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Sample records for actin network filaments

  1. Talin can crosslink actin filaments into both networks and bundles.

    PubMed

    Zhang, J; Robson, R M; Schmidt, J M; Stromer, M H

    1996-01-17

    The talin-actin interaction was examined by using negative staining and cosedimentation assays. At pH 6.4 and low ionic strength, talin extensively crosslinked actin filaments into both networks and bundles. The bundles consist of parallel actin filaments with a center-to-center distance of 13 nm, and talin crossbridges spaced at 36-nm intervals along the bundles. As pH was increased stepwise from 6.4 to 7.3, talin's bundling activity was decreased first, then its networking activity. Qualitatively similar results were obtained at pH 6.4 by increasing ionic strength. Chemical crosslinking indicated talin was present as a dimer from pH 6.4 to 7.3, with or without added KC1. The results show that talin can interact directly with actin filaments by formation of actin filament networks and bundles, with the bundles more sensitive to dissolution by increase in pH or ionic strength. PMID:8561791

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2013-01-28

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

  4. Characterization of actin filament deformation in response to actively driven microspheres propagated through entangled actin networks

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

    The semi-flexible biopolymer actin is a ubiquitous component of nearly all biological organisms, playing an important role in many biological processes such as cell structure and motility, cancer invasion and metastasis, muscle contraction, and cell signaling. Concentrated actin networks possess unique viscoelastic properties that have been the subject of much theoretical and experimental work. However, much is still unknown regarding the correlation of the applied stress on the network to the induced filament strain at the molecular level. Here, we use dual optical traps alongside fluorescence microscopy to carry out active microrheology measurements that link mechanical stress to structural response at the micron scale. Specifically, we actively drive microspheres through entangled actin networks while simultaneously measuring the force the surrounding filaments exert on the sphere and visualizing the deformation and subsequent relaxation of fluorescent labeled filaments within the network. These measurements, which provide much needed insight into the link between stress and strain in actin networks, are critical for clarifying our theoretical understanding of the complex viscoelastic behavior exhibited in actin networks.

  5. Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing.

    PubMed

    Miyoshi, Takushi; Tsuji, Takahiro; Higashida, Chiharu; Hertzog, Maud; Fujita, Akiko; Narumiya, Shuh; Scita, Giorgio; Watanabe, Naoki

    2006-12-18

    Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays. PMID:17178911

  6. 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

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

    NASA Astrophysics Data System (ADS)

    Stam, Samantha; Gardel, Margaret

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

  8. 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.

  9. Shortening actin filaments cause force generation in actomyosin network to change from contractile to extensile

    NASA Astrophysics Data System (ADS)

    Kumar, Nitin; Gardel, Margaret

    Motor proteins in conjunction with filamentous proteins convert biochemical energy into mechanical energy which serves a number of cellular processes including cell motility, force generation and intracellular cargo transport. In-vitro experiments suggest that the forces generated by kinesin motors on microtubule bundles are extensile in nature whereas myosin motors on actin filaments are contractile. It is not clear how qualitatively similar systems can show completely different behaviors in terms of the nature of force generation. In order to answer this question, we carry out in vitro experiments where we form quasi 2D filamentous actomyosin networks and vary the length of actin filaments by adding capping protein. We show that when filaments are much shorter than their typical persistence length (approximately 10 microns), the forces generated are extensile and we see active nematic defect propagation, as seen in the microtubule-kinesin system. Based on this observation, we claim that the rigidity of rods plays an important role in dictating the nature of force generation in such systems. In order to understand this transition, we selectively label individual filaments and find that longer filaments show considerable bending and buckling, making them difficult to slide and extend along their length.

  10. 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.

  11. Two approaches to glassy dynamics and diffusion on actin filament networks

    NASA Astrophysics Data System (ADS)

    Snider, Joseph

    In spite of mass effort to understand glasses, basic features are still not completely known. Even whether or not glasses, as in windows, bottles, etc., are solids or liquids is not settled, let alone their thermodynamics. To make some headway in understanding glasses, this dissertation will take two distinct approaches. First, a direct simulation of a glassy system will be performed and compared to experiments, and from this the thermodynamics will be found. Second, rather than looking directly at a specific system, a general energy landscape appropriate for glass will be considered, and a new numeric technique to exactly calculate thermodynamic quantities will be presented and applied. The second part of this thesis will study diffusion on actin filament networks. Intracellular molecular motor-driven transport is essential for such diverse processes as mitosis, neuronal function, and mitochondrial transport. In vitro studies clarify these motors' function at the single molecule level but fail to elucidate how effective transport emerges from the collective behavior of multiple motors on a filamentary network. We investigate how the combined system of Myosin-V (MV) motors plus actin filaments is used to transport pigment granules in Xenopus melanophores. By analyzing single particle tracking data, we construct simulations and test a hypothesis that cells regulate transport by controlling how often granules switch from one filament to another, rather than, for example, altering motor activity at the single molecule level.

  12. 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.

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

    PubMed

    John, Karin; Stöter, Thomas; Misbah, Chaouqi

    2016-09-21

    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. PMID:27420637

  14. Mechanical properties of branched actin filaments.

    PubMed

    Razbin, Mohammadhosein; Falcke, Martin; Benetatos, Panayotis; Zippelius, Annette

    2015-07-01

    Cells moving on a two dimensional substrate generate motion by polymerizing actin filament networks inside a flat membrane protrusion. New filaments are generated by branching off existing ones, giving rise to branched network structures. We investigate the force-extension relation of branched filaments, grafted on an elastic structure at one end and pushing with the free ends against the leading edge cell membrane. Single filaments are modeled as worm-like chains, whose thermal bending fluctuations are restricted by the leading edge cell membrane, resulting in an effective force. Branching can increase the stiffness considerably; however the effect depends on branch point position and filament orientation, being most pronounced for intermediate tilt angles and intermediate branch point positions. We describe filament networks without cross-linkers to focus on the effect of branching. We use randomly positioned branch points, as generated in the process of treadmilling, and orientation distributions as measured in lamellipodia. These networks reproduce both the weak and strong force response of lamellipodia as measured in force-velocity experiments. We compare properties of branched and unbranched networks. The ratio of the network average of the force per branched filament to the average force per unbranched filament depends on the orientation distribution of the filaments. The ratio exhibits compression dependence and may go up to about 4.5 in networks with a narrow orientation distribution. With orientation distributions measured in lamellipodia, it is about two and essentially independent from network compression, graft elasticity and filament persistence length. PMID:26040560

  15. Mechanical properties of branched actin filaments

    NASA Astrophysics Data System (ADS)

    Razbin, Mohammadhosein; Falcke, Martin; Benetatos, Panayotis; Zippelius, Annette

    2015-07-01

    Cells moving on a two dimensional substrate generate motion by polymerizing actin filament networks inside a flat membrane protrusion. New filaments are generated by branching off existing ones, giving rise to branched network structures. We investigate the force-extension relation of branched filaments, grafted on an elastic structure at one end and pushing with the free ends against the leading edge cell membrane. Single filaments are modeled as worm-like chains, whose thermal bending fluctuations are restricted by the leading edge cell membrane, resulting in an effective force. Branching can increase the stiffness considerably; however the effect depends on branch point position and filament orientation, being most pronounced for intermediate tilt angles and intermediate branch point positions. We describe filament networks without cross-linkers to focus on the effect of branching. We use randomly positioned branch points, as generated in the process of treadmilling, and orientation distributions as measured in lamellipodia. These networks reproduce both the weak and strong force response of lamellipodia as measured in force-velocity experiments. We compare properties of branched and unbranched networks. The ratio of the network average of the force per branched filament to the average force per unbranched filament depends on the orientation distribution of the filaments. The ratio exhibits compression dependence and may go up to about 4.5 in networks with a narrow orientation distribution. With orientation distributions measured in lamellipodia, it is about two and essentially independent from network compression, graft elasticity and filament persistence length.

  16. Spontaneous polarization in an interfacial growth model for actin filament networks with a rigorous mechanochemical coupling.

    PubMed

    John, Karin; Caillerie, Denis; Misbah, Chaouqi

    2014-11-01

    Many processes in eukaryotic cells, including cell motility, rely on the growth of branched actin networks from surfaces. Despite its central role the mechanochemical coupling mechanisms that guide the growth process are poorly understood, and a general continuum description combining growth and mechanics is lacking. We develop a theory that bridges the gap between mesoscale and continuum limit and propose a general framework providing the evolution law of actin networks growing under stress. This formulation opens an area for the systematic study of actin dynamics in arbitrary geometries. Our framework predicts a morphological instability of actin growth on a rigid sphere, leading to a spontaneous polarization of the network with a mode selection corresponding to a comet, as reported experimentally. We show that the mechanics of the contact between the network and the surface plays a crucial role, in that it determines directly the existence of the instability. We extract scaling laws relating growth dynamics and network properties offering basic perspectives for new experiments on growing actin networks. PMID:25493815

  17. A Robust Actin Filaments Image Analysis Framework.

    PubMed

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-08-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a 'cartoon' part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the 'cartoon' image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts grown in

  18. A Robust Actin Filaments Image Analysis Framework

    PubMed Central

    Alioscha-Perez, Mitchel; Benadiba, Carine; Goossens, Katty; Kasas, Sandor; Dietler, Giovanni; Willaert, Ronnie; Sahli, Hichem

    2016-01-01

    The cytoskeleton is a highly dynamical protein network that plays a central role in numerous cellular physiological processes, and is traditionally divided into three components according to its chemical composition, i.e. actin, tubulin and intermediate filament cytoskeletons. Understanding the cytoskeleton dynamics is of prime importance to unveil mechanisms involved in cell adaptation to any stress type. Fluorescence imaging of cytoskeleton structures allows analyzing the impact of mechanical stimulation in the cytoskeleton, but it also imposes additional challenges in the image processing stage, such as the presence of imaging-related artifacts and heavy blurring introduced by (high-throughput) automated scans. However, although there exists a considerable number of image-based analytical tools to address the image processing and analysis, most of them are unfit to cope with the aforementioned challenges. Filamentous structures in images can be considered as a piecewise composition of quasi-straight segments (at least in some finer or coarser scale). Based on this observation, we propose a three-steps actin filaments extraction methodology: (i) first the input image is decomposed into a ‘cartoon’ part corresponding to the filament structures in the image, and a noise/texture part, (ii) on the ‘cartoon’ image, we apply a multi-scale line detector coupled with a (iii) quasi-straight filaments merging algorithm for fiber extraction. The proposed robust actin filaments image analysis framework allows extracting individual filaments in the presence of noise, artifacts and heavy blurring. Moreover, it provides numerous parameters such as filaments orientation, position and length, useful for further analysis. Cell image decomposition is relatively under-exploited in biological images processing, and our study shows the benefits it provides when addressing such tasks. Experimental validation was conducted using publicly available datasets, and in osteoblasts

  19. 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

  20. Actin filament curvature biases branching direction

    NASA Astrophysics Data System (ADS)

    Wang, Evan; Risca, Viviana; Chaudhuri, Ovijit; Chia, Jia-Jun; Geissler, Phillip; Fletcher, Daniel

    2012-02-01

    Actin filaments are key components of the cellular machinery, vital for a wide range of processes ranging from cell motility to endocytosis. Actin filaments can branch, and essential in this process is a protein complex known as the Arp2/3 complex, which nucleate new ``daughter'' filaments from pre-existing ``mother'' filaments by attaching itself to the mother filament. Though much progress has been made in understanding the Arp2/3-actin junction, some very interesting questions remain. In particular, F-actin is a dynamic polymer that undergoes a wide range of fluctuations. Prior studies of the Arp2/3-actin junction provides a very static notion of Arp2/3 binding. The question we ask is how differently does the Arp2/3 complex interact with a straight filament compared to a bent filament? In this study, we used Monte Carlo simulations of a surface-tethered worm-like chain to explore possible mechanisms underlying the experimental observation that there exists preferential branch formation by the Arp2/3 complex on the convex face of a curved filament. We show that a fluctuation gating model in which Arp2/3 binding to the actin filament is dependent upon a rare high-local-curvature shape fluctuation of the filament is consistent with the experimental data.

  1. VASP Governs Actin Dynamics by Modulating Filament Anchoring

    PubMed Central

    Trichet, Léa; Campàs, Otger; Sykes, Cécile; Plastino, Julie

    2007-01-01

    Actin filament dynamics at the cell membrane are important for cell-matrix and cell-cell adhesions and the protrusion of the leading edge. Since actin filaments must be connected to the cell membrane to exert forces but must also detach from the membrane to allow it to move and evolve, the balance between actin filament tethering and detachment at adhesion sites and the leading edge is key for cell shape changes and motility. How this fine tuning is performed in cells remains an open question, but possible candidates are the Drosophila enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins, which localize to dynamic actin structures in the cell. Here we study VASP-mediated actin-related proteins 2/3 (Arp2/3) complex-dependent actin dynamics using a substrate that mimics the fluid properties of the cell membrane: an oil-water interface. We show evidence that polymerization activators undergo diffusion and convection on the fluid surface, due to continual attachment and detachment to the actin network. These dynamics are enhanced in the presence of VASP, and we observe cycles of catastrophic detachment of the actin network from the surface, resulting in stop-and-go motion. These results point to a role for VASP in the modulation of filament anchoring, with implications for actin dynamics at cell adhesions and at the leading edge of the cell. PMID:17098798

  2. Effect of ATP on actin filament stiffness.

    PubMed

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

    1990-09-01

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

  3. Single Filaments to Reveal the Multiple Flavors of Actin.

    PubMed

    Jégou, Antoine; Romet-Lemonne, Guillaume

    2016-05-24

    A number of key cell processes rely on specific assemblies of actin filaments, which are all constructed from nearly identical building blocks: the abundant and extremely conserved actin protein. A central question in the field is to understand how different filament networks can coexist and be regulated. Discoveries in science are often related to technical advances. Here, we focus on the ongoing single filament revolution and discuss how these techniques have greatly contributed to our understanding of actin assembly. In particular, we highlight how they have refined our understanding of the many protein-based regulatory mechanisms that modulate actin assembly. It is now becoming apparent that other factors give filaments a specific identity that determines which proteins will bind to them. We argue that single filament techniques will play an essential role in the coming years as we try to understand the many ways actin filaments can take different flavors and unveil how these flavors modulate the action of regulatory proteins. We discuss different factors known to make actin filaments distinguishable by regulatory proteins and speculate on their possible consequences. PMID:27224479

  4. Visualization of prosomes (MCP-proteasomes), intermediate filament and actin networks by "instantaneous fixation" preserving the cytoskeleton.

    PubMed

    Arcangeletti, C; Sütterlin, R; Aebi, U; De Conto, F; Missorini, S; Chezzi, C; Scherrer, K

    1997-06-01

    A new "instantaneous" fixation/extraction procedure, yielding good preservation of intermediate filaments (IFs) and actin filaments when applied at 37 degrees C, has been explored to reexamine the relationships of the prosomes to the cytoskeleton. Prosomes are protein complexes of variable subunit composition, including occasionally a small RNA, which were originally observed as trans-acting factors in untranslated mRNPs. Constituting also the proteolytic core of the 26S proteasomes, they are also called "multicatalytic proteinase (MCP) complexes" or "20S-Proteasomes." In Triton X-100-extracted epithelial, fibroblastic, and muscle cells, prosome particles were found associated primarily with the IFs (Olink-Coux et al., 1994). Application of "instantaneous fixation" has now led to the new observation that a major fraction of prosome particles, composed of specific sets of subunits, is distributed in variable proportions between the IFs and the microfilament/ stress fiber system in PtK1 epithelial cells and human fibroblasts. Electron microscopy using gold-labeled antibodies confirms this dual localization on classical whole mounts and on cells exposed to instantaneous fixation. In contrast to the resistance of the prosome-IF association, a variable fraction of the prosome particles is released from the actin cytoskeleton by Triton X-100 when applied prior to fixation. Moreover, in vitro copolymerization of prosomes with G-actin made it possible to observe "ladder-like" filamentous structures in the electron microscope, in which the prosome particles, like the "rungs of a ladder," laterally crosslink two or more actin filaments in a regular pattern. These results demonstrate that prosomes are bound in the cell not only to IFs but also to the actin cytoskeleton and, furthermore, not only within large M(r) complexes (possibly mRNPs and/or 26S proteasomes), but also directly, as individual prosome particles. PMID:9216087

  5. 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.

  6. Actin Filament Segmentation Using Dynamic Programming

    PubMed Central

    Li, Hongsheng; Shen, Tian; Huang, Xiaolei

    2011-01-01

    We introduce a novel algorithm for actin filament segmentation in 2D TIRFM image sequences. This problem is difficult because actin filaments dynamically change shapes during their growth, and the TIRFM images are usually noisy. We ask a user to specify the two tips of a filament of interest in the first frame. We then model the segmentation problem in an image sequence as a temporal chain, where its states are tip locations; given candidate tip locations, actin filaments' body points are inferred by a dynamic programming method, which adaptively generates candidate solutions. Combining candidate tip locations and their inferred body points, the temporal chain model is efficiently optimized using another dynamic programming method. Evaluation on noisy TIRFM image sequences demonstrates the accuracy and robustness of this approach. PMID:21761674

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

    PubMed Central

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

    2013-01-01

    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.—Mok, K., Mruk, D. D., Lee, W. M., Cheng, C. Y. Rictor/mTORC2 regulates blood-testis barrier dynamics via its effects on gap junction communications and actin filament network. PMID:23288930

  8. How cofilin severs an actin filament.

    PubMed

    De La Cruz, Enrique M

    2009-05-15

    The actin regulatory protein, cofilin, promotes actin assembly dynamics by severing filaments and increasing the number of ends from which subunits add and dissociate. Recent studies provide biophysical descriptions of cooperative filament interactions in energetic, mechanical and structural terms. A one-dimensional Ising model with nearest-neighbor interactions permits thermodynamic analysis of cooperative binding and indicates that one or a few cofilin molecules can sever a filament. Binding and cooperative interactions are entropically driven. A significant fraction of the binding free energy results from the linked dissociation of filament-associated ions (polyelectrolyte effect), which modulate filament structure, stability and mechanics. The remaining binding free energy and essentially all of the cooperative free energy arise from the enhanced conformational dynamics of the cofilactin complex. Filament mechanics are modulated by cofilin such that cofilin-saturated filaments are approximately 10- to 20-fold more compliant in bending and twisting than bare filaments. Cofilin activity is well described by models in which discontinuities in topology, mechanics and conformational dynamics generate stress concentration and promote fracture at junctions of bare and decorated segments, analogous to the grain boundary fracture of crystalline materials and the thermally driven formation of shear transformation zones in colloidal glass. PMID:20700473

  9. Mechanism of Actin Filament Bundling by Fascin

    SciTech Connect

    Jansen, Silvia; Collins, Agnieszka; Yang, Changsong; Rebowski, Grzegorz; Svitkina, Tatyana; Dominguez, Roberto

    2013-03-07

    Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four {beta}-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in {beta}-trefoil domains 1 and 3. The site in {beta}-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in {beta}-trefoil-3 is related by pseudo-2-fold symmetry to that in {beta}-trefoil-1. The two sites are {approx}5 nm apart, resulting in a distance between actin filaments in the bundle of {approx}8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

  10. Ionic wave propagation along actin filaments.

    PubMed

    Tuszyński, J A; Portet, S; Dixon, J M; Luxford, C; Cantiello, H F

    2004-04-01

    We investigate the conditions enabling actin filaments to act as electrical transmission lines for ion flows along their lengths. We propose a model in which each actin monomer is an electric element with a capacitive, inductive, and resistive property due to the molecular structure of the actin filament and viscosity of the solution. Based on Kirchhoff's laws taken in the continuum limit, a nonlinear partial differential equation is derived for the propagation of ionic waves. We solve this equation in two different regimes. In the first, the maximum propagation velocity wave is found in terms of Jacobi elliptic functions. In the general case, we analyze the equation in terms of Fisher-Kolmogoroff modes with both localized and extended wave characteristics. We propose a new signaling mechanism in the cell, especially in neurons. PMID:15041636

  11. Arabidopsis CROLIN1, a Novel Plant Actin-binding Protein, Functions in Cross-linking and Stabilizing Actin Filaments*

    PubMed Central

    Jia, Honglei; Li, Jisheng; Zhu, Jingen; Fan, Tingting; Qian, Dong; Zhou, Yuelong; Wang, Jiaojiao; Ren, Haiyun; Xiang, Yun; An, Lizhe

    2013-01-01

    Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which the higher order cytoskeleton is formed in plants remains unknown. In this study, we identified a novel actin-cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom. There are six isovariants of CROLIN in the Arabidopsis genome, with CROLIN1 specifically expressed in pollen. In vitro biochemical analyses showed that CROLIN1 is a novel actin-cross-linking protein with binding and stabilizing activities. Remarkably, CROLIN1 can cross-link actin bundles into actin networks. CROLIN1 loss of function induces pollen germination and pollen tube growth hypersensitive to latrunculin B. All of these results demonstrate that CROLIN1 may play an important role in stabilizing and remodeling actin filaments by binding to and cross-linking actin filaments. PMID:24072702

  12. Actin Filaments Regulate Exocytosis at the Hair Cell Ribbon Synapse.

    PubMed

    Guillet, Marie; Sendin, Gaston; Bourien, Jérôme; Puel, Jean-Luc; Nouvian, Régis

    2016-01-20

    Exocytosis at the inner hair cell ribbon synapse is achieved through the functional coupling between calcium channels and glutamate-filled synaptic vesicles. Using membrane capacitance measurements, we investigated whether the actin network regulates the exocytosis of synaptic vesicles at the mouse auditory hair cell. Our results suggest that actin network disruption increases exocytosis and that actin filaments may spatially organize a subfraction of synaptic vesicles with respect to the calcium channels. Significance statement: Inner hair cells (IHCs), the auditory sensory cells of the cochlea, release glutamate onto the afferent auditory nerve fibers to encode sound stimulation. To achieve this task, the IHC relies on the recruitment of glutamate-filled vesicles that can be located in close vicinity to the calcium channels or more remotely from them. The molecular determinants responsible for organizing these vesicle pools are not fully identified. Using pharmacological tools in combination with membrane capacitance measurements, we show that actin filament disruption increases exocytosis in IHCs and that actin filaments most likely position a fraction of vesicles away from the calcium channels. PMID:26791198

  13. Supercoiling of f-actin filaments.

    PubMed

    Lednev, V V; Popp, D

    1990-05-01

    In the X-ray diffraction pattern from oriented gels of actin-containing filaments sampling of layer lines indicating the development of a well-ordered pseudo-hexagonal lattice within the gels at interfilament spacings as large as 13 nm is observed. This value exceeds by 3 nm the largest estimate of an external diameter of pure f-actin. The development of layer line sampling is always accompanied by: (i) the appearance of strong forbidden meridional reflections on the 5.9- and 5.1-nm layer lines; (ii) a drastic intensification of the first (expected) 2.75-nm meridional reflection by a factor of about 4; (iii) the appearance of streaks, connecting near-meridional reflections on the 5.9-, 5.1-, and 37-nm layer lines; and (iv) a slight decrease in the number of subunits per turn of the basic f-actin helix. All these features strongly indicate that f-actin filaments are supercoiled and make regular local contacts between themselves, which may lead to periodic distortions of the mobile external domain in the actin subunits. PMID:2261308

  14. 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

  15. Architecture and Connectivity Govern Actin Network Contractility.

    PubMed

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

    2016-03-01

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

  16. 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. PMID:26240174

  17. Single turnovers of fluorescent ATP bound to bipolar myosin filament during actin filaments sliding

    PubMed Central

    Maruta, Takahiro; Kobatake, Takahiro; Okubo, Hiroyuki; Chaen, Shigeru

    2013-01-01

    The nucleotide turnover rates of bipolar myosin thick filament along which actin filament slides were measured by the displacement of prebound fluorescent ATP analog 2′(3′)-O-[N-[2-[(Cy3)]amindo]ethyl] carbamoyl]-adenosine 5′ triphosphate (Cy3-EDA-ATP) upon flash photolysis of caged ATP. The fluorescence of the thick filament where actin filament slides decayed with two exponential processes. The slower rate constant was the same as that without actin filament. Along bipolar myosin thick filament, actin filaments slide at a fast speed towards the central bare zone (forward), but more slowly away from the bare zone (backward). The displacement rate constant of fluorescent ATP from the myosin filament where actin filament moved forward was 5.0 s−1, whereas the rate constant where the actin filament slid backward was 1.7 s−1. These findings suggest that the slow ADP release rate is responsible for the slow backward sliding movement.

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

    PubMed Central

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

    2014-01-01

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

  19. Mechanosensitive kinetic preference of actin-binding protein to actin filament

    NASA Astrophysics Data System (ADS)

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

    The kinetic preference of actin-binding proteins to actin filaments is altered by external forces on the filament. Such an altered kinetic preference is largely responsible for remodeling the actin cytoskeletal structure in response to intracellular forces. During remodeling, actin-binding proteins and actin filaments interact under isothermal conditions, because the cells are homeostatic. In such a temperature homeostatic state, we can rigorously and thermodynamically link the chemical potential of actin-binding proteins to stresses on the actin filaments. From this relationship, we can construct a physical model that explains the force-dependent kinetic preference of actin-binding proteins to actin filaments. To confirm the model, we have analyzed the mechanosensitive alternation of the kinetic preference of Arp2/3 and cofilin to actin filaments. We show that this model captures the qualitative responses of these actin-binding proteins to the forces, as observed experimentally. Moreover, our theoretical results demonstrate that, depending on the structural parameters of the binding region, actin-binding proteins can show different kinetic responses even to the same mechanical signal tension, in which the double-helix nature of the actin filament also plays a critical role in a stretch-twist coupling of the filament.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2002-05-17

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

  2. Tropomyosin diffusion over actin subunits facilitates thin filament assembly

    PubMed Central

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

    2016-01-01

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

  3. Quantifying morphological features of actin cytoskeletal filaments in plant cells based on mathematical morphology.

    PubMed

    Kimori, Yoshitaka; Hikino, Kazumi; Nishimura, Mikio; Mano, Shoji

    2016-01-21

    By quantifying the morphological properties of biological structures, we can better evaluate complex shapes and detect subtle morphological changes in organisms. In this paper, we propose a shape analysis method based on morphological image processing, and apply it to image analysis of actin cytoskeletal filaments in root hair cells of Arabidopsis thaliana. In plant cells, the actin cytoskeletal filaments have critical roles in various cellular processes such as vesicle trafficking and organelle motility. The dynamics of vesicles and organelles in plant cells depend on actin cytoskeletal filaments, regulating cell division and cell enlargement. To better understand the actin-dependent organelle motility, we attempted to quantify the organization of actin filaments in the root hair cells of the root hair defective 3 (rhd3) mutant. RHD3 is involved in actin organization, and its defect has been reported to affect the dynamics of various vesicles and organelles. We measured three shape features of the actin filaments in wild-type and mutant plants. One feature (thickness) was depicted on a grayscale; the others (describing the complexity of the filament network patterns in two-dimensional space) were depicted as binary features. The morphological phenotypes of the cytoskeletal filaments clearly differed between wild-type and mutant. Subtle variations of filament morphology among the mutants were detected and statistically quantified. PMID:26551157

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

    PubMed Central

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

    2008-01-01

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

  5. 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

  6. 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. PMID:24523293

  7. 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

  8. Visualization of actin filaments and monomers in somatic cell nuclei.

    PubMed

    Belin, Brittany J; Cimini, Beth A; Blackburn, Elizabeth H; Mullins, R Dyche

    2013-04-01

    In addition to its long-studied presence in the cytoplasm, actin is also found in the nuclei of eukaryotic cells. The function and form (monomer, filament, or noncanonical oligomer) of nuclear actin are hotly debated, and its localization and dynamics are largely unknown. To determine the distribution of nuclear actin in live somatic cells and evaluate its potential functions, we constructed and validated fluorescent nuclear actin probes. Monomeric actin probes concentrate in nuclear speckles, suggesting an interaction of monomers with RNA-processing factors. Filamentous actin probes recognize discrete structures with submicron lengths that are excluded from chromatin-rich regions. In time-lapse movies, these actin filament structures exhibit one of two types of mobility: 1) diffusive, with an average diffusion coefficient of 0.06-0.08 μm(2)/s, or (2) subdiffusive, with a mobility coefficient of 0.015 μm(2)/s. Individual filament trajectories exhibit features of particles moving within a viscoelastic mesh. The small size of nuclear actin filaments is inconsistent with a role in micron-scale intranuclear transport, and their localization suggests that they do not participate directly in chromatin-based processes. Our results instead suggest that actin filaments form part of a large, viscoelastic structure in the nucleoplasm and may act as scaffolds that help organize nuclear contents. PMID:23447706

  9. Myosin motors fragment and compact membrane-bound actin filaments

    PubMed Central

    Vogel, Sven K; Petrasek, Zdenek; Heinemann, Fabian; Schwille, Petra

    2013-01-01

    Cell cortex remodeling during cell division is a result of myofilament-driven contractility of the cortical membrane-bound actin meshwork. Little is known about the interaction between individual myofilaments and membrane-bound actin filaments. Here we reconstituted a minimal actin cortex to directly visualize the action of individual myofilaments on membrane-bound actin filaments using TIRF microscopy. We show that synthetic myofilaments fragment and compact membrane-bound actin while processively moving along actin filaments. We propose a mechanism by which tension builds up between the ends of myofilaments, resulting in compressive stress exerted to single actin filaments, causing their buckling and breakage. Modeling of this mechanism revealed that sufficient force (∼20 pN) can be generated by single myofilaments to buckle and break actin filaments. This mechanism of filament fragmentation and compaction may contribute to actin turnover and cortex reorganization during cytokinesis. DOI: http://dx.doi.org/10.7554/eLife.00116.001 PMID:23326639

  10. Bundling actin filaments from membranes: some novel players

    PubMed Central

    Thomas, Clément

    2012-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe

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

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

    PubMed Central

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

    2012-01-01

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

  13. Dynamics of active actin networks

    NASA Astrophysics Data System (ADS)

    Koehler, Simone

    2014-03-01

    Local mechanical and structural properties of a eukaryotic cell are determined by its cytoskeleton. To adapt to their environment, cells rely on constant self-organized rearrangement processes of their actin cytoskeleton. To shed light on the principles underlying these dynamic self-organization processes we investigate a minimal reconstituted active system consisting of actin filaments, crosslinking molecules and molecular motor filaments. Using quantitative fluorescence microscopy and image analysis, we show, that these minimal model systems exhibit a generic structure formation mechanism. The competition between force generation by molecular motors and the stabilization of the network by crosslinking proteins results in a highly dynamic reorganization process which is characterized by anomalous transport dynamics with a superdiffusive behavior also found in intracellular dynamics. In vitro, these dynamics are governed by chemical and physical parameters that alter the balance of motor and crosslinking proteins, such as pH. These findings can be expected to have broad implications in our understanding of cytoskeletal regulation in vivo.

