have wide-ranging implications for our understanding of the cytoskeleton, where cross-linking molecules are omnipresent and essential.

  13. 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.

  14. The Differential Formation of the LINC-Mediated Perinuclear Actin Cap in Pluripotent and Somatic Cells

    PubMed Central

    Khatau, Shyam B.; Kusuma, Sravanti; Hanjaya-Putra, Donny; Mali, Prashant; Cheng, Linzhao; Lee, Jerry S. H.; Gerecht, Sharon; Wirtz, Denis

    2012-01-01

    The actin filament cytoskeleton mediates cell motility and adhesion in somatic cells. However, whether the function and organization of the actin network are fundamentally different in pluripotent stem cells is unknown. Here we show that while conventional actin stress fibers at the basal surface of cells are present before and after onset of differentiation of mouse (mESCs) and human embryonic stem cells (hESCs), actin stress fibers of the actin cap, which wrap around the nucleus, are completely absent from undifferentiated mESCs and hESCs and their formation strongly correlates with differentiation. Similarly, the perinuclear actin cap is absent from human induced pluripotent stem cells (hiPSCs), while it is organized in the parental lung fibroblasts from which these hiPSCs are derived and in a wide range of human somatic cells, including lung, embryonic, and foreskin fibroblasts and endothelial cells. During differentiation, the formation of the actin cap follows the expression and proper localization of nuclear lamin A/C and associated linkers of nucleus and cytoskeleton (LINC) complexes at the nuclear envelope, which physically couple the actin cap to the apical surface of the nucleus. The differentiation of hESCs is accompanied by the progressive formation of a perinuclear actin cap while induced pluripotency is accompanied by the specific elimination of the actin cap, and that, through lamin A/C and LINC complexes, this actin cap is involved in progressively shaping the nucleus of hESCs undergoing differentiation. While, the localization of lamin A/C at the nuclear envelope is required for perinuclear actin cap formation, it is not sufficient to control nuclear shape. PMID:22574215

  15. Actin Automata: Phenomenology and Localizations

    NASA Astrophysics Data System (ADS)

    Adamatzky, Andrew; Mayne, Richard

    Actin is a globular protein which forms long filaments in the eukaryotic cytoskeleton, whose roles in cell function include structural support, contractile activity to intracellular signaling. We model actin filaments as two chains of one-dimensional binary-state semi-totalistic automaton arrays to describe hypothetical signaling events therein. Each node of the actin automaton takes state "0" (resting) or "1" (excited) and updates its state in discrete time depending on its neighbor's states. We analyze the complete rule space of actin automata using integral characteristics of space-time configurations generated by these rules and compute state transition rules that support traveling and mobile localizations. Approaches towards selection of the localization supporting rules using the global characteristics are outlined. We find that some properties of actin automata rules may be predicted using Shannon entropy, activity and incoherence of excitation between the polymer chains. We also show that it is possible to infer whether a given rule supports traveling or stationary localizations by looking at ratios of excited neighbors that are essential for generations of the localizations. We conclude by applying biomolecular hypotheses to this model and discuss the significance of our findings in context with cell signaling and emergent behavior in cellular computation.

  16. Effects of actin-binding proteins on the thermal stability of monomeric actin.

    PubMed

    Pivovarova, Anastasia V; Chebotareva, Natalia A; Kremneva, Elena V; Lappalainen, Pekka; Levitsky, Dmitrii I

    2013-01-08

    Differential scanning calorime