  14. Multiscale Modelling for investigating single molecule effects on the mechanics of actin filaments

    NASA Astrophysics Data System (ADS)

    A, Deriu Marco; C, Bidone Tamara; Laura, Carbone; Cristina, Bignardi; M, Montevecchi Franco; Umberto, Morbiducci

    2011-12-01

    This work presents a preliminary multiscale computational investigation of the effects of nucleotides and cations on the mechanics of actin filaments (F-actin). At the molecular level, Molecular Dynamics (MD) simulations are employed to characterize the rearrangements of the actin monomers (G-actin) in terms of secondary structures evolution in physiological conditions. At the mesoscale level, a coarse grain (CG) procedure is adopted where each monomer is represented by means of Elastic Network Modeling (ENM) technique. At the macroscale level, actin filaments up to hundreds of nanometers are assumed as isotropic and elastic beams and characterized via Rotation Translation Block (RTB) analysis. F-actin bound to adenosine triphosphate (ATP) shows a persistence length around 5 μm, while actin filaments bound to adenosine diphosphate (ADP) have a persistence length of about 3 μm. With magnesium bound to the high affinity binding site of G-actin, the persistence length of F-actin decreases to about 2 μm only in the ADP-bound form of the filament, while the same ion has no effects, in terms of stiffness variation, on the ATP-bound form of F-actin. The molecular mechanisms behind these changes in flexibility are herein elucidated. Thus, this study allows to analyze how the local binding of cations and nucleotides on G-actin induce molecular rearrangements that transmit to the overall F-actin, characterizing shifts of mechanical properties, that can be related with physiological and pathological cellular phenomena, as cell migration and spreading. Further, this study provides the basis for upcoming investigating of network and cellular remodelling at higher length scales.

  15. High Speed Depolymerization at Actin Filament Ends Jointly Catalyzed by Twinfilin and Srv2/CAP

    PubMed Central

    Johnston, Adam B.; Collins, Agnieszka; Goode, Bruce L.

    2015-01-01

    Purified actin filaments depolymerize slowly, and cytosolic conditions strongly favor actin assembly over disassembly, which has left our understanding of how actin filaments are rapidly turned over in vivo incomplete 1,2. One mechanism for driving filament disassembly is severing by factors such as Cofilin. However, even after severing, pointed end depolymerization remains slow and unable to fully account for observed rates of actin filament turnover in vivo. Here we describe a mechanism by which Twinfilin and Cyclase-associated protein work in concert to accelerate depolymerization of actin filaments by 3-fold and 17-fold at their barbed and pointed ends, respectively. This mechanism occurs even under assembly conditions, allowing reconstitution and direct visualization of individual filaments undergoing tunable, accelerated treadmilling. Further, we use specific mutations to demonstrate that this activity is critical for Twinfilin function in vivo. These findings fill a major gap in our knowledge of mechanisms, and suggest that depolymerization and severing may be deployed separately or together to control the dynamics and architecture of distinct actin networks. PMID:26458246

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

    PubMed Central

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

    2014-01-01

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

  17. Mechanical Heterogeneity Favors Fragmentation of Strained Actin Filaments

    PubMed Central

    De La Cruz, Enrique M.; Martiel, Jean-Louis; Blanchoin, Laurent

    2015-01-01

    We present a general model of actin filament deformation and fragmentation in response to compressive forces. The elastic free energy density along filaments is determined by their shape and mechanical properties, which were modeled in terms of bending, twisting, and twist-bend coupling elasticities. The elastic energy stored in filament deformation (i.e., strain) tilts the fragmentation-annealing reaction free-energy profile to favor fragmentation. The energy gradient introduces a local shear force that accelerates filament intersubunit bond rupture. The severing protein, cofilin, renders filaments more compliant in bending and twisting. As a result, filaments that are partially decorated with cofilin are mechanically heterogeneous (i.e., nonuniform) and display asymmetric shape deformations and energy profiles distinct from mechanically homogenous (i.e., uniform), bare actin, or saturated cofilactin filaments. The local buckling strain depends on the relative size of the compliant segment as well as the bending and twisting rigidities of flanking regions. Filaments with a single bare/cofilin-decorated boundary localize energy and force adjacent to the boundary, within the compliant cofilactin segment. Filaments with small cofilin clusters were predicted to fragment within the compliant cofilactin rather than at boundaries. Neglecting contributions from twist-bend coupling elasticity underestimates the energy density and gradients along filaments, and thus the net effects of filament strain to fragmentation. Spatial confinement causes compliant cofilactin segments and filaments to adopt higher deformation modes and store more elastic energy, thereby promoting fragmentation. The theory and simulations presented here establish a quantitative relationship between actin filament fragmentation thermodynamics and elasticity, and reveal how local discontinuities in filament mechanical properties introduced by regulatory proteins can modulate both the severing efficiency

  18. Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

    SciTech Connect

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

    2013-11-20

    Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin {beta}4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin {beta}4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.

  19. Drosophila quail, a villin-related protein, bundles actin filaments in apoptotic nurse cells.

    PubMed

    Matova, N; Mahajan-Miklos, S; Mooseker, M S; Cooley, L

    1999-12-01

    Drosophila Quail protein is required for the completion of fast cytoplasm transport from nurse cells to the oocyte, an event critical for the production of viable oocytes. The abundant network of cytoplasmic filamentous actin, established at the onset of fast transport, is absent in quail mutant egg chambers. Previously, we showed that Quail is a germline-specific protein with sequence homology to villin, a vertebrate actin-regulating protein. In this study, we combined biochemical experiments with observations in egg chambers to define more precisely the function of this protein in the regulation of actin-bundle assembly in nurse cells. We report that recombinant Quail can bind and bundle filamentous actin in vitro in a manner similar to villin at a physiological calcium concentration. In contrast to villin, Quail is unable to sever or cap filamentous actin, or to promote nucleation of new actin filaments at a high calcium concentration. Instead, Quail bundles the filaments regardless of the calcium concentration. In vivo, the assembly of nurse-cell actin bundles is accompanied by extensive perforation of the nurse-cell nuclear envelopes, and both of these phenomena are manifestations of nurse-cell apoptosis. To investigate whether free calcium levels are affected during apoptosis, we loaded egg chambers with the calcium indicator Indo-1. Our observations indicate a rise in free calcium in the nurse-cell cytoplasm coincident with the permeabilization of the nuclear envelopes. We also show that human villin expressed in the Drosophila germline could sense elevated cytoplasmic calcium; in nurse cells with reduced levels of Quail protein, villin interfered with actin-bundle stability. We conclude that Quail efficiently assembles actin filaments into bundles in nurse cells and maintains their stability under fluctuating free calcium levels. We also propose a developmental model for the fast phase of cytoplasm transport incorporating findings presented in this study

  20. Actin filaments in normal dermis and during wound healing.

    PubMed Central

    Doillon, C. J.; Hembry, R. M.; Ehrlich, H. P.; Burke, J. F.

    1987-01-01

    During wound healing, it has been suggested, modified fibroblasts rich in actin filaments are responsible for wound contraction. With the use of specific fluorescent probe (NBD-phallacidin), the distribution of actin filaments are compared in normal dermis and in several wound contraction models, including open and burn wounds and full and thin-thickness skin autografts. Fibroblasts of normal dermis are slightly stained with NBD-phallacidin. Fibroblasts with actin filaments are increased in autografts, particularly at Days 15 and 21 after grafting, and are prominent in open and burn wounds. The wound contraction rate is not directly related to the presence of actin-staining fibroblasts. After stabilization of the contraction of open or burn wounds, fibroblasts rich in actin filaments remain. The superficial layer of full-thickness skin graft contains a similar actin distribution without concomitant contraction. It is concluded that the distribution of actin-rich fibroblasts corresponds morphologically to previous areas of necrosis or injury. Images Figure 2 Figure 3 PMID:3544851

  1. Regulators of actin filament barbed ends at a glance.

    PubMed

    Shekhar, Shashank; Pernier, Julien; Carlier, Marie-France

    2016-03-15

    Cells respond to external stimuli by rapidly remodeling their actin cytoskeleton. At the heart of this function lies the intricately controlled regulation of individual filaments. The barbed end of an actin filament is the hotspot for the majority of the biochemical reactions that control filament assembly. Assays performed in bulk solution and with single filaments have enabled characterization of a plethora of barbed-end-regulating proteins. Interestingly, many of these regulators work in tandem with other proteins, which increase or decrease their affinity for the barbed end in a spatially and temporally controlled manner, often through simultaneous binding of two regulators at the barbed ends, in addition to standard mutually exclusive binding schemes. In this Cell Science at a Glance and the accompanying poster, we discuss key barbed-end-interacting proteins and the kinetic mechanisms by which they regulate actin filament assembly. We take F-actin capping protein, gelsolin, profilin and barbed-end-tracking polymerases, including formins and WH2-domain-containing proteins, as examples, and illustrate how their activity and competition for the barbed end regulate filament dynamics. PMID:26940918

  2. The kinetics underlying the velocity of smooth muscle myosin filament sliding on actin filaments in vitro.

    PubMed

    Haldeman, Brian D; Brizendine, Richard K; Facemyer, Kevin C; Baker, Josh E; Cremo, Christine R

    2014-07-25

    Actin-myosin interactions are well studied using soluble myosin fragments, but little is known about effects of myosin filament structure on mechanochemistry. We stabilized unphosphorylated smooth muscle myosin (SMM) and phosphorylated smooth muscle myosin (pSMM) filaments against ATP-induced depolymerization using a cross-linker and attached fluorescent rhodamine (XL-Rh-SMM). Electron micrographs showed that these side polar filaments are very similar to unmodified filaments. They are ~0.63 μm long and contain ~176 molecules. Rate constants for ATP-induced dissociation and ADP release from acto-myosin for filaments and S1 heads were similar. Actin-activated ATPases of SMM and XL-Rh-SMM were similarly regulated. XL-Rh-pSMM filaments moved processively on F-actin that was bound to a PEG brush surface. ATP dependence of filament velocities was similar to that for solution ATPases at high [actin], suggesting that both processes are limited by the same kinetic step (weak to strong transition) and therefore are attachment- limited. This differs from actin sliding over myosin monomers, which is primarily detachment-limited. Fitting filament data to an attachment-limited model showed that approximately half of the heads are available to move the filament, consistent with a side polar structure. We suggest the low stiffness subfragment 2 (S2) domain remains unhindered during filament motion in our assay. Actin-bound negatively displaced heads will impart minimal drag force because of S2 buckling. Given the ADP release rate, the velocity, and the length of S2, these heads will detach from actin before slack is taken up into a backwardly displaced high stiffness position. This mechanism explains the lack of detachment- limited kinetics at physiological [ATP]. These findings address how nonlinear elasticity in assemblies of motors leads to efficient collective force generation. PMID:24907276

  3. The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in Vitro*

    PubMed Central

    Haldeman, Brian D.; Brizendine, Richard K.; Facemyer, Kevin C.; Baker, Josh E.; Cremo, Christine R.

    2014-01-01

    Actin-myosin interactions are well studied using soluble myosin fragments, but little is known about effects of myosin filament structure on mechanochemistry. We stabilized unphosphorylated smooth muscle myosin (SMM) and phosphorylated smooth muscle myosin (pSMM) filaments against ATP-induced depolymerization using a cross-linker and attached fluorescent rhodamine (XL-Rh-SMM). Electron micrographs showed that these side polar filaments are very similar to unmodified filaments. They are ∼0.63 μm long and contain ∼176 molecules. Rate constants for ATP-induced dissociation and ADP release from acto-myosin for filaments and S1 heads were similar. Actin-activated ATPases of SMM and XL-Rh-SMM were similarly regulated. XL-Rh-pSMM filaments moved processively on F-actin that was bound to a PEG brush surface. ATP dependence of filament velocities was similar to that for solution ATPases at high [actin], suggesting that both processes are limited by the same kinetic step (weak to strong transition) and therefore are attachment-limited. This differs from actin sliding over myosin monomers, which is primarily detachment-limited. Fitting filament data to an attachment-limited model showed that approximately half of the heads are available to move the filament, consistent with a side polar structure. We suggest the low stiffness subfragment 2 (S2) domain remains unhindered during filament motion in our assay. Actin-bound negatively displaced heads will impart minimal drag force because of S2 buckling. Given the ADP release rate, the velocity, and the length of S2, these heads will detach from actin before slack is taken up into a backwardly displaced high stiffness position. This mechanism explains the lack of detachment-limited kinetics at physiological [ATP]. These findings address how nonlinear elasticity in assemblies of motors leads to efficient collective force generation. PMID:24907276

  4. Filament assembly by Spire: key residues and concerted actin binding.

    PubMed

    Rasson, Amy S; Bois, Justin S; Pham, Duy Stephen L; Yoo, Haneul; Quinlan, Margot E

    2015-02-27

    The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers. PMID:25234086

  5. Filament Assembly by Spire: Key Residues and Concerted Actin Binding

    PubMed Central

    Rasson, Amy S.; Bois, Justin S.; Pham, Duy Stephen L.; Yoo, Haneul; Quinlan, Margot E.

    2014-01-01

    The most recently identified class of actin nucleators, WASp Homology domain 2 (WH2) – nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or Sc), plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of Sc in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within Sc that are critical for its activity. Using this information we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that Sc binds actin filaments, in addition to monomers. PMID:25234086

  6. Staining Fission Yeast Filamentous Actin with Fluorescent Phalloidin Conjugates.

    PubMed

    Hagan, Iain M

    2016-01-01

    The Schizosaccharomyces pombe filamentous (F)-actin cytoskeleton drives cell growth, morphogenesis, endocytosis, and cytokinesis. The protocol described here reveals the distribution of F-actin in fixed cells through the use of fluorescently conjugated phalloidin. Simultaneous staining of cell wall landmarks (with calcofluor) and chromatin (with 4',6-diamidino-2-phenylindole, or DAPI) makes this rapid staining procedure highly effective for staging cell cycle progression, monitoring morphogenetic abnormalities, and assessing the impact of environmental and genetic changes on the integrity of the F-actin cytoskeleton. PMID:27250943

  7. Electrostatics control actin filament nucleation and elongation kinetics.

    PubMed

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

    2013-04-26

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

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

    PubMed

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

    2012-06-15

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

  9. 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.

  10. Liquid-like bundles of crosslinked actin filaments contract without motors

    NASA Astrophysics Data System (ADS)

    Weirich, Kimberly

    The actin cytoskeleton is a dynamic, structural material that drives cellular-scale deformations during processes such as cell migration and division. Motor proteins are responsible for actively driving many deformations by buckling and translocating actin filaments. However, there is evidence that deformations, such as the constriction of the actin bundle that drives the separation of cells during division, can occur without motors, mediated instead by crosslinker proteins. How might crosslinkers, independent of motors, drive contraction of a bundle? Using a model system of purified proteins, we show that crosslinkers, analogous to molecular cohesion, create an effective surface tension that induces bundle contraction. Crosslinked short actin filaments form micron-sized spindle-shaped bundles. Similar to tactoid granules found at the isotropic-nematic phase transition in liquid crystals, these bundles coarsen and coalesce like liquid droplets. In contrast, crosslinked long filaments coarsen into a steady state of bundles that are frozen in a solid-like network. Near the liquid-solid boundary, filaments of intermediate length initially form bundles that spontaneously contract into tactoid droplets. Our results, that crosslinked actin bundles are liquid-like with an effective surface tension, provide evidence for a mechanism of motor-independent contractility in biological materials.

  11. Astral microtubules physically redistribute cortical actin filaments to the incipient contractile ring.

    PubMed

    Tseng, Kuo-Fu; Foss, Margit; Zhang, Dahong

    2012-11-01

    Prior to cell cleavage, cytokinetic proteins are recruited into the nascent actomyosin contractile ring, paving the way for formation of a functional cleavage furrow. Interactions between spindle microtubules and the cell cortex may play a critical role in this recruitment, since microtubules have been shown to affect distribution and activation of cytokinetic proteins within the cortex. However, direct evidence for physical interaction between microtubules and the cortex has been lacking. Here, we probed the physical connection between astral microtubules and cortical actin filaments, by micromanipulating the fluorescently tagged cytoskeleton in living spermatocytes of the grasshopper Melanoplus femurrubrum. When microtubules were tugged with a microneedle, they in turn pulled on cortical actin filaments, interrupting the filaments' journey toward the equator. Further displacement of the actin dragged the cell membrane inward, demonstrating that the cortical actin network physically linked spindle microtubules to the cell membrane. Regional disruption of the connection by breaking spindle microtubules prevented actin accumulation in a segment of the ring, which locally inhibited furrowing. We propose a model in which dynamic astral microtubules physically redistribute cortical actin into the incipient contractile ring. PMID:23027710

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

    NASA Astrophysics Data System (ADS)

    Wang, Ruizhe; Carlsson, Anders E.

    2015-12-01

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

  13. 3D Actin Network Centerline Extraction with Multiple Active Contours

    PubMed Central

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2013-01-01

    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. PMID:24316442

  14. Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

    PubMed Central

    Persson, Malin; Gullberg, Maria; Tolf, Conny; Lindberg, A. Michael; Månsson, Alf; Kocer, Armagan

    2013-01-01

    Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments (“side-attached”) or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 µm) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10–50 streptavidin molecules, 1–10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy. PMID:23437074

  15. 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.

  16. 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

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

    PubMed Central

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

    2015-01-01

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

  18. The Stationary-Phase Cells of Saccharomyces cerevisiae Display Dynamic Actin Filaments Required for Processes Extending Chronological Life Span

    PubMed Central

    Lejskova, Renata; Malcova, Ivana

    2015-01-01

    Stationary-growth-phase Saccharomyces cerevisiae yeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. In S. cerevisiae stationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Our in vivo analyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficient cox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth. PMID:26351139

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

    PubMed

    Grosse, Robert; Vartiainen, Maria K

    2013-11-01

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

  20. 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.

  1. A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface*

    PubMed Central

    Wong, Wilson; Webb, Andrew I.; Olshina, Maya A.; Infusini, Giuseppe; Tan, Yan Hong; Hanssen, Eric; Catimel, Bruno; Suarez, Cristian; Condron, Melanie; Angrisano, Fiona; NebI, Thomas; Kovar, David R.; Baum, Jake

    2014-01-01

    Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing. PMID:24371134

  2. Arabidopsis FIM5 decorates apical actin filaments and regulates their organization in the pollen tube

    PubMed Central

    Zhang, Meng; Zhang, Ruihui; Qu, Xiaolu; Huang, Shanjin

    2016-01-01

    The actin cytoskeleton is increasingly recognized as a major regulator of pollen tube growth. Actin filaments have distinct distribution patterns and dynamic properties within different regions of the pollen tube. Apical actin filaments are highly dynamic and crucial for pollen tube growth. However, how apical actin filaments are generated and properly constructed remains an open question. Here we showed that Arabidopsis fimbrin5 (FIM5) decorates filamentous structures throughout the entire tube but is apically concentrated. Apical actin structures are disorganized to different degrees in the pollen tubes of fim5 loss-of-function mutants. Further observations suggest that apical actin structures are not constructed properly because apical actin filaments cannot be maintained at the cortex of fim5 pollen tubes. Actin filaments appeared to be more curved in fim5 pollen tubes and this was confirmed by measurements showing that the convolutedness and the rate of change of convolutedness of actin filaments was significantly increased in fim5 pollen tubes. This suggests that the rigidity of the actin filaments may be compromised in fim5 pollen tubes. Further, the apical cell wall composition is altered, implying that tip-directed vesicle trafficking events are impaired in fim5 pollen tubes. Thus, we found that FIM5 decorates apical actin filaments and regulates their organization in order to drive polarized pollen tube growth. PMID:27117336

  3. CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor.

    PubMed

    Zhao, Shuangshuang; Jiang, Yuxiang; Zhao, Yang; Huang, Shanjin; Yuan, Ming; Zhao, Yanxiu; Guo, Yan

    2016-06-01

    The opening and closing of stomata are crucial for plant photosynthesis and transpiration. Actin filaments undergo dynamic reorganization during stomatal closure, but the underlying mechanism for this cytoskeletal reorganization remains largely unclear. In this study, we identified and characterized Arabidopsis thaliana casein kinase 1-like protein 2 (CKL2), which responds to abscisic acid (ABA) treatment and participates in ABA- and drought-induced stomatal closure. Although CKL2 does not bind to actin filaments directly and has no effect on actin assembly in vitro, it colocalizes with and stabilizes actin filaments in guard cells. Further investigation revealed that CKL2 physically interacts with and phosphorylates actin depolymerizing factor 4 (ADF4) and inhibits its activity in actin filament disassembly. During ABA-induced stomatal closure, deletion of CKL2 in Arabidopsis alters actin reorganization in stomata and renders stomatal closure less sensitive to ABA, whereas deletion of ADF4 impairs the disassembly of actin filaments and causes stomatal closure to be more sensitive to ABA Deletion of ADF4 in the ckl2 mutant partially recues its ABA-insensitive stomatal closure phenotype. Moreover, Arabidopsis ADFs from subclass I are targets of CKL2 in vitro. Thus, our results suggest that CKL2 regulates actin filament reorganization and stomatal closure mainly through phosphorylation of ADF. PMID:27268429

  4. Length regulation of mechanosensitive stereocilia depends on very slow actin dynamics and filament-severing proteins.

    PubMed

    Narayanan, Praveena; Chatterton, Paul; Ikeda, Akihiro; Ikeda, Sakae; Corey, David P; Ervasti, James M; Perrin, Benjamin J

    2015-01-01

    Auditory sensory hair cells depend on stereocilia with precisely regulated lengths to detect sound. Since stereocilia are primarily composed of crosslinked, parallel actin filaments, regulated actin dynamics are essential for controlling stereocilia length. Here we assessed stereocilia actin turnover by monitoring incorporation of inducibly expressed β-actin-GFP in adult mouse hair cells in vivo and by directly measuring β-actin-GFP turnover in explants. Stereocilia actin incorporation is remarkably slow and restricted to filament barbed ends in a small tip compartment, with minimal accumulation in the rest of the actin core. Shorter rows of stereocilia, which have mechanically gated ion channels, show more variable actin turnover than the tallest stereocilia, which lack channels. Finally, the proteins ADF and AIP1, which both mediate actin filament severing, contribute to stereocilia length maintenance. Altogether, the data support a model whereby stereocilia actin cores are largely static, with dynamic regulation at the tips to maintain a critical length. PMID:25897778

  5. Mechanism of Actin Network Attachment to Moving Membranes

    PubMed Central

    Co, Carl; Wong, Derek T.; Gierke, Sarah; Chang, Vicky; Taunton, Jack

    2007-01-01

    Summary Actin filament networks exert protrusive and attachment forces on membranes and thereby drive membrane deformation and movement. Here, we show that N-WASP WH2 domains play a previously unanticipated role in vesicle movement by transiently attaching actin filament barbed ends to the membrane. To dissect the attachment mechanism, we reconstituted the propulsive motility of lipid-coated glass beads using purified soluble proteins. N-WASP WH2 mutants assembled actin comet tails and initiated movement, but the comet tails catastrophically detached from the membrane. When presented on the surface of a lipid-coated bead, WH2 domains were sufficient to maintain comet tail attachment. In v-Src-transformed fibroblasts, N-WASP WH2 mutants were severely defective in the formation of circular podosome arrays. In addition to creating an attachment force, interactions between WH2 domains and barbed ends may locally amplify signals for dendritic actin nucleation. PMID:17350575

  6. SWAP-70 Identifies a Transitional Subset of Actin Filaments in Motile CellsV⃞

    PubMed Central

    Hilpelä, Pirta; Oberbanscheidt, Pia; Hahne, Penelope; Hund, Martin; Kalhammer, Georg; Small, J. Victor; Bähler, Martin

    2003-01-01

    Functionally different subsets of actin filament arrays contribute to cellular organization and motility. We report the identification of a novel subset of loose actin filament arrays through regulated association with the widely expressed protein SWAP-70. These loose actin filament arrays were commonly located behind protruding lamellipodia and membrane ruffles. Visualization of these loose actin filament arrays was dependent on lamellipodial protrusion and the binding of the SWAP-70 PH-domain to a 3′-phosphoinositide. SWAP-70 with a functional pleckstrin homology-domain lacking the C-terminal 60 residues was targeted to the area of the loose actin filament arrays, but it did not associate with actin filaments. The C-terminal 60 residues were sufficient for actin filament association, but they provided no specificity for the subset of loose actin filament arrays. These results identify SWAP-70 as a phosphoinositide 3-kinase signaling-dependent marker for a distinct, hitherto unrecognized, array of actin filaments. Overexpression of SWAP-70 altered the actin organization and lamellipodial morphology. These alterations were dependent on a proper subcellular targeting of SWAP-70. We propose that SWAP-70 regulates the actincytoskeletonasaneffectororadaptorproteininresponsetoagoniststimulatedphosphatidylinositol (3,4)-bisphosphate production and cell protrusion. PMID:12925760

  7. EXTRACTION AND ANALYSIS OF ACTIN NETWORKS BASED ON OPEN ACTIVE CONTOUR MODELS

    PubMed Central

    Xu, Ting; Li, Hongsheng; Shen, Tian; Ojkic, Nikola; Vavylonis, Dimitrios; Huang, Xiaolei

    2011-01-01

    Network structures formed by actin filaments are present in many kinds of fluorescence microscopy images. In order to quantify the conformations and dynamics of such actin filaments, we propose a fully automated method to extract actin networks from images and analyze network topology. The method handles well intersecting filaments and, to some extent, overlapping filaments. First we automatically initialize a large number of Stretching Open Active Contours (SOACs) from ridge points detected by searching for plus-to-minus sign changes in the gradient map of the image. These initial SOACs then elongate simultaneously along the bright center-lines of filaments by minimizing an energy function. During their evolution, they may merge or stop growing, thus forming a network that represents the topology of the filament ensemble. We further detect junction points in the network and break the SOACs at junctions to obtain “SOAC segments”. These segments are then re-grouped using a graph-cut spectral clustering method to represent the configuration of actin filaments. The proposed approach is generally applicable to extracting intersecting curvilinear structures in noisy images. We demonstrate its potential using two kinds of data: (1) actin filaments imaged by Total Internal Reflection Fluorescence Microscopy (TIRFM) in vitro; (2) actin cytoskeleton networks in fission yeast imaged by spinning disk confocal microscopy. PMID:21822463

  8. EXTRACTION AND ANALYSIS OF ACTIN NETWORKS BASED ON OPEN ACTIVE CONTOUR MODELS.

    PubMed

    Xu, Ting; Li, Hongsheng; Shen, Tian; Ojkic, Nikola; Vavylonis, Dimitrios; Huang, Xiaolei

    2011-03-30

    Network structures formed by actin filaments are present in many kinds of fluorescence microscopy images. In order to quantify the conformations and dynamics of such actin filaments, we propose a fully automated method to extract actin networks from images and analyze network topology. The method handles well intersecting filaments and, to some extent, overlapping filaments. First we automatically initialize a large number of Stretching Open Active Contours (SOACs) from ridge points detected by searching for plus-to-minus sign changes in the gradient map of the image. These initial SOACs then elongate simultaneously along the bright center-lines of filaments by minimizing an energy function. During their evolution, they may merge or stop growing, thus forming a network that represents the topology of the filament ensemble. We further detect junction points in the network and break the SOACs at junctions to obtain "SOAC segments". These segments are then re-grouped using a graph-cut spectral clustering method to represent the configuration of actin filaments. The proposed approach is generally applicable to extracting intersecting curvilinear structures in noisy images. We demonstrate its potential using two kinds of data: (1) actin filaments imaged by Total Internal Reflection Fluorescence Microscopy (TIRFM) in vitro; (2) actin cytoskeleton networks in fission yeast imaged by spinning disk confocal microscopy. PMID:21822463

  9. 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.

  10. mDia1 and formins: screw cap of the actin filament

    PubMed Central

    Mizuno, Hiroaki; Watanabe, Naoki

    2012-01-01

    Formin homology proteins (formins) are actin nucleation factors which remain bound to the growing barbed end and processively elongate actin filament (F-actin). Recently, we have demonstrated that a mammalian formin mDia1 rotates along the long-pitch helix of F-actin during processive elongation (helical rotation) by single-molecule fluorescence polarization. We have also shown processive depolymerization of mDia1-bound F-actin during which helical rotation was visualized. In the cell where F-actins are highly cross-linked, formins should rotate during filament elongation. Therefore, when formins are tightly anchored to cellular structures, formins may not elongate F-actin. Adversely, helical rotation of formins might affect the twist of F-actin. Formins could thus control actin elongation and regulate stability of cellular actin filaments through helical rotation. On the other hand, ADP-actin elongation at the mDia1-bound barbed end turned out to become decelerated by profilin, in marked contrast to its remarkably positive effect on mDia1-mediated ATP-actin elongation. This deceleration is caused by enhancement of the off-rate of ADP-actin. While mDia1 and profilin enhance the ADP-actin off-rate, they do not apparently increase the ADP-actin on-rate at the barbed end. These results imply that G-actin-bound ATP and its hydrolysis may be part of the acceleration mechanism of formin-mediated actin elongation.

  11. 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.

  12. Coronin 1C-free primary mouse fibroblasts exhibit robust rearrangements in the orientation of actin filaments, microtubules and intermediate filaments.

    PubMed

    Behrens, Juliane; Solga, Roxana; Ziemann, Anja; Rastetter, Raphael H; Berwanger, Carolin; Herrmann, Harald; Noegel, Angelika A; Clemen, Christoph S

    2016-08-01

    Coronin 1C is an established modulator of actin cytoskeleton dynamics. It has been shown to be involved in protrusion formation, cell migration and invasion. Here, we report the generation of primary fibroblasts from coronin 1C knock-out mice in order to investigate the impact of the loss of coronin 1C on cellular structural organisation. We demonstrate that the lack of coronin 1C not only affects the actin system, but also the microtubule and the vimentin intermediate filament networks. In particular, we show that the knock-out cells exhibit a reduced proliferation rate, impaired cell migration and protrusion formation as well as an aberrant subcellular localisation and function of mitochondria. Moreover, we demonstrate that coronin 1C specifically interacts with the non-α-helical amino-terminal domain ("head") of vimentin. Our data suggest that coronin 1C acts as a cytoskeletal integrator of actin filaments, microtubules and intermediate filaments. PMID:27178841

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  15. Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

    PubMed Central

    Uyeda, Taro Q. P.; Iwadate, Yoshiaki; Umeki, Nobuhisa; Nagasaki, Akira; Yumura, Shigehiko

    2011-01-01

    To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli. PMID:22022566

  16. Effect of tensile force on the mechanical behavior of actin filaments.

    PubMed

    Matsushita, Shinji; Inoue, Yasuhiro; Hojo, Masaki; Sokabe, Masahiro; Adachi, Taiji

    2011-06-01

    Actin filaments are the most abundant components of the cellular cytoskeleton, and play critical roles in various cellular functions such as migration, division and shape control. In these activities, mechanical tension causes structural changes in the double-helical structure of the actin filament, which is a key modulator of cytoskeletal reorganization. This study performed large-scale molecular dynamics (MD) and steered MD simulations to quantitatively analyze the effects of tensile force on the mechanical behavior of actin filaments. The results revealed that when a tensile force of 200pN was applied to a filament consisting of 14 actin subunits, the twist angle of the filament decreased by approximately 20°, corresponding to a rotation of approximately -2° per subunit, representing a critical structural change in actin filaments. Based on these structural changes, the variance in filament length and twist angle was found to decrease, leading to increases in extensional and torsional stiffness. Torsional stiffness increased significantly under the tensile condition, and the ratio of filament stiffness under tensile force to that under no external force increased significantly on longer temporal scales. The results obtained from this study contribute to the understanding of mechano-chemical interactions concerning actin dynamics, showing that increased tensile force in the filament prevents actin regulatory proteins from binding to the filament. PMID:21536289

  17. 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

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

    PubMed

    Takaine, Masak; Numata, Osamu; Nakano, Kentaro

    2009-10-21

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

  19. 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

  20. Capping of the barbed ends of actin filaments by a high-affinity profilin-actin complex.

    PubMed

    DiNubile, M J; Huang, S

    1997-01-01

    Profilin, a ubiquitous 12 to 15-kDa protein, serves many functions, including sequestering monomeric actin, accelerating nucleotide exchange on actin monomers, decreasing the critical concentration of the barbed end of actin filaments, and promoting actin polymerization when barbed ends are free. Most previous studies have focused on profilin itself rather than its complex with actin. A high-affinity profilin-actin complex (here called profilactin) can be isolated from a poly-(L)-proline (PLP) column by sequential elution with 3 M and 7 M urea. Profilactin inhibited the elongation rate of pyrenyl-G-actin from filament seeds in a concentration- and time-dependent manner. Much greater inhibition of elongation was observed with spectrin-F-actin than gelsolin-F-actin seeds, suggesting that the major effect of profilactin was due to capping the barbed ends of actin filaments. Its dissociation constant for binding to filament ends was 0.3 microM; the on- and off-rate constants were estimated to be 1.7 x 10(3) M-1 s-1 and 4.5 x 10(-4) s-1, respectively. Purified profilin (obtained by repetitive applications to a PLP column and assessed by silver-stained polyacylamide gels) did not slow the elongation rate of pyrenyl-G-actin from filament seeds. Capping protein could not be detected by Western blotting in the profilactin preparation, but low concentrations of gelsolin did contaminate our preparation. However, prolonged incubation with either calcium or EGTA did not affect capping activity, implying that contaminating gelsolin-actin complexes were not primarily responsible for the observed capping activity. Reapplication of the profilactin preparation to PLP-coupled Sepharose removed both profilin and actin and concurrently eliminated its capping activity. Profilactin that was reapplied to uncoupled Sepharose retained its capping activity. Phosphatidylinositol-4,5-bisphosphate (PIP2) was the most potent phosphoinositol in reducing the capping activity of profilactin

  1. Regional orientation of actin filaments in the pericanalicular cytoplasm of rat hepatocytes.

    PubMed

    Ishii, M; Washioka, H; Tonosaki, A; Toyota, T

    1991-12-01

    To elucidate how actin filaments participate in bile formation, polarity of actin filaments in the pericanalicular cytoplasm was determined with myosin subfragment 1 by transmission electron microscopy of ultrathin sections and deep-etching replicas. Densely concentrated actin filaments were identified around the bile canaliculi in the forms of microvillous core filaments, pericanalicular web filaments, and filaments on the junctional complex. They bound subfragment 1 to form double-helical strands on the deep-etching replica or typical arrowheads on the ultrathin section. All microvillous core filaments showed their arrowheads pointing basally, suggesting the molecular growth occurring at their apical ends. In contrast, filaments of the pericanalicular web, running in parallel to the cell surface, showed unfixed polarities as indicated by their arrowheads. Furthermore, neighboring filament pairs often showed opposite polarities, an alignment necessary for filament sliding. The junctional complex had filaments with arrowheads pointed mostly at the cell center with a small number in opposite direction. In addition, a group of sporadic filaments appeared to be installed to link to both the canalicular membrane and coated vesicles. Such regionally specialized actin filaments are considered inclusively to form a cytoskeletal system that is in charge of (a) maintenance of length of the microvilli, (b) contraction of the canalicular walls, and (c) translocation of coated vesicles in the pericanalicular cytoplasm. PMID:1955131

  2. Electrostatic Interactions Between the Bni1p Formin FH2 Domain and Actin Influence Actin Filament Nucleation

    PubMed Central

    Baker, Joseph L.; Courtemanche, Naomi; Parton, Daniel L.; McCullagh, Martin; Pollard, Thomas D.; Voth, Gregory A.

    2014-01-01

    SUMMARY Formins catalyze nucleation and growth of actin filaments. Here we study the structure and interactions of actin with the FH2 domain of budding yeast formin Bni1p. We built an all-atom model of the formin dimer on an Oda actin filament 7-mer and studied structural relaxation and inter-protein interactions by molecular dynamics simulations. These simulations produced a refined model for the FH2 dimer associated with the barbed end of the filament and revealed electrostatic interactions between the formin knob and actin target-binding cleft. Mutations of two formin residues contributing to these interactions (R1423N, K1467L or both) reduced the interaction energies between the proteins, and in coarse-grained simulations the formin lost more inter-protein contacts with an actin dimer than with an actin 7-mer. Biochemical experiments confirmed a strong influence of these mutations on Bni1p-mediated actin filament nucleation, but not elongation, suggesting that different interactions contribute to these two functions of formins. PMID:25482541

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

    PubMed Central

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

    2016-01-01

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

  4. 3D Filament Network Segmentation with Multiple Active Contours

    NASA Astrophysics Data System (ADS)

    Xu, Ting; Vavylonis, Dimitrios; Huang, Xiaolei

    2014-03-01

    Fluorescence microscopy is frequently used to study two and three dimensional network structures formed by cytoskeletal polymer fibers such as actin filaments and microtubules. 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 developed 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 TIRF Microscopy images, and 3D actin cable meshworks of live fission yeast cells imaged by spinning disk confocal microscopy.

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

    PubMed Central

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

    2016-01-01

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

  6. Mechanical Detection of a Long-Range Actin Network Emanating from a Biomimetic Cortex

    PubMed Central

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

    2014-01-01

    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. PMID:25140420

  7. Cofilin Increases the Bending Flexibility of Actin Filaments: Implications for Severing and Cell Mechanics

    PubMed Central

    McCullough, Brannon R.; Blanchoin, Laurent; Martiel, Jean-Louis; De La Cruz, Enrique M.

    2009-01-01

    We determined the flexural (bending) rigidities of actin and cofilactin filaments from a cosine correlation function analysis of their thermally driven, two-dimensional fluctuations in shape. The persistence length of actin filaments is 9.8 µm, corresponding to a flexural rigidity of 0.040 pN µm2. Cofilin binding lowers the persistence length ∼5-fold to a value of 2.2 µm and the filament flexural rigidity to 0.0091 pN µm2. That cofilin-decorated filaments are more flexible than native filaments despite an increased mass indicates that cofilin binding weakens and redistributes stabilizing subunit interactions of filaments. We favor a mechanism in which the increased flexibility of cofilin-decorated filaments results from the linked dissociation of filament-stabilizing ions and reorganization of actin subdomain 2 and as a consequence promotes severing due to a mechanical asymmetry. Knowledge of the effects of cofilin on actin filament bending mechanics, together with our previous analysis of torsional stiffness, provide a quantitative measure of the mechanical changes in actin filaments associated with cofilin binding, and suggest that the overall mechanical and force-producing properties of cells can be modulated by cofilin activity. PMID:18617188

  8. Unidirectional movement of an actin filament taking advantage of temperature gradients.

    PubMed

    Kawaguchi, Tomoaki; Honda, Hajime

    2007-01-01

    An actin filament with heat acceptors attached to its Cys374 residue in each actin monomer could move unidirectionally even under heat pulsation alone, while in the total absence of both ATP and myosin. The prime driver for the movement was temperature gradients operating between locally heated portions on an actin filament and its cooler surroundings. In this report, we investigated how the mitigation of the temperature gradients induces a unidirectional movement of an actin filament. We then observed the transversal fluctuations of the filament in response to heat pulsation and their transition into longitudinally unidirectional movement. The transition was significantly accelerated when Cys374 and Lys336 were simultaneously excited within an actin monomer. These results suggest that the mitigation of the temperature gradients within each actin monomer first went through the energy transformation to transversal fluctuations of the filament, and then followed by the transformation further down to longitudinal movements of the filament. The faster mitigation of temperature gradients within actin monomer helps build up the transition from the transversal to longitudinal movements of the filament by coordinating the interaction between the neighboring monomers. PMID:17030086

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

    PubMed

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

    2016-06-14

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

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

    PubMed

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

    2006-08-01

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

  11. Kinetics and thermodynamics of phalloidin binding to actin filaments from three divergent species.

    PubMed

    De La Cruz, E M; Pollard, T D

    1996-11-12

    We compared the kinetics and thermodynamics of rhodamine phalloidin binding to actin purified from rabbit skeletal muscle, Acanthamoeba castellanii, and Saccharomyces cerevisiae in 50 mM KCl, 1 mM MgCl2, and pH 7.0 buffer at 22 degrees C. Filaments of S. cerevisiae actin bind rhodamine phalloidin more weakly than Acanthamoeba and rabbit skeletal muscle actin filaments due to a more rapid dissociation rate in spite of a significantly faster association rate constant. The higher dissociation rate constant and lower binding affinity of rhodamine phalloidin for S. cerevisiae actin filaments provide a quantitative explanation for the inefficient staining of yeast actin filaments, compared with that of rabbit skeletal muscle actin filaments [Kron et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4466-4470]. The temperature dependence of the rate constants was interpreted according to transition state theory. There is a small enthalpic difference (delta H++) between the ground states and the transition state. Consequently, the free energy of activation (delta G++) for association and dissociation of rhodamine phalloidin is dominated by entropic changes (delta S++). At equilibrium, rhodamine phalloidin binding generates a positive entropy change (delta S0). The rates of rhodamine phalloidin binding are independent of the pH, ionic strength, and filament length. Rhodamine covalently bound decreases the association rate and affinity of phalloidin for actin. The association rate constant is low for both phalloidin and rhodamine phalloidin because the filaments must undergo conformational changes (i.e. "breathe") to expose the phalloidin binding site [De La Cruz, E. M., & Pollard, T. D. (1994) Biochemistry 33, 14387-14392]. Raising the solvent microviscosity, but not the macroviscosity, dampens these conformational fluctuations, and phalloidin binding kinetics are inhibited. Yeast actin filaments bind rhodamine phalloidin more rapidly, suggesting that perhaps they are more

  12. A microstructurally informed model for the mechanical response of three-dimensional actin networks

    PubMed Central

    KWON, R.Y.; LEW, A.J.; JACOBS, C.R.

    2008-01-01

    We propose a class of microstructurally informed models for the linear elastic mechanical behavior of cross-linked polymer networks such as the actin cytoskeleton. Salient features of the models include the possibility to represent anisotropic mechanical behavior resulting from anisotropic filament distributions, and a power-law scaling of the mechanical properties with the filament density. Mechanical models within the class are parameterized by seven different constants. We demonstrate a procedure for determining these constants using finite element models of three-dimensional actin networks. Actin filaments and cross-links were modeled as elastic rods, and the networks were constructed at physiological volume fractions and at the scale of an image voxel. We show the performance of the model in estimating the mechanical behavior of the networks over a wide range of filament densities and degrees of anisotropy. PMID:18568835

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

    PubMed Central

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

    2014-01-01

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

  14. 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.

  15. Arabidopsis Actin Depolymerizing Factor4 Modulates the Stochastic Dynamic Behavior of Actin Filaments in the Cortical Array of Epidermal Cells[C][W

    PubMed Central

    Henty, Jessica L.; Bledsoe, Samuel W.; Khurana, Parul; Meagher, Richard B.; Day, Brad; Blanchoin, Laurent; Staiger, Christopher J.

    2011-01-01

    Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells. PMID:22010035

  16. 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

  17. Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation

    PubMed Central

    Yamagishi, Yuka; Abe, Hiroshi

    2015-01-01

    We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation. PMID:26424802

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

    PubMed Central

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

    2001-01-01

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

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

    NASA Astrophysics Data System (ADS)

    van Oudenaarden, Alexander

    2000-03-01

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

  20. The Actin and Myosin Filaments of Human and Bovine Blood Platelets

    PubMed Central

    Zucker-Franklin, Dorothea; Grusky, George

    1972-01-01

    The contractility of platelets has been attributed to an actomyosin-like protein which has been well defined on a physicochemical basis. Moreover, platelets contain ±80 A filaments which resemble actin filaments in smooth muscle. Studies were undertaken on human and bovine platelets to better define the morphologic structures which may subserve this contractile function. In order to identify actin, the ability of the filaments to react with heavy meromyosin (HMM) was tested. Accordingly, platelets were glycerinated and treated with HMM. In addition, platelet actin was extracted, reacted with HMM, and examined by negative staining. In both instances typical arrowhead structures with clearly defined polarity and a periodicity of ±360 A formed. As is the case with purified muscle actin, the complexes were dissociable with Mg-ATP. The formation of myosin-like filaments was observed when osmotically shocked platelets were incubated with MgCl2 and excess ATP. These “thick” filaments measured 250-300 A in width, tapered at both ends and often occurred in clumps. They resembled aggregates of thick filaments described in contracted smooth muscle. Extraction of platelets by methods suitable for the demonstration of myosin showed filaments with an average length of 0.3 μ, a smooth shaft, and frayed or bulbous ends. These appeared identical to those seen in synthetically prepared myosin of striated muscle. It is suggested that the filaments described here represent the actin and myosin of platelets. Images PMID:4333023

  1. Isoform diversity in the Arp2/3 complex determines actin filament dynamics.

    PubMed

    Abella, Jasmine V G; Galloni, Chiara; Pernier, Julien; Barry, David J; Kjær, Svend; Carlier, Marie-France; Way, Michael

    2016-01-01

    The Arp2/3 complex consists of seven evolutionarily conserved subunits (Arp2, Arp3 and ARPC1-5) and plays an essential role in generating branched actin filament networks during many different cellular processes. In mammals, however, the ARPC1 and ARPC5 subunits are each encoded by two isoforms that are 67% identical. This raises the possibility that Arp2/3 complexes with different properties may exist.  We found that Arp2/3 complexes containing ARPC1B and ARPC5L are significantly better at promoting actin assembly than those with ARPC1A and ARPC5, both in cells and in vitro. Branched actin networks induced by complexes containing ARPC1B or ARPC5L are also disassembled ∼2-fold slower than those formed by their counterparts. This difference reflects the ability of cortactin to stabilize ARPC1B- and ARPC5L- but not ARPC1A- and ARPC5-containing complexes against coronin-mediated disassembly. Our observations demonstrate that the Arp2/3 complex in higher eukaryotes is actually a family of complexes with different properties. PMID:26655834

  2. Structural characterization of a capping protein interaction motif defines a family of actin filament regulators

    PubMed Central

    Hernandez-Valladares, Maria; Kim, Taekyung; Kannan, Balakrishnan; Tung, Alvin; Aguda, Adeleke H; Larsson, Mårten; Cooper, John A; Robinson, Robert C

    2011-01-01

    Capping protein (CP) regulates actin dynamics by binding the barbed ends of actin filaments. Removal of CP may be one means to harness actin polymerization for processes such as cell movement and endocytosis. Here we structurally and biochemically investigated a CP interaction (CPI) motif present in the otherwise unrelated proteins CARMIL and CD2AP. The CPI motif wraps around the stalk of the mushroom-shaped CP at a site distant from the actin-binding interface, which lies on the top of the mushroom cap. We propose that the CPI motif may act as an allosteric modulator, restricting CP to a low-affinity, filament-binding conformation. Structure-based sequence alignments extend the CPI motif–containing family to include CIN85, CKIP-1, CapZIP and a relatively uncharacterized protein, WASHCAP (FAM21). Peptides comprising these CPI motifs are able to inhibit CP and to uncap CP-bound actin filaments. PMID:20357771

  3. 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.

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

    PubMed Central

    Deshpande, Siddharth; Pfohl, Thomas

    2015-01-01

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

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

    PubMed

    Yang, Qing; Zhang, Xiao-Feng; Pollard, Thomas D; Forscher, Paul

    2012-06-25

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

  6. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases

    PubMed Central

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W.; Gelfand, Vladimir I.

    2014-01-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.—Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases. PMID:24652946

  7. Dendritic Actin Filament Nucleation Causes Traveling Waves and Patches

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders E.

    2010-06-01

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

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

    PubMed

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

    2016-06-01

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

  9. Dynamics of Actin Cables in Polarized Growth of the Filamentous Fungus Aspergillus nidulans

    PubMed Central

    Bergs, Anna; Ishitsuka, Yuji; Evangelinos, Minoas; Nienhaus, G. U.; Takeshita, Norio

    2016-01-01

    Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although, specific marker proteins have been developed to visualize actin cables in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here, we observed actin cables using tropomyosin (TpmA) and Lifeact fused to fluorescent proteins in living Aspergillus nidulans hyphae and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules. PMID:27242709

  10. Dynamics of Actin Cables in Polarized Growth of the Filamentous Fungus Aspergillus nidulans.

    PubMed

    Bergs, Anna; Ishitsuka, Yuji; Evangelinos, Minoas; Nienhaus, G U; Takeshita, Norio

    2016-01-01

    Highly polarized growth of filamentous fungi requires a continuous supply of proteins and lipids to the hyphal tip. This transport is managed by vesicle trafficking via the actin and microtubule cytoskeletons and their associated motor proteins. Particularly, actin cables originating from the hyphal tip are essential for hyphal growth. Although, specific marker proteins have been developed to visualize actin cables in filamentous fungi, the exact organization and dynamics of actin cables has remained elusive. Here, we observed actin cables using tropomyosin (TpmA) and Lifeact fused to fluorescent proteins in living Aspergillus nidulans hyphae and studied the dynamics and regulation. GFP tagged TpmA visualized dynamic actin cables formed from the hyphal tip with cycles of elongation and shrinkage. The elongation and shrinkage rates of actin cables were similar and approximately 0.6 μm/s. Comparison of actin markers revealed that high concentrations of Lifeact reduced actin dynamics. Simultaneous visualization of actin cables and microtubules suggests temporally and spatially coordinated polymerization and depolymerization between the two cytoskeletons. Our results provide new insights into the molecular mechanism of ordered polarized growth regulated by actin cables and microtubules. PMID:27242709

  11. Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits.

    PubMed

    Hocky, Glen M; Baker, Joseph L; Bradley, Michael J; Sinitskiy, Anton V; De La Cruz, Enrique M; Voth, Gregory A

    2016-05-26

    Ions regulate the assembly and mechanical properties of actin filaments. Recent work using structural bioinformatics and site-specific mutagenesis favors the existence of two discrete and specific divalent cation binding sites on actin filaments, positioned in the long axis between actin subunits. Cation binding at one site drives polymerization, while the other modulates filament stiffness and plays a role in filament severing by the regulatory protein, cofilin. Existing structural methods have not been able to resolve filament-associated cations, and so in this work we turn to molecular dynamics simulations to suggest a candidate binding pocket geometry for each site and to elucidate the mechanism by which occupancy of the "stiffness site" affects filament mechanical properties. Incorporating a magnesium ion in the "polymerization site" does not seem to require any large-scale change to an actin subunit's conformation. Binding of a magnesium ion in the "stiffness site" adheres the actin DNase-binding loop (D-loop) to its long-axis neighbor, which increases the filament torsional stiffness and bending persistence length. Our analysis shows that bound D-loops occupy a smaller region of accessible conformational space. Cation occupancy buries key conserved residues of the D-loop, restricting accessibility to regulatory proteins and enzymes that target these amino acids. PMID:27146246

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

    PubMed

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

    2016-01-14

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

  13. 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

  14. Disruption of the keratin filament network during epithelial cell division.

    PubMed Central

    Lane, E B; Goodman, S L; Trejdosiewicz, L K

    1982-01-01

    The behaviour of keratin filaments during cell division was examined in a wide range of epithelial lines from several species. Almost half of them show keratin disruption as described previously: by immunofluorescence, filaments are replaced during mitosis by a 'speckled' pattern of discrete cytoplasmic dots. In the electron microscope these ' speckles ' are seen as granules around the cell periphery, just below the actin cortical mesh, with no detectable 10 nm filament structure inside them and no keratin filament bundles in the rest of the cytoplasm. A time course of the filament reorganization was constructed from double immunofluorescence data; filaments are disrupted in prophase, and the filament network is intact again by cytokinesis. The phenomenon is restricted to cells rich in keratin filaments, such as keratinocytes; it is unrelated to the co-existence of vimentin in many of these cells, and vimentin is generally maintained as filaments while the keratin is restructured. Some resistance to the effect may be conferred by an extended cycle time. Filament reorganization takes place within minutes, so that a reversible mechanism seems more likely than one involving de novo protein synthesis, at this metabolically quiet stage of the cell cycle. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6202508

  15. Regulation of water flow by actin-binding protein-induced actin gelatin.

    PubMed Central

    Ito, T; Suzuki, A; Stossel, T P

    1992-01-01

    Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by actin filaments corresponds to the gel point measured viscometrically and because gelsolin, which noncovalently severs actin filaments, solates actin gels and restores water flow in a solution of actin cross-linked by ABP. Since ABP gels actin filaments in the periphery of many eukaryotic cells, such actin networks may contribute to physiological cell volume regulation. PMID:1318095

  16. Broken Detailed Balance of Filament Dynamics in Active Networks

    NASA Astrophysics Data System (ADS)

    Gladrow, J.; Fakhri, N.; MacKintosh, F. C.; Schmidt, C. F.; Broedersz, C. P.

    2016-06-01

    Myosin motor proteins drive vigorous steady-state fluctuations in the actin cytoskeleton of cells. Endogenous embedded semiflexible filaments such as microtubules, or added filaments such as single-walled carbon nanotubes are used as novel tools to noninvasively track equilibrium and nonequilibrium fluctuations in such biopolymer networks. Here, we analytically calculate shape fluctuations of semiflexible probe filaments in a viscoelastic environment, driven out of equilibrium by motor activity. Transverse bending fluctuations of the probe filaments can be decomposed into dynamic normal modes. We find that these modes no longer evolve independently under nonequilibrium driving. This effective mode coupling results in nonzero circulatory currents in a conformational phase space, reflecting a violation of detailed balance. We present predictions for the characteristic frequencies associated with these currents and investigate how the temporal signatures of motor activity determine mode correlations, which we find to be consistent with recent experiments on microtubules embedded in cytoskeletal networks.

  17. Broken Detailed Balance of Filament Dynamics in Active Networks.

    PubMed

    Gladrow, J; Fakhri, N; MacKintosh, F C; Schmidt, C F; Broedersz, C P

    2016-06-17

    Myosin motor proteins drive vigorous steady-state fluctuations in the actin cytoskeleton of cells. Endogenous embedded semiflexible filaments such as microtubules, or added filaments such as single-walled carbon nanotubes are used as novel tools to noninvasively track equilibrium and nonequilibrium fluctuations in such biopolymer networks. Here, we analytically calculate shape fluctuations of semiflexible probe filaments in a viscoelastic environment, driven out of equilibrium by motor activity. Transverse bending fluctuations of the probe filaments can be decomposed into dynamic normal modes. We find that these modes no longer evolve independently under nonequilibrium driving. This effective mode coupling results in nonzero circulatory currents in a conformational phase space, reflecting a violation of detailed balance. We present predictions for the characteristic frequencies associated with these currents and investigate how the temporal signatures of motor activity determine mode correlations, which we find to be consistent with recent experiments on microtubules embedded in cytoskeletal networks. PMID:27367410

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

    NASA Astrophysics Data System (ADS)

    Vicker, Michael G.

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

  19. The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

    PubMed Central

    Bane, Kartik S.; Singer, Mirko; Reinig, Miriam; Klug, Dennis; Heiss, Kirsten; Baum, Jake; Mueller, Ann-Kristin; Frischknecht, Friedrich

    2016-01-01

    Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics. PMID:27409081

  20. An Actin Filament Population Defined by the Tropomyosin Tpm3.1 Regulates Glucose Uptake

    PubMed Central

    Kee, Anthony J.; Yang, Lingyan; Lucas, Christine A.; Greenberg, Michael J.; Martel, Nick; Leong, Gary M.; Hughes, William E.; Cooney, Gregory J.; James, David E.; Ostap, E. Michael; Han, Weiping; Gunning, Peter W.; Hardeman, Edna C.

    2016-01-01

    Actin has an ill-defined role in the trafficking of GLUT4 glucose transporter vesicles to the plasma membrane (PM). We have identified novel actin filaments defined by the tropomyosin Tpm3.1 at glucose uptake sites in white adipose tissue (WAT) and skeletal muscle. In Tpm 3.1-overexpressing mice, insulin-stimulated glucose uptake was increased; while Tpm3.1-null mice they were more sensitive to the impact of high-fat diet on glucose uptake. Inhibition of Tpm3.1 function in 3T3-L1 adipocytes abrogates insulin-stimulated GLUT4 translocation and glucose uptake. In WAT, the amount of filamentous actin is determined by Tpm3.1 levels and is paralleled by changes in exocyst component (sec8) and Myo1c levels. In adipocytes, Tpm3.1 localizes with MyoIIA, but not Myo1c, and it inhibits Myo1c binding to actin. We propose that Tpm3.1 determines the amount of cortical actin that can engage MyoIIA and generate contractile force, and in parallel limits the interaction of Myo1c with actin filaments. The balance between these actin filament populations may determine the efficiency of movement and/or fusion of GLUT4 vesicles with the PM. PMID:25783006

  1. The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model.

    PubMed

    Bremer, A; Millonig, R C; Sütterlin, R; Engel, A; Pollard, T D; Aebi, U

    1991-11-01

    Three-dimensional (3-D) helical reconstructions computed from electron micrographs of negatively stained dispersed F-actin filaments invariably revealed two uninterrupted columns of mass forming the "backbone" of the double-helical filament. The contact between neighboring subunits along the thus defined two long-pitch helical strands was spatially conserved and of high mass density, while the intersubunit contact between them was of lower mass density and varied among reconstructions. In contrast, phalloidinstabilized F-actin filaments displayed higher and spatially more conserved mass density between the two long-pitch helical strands, suggesting that this bicyclic hepta-peptide toxin strengthens the intersubunit contact between the two strands. Consistent with this distinct intersubunit bonding pattern, the two long-pitch helical strands of unstabilized filaments were sometimes observed separated from each other over a distance of two to six subunits, suggesting that the intrastrand intersubunit contact is also physically stronger than the interstrand contact. The resolution of the filament reconstructions, extending to 2.5 nm axially and radially, enabled us to reproducibly "cut out" the F-actin subunit which measured 5.5 nm axially by 6.0 nm tangentially by 3.2 nm radially. The subunit is distinctly polar with a massive "base" pointing towards the "barbed" end of the filament, and a slender "tip" defining its "pointed" end (i.e., relative to the "arrowhead" pattern revealed after stoichiometric decoration of the filaments with myosin subfragment 1). Concavities running approximately parallel to the filament axis both on the inner and outer face of the subunit define a distinct cleft separating the subunit into two domains of similar size: an inner domain confined to radii less than or equal to 2.5-nm forms the uninterrupted backbone of the two long-pitch helical strands, and an outer domain placed at radii of 2-5-nm protrudes radially and thus predominantly

  2. Antibodies covalently immobilized on actin filaments for fast myosin driven analyte transport.

    PubMed

    Kumar, Saroj; ten Siethoff, Lasse; Persson, Malin; Lard, Mercy; te Kronnie, Geertruy; Linke, Heiner; Månsson, Alf

    2012-01-01

    Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm(-1)). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments. PMID:23056279

  3. Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport

    PubMed Central

    Kumar, Saroj; ten Siethoff, Lasse; Persson, Malin; Lard, Mercy; te Kronnie, Geertruy; Linke, Heiner; Månsson, Alf

    2012-01-01

    Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm−1). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments. PMID:23056279

  4. Three-dimensional architecture of actin filaments in Listeria monocytogenes comet tails

    PubMed Central

    Jasnin, Marion; Asano, Shoh; Gouin, Edith; Hegerl, Reiner; Plitzko, Jürgen M.; Villa, Elizabeth; Cossart, Pascale; Baumeister, Wolfgang

    2013-01-01

    The intracellular bacterial pathogen Listeria monocytogenes is capable of remodelling the actin cytoskeleton of its host cells such that “comet tails” are assembled powering its movement within cells and enabling cell-to-cell spread. We used cryo-electron tomography to visualize the 3D structure of the comet tails in situ at the level of individual filaments. We have performed a quantitative analysis of their supramolecular architecture revealing the existence of bundles of nearly parallel hexagonally packed filaments with spacings of 12–13 nm. Similar configurations were observed in stress fibers and filopodia, suggesting that nanoscopic bundles are a generic feature of actin filament assemblies involved in motility; presumably, they provide the necessary stiffness. We propose a mechanism for the initiation of comet tail assembly and two scenarios that occur either independently or in concert for the ensuing actin-based motility, both emphasizing the role of filament bundling. PMID:24306931

  5. Molecular dynamics simulation of a myosin subfragment-1 docking with an actin filament.

    PubMed

    Masuda, Tadashi

    2013-09-01

    Myosins are typical molecular motor proteins, which convert the chemical energy of ATP into mechanical work. The fundamental mechanism of this energy conversion is still unknown. To explain the experimental results observed in molecular motors, Masuda has proposed a theory called the "Driven by Detachment (DbD)" mechanism for the working principle of myosins. Based on this theory, the energy used during the power stroke of the myosins originates from the attractive force between a detached myosin head and an actin filament, and does not directly arise from the energy of ATP. According to this theory, every step in the myosin working process may be reproduced by molecular dynamics (MD) simulations, except for the ATP hydrolysis step. Therefore, MD simulations were conducted to reproduce the docking process of a myosin subfragment-1 (S1) against an actin filament. A myosin S1 directed toward the barbed end of an actin filament was placed at three different positions by shifting it away from the filament axis. After 30 ns of MD simulations, in three cases out of ten trials on average, the myosin made a close contact with two actin monomers by changing the positions and the orientation of both the myosin and the actin as predicted in previous studies. Once the docking was achieved, the distance between the myosin and the actin showed smaller fluctuations, indicating that the docking is stable over time. If the docking was not achieved, the myosin moved randomly around the initial position or moved away from the actin filament. MD simulations thus successfully reproduced the docking of a myosin S1 with an actin filament. By extending the similar MD simulations to the other steps of the myosin working process, the validity of the DbD theory may be computationally demonstrated. PMID:23791790

  6. Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells.

    PubMed

    Yamanaka, Daisuke; Akama, Takeshi; Chida, Kazuhiro; Minami, Shiro; Ito, Koichi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830-840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250-1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and plays

  7. Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells

    PubMed Central

    Yamanaka, Daisuke; Akama, Takeshi; Chida, Kazuhiro; Minami, Shiro; Ito, Koichi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830–840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250–1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and

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

    PubMed

    Lappalainen, Pekka

    2016-08-15

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

  9. Synthetic Chondramide A Analogues Stabilize Filamentous Actin and Block Invasion by Toxoplasma gondii

    PubMed Central

    2013-01-01

    Apicomplexan parasites such as Toxoplasma gondii rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of T. gondii in vitro. Structural modeling of chondramide A (2) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (2b–k) with substitutions in the β-tyrosine moiety blocked parasite growth on host cell monolayers with EC50 values that ranged from 0.3 to 1.3 μM. In vitro polymerization assays using highly purified recombinant actin from T. gondii verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites. PMID:24020843

  10. Probing the Flexibility of Tropomyosin and Its Binding to Filamentous Actin Using Molecular Dynamics Simulations

    PubMed Central

    Zheng, Wenjun; Barua, Bipasha; Hitchcock-DeGregori, Sarah E.

    2013-01-01

    Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments. PMID:24138864

  11. Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin.

    PubMed Central

    Kron, S J; Drubin, D G; Botstein, D; Spudich, J A

    1992-01-01

    The yeast Saccharomyces cerevisiae has been used to study the function of components of the actin cytoskeleton in vivo, mainly because it is easy to derive and characterize mutations affecting these proteins. In contrast, biochemical studies have generally used proteins derived from higher eukaryotes. We have devised a simple procedure to prepare, in high yield, homogeneous native actin from wild-type and act1 mutant yeast. Using intensified video fluorescence microscopy, we found that actin filaments polymerized from these preparations exhibit ATP-dependent sliding movement over surfaces coated with rabbit skeletal muscle myosin. The rates of sliding movement of the wild-type and mutant yeast actins were each about half that of rabbit skeletal muscle actin under similar conditions. We conclude that over the large evolutionary distance between yeast and mammals there has been significant conservation of actin function, specifically the ability to be moved by interaction with myosin. Images PMID:1533933

  12. Aluminum Induces Rigor within the Actin Network of Soybean Cells.

    PubMed Central

    Grabski, S.; Schindler, M.

    1995-01-01

    Aluminum is toxic to both plants and animals. Root growth and pollen-tube extension are inhibited after aluminum stress in acidic environments. Incubation of cultured neurons with aluminum results in the formation of neurofibrillar tangles reminiscent of the neural pathology observed in Alzheimer's disease. The present communication demonstrates that aluminum induces a rapid and dramatic increase in the rigidity of the actin network in soybean (Glycine max) root cells. This rigidity can be prevented by either co-incubation with sodium fluoride or magnesium, or pretreatment with cytochalasin D. It is proposed that the growth-inhibitory activity and cytotoxicity of aluminum in plants may be a consequence of a global rigor that is induced within the actin network. This rigor may result from the formation of nonhydrolyzable [Al3+-ADP] or [Al3+-ATP] complexes whose binding to actin/myosin can modify contraction. Additionally, Al3+-mediated interference with the normal kinetics of F-actin filament assembly/disassembly could precipitate subsequent disorganization of associated cytoskeletal structures and promote altered expression of cytoskeletal proteins. PMID:12228515

  13. 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.

  14. 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. PMID:19883801

  15. Transport of ER vesicles on actin filaments in neurons by myosin V.

    PubMed

    Tabb, J S; Molyneaux, B J; Cohen, D L; Kuznetsov, S A; Langford, G M

    1998-11-01

    Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was tightly bound and not extracted during isolation. These vesicles were identified as smooth ER by use of an antibody to an ER-resident protein, ERcalcistorin/protein disulfide isomerase (EcaSt/PDI). Furthermore, an antibody to squid myosin V was used in immunogold EM studies to show that myosin V localized to these vesicles. The antibody was generated to a squid brain myosin (p196) that was classified as myosin V based on comparisons of amino acid sequences of tryptic peptides of this myosin with those of other known members of the myosin V family. Dual labeling with the squid myosin V antibody and a kinesin heavy chain antibody showed that the two motors colocalized on the same vesicles. Finally, antibody inhibition experiments were performed with two myosin V-specific antibodies to show that myosin V motor activity is required for transport of vesicles on actin filaments in axoplasm. One antibody was made to a peptide in the globular tail domain and the other to the globular head fragment of myosin V. Both antibodies inhibited vesicle transport on actin filaments by greater than 90% compared to controls. These studies provide the first direct evidence that ER vesicles are transported on actin filaments by myosin V. These data confirm the role of actin filaments in fast axonal transport and provide support for

  16. Orientational order of the lamellipodial actin network as demonstrated in living motile cells.

    PubMed

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

    2003-11-01

    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

  17. 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

  18. Formation of an actin-like filament concurrent with the enzymatic synthesis of inorganic polyphosphate

    PubMed Central

    Gómez-García, María R.; Kornberg, Arthur

    2004-01-01

    Inorganic polyphosphate (poly P), a chain of hundreds of phosphate residues linked by ATP-like bonds, is found in every cell in nature and is commonly produced from ATP by poly P kinases (e.g., PPK1). Dictyostelium discoideum, the social slime mold, possesses a PPK activity (DdPPK1) with sequence similarity to bacterial PPKs. We find here a previously unrecognized PPK (DdPPK2) in D. discoideum with the sequences and properties of actin-related proteins (Arps) that are similar to muscle actins in size, properties, and globular-filamentous structural transitions. Significantly, the unique actin inhibitors, phalloidin and DNase I, also inhibit synthesis of poly P by DdPPK2. Thus, this particular Arp complex is an enzyme that can polymerize into an actin-like filament concurrent with its synthesis of a poly P chain in a fully reversible reaction. PMID:15496465

  19. Single-filament kinetic studies provide novel insights into regulation of actin-based motility

    PubMed Central

    Shekhar, Shashank; Carlier, Marie-France

    2016-01-01

    Polarized assembly of actin filaments forms the basis of actin-based motility and is regulated both spatially and temporally. Cells use a variety of mechanisms by which intrinsically slower processes are accelerated, and faster ones decelerated, to match rates observed in vivo. Here we discuss how kinetic studies of individual reactions and cycles that drive actin remodeling have provided a mechanistic and quantitative understanding of such processes. We specifically consider key barbed-end regulators such as capping protein and formins as illustrative examples. We compare and contrast different kinetic approaches, such as the traditional pyrene-polymerization bulk assays, as well as more recently developed single-filament and single-molecule imaging approaches. Recent development of novel biophysical methods for sensing and applying forces will in future allow us to address the very important relationship between mechanical stimulus and kinetics of actin-based motility. PMID:26715420

  20. Hierarchical Cross-linked F-actin Networks: Understanding Structure and Assembly

    NASA Astrophysics Data System (ADS)

    Hirst, Linda; Nguyen, Lam

    2009-11-01

    The protein, F-actin provides us with an interesting system in which to investigate the assembly properties of semi-flexible filaments in the presence of cross-linkers. Recently it was observed that F-actin, in the presence of the cross-linker alpha-actinin at high molar ratios will generate a novel hierarchical network of filament bundles. We investigate this system using coarse-grained molecular dynamics (MD) simulation, confocal microscopy and x-ray scattering. We have studied the F-actin/alpha-actinin system in detail with different actin conc. (C) and alpha-actinin/actin molar ratios (gamma). Confocal microscopy and analysis shows that the assembled systems fall into one of 3 phases depending on C and gamma: (1) loosely connected network of F-actin and bundles, (2) loosely connected network of dense domains and (3) uniform network of bundles. This can be explained and replicated using MD simulation. We have also examined different types of cross-linkers to represent the proteins, fascin and filamin. Results show that phase formation is related to the flexibility in binding between F-actin and cross-linkers. This degree of freedom, possible with longer cross-linkers allows the formation of branch points and thus bundle networks.

  1. Filamentous actin is a substrate for protealysin, a metalloprotease of invasive Serratia proteamaculans.

    PubMed

    Tsaplina, Olga; Efremova, Tatiana; Demidyuk, Ilya; Khaitlina, Sofia

    2012-01-01

    Homologous bacterial metalloproteases ECP32/grimelysin from Serratia grimesii and protealysin from Serratia proteamaculans are involved in the invasion of the nonpathogenic bacteria in eukaryotic cells and are suggested to translocate into the cytoplasm [Bozhokina ES et al. (2011) Cell Biol Int35, 111-118]. The proteases have been characterized as actin-hydrolyzing enzymes with a narrow specificity toward intact cell proteins. However, cleavage of filamentous actin (F-actin) (i.e. the main actin species in the cell) and the properties of the cleaved F-actin have not been investigated previously. In the present study, we revealed the presence of protealysin in the cytoplasm of 3T3-SV40 cells infected with S. proteamaculans or recombinant Escherichia coli expressing the protealysin gene. We also show for the first time that purified protealysin and the lysates of the recombinant E. coli producing protealysin cleave 20-40% of F-actin. Cleavage limited predominantly to the bond Gly42-Val43 efficiently increases the steady-state ATPase activity (dynamics) of F-actin. abolishes this effect and promotes the nucleation of protealysin-cleaved Mg-globular-actin even in the absence of 0.1 m KCl, most likely as a result of the stabilization of lateral intermonomer contacts of actin subunits. The results obtained in the present study suggest that F-actin can be a target for protealysin upon its translocation into the host cell. PMID:22077798

  2. Novel actin filaments from Bacillus thuringiensis form nanotubules for plasmid DNA segregation

    PubMed Central

    Jiang, Shimin; Narita, Akihiro; Popp, David; Ghoshdastider, Umesh; Lee, Lin Jie; Srinivasan, Ramanujam; Balasubramanian, Mohan K.; Oda, Toshiro; Koh, Fujiet; Larsson, Mårten; Robinson, Robert C.

    2016-01-01

    Here we report the discovery of a bacterial DNA-segregating actin-like protein (BtParM) from Bacillus thuringiensis, which forms novel antiparallel, two-stranded, supercoiled, nonpolar helical filaments, as determined by electron microscopy. The BtParM filament features of supercoiling and forming antiparallel double-strands are unique within the actin fold superfamily, and entirely different to the straight, double-stranded, polar helical filaments of all other known ParMs and of eukaryotic F-actin. The BtParM polymers show dynamic assembly and subsequent disassembly in the presence of ATP. BtParR, the DNA-BtParM linking protein, stimulated ATP hydrolysis/phosphate release by BtParM and paired two supercoiled BtParM filaments to form a cylinder, comprised of four strands with inner and outer diameters of 57 Å and 145 Å, respectively. Thus, in this prokaryote, the actin fold has evolved to produce a filament system with comparable features to the eukaryotic chromosome-segregating microtubule. PMID:26873105

  3. Capping Protein Increases the Rate of Actin-based Motility by Promoting Filament Nucleation by the Arp2/3 Complex

    PubMed Central

    Akin, Orkun; Mullins, R. Dyche

    2008-01-01

    Summary Capping protein is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between capping protein and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex, and not by increasing the rate of filament elongation as previously suggested. One consequence of this coupling between capping and nucleation is that, while the rate of motility depends strongly on the concentration of capping protein and Arp2/3, the net rate of actin assembly is insensitive to changes in either factor. By reorganizing their architecture, dendritic actin networks harness the same assembly kinetics to drive different rates of motility. PMID:18510928

  4. Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments[W

    PubMed Central

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca2+, in vitro. Analysis of a mutant that bears a point mutation at the Ca2+ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca2+ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth. PMID:24424096

  5. Analysis of flexural rigidity of actin filaments propelled by surface adsorbed myosin motors.

    PubMed

    Bengtsson, Elina; Persson, Malin; Månsson, Alf

    2013-11-01

    Actin filaments are central components of the cytoskeleton and the contractile machinery of muscle. The filaments are known to exist in a range of conformational states presumably with different flexural rigidity and thereby different persistence lengths. Our results analyze the approaches proposed previously to measure the persistence length from the statistics of the winding paths of actin filaments that are propelled by surface-adsorbed myosin motor fragments in the in vitro motility assay. Our results suggest that the persistence length of heavy meromyosin propelled actin filaments can be estimated with high accuracy and reproducibility using this approach provided that: (1) the in vitro motility assay experiments are designed to prevent bias in filament sliding directions, (2) at least 200 independent filament paths are studied, (3) the ratio between the sliding distance between measurements and the camera pixel-size is between 4 and 12, (4) the sliding distances between measurements is less than 50% of the expected persistence length, and (5) an appropriate cut-off value is chosen to exclude abrupt large angular changes in sliding direction that are complications, e.g., due to the presence of rigor heads. If the above precautions are taken the described method should be a useful routine part of in vitro motility assays thus expanding the amount of information to be gained from these. PMID:24039103

  6. 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

  7. Velocity of movement of actin filaments in in vitro motility assay. Measured by fluorescence correlation spectroscopy.

    PubMed Central

    Borejdo, J; Burlacu, S

    1992-01-01

    We have measured the velocity of actin filaments in in vitro motility assay by fluorescence correlation spectroscopy. In this method, one measures fluctuations in the number of filaments in an open sample volume. The number of filaments was calculated from measurements of fluorescence of rhodamine-phalloidin bound to F-actin. Sample volume was defined by a diaphragm placed in front of the photomultiplier. Fluctuations arise when actin filaments enter and leave the sample volume due to translations driven by mechanochemical interactions with myosin heads which are immobilized on a glass surface. The average velocity of the translation of filaments determined by the correlation method, (Vc), was equal to the diameter of the diaphragm divided by the half-time of the relaxation of fluctuations. The average number of moving filaments determined by correlation method, (Nc), was inversely proportional to the relative fluctuations. By the fluctuation method it was possible to determine the average velocity of over 800 moving filaments in less than 4 min. There was good agreement between (Vc) and (Nc) and the average velocity and the average number of moving filaments determined manually. To be able to apply correlation measurements to an experimental problem, neither (Vc) nor (Nc) must depend on the position of observation of filaments. We first confirmed that this was indeed the case. We then applied the method to investigate the dependence of motility on the ATPase activity of myosin heads. ATPase activity was varied by mixing intact heads with heads which were labeled with different thiol reagents. It was found that the motion was drastically influenced by the reagent used for modification. When the reagent was N-ethyl-maleimide, 1.5% modification was sufficient to completely inhibit the motion. When the reagent was 5-iodoacetamidofluorescein, motion declined hyperbolically with the fraction of modified heads. Images FIGURE 2 FIGURE 4 FIGURE 11 PMID:1534696

  8. Isoforms of α-Actinin from Cardiac, Smooth, and Skeletal Muscle Form Polar Arrays of Actin Filaments

    PubMed Central

    Taylor, Kenneth A.; Taylor, Dianne W.; Schachat, Fred

    2000-01-01

    We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by α-actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using α-actinin isoforms isolated from the three types of vertebrate muscle, i.e., cardiac, skeletal, and smooth, we have observed almost exclusively cross-linking between polar arrays of filaments, i.e., actin filaments with their plus ends oriented in the same direction. One type of bundle can be classified as an Archimedian spiral consisting of a single actin filament that spirals inward as the filament grows and the bundle is formed. These spirals have a consistent hand and grow to a limiting internal diameter of 0.4–0.7 μm, where the filaments appear to break and spiral formation ceases. These results, using isoforms usually characterized as cross-linkers of bipolar actin filament bundles, suggest that α-actinin is capable of cross-linking actin filaments in any orientation. Formation of specifically bipolar or polar filament arrays cross-linked by α-actinin may require additional factors that either determine the filament orientation or restrict the cross-linking capabilities of α-actinin. PMID:10791977

  9. Actin filament tracking based on particle filters and stretching open active contour models.

    PubMed

    Li, Hongsheng; Shen, Tian; Vavylonis, Dimitrios; Huang, Xiaolei

    2009-01-01

    We introduce a novel algorithm for actin filament tracking and elongation measurement. Particle Filters (PF) and Stretching Open Active Contours (SOAC) work cooperatively to simplify the modeling of PF in a one-dimensional state space while naturally integrating filament body constraints to tip estimation. Our algorithm reduces the PF state spaces to one-dimensional spaces by tracking filament bodies using SOAC and probabilistically estimating tip locations along the curve length of SOACs. Experimental evaluation on TIRFM image sequences with very low SNRs demonstrates the accuracy and robustness of this approach. PMID:20426170

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  12. Movement of scallop myosin on Nitella actin filaments: regulation by calcium.

    PubMed Central

    Vale, R D; Szent-Gyorgyi, A G; Sheetz, M P

    1984-01-01

    In order to determine if Ca2+ regulates scallop myosin movement on actin, we have measured motility of scallop myosin along actin filaments using a direct visual assay. This procedure consists of covalently linking myosin to 1-micron beads and pipetting them onto a parallel array of actin filaments located on the cytoplasmic face of a Nitella internodal cell. In the absence of Ca2+, scallop myosin-coated beads exhibit no directed motion; however, in the presence of pCa2+ of greater than 5.84, these beads undergo linear translocations with average velocities of 2.0 micron/s. This Ca2+ -sensitive motility requires the presence of regulatory light chains on the scallop myosin. Removal of regulatory light chains with 10 mM EDTA produces a "desensitized" myosin, no longer sensitive to Ca2+, which moves at rates of 0.09-0.3 micron in the presence or absence of Ca2+. Readdition of regulatory light chains to preparations of desensitized myosin once again confers Ca2+-sensitive motility. The Ca2+ dependence of scallop-myosin motility shows a sharp transition, consistent with the Ca2+ activation sensitivity of the actin-activated ATPase. Furthermore, relative rates of movement of calcium-regulated myosins from various molluscan species are consistent with their respective rates of ATP hydrolysis. Thus, myosin motility along actin filaments provides a sensitive and direct assay of myosin activity and is suitable for studying myosin regulation. PMID:6238334

  13. Arabidopsis VILLIN5, an Actin Filament Bundling and Severing Protein, Is Necessary for Normal Pollen Tube Growth[W

    PubMed Central

    Zhang, Hua; Qu, Xiaolu; Bao, Chanchan; Khurana, Parul; Wang, Qiannan; Xie, Yurong; Zheng, Yiyan; Chen, Naizhi; Blanchoin, Laurent; Staiger, Christopher J.; Huang, Shanjin

    2010-01-01

    A dynamic actin cytoskeleton is essential for pollen germination and tube growth. However, the molecular mechanisms underlying the organization and turnover of the actin cytoskeleton in pollen remain poorly understood. Villin plays a key role in the formation of higher-order structures from actin filaments and in the regulation of actin dynamics in eukaryotic cells. It belongs to the villin/gelsolin/fragmin superfamily of actin binding proteins and is composed of six gelsolin-homology domains at its core and a villin headpiece domain at its C terminus. Recently, several villin family members from plants have been shown to sever, cap, and bundle actin filaments in vitro. Here, we characterized a villin isovariant, Arabidopsis thaliana VILLIN5 (VLN5), that is highly and preferentially expressed in pollen. VLN5 loss-of-function retarded pollen tube growth and sensitized actin filaments in pollen grains and tubes to latrunculin B. In vitro biochemical analyses revealed that VLN5 is a typical member of the villin family and retains a full suite of activities, including barbed-end capping, filament bundling, and calcium-dependent severing. The severing activity was confirmed with time-lapse evanescent wave microscopy of individual actin filaments in vitro. We propose that VLN5 is a major regulator of actin filament stability and turnover that functions in concert with oscillatory calcium gradients in pollen and therefore plays an integral role in pollen germination and tube growth. PMID:20807879

  14. Capping Protein Modulates the Dynamic Behavior of Actin Filaments in Response to Phosphatidic Acid in Arabidopsis[C][W

    PubMed Central

    Li, Jiejie; Henty-Ridilla, Jessica L.; Huang, Shanjin; Wang, Xia; Blanchoin, Laurent; Staiger, Christopher J.

    2012-01-01

    Remodeling of actin filament arrays in response to biotic and abiotic stimuli is thought to require precise control over the generation and availability of filament ends. Heterodimeric capping protein (CP) is an abundant filament capper, and its activity is inhibited by membrane signaling phospholipids in vitro. How exactly CP modulates the properties of filament ends in cells and whether its activity is coordinated by phospholipids in vivo is not well understood. By observing directly the dynamic behavior of individual filament ends in the cortical array of living Arabidopsis thaliana epidermal cells, we dissected the contribution of CP to actin organization and dynamics in response to the signaling phospholipid, phosphatidic acid (PA). Here, we examined three cp knockdown mutants and found that reduced CP levels resulted in more dynamic activity at filament ends, and this significantly enhanced filament-filament annealing and filament elongation from free ends. The cp mutants also exhibited more dense actin filament arrays. Treatment of wild-type cells with exogenous PA phenocopied the actin-based defects in cp mutants, with an increase in the density of filament arrays and enhanced annealing frequency. These cytoskeletal responses to exogenous PA were completely abrogated in cp mutants. Our data provide compelling genetic evidence that the end-capping activity of CP is inhibited by membrane signaling lipids in eukaryotic cells. Specifically, CP acts as a PA biosensor and key transducer of fluxes in membrane signaling phospholipids into changes in actin cytoskeleton dynamics. PMID:22960908

  15. 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.

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

    PubMed

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

    2016-08-01

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

  17. Direct interaction of actin filaments with F-BAR protein pacsin2

    PubMed Central

    Kostan, Julius; Salzer, Ulrich; Orlova, Albina; Törö, Imre; Hodnik, Vesna; Senju, Yosuke; Zou, Juan; Schreiner, Claudia; Steiner, Julia; Meriläinen, Jari; Nikki, Marko; Virtanen, Ismo; Carugo, Oliviero; Rappsilber, Juri; Lappalainen, Pekka; Lehto, Veli-Pekka; Anderluh, Gregor; Egelman, Edward H; Djinović-Carugo, Kristina

    2014-01-01

    Two mechanisms have emerged as major regulators of membrane shape: BAR domain-containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F-BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N-BAR and F-BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes. PMID:25216944

  18. Extracellular Inhibitors, Repellents, and Semaphorin/Plexin/MICAL-mediated Actin Filament Disassembly

    PubMed Central

    Hung, Ruei-Jiun; Terman, Jonathan R.

    2011-01-01

    Multiple extracellular signals have been identified that regulate actin dynamics within motile cells, but how these instructive cues present on the cell surface exert their precise effects on the internal actin cytoskeleton is still poorly understood. One particularly interesting class of these cues is a group of extracellular proteins that negatively alter the movement of cells and their processes. Over the years, these types of events have been described using a variety of terms and herein we provide an overview of inhibitory/repulsive cellular phenomena and highlight the largest known protein family of repulsive extracellular cues, the Semaphorins. Specifically, the Semaphorins (Semas) utilize Plexin cell-surface receptors to dramatically collapse the actin cytoskeleton and we summarize what is known of the direct molecular and biochemical mechanisms of Sema-triggered actin filament (F-actin) disassembly. We also discuss new observations from our lab that reveal that the multi-domain oxidoreductase (Redox) enzyme MICAL, an important mediator of Sema/Plexin repulsion, is a novel F-actin disassembly factor. Our results indicate that MICAL triggers Sema/Plexin-mediated reorganization of the F-actin cytoskeleton and suggest a role for specific Redox signaling events in regulating actin dynamics. PMID:21800438

  19. Power transduction of actin filaments ratcheting in vitro against a load.

    PubMed

    Démoulin, Damien; Carlier, Marie-France; Bibette, Jérôme; Baudry, Jean

    2014-12-16

    The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been extensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime. PMID:25453075

  20. 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.

  1. A Processive Arabidopsis Formin Modulates Actin Filament Dynamics in Association with Profilin.

    PubMed

    Zhang, Sha; Liu, Chang; Wang, Jiaojiao; Ren, Zhanhong; Staiger, Christopher J; Ren, Haiyun

    2016-06-01

    Formins are conserved regulators of actin cytoskeletal organization and dynamics that have been implicated to be important for cell division and cell polarity. The mechanism by which diverse formins regulate actin dynamics in plants is still not well understood. Using in vitro single-molecule imaging technology, we directly observed that the FH1-FH2 domain of an Arabidopsis thaliana formin, AtFH14, processively attaches to the barbed end of actin filaments as a dimer and slows their elongation rate by 90%. The attachment persistence of FH1-FH2 is concentration dependent. Furthermore, by use of the triple-color total internal reflection fluorescence microscopy, we found that ABP29, a barbed-end capping protein, competes with FH1-FH2 at the filament barbed end, where its binding is mutually exclusive with AtFH14. In the presence of different plant profilin isoforms, FH1-FH2 enhances filament elongation rates from about 10 to 42 times. Filaments buckle when FH1-FH2 is anchored specifically to cover slides, further indicating that AtFH14 moves processively on the elongating barbed end. At high concentration, AtFH14 bundles actin filaments randomly into antiparallel or parallel spindle-like structures; however, the FH1-FH2-mediated bundles become thinner and longer in the presence of plant profilins. This is the direct demonstration of a processive formin from plants. Our results also illuminate the molecular mechanism of AtFH14 in regulating actin dynamics via association with profilin. PMID:26996265

  2. The Role of Formin Tails in Actin Nucleation, Processive Elongation, and Filament Bundling*

    PubMed Central

    Vizcarra, Christina L.; Bor, Batbileg; Quinlan, Margot E.

    2014-01-01

    Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements. PMID:25246531

  3. 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.

  4. Modulation of nuclear localization of the influenza virus nucleoprotein through interaction with actin filaments.

    PubMed

    Digard, P; Elton, D; Bishop, K; Medcalf, E; Weeds, A; Pope, B

    1999-03-01

    The influenza virus genome is transcribed in the nuclei of infected cells but assembled into progeny virions in the cytoplasm. This is reflected in the cellular distribution of the virus nucleoprotein (NP), a protein which encapsidates genomic RNA to form ribonucleoprotein structures. At early times postinfection NP is found in the nucleus, but at later times it is found predominantly in the cytoplasm. NP contains several sequences proposed to act as nuclear localization signals (NLSs), and it is not clear how these are overridden to allow cytoplasmic accumulation of the protein. We find that NP binds tightly to filamentous actin in vitro and have identified a cluster of residues in NP essential for the interaction. Complexes containing RNA, NP, and actin could be formed, suggesting that viral ribonucleoproteins also bind actin. In cells, exogenously expressed NP when expressed at a high level partitioned to the cytoplasm, where it associated with F-actin stress fibers. In contrast, mutants unable to bind F-actin efficiently were imported into the nucleus even under conditions of high-level expression. Similarly, nuclear import of NLS-deficient NP molecules was restored by concomitant disruption of F-actin binding. We propose that the interaction of NP with F-actin causes the cytoplasmic retention of influenza virus ribonucleoproteins. PMID:9971805

  5. Mechanism of actin filament nucleation by the bacterial effector VopL

    SciTech Connect

    Yu, Bingke; Cheng, Hui-Chun; Brautigam, Chad A.; Tomchick, Diana R.; Rosen, Michael K.

    2012-05-02

    Vibrio parahaemolyticus protein L (VopL) is an actin nucleation factor that induces stress fibers when injected into eukaryotic host cells. VopL contains three N-terminal Wiskott-Aldrich homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). We describe crystallographic and biochemical analyses of filament nucleation by VopL. The WH2 element of VopL does not nucleate on its own and requires the VCD for activity. The VCD forms a U-shaped dimer in the crystal, stabilized by a terminal coiled coil. Dimerization of the WH2 motifs contributes strongly to nucleation activity, as do contacts of the VCD to actin. Our data lead to a model in which VopL stabilizes primarily lateral (short-pitch) contacts between actin monomers to create the base of a two-stranded filament. Stabilization of lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.

  6. Dense granule trafficking in Toxoplasma gondii requires a unique class 27 myosin and actin filaments.

    PubMed

    Heaslip, Aoife T; Nelson, Shane R; Warshaw, David M

    2016-07-01

    The survival of Toxoplasma gondii within its host cell requires protein release from secretory vesicles, called dense granules, to maintain the parasite's intracellular replicative niche. Despite the importance of DGs, nothing is known about the mechanisms underlying their transport. In higher eukaryotes, secretory vesicles are transported to the plasma membrane by molecular motors moving on their respective cytoskeletal tracks (i.e., microtubules and actin). Because the organization of these cytoskeletal structures differs substantially in T. gondii, the molecular motor dependence of DG trafficking is far from certain. By imaging the motions of green fluorescent protein-tagged DGs in intracellular parasites with high temporal and spatial resolution, we show through a combination of molecular genetics and chemical perturbations that directed DG transport is independent of microtubules and presumably their kinesin/dynein motors. However, directed DG transport is dependent on filamentous actin and a unique class 27 myosin, TgMyoF, which has structural similarity to myosin V, the prototypical cargo transporter. Actomyosin DG transport was unexpected, since filamentous parasite actin has yet to be visualized in vivo due in part to the prevailing model that parasite actin forms short, unstable filaments. Thus our data uncover new critical roles for these essential proteins in the lytic cycle of this devastating pathogen. PMID:27146112

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

    NASA Astrophysics Data System (ADS)

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

    2003-04-01

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

  8. Rho GTPases have diverse effects on the organization of the actin filament system.

    PubMed Central

    Aspenström, Pontus; Fransson, Asa; Saras, Jan

    2004-01-01

    The Rho GTPases are related to the Ras proto-oncogenes and consist of 22 family members. These proteins have important roles in regulating the organization of the actin filament system, and thereby the morphogenesis of vertebrate cells as well as their ability to migrate. In an effort to compare the effects of all members of the Rho GTPase family, active Rho GTPases were transfected into porcine aortic endothelial cells and the effects on the actin filament system were monitored. Cdc42, TCL (TC10-like), Rac1-Rac3 and RhoG induced the formation of lamellipodia, whereas Cdc42, Rac1 and Rac2 also induced the formation of thick bundles of actin filaments. In contrast, transfection with TC10 or Chp resulted in the formation of focal adhesion-like structures, whereas Wrch-1 induced long and thin filopodia. Transfection with RhoA, RhoB or RhoC induced the assembly of stress fibres, whereas Rnd1-Rnd3 resulted in the loss of stress fibres, but this effect was associated with the formation of actin- and ezrin-containing dorsal microvilli. Cells expressing RhoD and Rif had extremely long and flexible filopodia. None of the RhoBTB or Miro GTPases had any major influence on the organization of the actin filament system; instead, RhoBTB1 and RhoBTB2 were present in vesicular structures, and Miro-1 and Miro-2 were present in mitochondria. Collectively, the data obtained in this study to some extent confirm earlier observations, but also allow the identification of previously undetected roles of the different members of the Rho GTPases. PMID:14521508

  9. 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

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

    PubMed Central

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

    2015-01-01

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

  11. CryoEM reveals different coronin binding modes for ADP- and ADP-BeFx- actin filaments

    PubMed Central

    Ge, Peng; Oztug Durer, Zeynep A.; Kudryashov, Dmitri; Zhou, Z. Hong; Reisler, Emil

    2015-01-01

    Essential cellular processes involving the actin cytoskeleton are regulated by auxiliary proteins which can sense the nucleotide state of actin. Here we report cryo electron microscopy (cryoEM) structures at 8.6 Å resolution for ADP- and ADP-BeFx- (mimicking ADP-Pi) bound actin filaments in complex with the β-propeller domain (residues 1–600) of yeast coronin 1 (crn1). Our structures identify the main differences in the interaction of coronin with the two nucleotide states of F-actin. We derived pseudo-atomic models by fitting the atomic structures of actin and coronin into these structures. The identified binding interfaces on actin were confirmed by chemical crosslinking, fluorescence spectroscopy and actin mutagenesis. Importantly, the structures of actin and coronin mapped in this study offer a structural explanation for the nucleotide-dependent effects of coronin on cofilin-assisted remodeling of F-actin. PMID:25362487

  12. 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.

  13. DeFiNe: an optimisation-based method for robust disentangling of filamentous networks

    NASA Astrophysics Data System (ADS)

    Breuer, David; Nikoloski, Zoran

    2015-12-01

    Thread-like structures are pervasive across scales, from polymeric proteins to root systems to galaxy filaments, and their characteristics can be readily investigated in the network formalism. Yet, network links usually represent only parts of filaments, which, when neglected, may lead to erroneous conclusions from network-based analyses. The existing alternatives to detect filaments in network representations require tuning of parameters over a large range of values and treat all filaments equally, thus, precluding automated analysis of diverse filamentous systems. Here, we propose a fully automated and robust optimisation-based approach to detect filaments of consistent intensities and angles in a given network. We test and demonstrate the accuracy of our solution with contrived, biological, and cosmic filamentous structures. In particular, we show that the proposed approach provides powerful automated means to study properties of individual actin filaments in their network context. Our solution is made publicly available as an open-source tool, “DeFiNe”, facilitating decomposition of any given network into individual filaments.

  14. DeFiNe: an optimisation-based method for robust disentangling of filamentous networks

    PubMed Central

    Breuer, David; Nikoloski, Zoran

    2015-01-01

    Thread-like structures are pervasive across scales, from polymeric proteins to root systems to galaxy filaments, and their characteristics can be readily investigated in the network formalism. Yet, network links usually represent only parts of filaments, which, when neglected, may lead to erroneous conclusions from network-based analyses. The existing alternatives to detect filaments in network representations require tuning of parameters over a large range of values and treat all filaments equally, thus, precluding automated analysis of diverse filamentous systems. Here, we propose a fully automated and robust optimisation-based approach to detect filaments of consistent intensities and angles in a given network. We test and demonstrate the accuracy of our solution with contrived, biological, and cosmic filamentous structures. In particular, we show that the proposed approach provides powerful automated means to study properties of individual actin filaments in their network context. Our solution is made publicly available as an open-source tool, “DeFiNe”, facilitating decomposition of any given network into individual filaments. PMID:26666975

  15. Microtubules and actin filaments are not critically involved in the biogenesis of epithelial cell surface polarity.

    PubMed

    Salas, P J; Misek, D E; Vega-Salas, D E; Gundersen, D; Cereijido, M; Rodriguez-Boulan, E

    1986-05-01

    We have studied the role of microtubules and actin filaments in the biogenesis of epithelial cell surface polarity, using influenza hemagglutinin and vesicular stomatitis G protein as model apical and basolateral proteins in infected Madin-Darby canine kidney cells. Addition of colchicine or nocodazole to confluent monolayers at concentrations sufficient to completely disassemble microtubules did not affect the asymmetric budding of influenza or vesicular stomatitis virus and only slightly reduced the typical asymmetric surface distribution of their envelope proteins, despite extensive cytoplasmic redistribution of the Golgi apparatus. Alteration of microtubular function by taxol or dissociation of actin filaments by cytochalasin D also failed to have a significant effect. Furthermore, neither colchicine nor cytochalasin D pretreatment blocked the ability of subconfluent Madin-Darby canine kidney cells to sustain polarized budding of influenza virus a few hours after attachment to the substrate. Our results indicate that domain-specific microtubule or actin filament "tracks" are not responsible for the vectorial delivery of apically or basolaterally directed transport vesicles. In conjunction with currently available evidence, they are compatible with a model in which receptors in the cytoplasmic aspect of apical or basolateral regions provide vectoriality to the transport of vesicles carrying plasma membrane proteins to their final surface localization. PMID:2871031

  16. Microtubules continuously dictate distribution of actin filaments and positioning of cell cleavage in grasshopper spermatocytes.

    PubMed

    Alsop, G Bradley; Zhang, Dahong

    2004-03-15

    We systematically examined the impact of microtubules on distribution of actin filaments and positioning of cell cleavage using micromanipulation to progressively alter the symmetric distribution of spindle microtubules in grasshopper spermatocytes. The initial microtubule asymmetry was induced by placing a single chromosome at one spindle pole using a microneedle, which facilitates regional assembly of spindle microtubules. We augmented chromosome-induced microtubule asymmetry by further removing the aster from the achromosomal pole, producing unichromosome-bearing monopolar spindles. We created the highest spindle asymmetry by cutting early anaphase cells in two, each containing a full set of segregating chromosomes in a half-spindle. We demonstrate that the location of the spindle midzone, distribution of actin filaments, and position of cell cleavage depend on the amount of microtubule asymmetry generated, shifting up to 48.6+/-3.8% away from the spindle equator in cut cells. The positional shift is dynamic, changing incessantly as spindle microtubules reorganize during cytokinesis. These results suggest that microtubules continuously dictate the distribution of actin filaments and positioning of cell cleavage in grasshopper spermatocytes. PMID:15020685

  17. Capping complex formation at the slow-growing end of the actin filament.

    PubMed

    Kostyukova, A S

    2008-12-01

    Actin filaments are polar; their barbed (fast-growing) and pointed (slow-growing) ends differ in structure and dynamic properties. The slow-growing end is regulated by tropomodulins, a family of capping proteins that require tropomyosins for optimal function. There are four tropomodulin isoforms; their distributions vary depending on tissue type and change during development. The C-terminal half of tropomodulin contains one compact domain represented by alternating alpha-helices and beta-structures. The tropomyosin-independent actin-capping site is located at the C-terminus. The N-terminal half has no regular structure; however, it contains a tropomyosin-dependent actin-capping site and two tropomyosin-binding sites. One tropomodulin molecule can bind two tropomyosin molecules. Effectiveness of tropomodulin binding to tropomyosin depends on the tropomyosin isoform. Regulation of tropomodulin binding at the pointed end as well as capping effectiveness in the presence of specific tropomyosins may affect formation of local cytoskeleton and dynamics of actin filaments in cells. PMID:19216712

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

    PubMed Central

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

    1998-01-01

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

  19. Analysis of the local organization and dynamics of cellular actin networks.

    PubMed

    Luo, Weiwei; Yu, Cheng-han; Lieu, Zi Zhao; Allard, Jun; Mogilner, Alex; Sheetz, Michael P; Bershadsky, Alexander D

    2013-09-30

    Actin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm. PMID:24081490

  20. Actin Filament Tracking Based on Particle Filters and Stretching Open Active Contour Models

    PubMed Central

    Li, Hongsheng; Shen, Tian; Vavylonis, Dimitrios; Huang, Xiaolei

    2010-01-01

    We introduce a novel algorithm for actin filament tracking and elongation measurement. Particle Filters (PF) and Stretching Open Active Contours (SOAC) work cooperatively to simplify the modeling of PF in a one-dimensional state space while naturally integrating filament body constraints to tip estimation. Existing microtubule (MT) tracking methods track either MT tips or entire bodies in high-dimensional state spaces. In contrast, our algorithm reduces the PF state spaces to one-dimensional spaces by tracking filament bodies using SOAC and probabilistically estimating tip locations along the curve length of SOACs. Experimental evaluation on TIRFM image sequences with very low SNRs demonstrates the accuracy and robustness of the proposed approach. PMID:20426170

  1. The More the Tubular: Dynamic Bundling of Actin Filaments for Membrane Tube Formation

    PubMed Central

    Weichsel, Julian; Geissler, Phillip L.

    2016-01-01

    Tubular protrusions are a common feature of living cells, arising from polymerization of stiff protein filaments against a comparably soft membrane. Although this process involves many accessory proteins in cells, in vitro experiments indicate that similar tube-like structures can emerge without them, through spontaneous bundling of filaments mediated by the membrane. Using theory and simulation of physical models, we have elaborated how nonequilibrium fluctuations in growth kinetics and membrane shape can yield such protrusions. Enabled by a new grand canonical Monte Carlo method for membrane simulation, our work reveals a cascade of dynamical transitions from individually polymerizing filaments to highly cooperatively growing bundles as a dynamical bottleneck to tube formation. Filament network organization as well as adhesion points to the membrane, which bias filament bending and constrain membrane height fluctuations, screen the effective attractive interactions between filaments, significantly delaying bundling and tube formation. PMID:27384915

  2. The More the Tubular: Dynamic Bundling of Actin Filaments for Membrane Tube Formation.

    PubMed

    Weichsel, Julian; Geissler, Phillip L

    2016-07-01

    Tubular protrusions are a common feature of living cells, arising from polymerization of stiff protein filaments against a comparably soft membrane. Although this process involves many accessory proteins in cells, in vitro experiments indicate that similar tube-like structures can emerge without them, through spontaneous bundling of filaments mediated by the membrane. Using theory and simulation of physical models, we have elaborated how nonequilibrium fluctuations in growth kinetics and membrane shape can yield such protrusions. Enabled by a new grand canonical Monte Carlo method for membrane simulation, our work reveals a cascade of dynamical transitions from individually polymerizing filaments to highly cooperatively growing bundles as a dynamical bottleneck to tube formation. Filament network organization as well as adhesion points to the membrane, which bias filament bending and constrain membrane height fluctuations, screen the effective attractive interactions between filaments, significantly delaying bundling and tube formation. PMID:27384915

  3. On the properties of a bundle of flexible actin filaments in an optical trap

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

    We establish the statistical mechanics framework for a bundle of Nf living and uncrosslinked actin filaments in a supercritical solution of free monomers pressing against a mobile wall. The filaments are anchored normally to a fixed planar surface at one of their ends and, because of their limited flexibility, they grow almost parallel to each other. Their growing ends hit a moving obstacle, depicted as a second planar wall, parallel to the previous one and subjected to a harmonic compressive force. The force constant is denoted as the trap strength while the distance between the two walls as the trap length to make contact with the experimental optical trap apparatus. For an ideal solution of reactive filaments and free monomers at fixed free monomer chemical potential μ1, we obtain the general expression for the grand potential from which we derive averages and distributions of relevant physical quantities, namely, the obstacle position, the bundle polymerization force, and the number of filaments in direct contact with the wall. The grafted living filaments are modeled as discrete Wormlike chains, with F-actin persistence length ℓp, subject to discrete contour length variations ±d (the monomer size) to model single monomer (de)polymerization steps. Rigid filaments (ℓp = ∞), either isolated or in bundles, all provide average values of the stalling force in agreement with Hill's predictions Fs H = N f k B T ln ( ρ 1 / ρ 1 c) / d , independent of the average trap length. Here ρ1 is the density of free monomers in the solution and ρ1c its critical value at which the filament does not grow nor shrink in the absence of external forces. Flexible filaments (ℓp < ∞) instead, for values of the trap strength suitable to prevent their lateral escape, provide an average bundle force and an average trap length slightly larger than the corresponding rigid cases (few percents). Still the stalling force remains nearly independent on the average trap length, but

  4. Emergence of Large-Scale Cell Morphology and Movement from Local Actin Filament Growth Dynamics

    PubMed Central

    Lacayo, Catherine I; Pincus, Zachary; VanDuijn, Martijn M; Wilson, Cyrus A; Fletcher, Daniel A; Gertler, Frank B; Mogilner, Alex; Theriot, Julie A

    2007-01-01

    Variations in cell migration and morphology are consequences of changes in underlying cytoskeletal organization and dynamics. We investigated how these large-scale cellular events emerge as direct consequences of small-scale cytoskeletal molecular activities. Because the properties of the actin cytoskeleton can be modulated by actin-remodeling proteins, we quantitatively examined how one such family of proteins, enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), affects the migration and morphology of epithelial fish keratocytes. Keratocytes generally migrate persistently while exhibiting a characteristic smooth-edged “canoe” shape, but may also exhibit less regular morphologies and less persistent movement. When we observed that the smooth-edged canoe keratocyte morphology correlated with enrichment of Ena/VASP at the leading edge, we mislocalized and overexpressed Ena/VASP proteins and found that this led to changes in the morphology and movement persistence of cells within a population. Thus, local changes in actin filament dynamics due to Ena/VASP activity directly caused changes in cell morphology, which is coupled to the motile behavior of keratocytes. We also characterized the range of natural cell-to-cell variation within a population by using measurable morphological and behavioral features—cell shape, leading-edge shape, filamentous actin (F-actin) distribution, cell speed, and directional persistence—that we have found to correlate with each other to describe a spectrum of coordinated phenotypes based on Ena/VASP enrichment at the leading edge. This spectrum stretched from smooth-edged, canoe-shaped keratocytes—which had VASP highly enriched at their leading edges and migrated fast with straight trajectories—to more irregular, rounder cells migrating slower with less directional persistence and low levels of VASP at their leading edges. We developed a mathematical model that accounts for these coordinated cell-shape and behavior

  5. Nonlinear elasticity of semiflexible filament networks.

    PubMed

    Meng, Fanlong; Terentjev, Eugene M

    2016-08-10

    We develop a continuum theory for equilibrium elasticity of a network of crosslinked semiflexible filaments, spanning the full range between flexible entropy-driven chains to stiff athermal rods. We choose the 3-chain constitutive model of network elasticity over several plausible candidates, and derive analytical expressions for the elastic energy at arbitrary strain, with the corresponding stress-strain relationship. The theory fits well to a wide range of experimental data on simple shear in different filament networks, quantitatively matching the differential shear modulus variation with stress, with only two adjustable parameters (which represent the filament stiffness and the pre-tension in the network, respectively). The general theory accurately describes the crossover between the positive and negative Poynting effect (normal stress on imposed shear) on increasing the stiffness of filaments forming the network. We discuss the network stability (the point of marginal rigidity) and the phenomenon of tensegrity, showing that filament pre-tension on crosslinking into the network determines the magnitude of linear modulus G0. PMID:27444846

  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. PMID:26004635

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

    NASA Astrophysics Data System (ADS)

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

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

  8. He-Ne laser influenced actin filaments alleviate the damage of UV-B in wheat

    NASA Astrophysics Data System (ADS)

    Chen, Huize; Han, Rong

    2015-01-01

    This work investigated the use of a He-Ne laser in alleviating the damaging effects of ultraviolet-B (UV-B) radiation on wheat seedlings by influenced actin filaments. Triticum aestivum seedlings were irradiated with either enhanced UV-B (10.08 KJ m-2 d-1) or a combination of UV-B light and the He-Ne laser. Plants were also exposed to the He-Ne laser alone. In order to compare the effect of the He-Ne laser, red light (same power and wavelength as the He-Ne laser) treatment and the combined UV-B and red light treatment were added. Moreover, wheat seedlings were treated with actin special drugs, including cytochalasin B (CB) and jasplakinolide (JAS). We analyzed the growth of the seedlings, the distribution of actin filaments (AFs), DNA laddering and ACTIN expression in the different groups. The results showed that enhanced UV-B produced negative effects on the growth of wheat seedlings while implementing the He-Ne laser partially alleviated the injury. With the red light treatment, there are no positive effects. The ACTIN expression stayed the same in the different treatments, while the distribution and the protein content are different. The Fourier transform infrared (FTIR) microspectroscopic results further established significant changes in the chemical composition of the wall material. These results suggested that the He-Ne laser alleviated the damaging effects of UV-B radiation in wheat seedlings by changing the characteristics of the AFs.

  9. A technique for simultaneous measurement of force and overlap between single muscle filaments of myosin and actin.

    PubMed

    Kalganov, Albert; Novinger, Rowan; Rassier, Dilson E

    2010-12-17

    In this study, we show a method for direct measurements of force and simultaneous visualization of isolated muscle filaments. Single actin filaments isolated from chicken skeletal muscle and single thick filaments isolated from Mussels were imaged using fluorescence and dark field microscopy, respectively. Force generated by the filaments was measured using micro-fabricated cantilevers. Force values were in the range observed previously with myosin filaments and molecules. The results suggest that the technique can be used to investigate many issues of interest and debate in the field of muscle biophysics. PMID:21081114

  10. The Plant-Specific Actin Binding Protein SCAB1 Stabilizes Actin Filaments and Regulates Stomatal Movement in Arabidopsis[C][W

    PubMed Central

    Zhao, Yang; Zhao, Shuangshuang; Mao, Tonglin; Qu, Xiaolu; Cao, Wanhong; Zhang, Li; Zhang, Wei; He, Liu; Li, Sidi; Ren, Sulin; Zhao, Jinfeng; Zhu, Guoli; Huang, Shanjin; Ye, Keqiong; Yuan, Ming; Guo, Yan

    2011-01-01

    Microfilament dynamics play a critical role in regulating stomatal movement; however, the molecular mechanism underlying this process is not well understood. We report here the identification and characterization of STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1), an Arabidopsis thaliana actin binding protein. Plants lacking SCAB1 were hypersensitive to drought stress and exhibited reduced abscisic acid-, H2O2-, and CaCl2-regulated stomatal movement. In vitro and in vivo analyses revealed that SCAB1 binds, stabilizes, and bundles actin filaments. SCAB1 shares sequence similarity only with plant proteins and contains a previously undiscovered actin binding domain. During stomatal closure, actin filaments switched from a radial orientation in open stomata to a longitudinal orientation in closed stomata. This switch took longer in scab1 plants than in wild-type plants and was correlated with the delay in stomatal closure seen in scab1 mutants in response to drought stress. Our results suggest that SCAB1 is required for the precise regulation of actin filament reorganization during stomatal closure. PMID:21719691

  11. Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction.

    PubMed

    Butkevich, Eugenia; Bodensiek, Kai; Fakhri, Nikta; von Roden, Kerstin; Schaap, Iwan A T; Majoul, Irina; Schmidt, Christoph F; Klopfenstein, Dieter R

    2015-01-01

    Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling. PMID:26146072

  12. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments

    PubMed Central

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C.; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-01-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H+-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation. PMID:26272553

  13. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments.

    PubMed

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-10-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H(+)-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation. PMID:26272553

  14. Closed membrane shapes with attached BAR domains subject to external force of actin filaments.

    PubMed

    Mesarec, Luka; Góźdź, Wojciech; Iglič, Veronika Kralj; Kralj, Samo; Iglič, Aleš

    2016-05-01

    Membrane deformations induced by attached BAR superfamily domains could trigger or facilitate the growth of plasma membrane protrusions. The BAR domain family consists of BAR, F-BAR and I-BAR domains, each enforcing a different local curvature when attached to the membrane surface. Our theoretical study mainly focuses on the role of I-BAR in the membrane tubular deformations generated or stabilised by actin filaments. The influence of the area density of membrane attached BAR domains and their intrinsic curvature on the closed membrane shapes (vesicles) was investigated numerically. We derived an analytical approximative expression for the critical relative area density of BARs at which the membrane tubular protrusions on vesicles are most prominent. We have shown that the BARs with a higher intrinsic curvature induce thinner and longer cylindrical protrusions. The average orientation of the membrane attached BARs is altered when the vesicle shape is subjected to external force of growing actin rod-like structure inside a vesicle. The average orientation angle of membrane attached BARs may indicate whether the actin filaments are just stabilising the protrusion or generating it by stretching the vesicle. PMID:26854580

  15. Branching and capping determine the force-velocity relationships of branching actin networks.

    PubMed

    Smith, Daniel B; Liu, Jian

    2013-02-01

    A branching actin network is the major engine that drives cell motility. A measure of the effectiveness of an engine is the velocity the engine is able to produce at a given resistance-the force-velocity relationship. Concave force-velocity relationships consist of a force-insensitive region, indicative of an adaptive response. In contrast, convex force-velocity relationships would reflect a passive response. Even in in vitro experiments, branching actin networks can exhibit both concave and convex force-velocity curves. However, the exact mechanism that can explain both force-velocity curves is not yet known. We carried out an agent-based stochastic simulation to explore such a mechanism. We discovered an emergent behavior of a branching actin network: Upon resistance, it remodels itself by increasing the number of filaments growing in contact with the load. The remodeling is favored by branching events and limited by capping. The force-velocity relationship hinges on the relative time-scale between the intrinsic kinetics of the branching actin network and the loading. Shortly after encountering resistance (∼seconds), the force-velocity relationship of the actin network is always convex, as it does not have enough time to remodel itself. A concave force-velocity relationship requires network remodeling at longer time-scales (∼tens of seconds to minutes) and the faster branching event relative to capping. Furthermore, our model explains the observed hysteresis in the force-velocity relationship of actin networks. Our model thus establishes a unified mechanism that can account for both convex and concave force-velocity relationships observed in branching actin networks. PMID:23358606

  16. Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein

    PubMed Central

    Brzoska, Anthony J.; Jensen, Slade O.; Barton, Deborah A.; Davies, Danielle S.; Overall, Robyn L.; Skurray, Ronald A.; Firth, Neville

    2016-01-01

    Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments. PMID:27310470

  17. Analysis of the local organization and dynamics of cellular actin networks

    PubMed Central

    Luo, Weiwei; Yu, Cheng-han; Lieu, Zi Zhao; Allard, Jun; Mogilner, Alex; Sheetz, Michael P.

    2013-01-01

    A ctin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm. PMID:24081490

  18. Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    SciTech Connect

    Stall, Richard; Ramos, Joseph; Kent Fulcher, F.; Patel, Yashomati M.

    2014-03-10

    Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated that MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin.

  19. In vitro modulation of filament bundling in F-actin and keratins by annexin II and calcium.

    PubMed

    Ma, A S; Bystol, M E; Tranvan, A

    1994-05-01

    In our preliminary subcellular localization experiment we demonstrated that annexin II co-localized with submembranous actin in subpopulations of both cultured fibroblasts and keratinocytes. To investigate the physical interaction between annexin II and actin at the cell periphery, in vitro reconstitution experiments were carried out with keratins used as a control. Annexin II, isolated by immunoaffinity column chromatography, was found to exist as globular structures measuring 10 to 25 nm in diameter by rotary shadowing, similar to a previous report. We believe that these structures represent its polymeric forms. By negative staining, monomeric annexin II was detectable as tapered rods, measuring 6 nm in length and 1 to 2 nm in diameter. When annexin II was mixed with actin in 3 mM piperazine-N, N-bis-2-ethanesulfonic acid (PIPES) buffer with 10 mM NaCl2, 2 mM MgCl2 and 0.1 mM CaCl2, thick twisting actin bundles formed, confirming previous reports. This bundling was much reduced when calcium was removed. In the presence of 5 mM ethylenediamine tetra-acetic acid (EDTA) in 5 mM tris, pH 7.2, keratins were found to form a network of filaments, which began to disassemble when the chelator was removed and became fragmented when 0.1 mM CaCl2 was added. Keratins under the same conditions did not fragment when annexin II was present. These results suggest that annexin II, in conjunction with Ca2+, may be involved in a flexible system accommodating changes in the membrane cytoskeletal framework at the cell periphery in keratinocytes. PMID:7520812

  20. Proposed modification of the Huxley-Simmons model for myosin head motion along an actin filament.

    PubMed

    Mitsui, T; Chiba, H

    1996-09-21

    A model is proposed for myosin head motion along an actin filament which accommodates recent experimental data. The model includes three attached states of a myosin head and is thus similar to the classical Huxley & Simmons (1971) model, but differs in that an explicit expression is given for the spatial distribution of potential energy wells for the myosin head. Our model also differs from the classical model, in that it assumes that the proportion of myosin heads attached to actin filament is constant and independent of shortening velocity, as suggested by X-ray diffraction data. Furthermore, it posits that the crossbridge is string-like rather than spring-like. This modified model fits well to the experimental data in the following respects. (1) The calculated tension dependence of muscle stiffness agrees with the observation by Ford et al. (1985 J. Physiol. 361, 131-150). (2) A myosin head under low load can move as far as 60 nm along an actin filament during one ATP hydrolysis cycle in muscle, in agreement with the results by Yanagida et al. (1985 Nature 316, 366-369) and others. (3) The model predicts that such movements consist of a series of elementary steps of 11 nm. (4) A single myosin head hardly moves after the first step of 11 nm under the condition of in vitro experiment carried out by Finer et al. (1994 Nature 368, 113-119), in agreement with their observation. (5) The calculated energy liberation rate reproduces the characteristics of Hill's equation. (6) The "double-hyperbolic force-velocity relation" reported by Edman (1988 J. Physiol. 404, 301-321) can be understood in terms of a potential barrier against movement of a potential well in which a myosin head is trapped. PMID:8944146

  1. Effects of asymmetric nanostructures on the extinction difference properties of actin biomolecules and filaments

    PubMed Central

    Khoo, E. H.; Leong, Eunice S. P.; Wu, S. J.; Phua, W. K.; Hor, Y. L.; Liu, Y. J.

    2016-01-01

    In this paper, symmetric and asymmetric tapering on the arms of the gammadion nanostructure is proposed to enhance both local field distribution and extinction difference (ED). The asymmetric tapered gammadion with tapering fraction (TF) of 0.67 is seen to have the largest ED and spatial local field distribution, producing a large wavelength shift of more than 50 percent as compared to the untapered gammadion nanostructures when immersed in a solution of actin molecules and filaments. The optical chirality, ζ shows that the larger local field amplitudes produced by the asymmetric designs increases the rate of chiral molecules excitation. This enhanced field is strongly rotating and highly sensitive to single molecules and larger filaments. Here, we show that the ED, optical chirality, sensitivity and rate of chiral molecules excitation can be improved by incorporating asymmetric designs into chiral gammadion nanostructures through tapering. PMID:26792371

  2. Effects of asymmetric nanostructures on the extinction difference properties of actin biomolecules and filaments

    NASA Astrophysics Data System (ADS)

    Khoo, E. H.; Leong, Eunice S. P.; Wu, S. J.; Phua, W. K.; Hor, Y. L.; Liu, Y. J.

    2016-01-01

    In this paper, symmetric and asymmetric tapering on the arms of the gammadion nanostructure is proposed to enhance both local field distribution and extinction difference (ED). The asymmetric tapered gammadion with tapering fraction (TF) of 0.67 is seen to have the largest ED and spatial local field distribution, producing a large wavelength shift of more than 50 percent as compared to the untapered gammadion nanostructures when immersed in a solution of actin molecules and filaments. The optical chirality, ζ shows that the larger local field amplitudes produced by the asymmetric designs increases the rate of chiral molecules excitation. This enhanced field is strongly rotating and highly sensitive to single molecules and larger filaments. Here, we show that the ED, optical chirality, sensitivity and rate of chiral molecules excitation can be improved by incorporating asymmetric designs into chiral gammadion nanostructures through tapering.

  3. Regulation of structure and function of sarcomeric actin filaments in striated muscle of the nematode Caenorhabditis elegans

    PubMed Central

    Ono, Shoichiro

    2014-01-01

    The nematode Caenorhabditis elegans has been used as a valuable system to study structure and function of striated muscle. The body wall muscle of C. elegans is obliquely striated muscle with highly organized sarcomeric assembly of actin, myosin, and other accessary proteins. Genetic and molecular biological studies in C. elegans have identified a number of genes encoding structural and regulatory components for the muscle contractile apparatuses, and many of them have counterparts in mammalian cardiac and skeletal muscles or striated muscles in other invertebrates. Applicability of genetics, cell biology, and biochemistry has made C. elegans an excellent system to study mechanisms of muscle contractility and assembly and maintenance of myofibrils. This review focuses on the regulatory mechanisms of structure and function of actin filaments in the C. elegans body wall muscle. Sarcomeric actin filaments in C. elegans muscle are associated with the troponin-tropomyosin system that regulates the actin-myosin interaction. Proteins that bind to the side and ends of actin filaments support ordered assembly of thin filaments. Furthermore, regulators of actin dynamics play important roles in initial assembly, growth, and maintenance of sarcomeres. The knowledge acquired in C. elegans can serve as bases to understand the basic mechanisms of muscle structure and function. PMID:25125169

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

    PubMed Central

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

    2010-01-01

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

  5. A CapG gain-of-function mutant reveals critical structural and functional determinants for actin filament severing

    PubMed Central

    Zhang, Y; Vorobiev, Sergey M; Gibson, Bruce G; Hao, Binghua; Sidhu, Gurjit S; Mishra, Vishnu S; Yarmola, Elena G; Bubb, Michael R; Almo, Steven C; Southwick, Frederick S

    2006-01-01

    CapG is the only member of the gelsolin family unable to sever actin filaments. Changing amino acids 84–91 (severing domain) and 124–137 (WH2-containing segment) simultaneously to the sequences of gelsolin results in a mutant, CapG-sev, capable of severing actin filaments. The gain of severing function does not alter actin filament capping, but is accompanied by a higher affinity for monomeric actin, and the capacity to bind and sequester two actin monomers. Analysis of CapG-sev crystal structure suggests a more loosely folded inactive conformation than gelsolin, with a shorter S1–S2 latch. Calcium binding to S1 opens this latch and S1 becomes separated from a closely interfaced S2–S3 complex by an extended arm consisting of amino acids 118–137. Modeling with F-actin predicts that the length of this WH2-containing arm is critical for severing function, and the addition of a single amino acid (alanine or histidine) eliminates CapG-sev severing activity, confirming this prediction. We conclude that efficient severing utilizes two actin monomer-binding sites, and that the length of the WH2-containing segment is a critical functional determinant for severing. PMID:16977317

  6. An affine continuum mechanical model for cross-linked F-actin networks with compliant linker proteins.

    PubMed

    Holzapfel, Gerhard A; Unterberger, Michael J; Ogden, Ray W

    2014-10-01

    Cross-linked actin networks are important building blocks of the cytoskeleton. In order to gain deeper insight into the interpretation of experimental data on actin networks, adequate models are required. In this paper we introduce an affine constitutive network model for cross-linked F-actin networks based on nonlinear continuum mechanics, and specialize it in order to reproduce the experimental behavior of in vitro reconstituted model networks. The model is based on the elastic properties of single filaments embedded in an isotropic matrix such that the overall properties of the composite are described by a free-energy function. In particular, we are able to obtain the experimentally determined shear and normal stress responses of cross-linked actin networks typically observed in rheometer tests. In the present study an extensive analysis is performed by applying the proposed model network to a simple shear deformation. The single filament model is then extended by incorporating the compliance of cross-linker proteins and further extended by including viscoelasticity. All that is needed for the finite element implementation is the constitutive model for the filaments, the linkers and the matrix, and the associated elasticity tensor in either the Lagrangian or Eulerian formulation. The model facilitates parameter studies of experimental setups such as micropipette aspiration experiments and we present such studies to illustrate the efficacy of this modeling approach. PMID:25043658

  7. Characterization of Actin Filament Dynamics during Mitosis in Wheat Protoplasts under UV-B Radiation

    PubMed Central

    Chen, Huize; Han, Rong

    2016-01-01

    Enhanced ultraviolet-B (UV-B) radiation is caused by the thinning ozone and affects photosynthesis and crop yield. Recently, UV-B radiation has been considered as an environmental signal that regulates plant growth. Elucidating the downstream effectors in UV-B-triggered pathways is of particular interest. Previous studies have shown that actin filaments (AFs) play many roles during cell physiological processes. However, the underlying response of AFs to UV-B radiation remains unclear. In this study, wheat protoplasts were isolated from 7-d-old leaves. The dynamics of AFs during mitosis were observed under different treatments. The protoplasts were treated with UV-B radiation, cytochalasin B (CB) and jasplakinolide (JAS). Ph-FITC labelling results revealed typical actin filament structures in the control group; AFs were rearranged under UV-B radiation. AFs polymerized into bundles during interphase, the preprophase band (PPB) structure was destroyed during prophase, and the AFs gathered into plaques during metaphase in response to UV-B radiation. During anaphase and telophase, the distribution of AFs was dispersed. Pharmacologic experiments revealed that CB induced apoptosis and JAS induced nuclear division without cytokinesis in wheat protoplasts. These results indicated that AFs respond to UV-B radiation during mitosis, supplying evidence of UV-B signal transduction in plants. PMID:26823006

  8. Characterization of Actin Filament Dynamics during Mitosis in Wheat Protoplasts under UV-B Radiation.

    PubMed

    Chen, Huize; Han, Rong

    2016-01-01

    Enhanced ultraviolet-B (UV-B) radiation is caused by the thinning ozone and affects photosynthesis and crop yield. Recently, UV-B radiation has been considered as an environmental signal that regulates plant growth. Elucidating the downstream effectors in UV-B-triggered pathways is of particular interest. Previous studies have shown that actin filaments (AFs) play many roles during cell physiological processes. However, the underlying response of AFs to UV-B radiation remains unclear. In this study, wheat protoplasts were isolated from 7-d-old leaves. The dynamics of AFs during mitosis were observed under different treatments. The protoplasts were treated with UV-B radiation, cytochalasin B (CB) and jasplakinolide (JAS). Ph-FITC labelling results revealed typical actin filament structures in the control group; AFs were rearranged under UV-B radiation. AFs polymerized into bundles during interphase, the preprophase band (PPB) structure was destroyed during prophase, and the AFs gathered into plaques during metaphase in response to UV-B radiation. During anaphase and telophase, the distribution of AFs was dispersed. Pharmacologic experiments revealed that CB induced apoptosis and JAS induced nuclear division without cytokinesis in wheat protoplasts. These results indicated that AFs respond to UV-B radiation during mitosis, supplying evidence of UV-B signal transduction in plants. PMID:26823006

  9. Actin network disassembly powers dissemination of Listeria monocytogenes.

    PubMed

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

    2014-01-01

    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

  10. Actin Filaments in Mature Guard Cells Are Radially Distributed and Involved in Stomatal Movement.

    PubMed Central

    Kim, M.; Hepler, P. K.; Eun, S. O.; Ha, K. S.; Lee, Y.

    1995-01-01

    Stomatal movements, which regulate gas exchange in plants, involve pronounced changes in the shape and volume of the guard cell. To test whether the changes are regulated by actin filaments, we visualized microfilaments in mature guard cells and examined the effects of actin antagonists on stomatal movements. Immunolocalization on fixed cells and microinjection of fluorescein isothiocyanate-phalloidin into living guard cells of Commelina communis L. showed that cortical microfilaments were radially distributed, fanning out from the stomatal pore site, resembling the known pattern of microtubules. Treatment of epidermal peels with phalloidin prior to stabilizing microfilaments with m-maleimidobenzoyl N-hydroxysuccimimide caused dense packing of radial microfilaments and an accumulation of actin around many organelles. Both stomatal closing induced by abscisic acid and opening under light were inhibited. Treatment of guard cells with cytochalasin D abolished the radial pattern of microfilaments; generated sparse, poorly oriented arrays; and caused partial opening of dark-closed stomata. These results suggest that microfilaments participate in stomatal aperture regulation. PMID:12228654

  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. Pseudorabies virus US3 leads to filamentous actin disassembly and contributes to viral genome delivery to the nucleus.

    PubMed

    Jacob, Thary; Van den Broeke, Céline; Grauwet, Korneel; Baert, Kim; Claessen, Christophe; De Pelsmaeker, Steffi; Van Waesberghe, Cliff; Favoreel, Herman W

    2015-06-12

    The conserved alphaherpesvirus US3 tegument protein induces rearrangements of the actin cytoskeleton, consisting of protrusion formation and stress fiber breakdown. Although US3 does not affect levels of total actin protein, it remains unclear whether US3 modulates the total levels of filamentous (F) actin. In this report, we show that the pseudorabies virus (PRV) US3 protein, via its kinase activity, leads to disassembly of F-actin in porcine ST cells. F-actin disassembly has been reported before to contribute to host cell entry of HIV. In line with this, in the current study, we report that US3 has a previously uncharacterized role in viral genome delivery to the nucleus, since quantitative polymerase chain reaction (qPCR) assays on nuclear fractions demonstrated a reduced nuclear delivery of US3null PRV compared to wild type PRV genomes. Treatment of cells with the actin depolymerizing drug cytochalasin D enhanced virus genome delivery to the nucleus, particularly of US3null PRV, supporting a role for F-actin disassembly during certain aspects of viral entry. In conclusion, the US3 kinase of PRV leads to F-actin depolymerization, and US3 and F-actin disassembly contribute to viral genome delivery to the nucleus. PMID:25869795

  13. Saturable binding of the echinoderm microtubule-associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments.

    PubMed

    Eichenmüller, B; Ahrens, D P; Li, Q; Suprenant, K A

    2001-11-01

    The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus. To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components. To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells. After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture. EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography. In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP. EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer. In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain. Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM. Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin. PMID:11807937

  14. A novel multitarget tracking algorithm for Myosin VI protein molecules on actin filaments in TIRFM sequences.

    PubMed

    Li, G; Sanchez, V; Nagaraj, P C S B; Khan, S; Rajpoot, N

    2015-12-01

    We propose a novel multitarget tracking framework for Myosin VI protein molecules in total internal reflection fluorescence microscopy sequences which integrates an extended Hungarian algorithm with an interacting multiple model filter. The extended Hungarian algorithm, which is a linear assignment problem based method, helps to solve measurement assignment and spot association problems commonly encountered when dealing with multiple targets, although a two-motion model interacting multiple model filter increases the tracking accuracy by modelling the nonlinear dynamics of Myosin VI protein molecules on actin filaments. The evaluation of our tracking framework is conducted on both real and synthetic total internal reflection fluorescence microscopy sequences. The results show that the framework achieves higher tracking accuracies compared to the state-of-the-art tracking methods, especially for sequences with high spot density. PMID:26259144

  15. 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.

  16. Specific Transformation of Assembly with Actin Filaments and Molecular Motors in a Cell-Sized Self-Emerged Liposome

    NASA Astrophysics Data System (ADS)

    Takiguchi, Kingo; Negishi, Makiko; Tanaka-Takiguchi, Yohko; Hayashi, Masahito; Yoshikawa, Kenichi

    2014-12-01

    Eukaryotes, by the same combination of cytoskeleton and molecular motor, for example actin filament and myosin, can generate a variety of movements. For this diversity, the organization of biological machineries caused by the confinement and/or crowding effects of internal living cells, may play very important roles.

  17. The IQGAP1 Protein Is a Calmodulin-regulated Barbed End Capper of Actin Filaments

    PubMed Central

    Pelikan-Conchaudron, Andrea; Le Clainche, Christophe; Didry, Dominique; Carlier, Marie-France

    2011-01-01

    IQGAP1 is a large modular protein that displays multiple partnership and is thought to act as a scaffold in coupling cell signaling to the actin and microtubule cytoskeletons in cell migration, adhesion, and cytokinesis. However the molecular mechanisms underlying the activities of IQGAP1 are poorly understood in part because of its large size, poor solubility and lack of functional assays to challenge biochemical properties in various contexts. We have purified bacterially expressed recombinant human IQGAP1. The protein binds Cdc42, Rac1, and the CRIB domain of N-WASP in a calmodulin-sensitive fashion. We further show that in addition to bundling of filaments via a single N-terminal calponin-homology domain, IQGAP1 actually regulates actin assembly. It caps barbed ends, with a higher affinity for ADP-bound terminal subunits (KB = 4 nm). The barbed end capping activity is inhibited by calmodulin, consistent with calmodulin binding to IQGAP1 with a KC of 40 nm, both in the absence and presence of Ca2+ ions. The barbed end capping activity resides in the C-terminal half of IQGAP1. It is possible that the capping activity of IQGAP1 accounts for its stimulation of cell migration. We further find that bacterially expressed recombinant IQGAP1 fragments easily co-purify with nucleic acids that turn out to activate N-WASP protein to branch filaments with Arp2/3 complex. The present results open perspectives for tackling the function of IQGAP1 in more complex reconstituted systems. PMID:21730051

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  19. Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells

    PubMed Central

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

    2015-01-01

    Leiomodin (Lmod) is a class of potent tandem-G-actin–binding nucleators in muscle cells. Lmod mutations, deletion, or instability are linked to lethal nemaline myopathy. However, the lack of high-resolution structures of Lmod nucleators in action severely hampered our understanding of their essential cellular functions. Here we report the crystal structure of the actin–Lmod2162–495 nucleus. The structure contains two actin subunits connected by one Lmod2162–495 molecule in a non–filament-like conformation. Complementary functional studies suggest that the binding of Lmod2 stimulates ATP hydrolysis and accelerates actin nucleation and polymerization. The high level of conservation among Lmod proteins in sequence and functions suggests that the mechanistic insights of human Lmod2 uncovered here may aid in a molecular understanding of other Lmod proteins. Furthermore, our structural and mechanistic studies unraveled a previously unrecognized level of regulation in mammalian signal transduction mediated by certain tandem-G-actin–binding nucleators. PMID:26417072

  20. Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly.

    PubMed

    Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

    2015-01-01

    Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells. PMID:26652273

  1. Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly

    PubMed Central

    Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

    2015-01-01

    Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells. DOI: http://dx.doi.org/10.7554/eLife.06126.001 PMID:26652273

  2. Crystal structure of the C-terminal half of tropomodulin and structural basis of actin filament pointed-end capping.

    PubMed Central

    Krieger, Inna; Kostyukova, Alla; Yamashita, Atsuko; Nitanai, Yasushi; Maéda, Yuichiro

    2002-01-01

    Tropomodulin is the unique pointed-end capping protein of the actin-tropomyosin filament. By blocking elongation and depolymerization, tropomodulin regulates the architecture and the dynamics of the filament. Here we report the crystal structure at 1.45-A resolution of the C-terminal half of tropomodulin (C20), the actin-binding moiety of tropomodulin. C20 is a leucine-rich repeat domain, and this is the first actin-associated protein with a leucine-rich repeat. Binding assays suggested that C20 also interacts with the N-terminal fragment, M1-M2-M3, of nebulin. Based on the crystal structure, we propose a model for C20 docking to the actin subunit at the pointed end. Although speculative, the model is consistent with the idea that a tropomodulin molecule competes with an actin subunit for a pointed end. The model also suggests that interactions with tropomyosin, actin, and nebulin are all possible sources of influences on the dynamic properties of pointed-end capping by tropomodulin. PMID:12414704

  3. Dynamic light-scattering study on changes in mobility of chromaffin granules in actin network with its assembly and Ca2+-dependent disassembly by gelsolin

    NASA Astrophysics Data System (ADS)

    Fujime, Satoru; Miyamoto, Shigeaki; Funatsu, Takashi; Ishiwata, S.

    1993-06-01

    As a final stage of cell signal transduction, secretory cells release hormones by exocytosis. Before secretory granules contact with the cell membrane for fusion, an actin network barrier must dissociate as a prelude. In order to elucidate dynamical behaviors of secretory granules in actin network, in vitro assembly and disassembly processes of actin networks were examined by means of dynamic light-scattering spectroscopy. We studied actin polymerization in the presence of chromaffin granules isolated from bovine adrenal medullae, and found that the entanglement of actin filaments rapidly formed cages which confined granules in them. We also studied the effect of gelsolin, one of the actin-severing proteins, on the network of actin filaments performed in the presence of chromaffin granules. It turned out that the cages which confined granules rapidly disappeared when gelsolin was added in the presence of free Ca2+ ions. Semiquantitative analyses of dynamic light-scattering spectra permitted us to estimate the changes in the mobility (or translational diffusion coefficient) of chromaffin granules in the actin network with its assembly and Ca2+-dependent disassembly by gelsolin. Based on the present results and some pieces of evidence in literature, a model is proposed for biophysical situations before, during, and after an exocytotic event.

  4. In vivo visualization of type II plasmid segregation: bacterial actin filaments pushing plasmids

    PubMed Central

    Campbell, Christopher S.; Mullins, R. Dyche

    2007-01-01

    Type II par operons harness polymerization of the dynamically unstable actin-like protein ParM to segregate low-copy plasmids in rod-shaped bacteria. In this study, we use time-lapse fluorescence microscopy to follow plasmid dynamics and ParM assembly in Escherichia coli. Plasmids lacking a par operon undergo confined diffusion with a diffusion constant of 5 × 10−5 μm2/s and a confinement radius of 0.28 μm. Single par-containing plasmids also move diffusively but with a larger diffusion constant (4 × 10−4 μm2/s) and confinement radius (0.42 μm). ParM filaments are dynamically unstable in vivo and form spindles that link pairs of par-containing plasmids and drive them rapidly (3.1 μm/min) toward opposite poles of the cell. After reaching the poles, ParM filaments rapidly and completely depolymerize. After ParM disassembly, segregated plasmids resume diffusive motion, often encountering each other many times and undergoing multiple rounds of ParM-dependent segregation in a single cell cycle. We propose that in addition to driving segregation, the par operon enables plasmids to search space and find sister plasmids more effectively. PMID:18039937

  5. 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

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

    NASA Astrophysics Data System (ADS)

    Blanpied, Thomas A.

    2013-03-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  9. Motion in partially and fully cross-linked F-actin networks

    NASA Astrophysics Data System (ADS)

    Morris, Eliza; Ehrlicher, Allen; Weitz, David

    2012-02-01

    Single molecule experiments have measured stall forces and procession rates of molecular motors on isolated cytoskeletal fibers in Newtonian fluids. But in the cell, these motors are transporting cargo through a highly complex cytoskeletal network. To compare these single molecule results to the forces exerted by motors within the cell, an evaluation of the response of the cytoskeletal network is needed. Using magnetic tweezers and fluorescence confocal microscopy we observe and quantify the relationship between bead motion and filament response in F-actin networks both partially and fully cross-linked with filamin We find that when the transition from full to partial cross-linking is brought about by a decrease in cross-linker concentration there is a simultaneous decline in the elasticity of the network, but the response of the bead remains qualitatively similar. However, when the cross-linking is reduced through a shortening of the F-actin filaments the bead response is completely altered. The characteristics of the altered bead response will be discussed here.

  10. 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.

  11. Formin and capping protein together embrace the actin filament in a ménage à trois

    PubMed Central

    Shekhar, Shashank; Kerleau, Mikael; Kühn, Sonja; Pernier, Julien; Romet-Lemonne, Guillaume; Jégou, Antoine; Carlier, Marie-France

    2015-01-01

    Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed. PMID:26564775

  12. Purification and characterization of caldesmon77: a calmodulin-binding protein that interacts with actin filaments from bovine adrenal medulla.

    PubMed Central

    Sobue, K; Tanaka, T; Kanda, K; Ashino, N; Kakiuchi, S

    1985-01-01

    Caldesmon150, a protein composed of the Mr 150,000/147,000 doublet, alternately binds to calmodulin and actin filaments in a Ca2+-dependent "flip-flop" fashion. In all fibroblast cell lines examined, we also found a Mr 77,000 protein that crossreacts with anti-caldesmon150 antibody by using an immunoprecipitation technique [Owada, M.K., Hakura, A., Iida, K., Yahara, I., Sobue, K. & Kakiuchi, S. (1984) Proc. Natl. Acad. Sci. USA 81, 3133-3137]. In this report, we examine the tissue distribution of caldesmon by the method of immunoblotting, using caldesmon-specific antibody. Both caldesmon150 and caldesmon77 show widespread distribution in the tissues examined. Caldesmon77 is more widely distributed than caldesmon150, and we have purified caldesmon77 from bovine adrenal medulla. Its molecular weight estimated by NaDodSO4/polyacrylamide gel electrophoresis was 77,000, and a tetramer of this polypeptide may constitute the native molecule (Mr, 300,000). Caldesmon77 possesses a number of features in common with caldesmon150, including flip-flop binding to calmodulin and actin filaments depending on the concentration of Ca2+ and crossreactivity with caldesmon150-specific antibody. Analysis of caldesmon77-F actin interaction by sedimentation and electrophoresis revealed that 0.5 mg of caldesmon77 bound to 1 mg of F actin. This indicated that the molar ratio between caldesmon77 (tetramer) and actin monomer was calculated to be 1:12-14. In addition, caldesmon77 regulated the actin-myosin interaction in Ca2+-sensitive actomyosin obtained from adrenal medulla. These results suggest that caldesmon77 might be a ubiquitous actin-linked regulator of nonmuscle contractile processes, including those in adrenal medulla. Images PMID:2991905

  13. Arabidopsis VILLIN2 and VILLIN3 are required for the generation of thick actin filament bundles and for directional organ growth.

    PubMed

    van der Honing, Hannie S; Kieft, Henk; Emons, Anne Mie C; Ketelaar, Tijs

    2012-03-01

    In plant cells, actin filament bundles serve as tracks for myosin-dependent organelle movement and play a role in the organization of the cytoplasm. Although virtually all plant cells contain actin filament bundles, the role of the different actin-bundling proteins remains largely unknown. In this study, we investigated the role of the actin-bundling protein villin in Arabidopsis (Arabidopsis thaliana). We used Arabidopsis T-DNA insertion lines to generate a double mutant in which VILLIN2 (VLN2) and VLN3 transcripts are truncated. Leaves, stems, siliques, and roots of vln2 vln3 double mutant plants are twisted, which is caused by local differences in cell length. Microscopy analysis of the actin cytoskeleton showed that in these double mutant plants, thin actin filament bundles are more abundant while thick actin filament bundles are virtually absent. In contrast to full-length VLN3, truncated VLN3 lacking the headpiece region does not rescue the phenotype of the vln2 vln3 double mutant. Our results show that villin is involved in the generation of thick actin filament bundles in several cell types and suggest that these bundles are involved in the regulation of coordinated cell expansion. PMID:22209875

  14. Arabidopsis VILLIN2 and VILLIN3 Are Required for the Generation of Thick Actin Filament Bundles and for Directional Organ Growth[C][W

    PubMed Central

    van der Honing, Hannie S.; Kieft, Henk; Emons, Anne Mie C.; Ketelaar, Tijs

    2012-01-01

    In plant cells, actin filament bundles serve as tracks for myosin-dependent organelle movement and play a role in the organization of the cytoplasm. Although virtually all plant cells contain actin filament bundles, the role of the different actin-bundling proteins remains largely unknown. In this study, we investigated the role of the actin-bundling protein villin in Arabidopsis (Arabidopsis thaliana). We used Arabidopsis T-DNA insertion lines to generate a double mutant in which VILLIN2 (VLN2) and VLN3 transcripts are truncated. Leaves, stems, siliques, and roots of vln2 vln3 double mutant plants are twisted, which is caused by local differences in cell length. Microscopy analysis of the actin cytoskeleton showed that in these double mutant plants, thin actin filament bundles are more abundant while thick actin filament bundles are virtually absent. In contrast to full-length VLN3, truncated VLN3 lacking the headpiece region does not rescue the phenotype of the vln2 vln3 double mutant. Our results show that villin is involved in the generation of thick actin filament bundles in several cell types and suggest that these bundles are involved in the regulation of coordinated cell expansion. PMID:22209875

  15. Single-molecule visualization of a formin-capping protein ‘decision complex' at the actin filament barbed end

    PubMed Central

    Bombardier, Jeffrey P.; Eskin, Julian A.; Jaiswal, Richa; Corrêa, Ivan R.; Xu, Ming-Qun; Goode, Bruce L.; Gelles, Jeff

    2015-01-01

    Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived ‘decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells. PMID:26566078

  16. Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo {sup 1}H NMR and fluorescence spectroscopy

    SciTech Connect

    Wazawa, Tetsuichi; Sagawa, Takashi; Ogawa, Tsubasa; Morimoto, Nobuyuki; Kodama, Takao; Suzuki, Makoto

    2011-01-28

    Research highlights: {yields} Translationally hyper-mobile water has been detected around actin filaments. {yields} Translationally hyper-mobile water is formed upon polymerization of actin. {yields} Low water viscosity was found around F-actin using fluorescence anisotropy. {yields} Formation of hyper-mobile water may explain endothermic actin polymerization. -- Abstract: This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo {sup 1}H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by {approx}5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1 ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.

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

    PubMed Central

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

    2011-01-01

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

  18. Actomyosin contractility spatiotemporally regulates actin network dynamics in migrating cells.

    PubMed

    Okeyo, Kennedy Omondi; Adachi, Taiji; Sunaga, Junko; Hojo, Masaki

    2009-11-13

    Coupling interactions among mechanical and biochemical factors are important for the realization of various cellular processes that determine cell migration. Although F-actin network dynamics has been the focus of many studies, it is not yet clear how mechanical forces generated by actomyosin contractility spatiotemporally regulate this fundamental aspect of cell migration. In this study, using a combination of fluorescent speckle microscopy and particle imaging velocimetry techniques, we perturbed the actomyosin system and examined quantitatively the consequence of actomyosin contractility on F-actin network flow and deformation in the lamellipodia of actively migrating fish keratocytes. F-actin flow fields were characterized by retrograde flow at the front and anterograde flow at the back of the lamellipodia, and the two flows merged to form a convergence zone of reduced flow intensity. Interestingly, activating or inhibiting actomyosin contractility altered network flow intensity and convergence, suggesting that network dynamics is directly regulated by actomyosin contractility. Moreover, quantitative analysis of F-actin network deformation revealed that the deformation was significantly negative and predominant in the direction of cell migration. Furthermore, perturbation experiments revealed that the deformation was a function of actomyosin contractility. Based on these results, we suggest that the actin cytoskeletal structure is a mechanically self-regulating system, and we propose an elaborate pathway for the spatiotemporal self-regulation of the actin cytoskeletal structure during cell migration. In the proposed pathway, mechanical forces generated by actomyosin interactions are considered central to the realization of the various mechanochemical processes that determine cell motility. PMID:19665125

  19. Preparation of bead-tailed actin filaments: estimation of the torque produced by the sliding force in an in vitro motility assay.

    PubMed Central

    Suzuki, N; Miyata, H; Ishiwata, S; Kinosita, K

    1996-01-01

    By coating covalently the surface of a polystyrene bead (diameter = 1 micron) with gelsolin, we have succeeded in attaching the bead selectively at the barbed end of an actin filament and forming a 1:1 bead-actin filament complex. On a layer of heavy meromyosin on a nitrocellulose-coated coverglass, this bead-actin filament complex slid smoothly, trailing the bead at its end. Therefore we called this preparation "bead-tailed" actin filaments. The sliding velocity was indistinguishable from that of nonbeaded filaments. With use of this system, we tried to detect the axial rotation (rotation around the filament axis) in a sliding actin filament. Although a single bead at the tail end did not serve as the marker for the axial rotation, we occasionally found another bead bound to the tail bead. In this case, the orientation of the bead-aggregate could be followed continuously with a video monitor while the filament was sliding over heavy meromyosin. We observed that actin filaments slid over distances of many tens of micrometers without showing a complete turn of the bead-aggregates. On the basis of the calculation of rotational friction drag on the bead-aggregate, we estimate that the rotational component of the sliding force and the torque produced on a sliding actin filament (length < or = 10 microns) did not accumulate > 1 pN and 5 pN.nm, respectively. In the present system of randomly oriented heavy meromyosin lying on a nitrocellulose film without an external load. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 6 PMID:8770216

  20. Thrombospondin-1 Modulates Actin Filament Remodeling and Cell Motility in Mouse Mammary Tumor cells in Vitro

    PubMed Central

    Ndishabandi, Dorothy; Duquette, Cameron; Billah, Ghita El-Moatassim; Reyes, Millys; Duquette, Mark; Lawler, Jack; Kazerounian, Shideh

    2015-01-01

    It is well established that the secretion of thrombospondin-1 (TSP-1) by activated stromal cells and its accumulation in the tumor microenvironment during dysplasia inhibits primary tumor growth through inhibition of angiogenesis. This inhibitory function of TSP-1 is actuated either by inhibiting MMP9 activation and the release of VEGF from extracellular matrix or by an interaction with CD36 on the surface of endothelial cells resulting in an increase in apoptosis. In contrast, several published articles have also shown that as tumor cells become more invasive and enter the early stage of carcinoma, they up-regulate TSP-1 expression, which may promote invasion and migration. In our in vivo studies using the polyoma middle T antigen (PyT) transgenic mouse model of breast cancer, we observed that the absence of TSP-1 significantly increased the growth of primary tumors, but delayed metastasis to the lungs. In this study, we propose a mechanism for the promigratory function of TSP-1 in mouse mammary tumor cells in vitro. We demonstrate the correlations between expression of TSP-1 and its receptor integrin α3β1, which is considered a promigratory protein in cancer cells. In addition we propose that binding of TSP-1 to integrin α3β1 is important for mediating actin filament polymerization and therefore, cell motility. These findings can help explain the dual functionality of TSP-1 in cancer progression. PMID:26273699

  1. Cortical actin filament organization in developing and functioning stomatal complexes of Zea mays and Triticum turgidum.

    PubMed

    Panteris, Emmanuel; Galatis, Basil; Quader, Hartmut; Apostolakos, Panagiotis

    2007-07-01

    Cortical actin filament (AF) organization was studied in detail in developing stomatal complexes of the grasses Zea mays and Triticum turgidum. AF arrays during the whole stomatal complex development are dynamic, partly following the pattern of cortical microtubule (MT) organization. They also exhibit particular patterns of organization, spatially and temporarily restricted. Among AF arrays, the radial ones that underlie young guard cell (GC) periclinal walls, those that line the bulbous GC ends and the AF ring at the junction between subsidiary cells (SCs) and GCs are described here for the first time. Although many similarities in cortical AF organization exist among the stomatal cells of both plants studied, considerable differences have also been observed between them. Our data reveal that the expanding areas of stomatal cell walls are lined by distinct cortical AF aggregations that probably protect the plasmalemma against mechanical stresses. Experimental AF disruption does not seem to affect detectably stomatal cell morphogenesis. Moreover, the structural and experimental data of this study revealed that, in contrast to the elliptical stomata, in the dumbbell-shaped ones the AFs and MTs seem not to be involved in the mechanism of opening and closing of the stomatal pore. PMID:17443701

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

    PubMed Central

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

    2007-01-01

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

  3. Osmotic tolerance of in vitro produced porcine blastocysts assessed by their morphological integrity and cellular actin filament organization.

    PubMed

    Men, Hongsheng; Agca, Yuksel; Mullen, Steven F; Critser, Elizabeth S; Critser, John K

    2005-10-01

    This experiment investigated the osmotic tolerance limits of the morphology and the cellular actin filament organization of porcine blastocysts. In vitro produced Day 6 blastocysts were subjected to osmotic treatments with sucrose solutions of different osmolalities (75, 150, 210, 600, 1200, and 2400 mOsm) and one isotonic solution (NCSU-23, 285 mOsm). Blastocysts were then either fixed immediately, or cultured for 18 h and subsequently fixed with formalin. The morphology of the treated blastocysts was examined under a stereomicroscope and the integrity of the cellular actin filaments of the blastocysts was examined by confocal microscopy after staining with Alexa Fluor 488 phalloidin. The results indicated that there was a significant relationship between the osmotic levels and the probability of blastocysts exhibiting disrupted cellular actin filaments. In addition, blastocysts also collapsed in proportion to the levels of osmotic treatments. The osmotic tolerance limits which would maintain 70% of the blastocysts with their original morphology immediately after the treatment were 90 and 170%, respectively, of isotonicity. After 18 h of culture, the osmotic tolerance limits were 61 and 163%, respectively, of isotonicity. Similarly, the osmotic conditions relative to isotonicity which would maintain the integrity of cellular actin filaments in 70% of treated blastocysts had to be within the range of 87 and 147% immediately after the treatment and 87 and 169% after 18 h of culture. Collectively, these data indicate that in vitro produced porcine blastocysts are very sensitive to osmotic stress. This information can be used to optimize cryopreservation procedures for porcine embryos. PMID:16024011

  4. Micromechanical properties of keratin intermediate filament networks.

    PubMed

    Sivaramakrishnan, Sivaraj; DeGiulio, James V; Lorand, Laszlo; Goldman, Robert D; Ridge, Karen M

    2008-01-22

    Keratin intermediate filaments (KIFs) form cytoskeletal KIF networks that are essential for the structural integrity of epithelial cells. However, the mechanical properties of the in situ network have not been defined. Particle-tracking microrheology (PTM) was used to obtain the micromechanical properties of the KIF network in alveolar epithelial cells (AECs), independent of other cytoskeletal components, such as microtubules and microfilaments. The storage modulus (G') at 1 Hz of the KIF network decreases from the perinuclear region (335 dyn/cm(2)) to the cell periphery (95 dyn/cm(2)), yielding a mean value of 210 dyn/cm(2). These changes in G' are inversely proportional to the mesh size of the network, which increases approximately 10-fold from the perinuclear region (0.02 microm(2)) to the cell periphery (0.3 microm(2)). Shear stress (15 dyn/cm(2) for 4 h) applied across the surface of AECs induces a more uniform distribution of KIF, with the mesh size of the network ranging from 0.02 microm(2) near the nucleus to only 0.04 microm(2) at the cell periphery. This amounts to a 40% increase in the mean G'. The storage modulus of the KIF network in the perinuclear region accurately predicts the shear-induced deflection of the cell nucleus to be 0.87 +/- 0.03 microm. The high storage modulus of the KIF network, coupled with its solid-like rheological behavior, supports the role of KIF as an intracellular structural scaffold that helps epithelial cells to withstand external mechanical forces. PMID:18199836

  5. Viscoelastic dynamics in a system of two actin filaments under stress

    NASA Astrophysics Data System (ADS)

    Boerma, Arjan Erik; van der Giessen, Erik; Papanikolaou, Stefanos

    The viscoelasticity of cytoskeleton networks is experimentally well-established but still lacks a consistent theoretical description. We present a novel minimal model that consists of two semi-flexible filaments coupled by cross-linkers, whose dynamics are described by Grand Canonical Monte Carlo. The mechanical properties are captured in the continuum and solved through an athermal finite-element approach. We discuss the phase diagram of the model and the emergence of viscoelastic behavior: the variation of the dynamic modulus as a function of loading frequency and density of cross-linkers, in thermodynamically and biologically realistic settings.

  6. RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling Endosomes.

    PubMed

    Wang, Peixiang; Liu, Hang; Wang, Yu; Liu, Ou; Zhang, Jing; Gleason, Adenrele; Yang, Zhenrong; Wang, Hui; Shi, Anbing; Grant, Barth D

    2016-06-01

    EHBP-1 (Ehbp1) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction. Furthermore, our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin microfilaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. We propose a mechanism whereby RAB-10 promotes the ability of endosome-bound EHBP-1 to also bind to the actin cytoskeleton, thereby promoting endosomal tubulation. PMID:27272733

  7. RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling Endosomes

    PubMed Central

    Wang, Yu; Liu, Ou; Zhang, Jing; Gleason, Adenrele; Yang, Zhenrong; Wang, Hui; Shi, Anbing; Grant, Barth D.

    2016-01-01

    EHBP-1 (Ehbp1) is a conserved regulator of endocytic recycling, acting as an effector of small GTPases including RAB-10 (Rab10). Here we present evidence that EHBP-1 associates with tubular endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] enriched membranes through an N-terminal C2-like (NT-C2) domain, and define residues within the NT-C2 domain that mediate membrane interaction. Furthermore, our results indicate that the EHBP-1 central calponin homology (CH) domain binds to actin microfilaments in a reaction that is stimulated by RAB-10(GTP). Loss of any aspect of this RAB-10/EHBP-1 system in the C. elegans intestinal epithelium leads to retention of basolateral recycling cargo in endosomes that have lost their normal tubular endosomal network (TEN) organization. We propose a mechanism whereby RAB-10 promotes the ability of endosome-bound EHBP-1 to also bind to the actin cytoskeleton, thereby promoting endosomal tubulation. PMID:27272733

  8. A tridimensional view of the organization of actin filaments in the central nervous system by use of fluorescent photooxidation.

    PubMed

    Capani, Francisco; Saraceno, Ezequiel; Boti, Valeria Romina; Aon-Bertolino, Laura; Fernández, Juan Carlos; Gato, Fernándo; Kruse, Maria Sol; Krause, Maria Sol; Giraldez, Lisandro; Ellisman, Mark H; Coirini, Héctor

    2008-04-01

    Cellular and subcellular organization and distribution of actin filaments have been studied with various techniques. The use of fluorescence photo-oxidation combined with phalloidin conjugates with eosin has allowed the examination of the precise cellular and subcellular location of F-actin. Correlative fluorescence light microscopy and transmission electron microscopy studies of F-actin distribution are facilitated with this method for morphological and physiological studies. Because phalloidin-eosin is smaller than other markers, this method allows the analysis of the three-dimensional location of F-actin with high-resolution light microscopy, three-d serial sections reconstructions, and electron tomography. The combination of selective staining and three-dimensional reconstructions provide a valuable tool for revealing aspects of the synaptic morphology that are not available when conventional electron microscopy is used. By applying this selective staining technique and three-dimensional imaging, we uncovered the structural organization of actin in the postsynaptic densities in physiological and pathological conditions. PMID:18669318

  9. Roles of actin filaments and three second-messenger systems in short-term regulation of chick dorsal root ganglion neurite outgrowth.

    PubMed

    Lankford, K L; Letourneau, P C

    1991-01-01

    In a previous study (J. Cell Biol. 109: 1229-1243, 1989), we reported that conditions which increased growth cone calcium levels and induced neurite retraction in cultured chick DRG neurons also resulted in an apparent loss of actin filaments in the growth cone periphery. We further showed that the actin-stabilizing drug phalloidin could block or reverse calcium-ionophore-induced neurite retraction, indicating that the behavioral changes were mediated, at least in part, by changes in actin filament stability. In this study, we have further characterized the calcium sensitivity of growth cone behavior to identify which features of calcium-induced behavioral effects can be attributed to effects on actin filaments alone, and to assess whether two other second-messenger systems, cAMP and protein kinase C, might influence neurite outgrowth by altering calcium levels or actin stability. The results indicated that growth cone behavior was highly sensitive to small changes in calcium concentrations. Neurite outgrowth was only observed in calcium-permeabilized cells when extracellular calcium concentrations were between 200 and 300 nM, and changes as small as 50 nM commonly produced detectable changes in behavior. Furthermore, low doses of cytochalasins mimicked all of the grossly observable features of growth cone responses to elevation of intracellular calcium, including the apparent preferential destruction of lamellipodial actin filaments and sparing of filopodial actin, suggesting that the behavioral effects of calcium elevation could be explained by loss of actin filaments alone. The effects of cAMP elevation and protein kinase C activation on growth cone behavior, ultrastructure, and fura2-AM-measured calcium levels indicated that the effects of cAMP manipulations could be partially explained by a cAMP-induced lowering of growth cone calcium levels and concomitant increased stabilization of actin filaments, but protein kinase C appeared to act through an independent

  10. Plant pathogenic bacteria target the actin microfilament network involved in the trafficking of disease defense components

    PubMed Central

    Jelenska, Joanna; Kang, Yongsung; Greenberg, Jean T

    2014-01-01

    Cells of infected organisms transport disease defense-related molecules along actin filaments to deliver them to their sites of action to combat the pathogen. To accommodate higher demand for intracellular traffic, plant F-actin density increases transiently during infection or treatment of Arabidopsis with pathogen-associated molecules. Many animal and plant pathogens interfere with actin polymerization and depolymerization to avoid immune responses. Pseudomonas syringae, a plant extracellular pathogen, injects HopW1 effector into host cells to disrupt the actin cytoskeleton and reduce vesicle movement in order to elude defense responses. In some Arabidopsis accessions, however, HopW1 is recognized and causes resistance via an actin-independent mechanism. HopW1 targets isoform 7 of vegetative actin (ACT7) that is regulated by phytohormones and environmental factors. We hypothesize that dynamic changes of ACT7 filaments are involved in plant immunity. PMID:25551177

  11. Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 is regulated by actin filaments and 4.1N in neuronal dendrites.

    PubMed

    Fukatsu, Kazumi; Bannai, Hiroko; Zhang, Songbai; Nakamura, Hideki; Inoue, Takafumi; Mikoshiba, Katsuhiko

    2004-11-19

    Inositol 1,4,5-trisphosphate receptor type1 (IP3R1) plays an important role in neuronal functions; however, the lateral diffusion of IP3R1 on the endoplasmic reticulum membrane and its regulation in the living neurons remain unknown. We expressed green fluorescent protein-tagged IP3R1 in cultured rat hippocampal neurons and observed the lateral diffusion by the fluorescence recovery after photobleaching technique. IP3R1 showed lateral diffusion with an effective diffusion constant of approximately 0.3 microm2/s. Depletion of actin filaments increased the diffusion constant of IP3R1, suggesting that the diffusion of IP3R1 is regulated negatively through actin filaments. We also found that protein 4.1N, which binds to IP3R1 and contains an actin-spectrin-binding region, was responsible for this actin regulation of the IP3R1 diffusion constant. Overexpression of dominant-negative 4.1N and blockade of 4.1N binding to IP3R1 increased the IP3R1 diffusion constant. The diffusion of IP3R type 3 (IP3R3), one of the isoforms of IP3Rs lacking the binding ability to 4.1N, was not dependent on actin filaments but became dependent on actin filaments after the addition of a 4.1N-binding sequence. These data suggest that 4.1N serves as a linker protein between IP3R1 and actin filaments. This actin filament-dependent regulation of IP3R1 diffusion may be important for the spatiotemporal regulation of intracellular Ca2+ signaling. PMID:15364918

  12. Drosophila Homologues of Adenomatous Polyposis Coli (APC) and the Formin Diaphanous Collaborate by a Conserved Mechanism to Stimulate Actin Filament Assembly*

    PubMed Central

    Jaiswal, Richa; Stepanik, Vince; Rankova, Aneliya; Molinar, Olivia; Goode, Bruce L.; McCartney, Brooke M.

    2013-01-01

    Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad range of animal phyla. PMID:23558679

  13. An easy-to-use single-molecule speckle microscopy enabling nanometer-scale flow and wide-range lifetime measurement of cellular actin filaments.

    PubMed

    Yamashiro, Sawako; Mizuno, Hiroaki; Watanabe, Naoki

    2015-01-01

    Single-molecule speckle (SiMS) microscopy has been a powerful method to analyze actin dynamics in live cells by tracking single molecule of fluorescently labeled actin. Recently we developed a new SiMS method, which is easy-to-use for inexperienced researchers and achieves high spatiotemporal resolution. In this method, actin labeled with fluorescent DyLight dye on lysines is employed as a probe. Electroporation-mediated delivery of DyLight-actin (DL-actin) into cells enables to label cells with 100% efficiency at the optimal density. DL-actin labels cellular actin filaments including formin-based structures with improved photostability and brightness compared to green fluorescent protein-actin. These favorable properties of DL-actin extend time window of the SiMS analysis. Furthermore, the new SiMS method enables nanometer-scale displacement analysis with a low localization error of ±8-8.5 nm. With these advantages, our new SiMS microscopy method will help researchers to investigate various actin remodeling processes. In this chapter, we introduce the methods for preparation of DL-actin probes, electroporation to deliver DL-actin, the SiMS imaging and data analysis. PMID:25640423

  14. Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth

    PubMed Central

    Zhou, Zhenzhen; Shi, Haifan; Chen, Binqing; Zhang, Ruihui; Huang, Shanjin; Fu, Ying

    2015-01-01

    Pollen tubes deliver sperms to the ovule for fertilization via tip growth. The rapid turnover of F-actin in pollen tube tips plays an important role in this process. In this study, we demonstrate that Arabidopsis thaliana RIC1, a member of the ROP-interactive CRIB motif-containing protein family, regulates pollen tube growth via its F-actin severing activity. Knockout of RIC1 enhanced pollen tube elongation, while overexpression of RIC1 dramatically reduced tube growth. Pharmacological analysis indicated that RIC1 affected F-actin dynamics in pollen tubes. In vitro biochemical assays revealed that RIC1 directly bound and severed F-actin in the presence of Ca2+ in addition to interfering with F-actin turnover by capping F-actin at the barbed ends. In vivo, RIC1 localized primarily to the apical plasma membrane (PM) of pollen tubes. The level of RIC1 at the apical PM oscillated during pollen tube growth. The frequency of F-actin severing at the apex was notably decreased in ric1-1 pollen tubes but was increased in pollen tubes overexpressing RIC1. We propose that RIC1 regulates F-actin dynamics at the apical PM as well as the cytosol by severing F-actin and capping the barbed ends in the cytoplasm, establishing a novel mechanism that underlies the regulation of pollen tube growth. PMID:25804540

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

    PubMed

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

    2016-10-01

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

  16. A simple method for measuring the relative force exerted by myosin on actin filaments in the in vitro motility assay: evidence that tropomyosin and troponin increase force in single thin filaments.

    PubMed Central

    Bing, W; Knott, A; Marston, S B

    2000-01-01

    We have studied the effect of an internal load on the movement of actin filaments over a bed of heavy meromyosin (HMM) in the in vitro motility assay. Immobilized alpha-actinin can bind to actin filaments reversibly and ultimately stop the filaments from moving. Above a critical concentration of alpha-actinin, thin filament velocity rapidly diminished to zero. The fraction of thin motile filaments decreased linearly to zero with increasing alpha-actinin concentration. The concentration of alpha-actinin needed to stop all filaments from moving (0.8 microg/ml with actin) was very consistent both within and between experiments. In the present study we have defined the 'index of retardation' as the concentration of alpha-actinin needed to stop all filament movement, and we propose that this index is a measure of the isometric force exerted by HMM on actin filaments. When we measured the effect of immobilized alpha-actinin on motility in the presence of 10 mM P(i) we found that the index of retardation was 0.62+/-0.07 (n=3) times that in the absence of P(i). This observation is in agreement with the reduction of isometric tension in chemically-skinned muscle due to P(i). In a series of comparative experiments we observed that tropomyosin and troponin increase the index of retardation and that the degree of increase depends upon the tropomyosin isoform studied. The index of retardation of actin is increased 1.8-fold by skeletal-muscle tropomyosin, and 3-fold by both cardiac-muscle and smooth-muscle tropomyosin. In the presence of troponin the index of retardation is 2.9-3.4-fold greater than that of actin with all tropomyosin isoforms. PMID:10970781

  17. Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an exemplary target

    PubMed Central

    2013-01-01

    Background Cucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology. Findings Cucurbitacin reacted with protein cysteine thiols as well as dithiothreitol, and we propose that the cucurbitacin mechanism of action is through broad protein thiol modifications that could result in inhibition of numerous protein targets. An example of such a target protein is Cofilin1, whose filamentous actin severing activity is inhibited by cucurbitacin conjugation. Conclusions The implications of these results are that cucurbitacins are unlikely to be improved for selectivity by medicinal chemistry and that their use as chemical biology probes to analyse the role of specific signalling pathways should be undertaken with caution. PMID:23945128

  18. Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton

    PubMed Central

    Jia, Yifeng; Wu, Shiaw-Lin; Isenberg, Jeff S.; Dai, Shujia; Sipes, John M.; Field, Lyndsay; Zeng, Bixi; Bandle, Russell W.; Ridnour, Lisa A.; Wink, David A.; Ramchandran, Ramani; Karger, Barry L.

    2009-01-01

    Thiolutin is a dithiole synthesized by Streptomyces sp. that inhibits endothelial cell adhesion and tumor growth. We show here that thiolutin potently inhibits developmental angiogenesis in zebrafish and vascular outgrowth from tissue explants in 3D cultures. Thiolutin is a potent and selective inhibitor of endothelial cell adhesion accompanied by rapid induction of HSPB1 (Hsp27) phosphorylation. The inhibitory effects of thiolutin on endothelial cell adhesion are transient, potentially due to a compensatory increase in Hsp27 protein levels. Accordingly, heat shock induction of Hsp27 limits the anti-adhesive activity of thiolutin. Thiolutin treatment results in loss of actin stress fibers, increased cortical actin as cells retract, and decreased cellular F-actin. Mass spectrometric analysis of Hsp27 binding partners following immunoaffinity purification identified several regulatory components of the actin cytoskeleton that associate with Hsp27 in a thiolutin-sensitive manner including several components of the Arp2/3 complex. Among these, ArpC1a is a direct binding partner of Hsp27. Thiolutin treatment induces peripheral localization of phosphorylated Hsp27 and Arp2/3. Hsp27 also associates with the intermediate filament components vimentin and nestin. Thiolutin treatment specifically ablates Hsp27 interaction with nestin and collapses nestin filaments. These results provide new mechanistic insights into regulation of cell adhesion and cytoskeletal dynamics by Hsp27. Electronic supplementary material The online version of this article (doi:10.1007/s12192-009-0130-0) contains supplementary material, which is available to authorized users. PMID:19579057

  19. A coat of filamentous actin prevents clustering of late-endosomal vacuoles in vivo.

    PubMed

    Drengk, Anja; Fritsch, Jürgen; Schmauch, Christian; Rühling, Harald; Maniak, Markus

    2003-10-14

    The endocytic pathway depends on the actin cytoskeleton. Actin contributes to internalization at the plasma membrane and to subsequent trafficking steps like propulsion through the cytoplasm, fusion of phagosomes with early endosomes, and transport from early to late endosomes. In vitro studies with mammalian endosomes and yeast vacuoles implicate actin in membrane fusion. Here, we investigate the function of the actin coat that surrounds late endosomes in Dictyostelium. Latrunculin treatment leads to aggregation of these endosomes into grape-like clusters and completely blocks progression of endocytic marker. In addition, the cells round up and stop moving. Because this drug treatment perturbs all actin assemblies in the cell simultaneously, we used a novel targeting approach to specifically study the function of the cytoskeleton in one subcellular location. To this end, we constructed a hybrid protein targeting cofilin, an actin depolymerizing protein, to late endosomes. As a consequence, the endosomal compartments lost their actin coats and aggregated, but these cells remained morphologically normal, and the kinetics of endocytic marker trafficking were unaltered. Therefore, the actin coat prevents the clustering of endosomes, which could be one safeguard mechanism precluding their docking and fusion. PMID:14561408

  20. Filament Recognition In Solar Images With The Neural Network Technique

    NASA Astrophysics Data System (ADS)

    Zharkova, V. V.; Schetinin, V.

    2005-05-01

    We describe a new technique developed for an automated recognition of solar filaments visible in Hα hydrogen line full-disk spectroheliograms. These filaments are difficult to recognize because of variability in the background caused by atmospheric conditions. The presented technique is based on an artificial neural network (ANN) consisting of two hidden neurons and one output neuron which learn to exclude the contribution of a changeable background to a filament. The ANN is trained on a single image fragment labeled manually to recognize the filament elements depicted on a local background. The background contribution is approximated with linear and parabolic functions. This technique applied to the filament recognition in 54 cropped images reveals better detection results for a parabolic approximation than for a linear one approaching an accuracy of about 82% of the total filament pixels.

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

    PubMed

    Holzinger, A; Lütz-Meindl, U

    2001-02-01

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

  2. Structural Evidence for Actin-like Filaments in Toxoplasma gondii Using High-Resolution Low-Voltage Field Emission Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Schatten, Heide; Sibley, L. David; Ris, Hans

    2003-08-01

    The protozoan parasite Toxoplasma gondii is representative of a large group of parasites within the phylum Apicomplexa, which share a highly unusual motility system that is crucial for locomotion and active host cell invasion. Despite the importance of motility in the pathology of these unicellular organisms, the motor mechanisms for locomotion remain uncertain, largely because only limited data exist about composition and organization of the cytoskeleton. By using cytoskeleton stabilizing protocols on membrane-extracted parasites and novel imaging with high-resolution low-voltage field emission scanning electron microscopy (LVFESEM), we were able to visualize for the first time a network of actin-sized filaments just below the cell membrane. A complex cytoskeletal network remained after removing the actin-sized fibers with cytochalasin D, revealing longitudinally arranged, subpellicular microtubules and intermediate-sized fibers of 10 nm, which, in stereo images, are seen both above and below the microtubules. These approaches open new possibilities to characterize more fully the largely unexplored and unconventional cytoskeletal motility complex in apicomplexan parasites.

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

    PubMed Central

    2016-01-01

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

  4. Actin Mechanics and Fragmentation*

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Limozin, Laurent; Sackmann, Erich

    2002-09-01

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

  6. Non-equilibrium States of Active Filament Networks

    NASA Astrophysics Data System (ADS)

    Blackwell, Robert A.; Betterton, Meredith D.; Sweezy, Oliver M.; Glaser, Matthew A.

    2014-03-01

    Active networks of filamentous proteins and crosslinking motor proteins play a critical role in many cellular processes. Among the most important active networks is the mitotic spindle, an assembly of microtubules and crosslinking motor proteins that forms during cell division and that ultimately separates chromosomes into two daughter cells. To evolve a better understanding of spindle formation, structure, and dynamics, we have developed course-grained models of active networks composed of filaments, modeled as hard spherocylinders, in diffusive equilibrium with a reservoir of crosslinking motors, modeled as Hookean springs that can adsorb to microtubules and translocate at finite velocity along the microtubule axis. We explore the phase diagram and other characteristics of this model in two and three dimensions as a function of filament packing fraction, and of crosslink concentration, velocity, and adsorption and desorption rates. We observe a variety of interesting emergent behaviors including sorting of filaments into polar domains, generation of extensile stress, and superdiffusive transport. DMR-0820579

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

    PubMed

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

    2015-08-01

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

  8. Observation of Actin Filaments in Leydig Cells with a Contact-type Soft X-ray Microscope with Laser Plasma X-ray Source

    NASA Astrophysics Data System (ADS)

    Kado, Masataka; Ishino, Masahiko; Tamotsu, Satoshi; Yasuda, Keiko; Kishimoto, Maki; Nishikino, Masaharu; Kinjo, Yasuhito; Shinohara, Kunio

    Actin filaments in Leydig cells from mouse testes have been observed with a contact-type soft x-ray microscope with laser plasma x-ray source. The Leydig cells were fixed with paraformaldehyde, stained with Phalloidin, and observed with a confocal laser microscope prior to the observation with x-ray microscope. Obtained images by both of the confocal laser microscopy and the x-ray microscopy were directly compared and revealed that not only position of actin filaments but also the shapes can be identified each other. The actin filaments in the x-ray images were clearly recognized and their structures were obtained in more detail compared to those in the confocal laser microscope images.

  9. 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

  10. The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis.

    PubMed

    Li, Li-Juan; Ren, Fei; Gao, Xin-Qi; Wei, Peng-Cheng; Wang, Xue-Chen

    2013-02-01

    The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin-related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin-D-induced depolymerization or phalloidin-induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild-type (WT) Arabidopsis plants. Light-induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening. PMID:22891733

  11. Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD

    PubMed Central

    Chen, Yujian; Liu, Yong; Guo, Jiayu; Tang, Tao; Gao, Jian; Huang, Tao; Wang, Bin; Liu, Shaojun

    2016-01-01

    Actin filament-associated protein-120kD (AFAP-120) is an alternatively spliced isoform of actin filament-associated protein-110kD (AFAP-110) and contains an additional neuronal insert (NINS) fragment in addition to identical domains to the AFAP-110. Unlike AFAP-110 widely expressed in tissues, AFAP-120 is specifically expressed in the nervous system and plays a role in organizing dynamic actin structures during neuronal differentiation. However, anti-AFAP-120 antibody is still commercially unavailable, and this may hinder the function research for AFAP-120. In this study, we simultaneously used the ABCpred online server and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of human AFAP-120 protein, and found that a 16aa-peptide sequence was the consensus epitope predicted by both tools. This peptide was chemically synthesized and used as an immunogen to develop polyclonal antibody against AFAP-120 (anti-AFAP-120). The sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, immunoprecipitation, and immunofluorescence assays. Our results indicated that anti-AFAP-120 could react with over-expressed and endogenous human AFAP-120 protein under denatured condition, but not with human AFAP-110 protein. Moreover, native human AFAP-120 protein could also be recognized by the anti-AFAP-120 antibody. These results suggested that the prepared anit-AFAP-120 antibody would be a useful tool for studying the biochemical and biological functions of AFAP-120. PMID:27322249

  12. Association of hepatitis C virus replication complexes with microtubules and actin filaments is dependent on the interaction of NS3 and NS5A.

    PubMed

    Lai, Chao-Kuen; Jeng, King-Song; Machida, Keigo; Lai, Michael M C

    2008-09-01

    The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A. PMID:18562541

  13. Invadosomes - shaping actin networks to follow mechanical cues.

    PubMed

    Kedziora, Katarzyna M; Isogai, Tadamoto; Jalink, Kees; Innocenti, Metello

    2016-01-01

    Invadosomes are actin-based protrusions formed by cells in response to obstacles in their microenvironment, especially basement membranes and dense interstitial matrices. A versatile set of proteins controls assembly and dynamics of the actin networks at invadosomes and adhesive molecules link them with the extracellular matrix. Furthermore, polarized delivery of proteases makes invadosomes degradative. Therefore, invadosomes have been classically viewed as specialized protrusions involved in cell migration and remodeling of the microenvironment. Recent discoveries have considerably broadened this picture by showing that invadosomes respond to traction forces and can self-organize into dynamic arrays capable of following the topography of the substrate. Although these findings suggest that invadosomes may function as mechanosensors, this possibility has not been critically evaluated. In this review, we first summarize the organization and dynamics of actin in invadosomes and their superstructures with emphasis on force-production mechanisms. Next, we outline our current understanding of how mechanical cues impinge on invadosomes and modify their behavior. From this perspective, we provide an outlook of the outstanding open questions and the main challenges in the field. PMID:27100494

  14. MamK, a bacterial actin, forms dynamic filaments in vivo that are regulated by the acidic proteins MamJ and LimJ

    PubMed Central

    Draper, Olga; Byrne, Meghan E.; Li, Zhuo; Keyhani, Sepehr; Cueto Barrozo, Joyce; Jensen, Grant; Komeili, Arash

    2011-01-01

    SUMMARY Bacterial actins, in contrast to their eukaryotic counterparts, are highly divergent proteins whose wide-ranging functions are thought to correlate with their evolutionary diversity. One clade, represented by the MamK protein of magnetotactic bacteria, is required for the subcellular organization of magnetosomes, membrane-bound organelles that aid in navigation along the earth’s magnetic field. Using a fluorescence recovery after photobleaching assay in Magnetospirillum magneticum AMB-1, we find that, like traditional actins, MamK forms dynamic filaments that require an intact NTPase motif for their turnover in vivo. We also uncover two proteins, MamJ and LimJ, which perform a redundant function to promote the dynamic behavior of MamK filaments in wildtype cells. The absence of both MamJ and LimJ leads to static filaments, a disrupted magnetosome chain, and an anomalous build-up of cytoskeletal filaments between magnetosomes. Our results suggest that MamK filaments, like eukaryotic actins, are intrinsically stable and rely on regulators for their dynamic behavior, a feature that stands in contrast to some classes of bacterial actins characterized to date. PMID:21883528

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

    PubMed

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

    2016-09-01

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

  16. CF2 represses Actin 88F gene expression and maintains filament balance during indirect flight muscle development in Drosophila.

    PubMed

    Gajewski, Kathleen M; Schulz, Robert A

    2010-01-01

    The zinc finger protein CF2 is a characterized activator of muscle structural genes in the body wall muscles of the Drosophila larva. To investigate the function of CF2 in the indirect flight muscle (IFM), we examined the phenotypes of flies bearing five homozygous viable mutations. The gross structure of the IFM was not affected, but the stronger hypomorphic alleles caused an increase of up to 1.5X in the diameter of the myofibrils. This size increase did not cause any disruption of the hexameric arrangement of thick and thin filaments. RT-PCR analysis revealed an increase in the transcription of several structural genes. Ectopic overexpression of CF2 in the developing IFM disrupts muscle formation. While our results indicate a role for CF2 as a direct negative regulator of the thin filament protein gene Actin 88F (Act88F), effects on levels of transcripts of myosin heavy chain (mhc) appear to be indirect. This role is in direct contrast to that described in the larval muscles, where CF2 activates structural gene expression. The variation in myofibril phenotypes of CF2 mutants suggest the CF2 may have separate functions in fine-tuning expression of structural genes to insure proper filament stoichiometry, and monitoring and/or controlling the final myofibril size. PMID:20520827

  17. Determination of the persistence length of actin filaments on microcontact printed myosin patterns

    NASA Astrophysics Data System (ADS)

    Hajne, Joanna; Hanson, Kristi L.; van Zalinge, Harm; Nicolau, Dan V.; Nicolau, Dan V.

    2015-03-01

    Protein molecular motors, which convert chemical energy into kinetic energy, are prime candidates for use in nanodevice in which active transport is required. To be able to design these devices it is essential that the properties of the cytoskeletal filaments propelled by the molecular motors are well established. Here we used micro-contact printed BSA to limit the amount of HMM that can adsorb creating a tightly confined pathway for the filaments to travel. Both the image and statistical analysis of the movement of the filaments through these structures have been used to new insights into the motility behaviour of actomyosin on topographically homogenous, but motor-heterogeneous planar systems. It will be shown that it is possible to determine the persistence length of the filaments and that it is related to the amount of locally adsorbed HMM. This provides a basis that can be used to optimize the design of future nanodevices incorporating the actomyosin system for the active transport.

  18. Attractive interactions among intermediate filaments determine network mechanics in vitro.

    PubMed

    Pawelzyk, Paul; Mücke, Norbert; Herrmann, Harald; Willenbacher, Norbert

    2014-01-01

    Mechanical and structural properties of K8/K18 and vimentin intermediate filament (IF) networks have been investigated using bulk mechanical rheometry and optical microrheology including diffusing wave spectroscopy and multiple particle tracking. A high elastic modulus G0 at low protein concentration c, a weak concentration dependency of G0 (G0 ∼ c(0.5 ± 0.1)) and pronounced strain stiffening are found for these systems even without external crossbridgers. Strong attractive interactions among filaments are required to maintain these characteristic mechanical features, which have also been reported for various other IF networks. Filament assembly, the persistence length of the filaments and the network mesh size remain essentially unaffected when a nonionic surfactant is added, but strain stiffening is completely suppressed, G0 drops by orders of magnitude and exhibits a scaling G0 ∼ c(1.9 ± 0.2) in agreement with microrheological measurements and as expected for entangled networks of semi-flexible polymers. Tailless K8Δ/K18ΔT and various other tailless filament networks do not exhibit strain stiffening, but still show high G0 values. Therefore, two binding sites are proposed to exist in IF networks. A weaker one mediated by hydrophobic amino acid clusters in the central rod prevents stretched filaments between adjacent cross-links from thermal equilibration and thus provides the high G0 values. Another strong one facilitating strain stiffening is located in the tail domain with its high fraction of hydrophobic amino acid sequences. Strain stiffening is less pronounced for vimentin than for K8/K18 due to electrostatic repulsion forces partly compensating the strong attraction at filament contact points. PMID:24690778

  19. Filamentous fungal-specific septin AspE is phosphorylated in vivo and interacts with actin, tubulin and other septins in the human pathogen Aspergillus fumigatus

    SciTech Connect

    Juvvadi, Praveen Rao; Belina, Detti; Soderblom, Erik J.; Moseley, M. Arthur; Steinbach, William J.

    2013-02-15

    Highlights: ► In vivo interactions of the novel septin AspE were identified by GFP-Trap® affinity purification. ► Septins AspA, AspB, AspC and AspD interacted with AspE in vivo. ► Actin and tubulin interacted with AspE in vivo. ► AspE is phosphorylated at six serine residues in vivo. -- Abstract: We previously analyzed the differential localization patterns of five septins (AspA–E), including a filamentous fungal-specific septin, AspE, in the human pathogen Aspergillus fumigatus. Here we utilized the A. fumigatus strain expressing an AspE–EGFP fusion protein and show that this novel septin with a tubular localization pattern in hyphae is phosphorylated in vivo and interacts with the other septins, AspA, AspB, AspC and AspD. The other major proteins interacting with AspE included the cytoskeletal proteins, actin and tubulin, which may be involved in the organization and transport of the septins. This is the first report analyzing the phosphorylation of AspE and localizing the sites of phosphorylation, and opens opportunities for further analysis on the role of post-translational modifications in the assembly and organization of A. fumigatus septins. This study also describes the previously unknown interaction of AspE with the actin-microtubule network. Furthermore, the novel GFP-Trap® affinity purification method used here complements widely-used GFP localization studies in fungal systems.

  20. Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy

    PubMed Central

    2013-01-01

    Background In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. Results We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. Conclusions Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities. PMID:23937664

  1. Mesoscopic model of actin-based propulsion.

    PubMed

    Zhu, Jie; Mogilner, Alex

    2012-01-01

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

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

    PubMed Central

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

    2008-01-01

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

  3. Regulation of cellular actin architecture by S100A10.

    PubMed

    Jung, M Juliane; Murzik, Ulrike; Wehder, Liane; Hemmerich, Peter; Melle, Christian

    2010-04-15

    Actin structures are involved in several biological processes and the disruption of actin polymerisation induces impaired motility of eukaryotic cells. Different factors are involved in regulation and maintenance of the cytoskeletal actin architecture. Here we show that S100A10 participates in the particular organisation of actin filaments. Down-regulation of S100A10 by specific siRNA triggered a disorganisation of filamentous actin structures without a reduction of the total cellular actin concentration. In contrast, the formation of cytoskeleton structures containing tubulin was unhindered in S100A10 depleted cells. Interestingly, the cellular distribution of annexin A2, an interaction partner of S100A10, was unaffected in S100A10 depleted cells. Cells lacking S100A10 showed an impaired migration activity and were unable to close a scratched wound. Our data provide first insights of S100A10 function as a regulator of the filamentous actin network. PMID:20100475

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

    PubMed

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

    2003-05-01

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

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

    SciTech Connect

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

    2009-06-09

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

  6. Cryo-EM structures of the actin:tropomyosin filament reveal the mechanism for the transition from C- to M-state.

    PubMed

    Sousa, Duncan R; Stagg, Scott M; Stroupe, M Elizabeth

    2013-11-15

    Tropomyosin (Tm) is a key factor in the molecular mechanisms that regulate the binding of myosin motors to actin filaments (F-Actins) in most eukaryotic cells. This regulation is achieved by the azimuthal repositioning of Tm along the actin (Ac):Tm:troponin (Tn) thin filament to block or expose myosin binding sites on Ac. In striated muscle, including involuntary cardiac muscle, Tm regulates muscle contraction by coupling Ca(2+) binding to Tn with myosin binding to the thin filament. In smooth muscle, the switch is the posttranslational modification of the myosin. Depending on the activation state of Tn and the binding state of myosin, Tm can occupy the blocked, closed, or open position on Ac. Using native cryogenic 3DEM (three-dimensional electron microscopy), we have directly resolved and visualized cardiac and gizzard muscle Tm on filamentous Ac in the position that corresponds to the closed state. From the 8-Å-resolution structure of the reconstituted Ac:Tm filament formed with gizzard-derived Tm, we discuss two possible mechanisms for the transition from closed to open state and describe the role Tm plays in blocking myosin tight binding in the closed-state position. PMID:24021812

  7. Pushing with actin: from cells to pathogens.

    PubMed

    Small, J Victor

    2015-02-01

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

  8. Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments.

    PubMed

    Montaville, Pierre; Kühn, Sonja; Compper, Christel; Carlier, Marie-France

    2016-02-12

    Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end. PMID:26668326

  9. Elasticity, adhesion and actin based propulsion

    NASA Astrophysics Data System (ADS)

    Gopinathan, Ajay

    2006-03-01

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

  10. 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

    2016-04-01

    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. PMID:26997484

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

    PubMed Central

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

    2015-01-01

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

  12. Distribution of Callose Synthase, Cellulose Synthase, and Sucrose Synthase in Tobacco Pollen Tube Is Controlled in Dissimilar Ways by Actin Filaments and Microtubules1[W

    PubMed Central

    Cai, Giampiero; Faleri, Claudia; Del Casino, Cecilia; Emons, Anne Mie C.; Cresti, Mauro

    2011-01-01

    Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules. PMID:21205616

  13. Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

    PubMed

    Fang, Kefeng; Gao, Sai; Zhang, Weiwei; Xing, Yu; Cao, Qingqin; Qin, Ling

    2016-01-01

    A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components. PMID:26886907

  14. Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture

    PubMed Central

    Fang, Kefeng; Gao, Sai; Zhang, Weiwei; Xing, Yu; Cao, Qingqin; Qin, Ling

    2016-01-01

    A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components. PMID:26886907

  15. Temperature control of the motility of actin filaments interacting with myosin molecules using an electrically conductive glass in the presence of direct current.

    PubMed

    Wada, Reito; Sato, Daisuke; Nakamura, Takao; Hatori, Kuniyuki

    2015-11-15

    The motility of actin filaments interacting with heavy meromyosin molecules was directly observed on indium tin oxide-coated glass (ITO-glass), over which a surface current flowed. Because the increase in surface current applied to ITO-glass increases the temperature, we focused on the temperature-dependence of the sliding velocity and the effect of the current flow on the orientation of filament motion. Using high precision fluorescence measurements, the displacement vectors of filaments were collected at intervals of 1/30 s. The direction of filament motion was independent to that of current flow up to 0.17 A (7.7 A/m of surface current density); however, the velocity increased by approximately 2-fold when the surface temperature increased from 25 °C to 37 °C. The moving actin filaments exhibited a broader velocity distribution at high temperature than at low temperature. Collectively, these data suggest that using ITO-glass with a surface current to generate a well-controlled temperature change may serve to evaluate temperature-dependent transient responses in protein activity under a microscope, without interference from electrical effects. PMID:26456400

  16. Myosins, Actin and Autophagy.

    PubMed

    Kruppa, Antonina J; Kendrick-Jones, John; Buss, Folma

    2016-08-01

    Myosin motor proteins working together with the actin cytoskeleton drive a wide range of cellular processes. In this review, we focus on their roles in autophagy - the pathway the cell uses to ensure homeostasis by targeting pathogens, misfolded proteins and damaged organelles for degradation. The actin cytoskeleton regulated by a host of nucleating, anchoring and stabilizing proteins provides the filament network for the delivery of essential membrane vesicles from different cellular compartments to the autophagosome. Actin networks have also been implicated in structurally supporting the expanding phagophore, moving autophagosomes and enabling efficient fusion with the lysosome. Only a few myosins have so far been shown to play a role in autophagy. Non-muscle myosin IIA functions in the early stages delivering membrane for the initial formation of the autophagosome, whereas myosin IC and myosin VI are involved in the final stages providing specific membranes for autophagosome maturation and its fusion with the lysosome. PMID:27146966

  17. Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets.

    PubMed Central

    Li, G; Rungger-Brändle, E; Just, I; Jonas, J C; Aktories, K; Wollheim, C B

    1994-01-01

    To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin. Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin. Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner. The toxin did not affect cellular insulin content or nonstimulated secretion. In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase). In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80%. Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin. Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed. In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin. C2 toxin dose-dependently reduced cellular F-actin content. Stimulation of insulin secretion was not associated with changes in F-actin content and organization. Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content. In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of

  18. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  1. 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

  2. Branching of keratin intermediate filaments.

    PubMed

    Nafeey, Soufi; Martin, Ines; Felder, Tatiana; Walther, Paul; Felder, Edward

    2016-06-01

    Keratin intermediate filaments (IFs) are crucial to maintain mechanical stability in epithelial cells. Since little is known about the network architecture that provides this stiffness and especially about branching properties of filaments, we addressed this question with different electron microscopic (EM) methods. Using EM tomography of high pressure frozen keratinocytes, we investigated the course of several filaments in a branching of a filament bundle. Moreover we found several putative bifurcations in individual filaments. To verify our observation we also visualized the keratin network in detergent extracted keratinocytes with scanning EM. Here bifurcations of individual filaments could unambiguously be identified additionally to bundle branchings. Interestingly, identical filament bifurcations were also found in purified keratin 8/18 filaments expressed in Escherichia coli which were reassembled in vitro. This excludes that an accessory protein contributes to the branch formation. Measurements of the filament cross sectional areas showed various ratios between the three bifurcation arms. This demonstrates that intermediate filament furcation is very different from actin furcation where an entire new filament is attached to an existing filament. Instead, the architecture of intermediate filament bifurcations is less predetermined and hence consistent with the general concept of IF formation. PMID:27039023

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

    NASA Astrophysics Data System (ADS)

    Burroughs, N. J.; Marenduzzo, D.

    2007-06-01

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

  4. 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.

  5. αT-Catenin Is a Constitutive Actin-binding α-Catenin That Directly Couples the Cadherin·Catenin Complex to Actin Filaments*

    PubMed Central

    Wickline, Emily D.; Dale, Ian W.; Merkel, Chelsea D.; Heier, Jonathon A.; Stolz, Donna B.

    2016-01-01

    α-Catenin is the primary link between the cadherin·catenin complex and the actin cytoskeleton. Mammalian αE-catenin is allosterically regulated: the monomer binds the β-catenin·cadherin complex, whereas the homodimer does not bind β-catenin but interacts with F-actin. As part of the cadherin·catenin complex, αE-catenin requires force to bind F-actin strongly. It is not known whether these properties are conserved across the mammalian α-catenin family. Here we show that αT (testes)-catenin, a protein unique to amniotes that is expressed predominantly in the heart, is a constitutive actin-binding α-catenin. We demonstrate that αT-catenin is primarily a monomer in solution and that αT-catenin monomer binds F-actin in cosedimentation assays as strongly as αE-catenin homodimer. The β-catenin·αT-catenin heterocomplex also binds F-actin with high affinity unlike the β-catenin·αE-catenin complex, indicating that αT-catenin can directly link the cadherin·catenin complex to the actin cytoskeleton. Finally, we show that a mutation in αT-catenin linked to arrhythmogenic right ventricular cardiomyopathy, V94D, promotes homodimerization, blocks β-catenin binding, and in cardiomyocytes disrupts localization at cell-cell contacts. Together, our data demonstrate that αT-catenin is a constitutively active actin-binding protein that can physically couple the cadherin·catenin complex to F-actin in the absence of tension. We speculate that these properties are optimized to meet the demands of cardiomyocyte adhesion. PMID:27231342

  6. Direct dynamin–actin interactions regulate the actin cytoskeleton

    PubMed Central

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

    2010-01-01

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

  7. Arabidopsis response to low-phosphate conditions includes active changes in actin filaments and PIN2 polarization and is dependent on strigolactone signalling

    PubMed Central

    Kumar, Manoj; Pandya-Kumar, Nirali; Dam, Anandamoy; Haor, Hila; Mayzlish-Gati, Einav; Belausov, Eduard; Wininger, Smadar; Abu-Abied, Mohamad; McErlean, Christopher S. P.; Bromhead, Liam J.; Prandi, Cristina; Kapulnik, Yoram; Koltai, Hinanit

    2015-01-01

    Strigolactones (SLs) are plant hormones that regulate the plant response to phosphate (Pi) growth conditions. At least part of SL-signalling execution in roots involves MAX2-dependent effects on PIN2 polar localization in the plasma membrane (PM) and actin bundling and dynamics. We examined PIN2 expression, PIN2 PM localization, endosome trafficking, and actin bundling under low-Pi conditions: a MAX2-dependent reduction in PIN2 trafficking and polarization in the PM, reduced endosome trafficking, and increased actin-filament bundling were detected in root cells. The intracellular protein trafficking that is related to PIN proteins but unassociated with AUX1 PM localization was selectively inhibited. Exogenous supplementation of the synthetic SL GR24 to a SL-deficient mutant (max4) led to depletion of PIN2 from the PM under low-Pi conditions. Accordingly, roots of mutants in MAX2, MAX4, PIN2, TIR3, and ACTIN2 showed a reduced low-Pi response compared with the wild type, which could be restored by auxin (for all mutants) or GR24 (for all mutants except max2-1). Changes in PIN2 polarity, actin bundling, and vesicle trafficking may be involved in the response to low Pi in roots, dependent on SL/MAX2 signalling. PMID:25609825

  8. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    SciTech Connect

    Purdy, Kirstin R.; Wong, Gerard C. L.; Bartles, James R.

    2007-02-02

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system's phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  9. Structural Polymorphism of the Actin-Espin System: A Prototypical System of Filaments and Linkers in Stereocilia

    NASA Astrophysics Data System (ADS)

    Purdy, Kirstin R.; Bartles, James R.; Wong, Gerard C. L.

    2007-02-01

    We examine the interaction between cytoskeletal F-actin and espin 3A, a prototypical actin bundling protein found in sensory cell microvilli, including ear cell stereocilia. Espin induces twist distortions in F-actin as well as facilitates bundle formation. Mutations in one of the two F-actin binding sites of espin, which have been implicated in deafness, can tune espin-actin interactions and radically transform the system’s phase behavior. These results are compared to recent theoretical work on the general phase behavior linker-rod systems.

  10. Coronin 1B antagonizes Cortactin and remodels Arp2/3-containing actin branches in lamellipodia

    PubMed Central

    Cai, Liang; Makhov, Alexander M.; Schafer, Dorothy A.; Bear, James E.

    2008-01-01

    Summary The dendritic actin network generated by Arp2/3 complex in lamellipodia underlies formation of protrusions, directional sensing and migration. While the generation of this network is well studied, the mechanisms regulating network disassembly are poorly understood. We report that Coronin 1B disassembles Arp2/3-containing actin filament branches by inducing Arp2/3 dissociation. This activity is antagonized by Cortactin, a filament branch stabilizer. Consistent with this biochemical competition, depletion of both proteins partially rescues defects in lamellipodial dynamics observed upon depletion of either protein alone. Coronin 1B targets actin branches in a manner that is mutually exclusive with Arp2/3 complex and alters the branch angle. We conclude that Coronin 1B replaces Arp2/3 complex at actin filament branches as the dendritic network matures and drives the turnover of branched actin networks. PMID:18775315

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-05-10

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

  13. 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.

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

    PubMed

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

    2015-02-01

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

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

    PubMed Central

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

    2011-01-01

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

  16. Role and structural mechanism of WASP-triggered conformational changes in branched actin filament nucleation by Arp2/3 complex.

    PubMed

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

    2016-07-01

    The Arp2/3 (Actin-related proteins 2/3) complex is activated by WASP (Wiskott-Aldrich syndrome protein) family proteins to nucleate branched actin filaments that are important for cellular motility. WASP recruits actin monomers to the complex and stimulates movement of Arp2 and Arp3 into a "short-pitch" conformation that mimics the arrangement of actin subunits within filaments. The relative contribution of these functions in Arp2/3 complex activation and the mechanism by which WASP stimulates the conformational change have been unknown. We purified budding yeast Arp2/3 complex held in or near the short-pitch conformation by an engineered covalent cross-link to determine if the WASP-induced conformational change is sufficient for activity. Remarkably, cross-linked Arp2/3 complex bypasses the need for WASP in activation and is more active than WASP-activated Arp2/3 complex. These data indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation. During activation, WASP limits nucleation rates by releasing slowly from nascent branches. The cross-linked complex is inhibited by WASP's CA region, even though CA potently stimulates cross-linking, suggesting that slow WASP detachment masks the activating potential of the short-pitch conformational switch. We use structure-based mutations and WASP-Arp fusion chimeras to determine how WASP stimulates movement toward the short-pitch conformation. Our data indicate that WASP displaces the autoinhibitory Arp3 C-terminal tail from a hydrophobic groove at Arp3's barbed end to destabilize the inactive state, providing a mechanism by which WASP stimulates the short-pitch conformation and activates Arp2/3 complex. PMID:27325766

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

    PubMed Central

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

    2015-01-01

    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. PMID:25609828

  18. 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. PMID:25609828

  19. Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex.

    PubMed

    Serra-Peinado, Carla; Sicart, Adrià; Llopis, Juan; Egea, Gustavo

    2016-04-01

    We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH. PMID:26872971

  20. 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.

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

    PubMed

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

    1998-06-01

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

  2. A chemo-mechanical constitutive model for transiently cross-linked actin networks and a theoretical assessment of their viscoelastic behaviour.

    PubMed

    Fallqvist, B; Kroon, M

    2013-04-01

    Biological materials can undergo large deformations and also show viscoelastic behaviour. One such material is the network of actin filaments found in biological cells, giving the cell much of its mechanical stiffness. A theory for predicting the relaxation behaviour of actin networks cross-linked with the cross-linker α-actinin is proposed. The constitutive model is based on a continuum approach involving a neo-Hookean material model, modified in terms of concentration of chemically activated cross-links. The chemical model builds on work done by Spiros (Doctoral thesis, University of British Columbia, Vancouver, Canada, 1998) and has been modified to respond to mechanical stress experienced by the network. The deformation is split into a viscous and elastic part, and a thermodynamically motivated rate equation is assigned for the evolution of viscous deformation. The model predictions were evaluated for stress relaxation tests at different levels of strain and found to be in good agreement with experimental results for actin networks cross-linked with α-actinin. PMID:22623110

  3. Buckling of Branched Cytoskeletal Filaments

    NASA Astrophysics Data System (ADS)

    Quint, D. A.; Schwarz, J. M.

    2011-03-01

    In vitro experiments of growing dendritic actin networks demonstrate reversible stress-softening at high loads, above some critical load. The transition to the stress-softening regime has been attributed to the elastic buckling of individual actin filaments. To estimate the critical load above which softening should occur, we extend the elastic theory of buckling of individual filaments embedded in a network to include the buckling of branched filaments, a signature trait of growing dendritic actin networks. Under certain assumptions, there will be approximately a seven-fold increase in the classical critical bucking load, when compared to the unbranched filament, which is entirely due to the presence of a branch. Moreover, we go beyond the classical buckling regime to investigate the effect of entropic fluctuations. The result of compressing the filament in this case leads to an increase in these fluctuations and eventually the harmonic approximation breaks down signifying the onset of the buckling transition. We compute corrections to the classical critical buckling load near this breakdown.

  4. Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements

    PubMed Central

    Buxa, Stefanie V.; Degola, Francesca; Polizzotto, Rachele; De Marco, Federica; Loschi, Alberto; Kogel, Karl-Heinz; di Toppi, Luigi Sanità; van Bel, Aart J. E.; Musetti, Rita

    2015-01-01

    Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by ‘Candidatus Phytoplasma solani,’ the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Western blot analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes. PMID:26347766

  5. VASP is a processive actin polymerase that requires monomeric actin for barbed end association

    PubMed Central

    Hansen, Scott D.

    2010-01-01

    Ena/VASP proteins regulate the actin cytoskeleton during cell migration and morphogenesis and promote assembly of both filopodial and lamellipodial actin networks. To understand the molecular mechanisms underlying their cellular functions we used total internal reflection fluorescence microscopy to visualize VASP tetramers interacting with static and growing actin filaments in vitro. We observed multiple filament binding modes: (1) static side binding, (2) side binding with one-dimensional diffusion, and (3) processive barbed end tracking. Actin monomers antagonize side binding but promote high affinity (Kd = 9 nM) barbed end attachment. In low ionic strength buffers, VASP tetramers are weakly processive (Koff = 0.69 s−1) polymerases that deliver multiple actin monomers per barbed end–binding event and effectively antagonize filament capping. In higher ionic strength buffers, VASP requires profilin for effective polymerase and anti-capping activity. Based on our observations, we propose a mechanism that accounts for all three binding modes and provides a model for how VASP promotes actin filament assembly. PMID:21041447

  6. 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.

  7. Intermediate filament mechanics in vitro and in the cell: From coiled coils to filaments, fibers and networks

    PubMed Central

    Köster, Sarah; Weitz, David; Goldman, Robert D.; Aebi, Ueli; Herrmann, Harald

    2015-01-01

    Summary Intermediate filament proteins form filaments, fibers and networks both in the cytoplasm and the nucleus of metazoan cells. Their general structural building plan accommodates highly varying amino acid sequences to yield extended dimeric α-helical coiled coils of highly conserved design. These “rod” particles are the basic building blocks of intrinsically flexible, filamentous structures that are able to resist high mechanical stresses, i.e. bending and stretching to a considerable degree, both in vitro and in the cell. Biophysical and computer modeling studies are beginning to unfold detailed structural and mechanical insights into these major supramolecular assemblies of cell architecture, not only in the “test tube” but also in the cellular and tissue context. PMID:25621895

  8. Quantitative fluorescent speckle microscopy (QFSM) to measure actin dynamics.

    PubMed

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

    2012-10-01

    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 meiotic/mitotic spindle. Here, focus is 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 the microinjection of fluorophore-labeled actin into cells, which generate a low ratio of fluorescently labeled to endogenously unlabeled actin monomers. Spinning disk confocal or wide-field imaging then visualizes fluorophore clusters (two to eight 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

  9. Actin filament barbed-end capping activity in neutrophil lysates: the role of capping protein-beta 2.

    PubMed

    DiNubile, M J; Cassimeris, L; Joyce, M; Zigmond, S H

    1995-12-01

    A barbed-end capping activity was found in high speed supernates of neutrophils lysed in submicromolar calcium. In dilute supernate (> or = 100-fold dilution of cytoplasm), this activity accounted for most of the inhibition of barbed-end elongation of pyrenyl-G-actin from spectrin-F-actin seeds. Pointed-end elongation from gelsolin-capped F-actin seeds was not inhibited at comparable concentrations of supernate, thus excluding actin monomer sequestration as a cause of the observed inhibition. Most of the capping activity was due to capping protein-beta 2 (a homologue of cap Z). Thus, while immunoadsorption of > or = 95% of the gelsolin in the supernate did not decrease capping activity, immunoadsorption of capping protein-beta 2 reduced capping activity proportionally to the amount of capping protein-beta 2 adsorbed. Depletion of > 90% of capping protein-beta 2 from the supernate removed 90% of its capping activity. The functional properties of the capping activity were defined. The dissociation constant for binding to barbed ends (determined by steady state and kinetic analyses) was approximately 1-2 nM; the on-rate of capping was between 7 x 10(5) and 5 x 10(6) M-1 s-1; and the off-rate was approximately 2 x 10(-3) s-1. The concentration of capper free in the intact cell (determined by adsorption of supernate with spectrin-actin seeds) was estimated to be approximately 1-2 microM. Thus, there appeared to be enough high affinity capper to cap all the barbed ends in vivo. Nevertheless, immediately after lysis with detergent, neutrophils contained sites that nucleate barbed-end elongation of pyrenyl-G-actin. These barbed ends subsequently become capped with a time course and concentration dependence similar to that of spectrin-F-actin seeds in high speed supernates. These observations suggest that, despite the excess of high affinity capper, some ends either are not capped in vivo or are transiently uncapped upon lysis and dilution. PMID:8590796

  10. Viruses that ride on the coat-tails of actin nucleation.

    PubMed

    Newsome, Timothy P; Marzook, N Bishara

    2015-10-01

    Actin nucleation drives a diversity of critical cellular processes and the motility of a select group of viral pathogens. Vaccinia virus and baculovirus, Autographa californica multiple nucleopolyhedrovirus, recruit and activate the cellular actin nucleator, the Arp2/3 complex, at the surface of virus particles thereby instigating highly localized actin nucleation. The extension of these filaments provides a mechanical force that bestows the ability to navigate the intracellular environment and promote their infectious cycles. This review outlines the viral and cellular proteins that initiate and regulate the signalling networks leading to viral modification of the actin cytoskeleton and summarizes recent insights into the role of actin-based virus transport. PMID:26459972

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

    PubMed

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

    2013-01-01

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

  12. Disarrangement of actin filaments and Ca²⁺ gradient by CdCl₂ alters cell wall construction in Arabidopsis thaliana root hairs by inhibiting vesicular trafficking.

    PubMed

    Fan, Jun-Ling; Wei, Xue-Zhi; Wan, Li-Chuan; Zhang, Ling-Yun; Zhao, Xue-Qin; Liu, Wei-Zhong; Hao, Huai-Qin; Zhang, Hai-Yan

    2011-07-15

    Cadmium (Cd), one of the most toxic heavy metals, inhibits many cellular and physiological processes in plants. Here, the involvement of cytoplasmic Ca²⁺ gradient and actin filaments (AFs) in vesicular trafficking, cell wall deposition and tip growth was investigated during root (hair) development of Arabidopsis thaliana in response to CdCl₂ treatment. Seed germination and root elongation were prevented in a dose- and time-dependent manner by CdCl₂ treatment. Fluorescence labelling and non-invasive detection showed that CdCl₂ inhibited extracellular Ca²⁺ influx, promoted intracellular Ca²⁺ efflux, and disturbed the cytoplasmic tip-focused Ca²⁺ gradient. In vivo labelling revealed that CdCl₂ modified actin organization, which subsequently contributed to vesicle trafficking. Transmission electron microscopy revealed that CdCl₂ induced cytoplasmic vacuolization and was detrimental to organelles such as mitochondria and endoplasmic reticulum (ER). Finally, immunofluorescent labelling and Fourier transform infrared (FTIR) analysis indicated that configuration/distribution of cell wall components such as pectins and cellulose was significantly altered in response to CdCl₂. Our results indicate that CdCl₂ induces disruption of Ca²⁺ gradient and AFs affects the distribution of cell wall components in root hairs by disturbing vesicular trafficking in A. thaliana. PMID:21497412

  13. The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts.

    PubMed Central

    Reinhard, M; Halbrügge, M; Scheer, U; Wiegand, C; Jockusch, B M; Walter, U

    1992-01-01

    Vasoactive agents which elevate either cGMP or cAMP inhibit platelet activation by pathways sharing at least one component, the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP). VASP is stoichiometrically phosphorylated by both cGMP-dependent and cAMP-dependent protein kinases in intact human platelets, and its phosphorylation correlates very well with platelet inhibition caused by cGMP- and cAMP-elevating agents. Here we report that in human platelets spread on glass, VASP is associated predominantly with the distal parts of radial microfilament bundles and with microfilaments outlining the periphery, whereas less VASP is associated with a central microfilamentous ring. VASP is also detectable in a variety of different cell types including fibroblasts and epithelial cells. In fibroblasts, VASP is concentrated at focal contact areas, along microfilament bundles (stress fibres) in a punctate pattern, in the periphery of protruding lamellae, and is phosphorylated by cGMP- and cAMP-dependent protein kinases in response to appropriate stimuli. Evidence for the direct binding of VASP to F-actin is also presented. The data demonstrate that VASP is a novel phosphoprotein associated with actin filaments and focal contact areas, i.e. transmembrane junctions between microfilaments and the extracellular matrix. Images PMID:1318192

  14. Fascin 1 is an actin filament-bundling protein that regulates ectoplasmic specialization dynamics in the rat testis

    PubMed Central

    Gungor-Ordueri, N. Ece; Celik-Ozenci, Ciler

    2014-01-01

    In the testis, spermatids are polarized cells, with their heads pointing toward the basement membrane during maturation. This polarity is crucial to pack the maximal number of spermatids in the seminiferous epithelium so that millions of sperms can be produced daily. A loss of spermatid polarity is detected after rodents are exposed to toxicants (e.g., cadmium) or nonhormonal male contraceptives (e.g., adjudin), which is associated with a disruption on the expression and/or localization of polarity proteins. In the rat testis, fascin 1, an actin-bundling protein found in mammalian cells, was expressed by Sertoli and germ cells. Fascin 1 was a component of the ectoplasmic specialization (ES), a testis-specific anchoring junction known to confer spermatid adhesion and polarity. Its expression in the seminiferous epithelium was stage specific. Fascin 1 was localized to the basal ES at the Sertoli cell-cell interface of the blood-testis barrier in all stages of the epithelial cycle, except it diminished considerably at late stage VIII. Fascin 1 was highly expressed at the apical ES at stage VII–early stage VIII and restricted to the step 19 spermatids. Its knockdown by RNAi that silenced fascin 1 by ∼70% in Sertoli cells cultured in vitro was found to perturb the tight junction-permeability barrier via a disruption of F-actin organization. Knockdown of fascin 1 in vivo by ∼60–70% induced defects in spermatid polarity, which was mediated by a mislocalization and/or downregulation of actin-bundling proteins Eps8 and palladin, thereby impeding F-actin organization and disrupting spermatid polarity. In summary, these findings provide insightful information on spermatid polarity regulation. PMID:25159326

  15. Fascin 1 is an actin filament-bundling protein that regulates ectoplasmic specialization dynamics in the rat testis.

    PubMed

    Gungor-Ordueri, N Ece; Celik-Ozenci, Ciler; Cheng, C Yan

    2014-11-01

    In the testis, spermatids are polarized cells, with their heads pointing toward the basement membrane during maturation. This polarity is crucial to pack the maximal number of spermatids in the seminiferous epithelium so that millions of sperms can be produced daily. A loss of spermatid polarity is detected after rodents are exposed to toxicants (e.g., cadmium) or nonhormonal male contraceptives (e.g., adjudin), which is associated with a disruption on the expression and/or localization of polarity proteins. In the rat testis, fascin 1, an actin-bundling protein found in mammalian cells, was expressed by Sertoli and germ cells. Fascin 1 was a component of the ectoplasmic specialization (ES), a testis-specific anchoring junction known to confer spermatid adhesion and polarity. Its expression in the seminiferous epithelium was stage specific. Fascin 1 was localized to the basal ES at the Sertoli cell-cell interface of the blood-testis barrier in all stages of the epithelial cycle, except it diminished considerably at late stage VIII. Fascin 1 was highly expressed at the apical ES at stage VII-early stage VIII and restricted to the step 19 spermatids. Its knockdown by RNAi that silenced fascin 1 by ~70% in Sertoli cells cultured in vitro was found to perturb the tight junction-permeability barrier via a disruption of F-actin organization. Knockdown of fascin 1 in vivo by ~60-70% induced defects in spermatid polarity, which was mediated by a mislocalization and/or downregulation of actin-bundling proteins Eps8 and palladin, thereby impeding F-actin organization and disrupting spermatid polarity. In summary, these findings provide insightful information on spermatid polarity regulation. PMID:25159326

  16. Mathematical modelling and numerical simulations of actin dynamics in the eukaryotic cell.

    PubMed

    George, Uduak Z; Stéphanou, Angélique; Madzvamuse, Anotida

    2013-02-01

    The aim of this article is to study cell deformation and cell movement by considering both the mechanical and biochemical properties of the cortical network of actin filaments and its concentration. Actin is a polymer that can exist either in filamentous form (F-actin) or in monometric form (G-actin) (Chen et al. in Trends Biochem Sci 25:19-23, 2000) and the filamentous form is arranged in a paired helix of two protofilaments (Ananthakrishnan et al. in Recent Res Devel Biophys 5:39-69, 2006). By assuming that cell deformations are a result of the cortical actin dynamics in the cell cytoskeleton, we consider a continuum mathematical model that couples the mechanics of the network of actin filaments with its bio-chemical dynamics. Numerical treatment of the model is carried out using the moving grid finite element method (Madzvamuse et al. in J Comput Phys 190:478-500, 2003). Furthermore, by assuming slow deformations of the cell, we use linear stability theory to validate the numerical simulation results close to bifurcation points. Far from bifurcation points, we show that the mathematical model is able to describe the complex cell deformations typically observed in experimental results. Our numerical results illustrate cell expansion, cell contraction, cell translation and cell relocation as well as cell protrusions. In all these results, the contractile tonicity formed by the association of actin filaments to the myosin II motor proteins is identified as a key bifurcation parameter. PMID:22434394

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

    PubMed

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

    2015-11-01

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

  18. 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

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

    PubMed

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

    2016-07-26

    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

  20. 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

  1. Treadmilling and length distributions of active polar filaments

    NASA Astrophysics Data System (ADS)

    Erlenkämper, C.; Kruse, K.

    2013-10-01

    The cytoskeleton is a network of filamentous proteins, notably, actin filaments and microtubules. These filaments are active as their assembly is driven by the hydrolysis of nucleotides bound to the constituting protomers. In addition, the assembly kinetics differs at the two respective ends, making them active polar filaments. Experimental evidence suggests, that, in vivo, actin filaments and microtubules can grow at one and shrink at the other end at the same rate, a state that is known as treadmilling. In this work, we use a generic discrete two-state model for active polar filaments to analyze the conditions leading to treadmilling. We find that a single filament can self-organize into the treadmilling state for a broad range of monomer concentrations. In this regime the corresponding length distribution has a pronounced maximum at a finite value. We then extend our description to consider specifically the dynamics of actin filaments. We show that actin treadmilling should be observable in vitro in the presence of appropriate depolymerization promoting factors.

  2. Plectin reinforces vascular integrity by mediating crosstalk between the vimentin and the actin networks

    PubMed Central

    Osmanagic-Myers, Selma; Rus, Stefanie; Wolfram, Michael; Brunner, Daniela; Goldmann, Wolfgang H.; Bonakdar, Navid; Fischer, Irmgard; Reipert, Siegfried; Zuzuarregui, Aurora; Walko, Gernot; Wiche, Gerhard

    2015-01-01

    ABSTRACT Mutations in the cytoskeletal linker protein plectin result in multisystemic diseases affecting skin and muscle with indications of additional vascular system involvement. To study the mechanisms underlying vascular disorders, we established plectin-deficient endothelial cell and mouse models. We show that apart from perturbing the vimentin cytoskeleton of endothelial cells, plectin deficiency leads to severe distortions of adherens junctions (AJs), as well as tight junctions, accompanied by an upregulation of actin stress fibres and increased cellular contractility. Plectin-deficient endothelial cell layers were more leaky and showed reduced mechanical resilience in fluid-shear stress and mechanical stretch experiments. We suggest that the distorted AJs and upregulated actin stress fibres in plectin-deficient cells are rooted in perturbations of the vimentin cytoskeleton, as similar phenotypes could be mimicked in wild-type cells by disruption of vimentin filaments. In vivo studies in endothelium-restricted conditional plectin-knockout mice revealed significant distortions of AJs in stress-prone aortic arch regions and increased pulmonary vascular leakage. Our study opens a new perspective on cytoskeleton-controlled vascular permeability, where a plectin-organized vimentin scaffold keeps actomyosin contractility ‘in-check’ and maintains AJ homeostasis. PMID:26519478

  3. Plectin reinforces vascular integrity by mediating crosstalk between the vimentin and the actin networks.

    PubMed

    Osmanagic-Myers, Selma; Rus, Stefanie; Wolfram, Michael; Brunner, Daniela; Goldmann, Wolfgang H; Bonakdar, Navid; Fischer, Irmgard; Reipert, Siegfried; Zuzuarregui, Aurora; Walko, Gernot; Wiche, Gerhard

    2015-11-15

    Mutations in the cytoskeletal linker protein plectin result in multisystemic diseases affecting skin and muscle with indications of additional vascular system involvement. To study the mechanisms underlying vascular disorders, we established plectin-deficient endothelial cell and mouse models. We show that apart from perturbing the vimentin cytoskeleton of endothelial cells, plectin deficiency leads to severe distortions of adherens junctions (AJs), as well as tight junctions, accompanied by an upregulation of actin stress fibres and increased cellular contractility. Plectin-deficient endothelial cell layers were more leaky and showed reduced mechanical resilience in fluid-shear stress and mechanical stretch experiments. We suggest that the distorted AJs and upregulated actin stress fibres in plectin-deficient cells are rooted in perturbations of the vimentin cytoskeleton, as similar phenotypes could be mimicked in wild-type cells by disruption of vimentin filaments. In vivo studies in endothelium-restricted conditional plectin-knockout mice revealed significant distortions of AJs in stress-prone aortic arch regions and increased pulmonary vascular leakage. Our study opens a new perspective on cytoskeleton-controlled vascular permeability, where a plectin-organized vimentin scaffold keeps actomyosin contractility 'in-check' and maintains AJ homeostasis. PMID:26519478

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

    PubMed

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

    2015-05-01

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

  5. Role of Turnover in Active Stress Generation in a Filament Network

    NASA Astrophysics Data System (ADS)

    Hiraiwa, Tetsuya; Salbreux, Guillaume

    2016-05-01

    We study the effect of turnover of cross-linkers, motors, and filaments on the generation of a contractile stress in a network of filaments connected by passive cross-linkers and subjected to the forces exerted by molecular motors. We perform numerical simulations where filaments are treated as rigid rods and molecular motors move fast compared to the time scale of an exchange of cross-linkers. We show that molecular motors create a contractile stress above a critical number of cross-linkers. When passive cross-linkers are allowed to turn over, the stress exerted by the network vanishes due to the formation of clusters. When both filaments and passive cross-linkers turn over, clustering is prevented and the network reaches a dynamic contractile steady state. A maximum stress is reached for an optimum ratio of the filament and cross-linker turnover rates. Taken together, our work reveals conditions for stress generation by molecular motors in a fluid isotropic network of rearranging filaments.

  6. Role of turnover in active stress generation in a filament network

    PubMed Central

    Hiraiwa, Tetsuya; Salbreux, Guillaume

    2016-01-01

    We study the effect of turnover of cross-linkers, motors, and filaments on the generation of a contractile stress in a network of filaments connected by passive cross-linkers and subjected to the forces exerted by molecular motors. We perform numerical simulations where filaments are treated as rigid rods and molecular motors move fast compared to the time scale of an exchange of cross-linkers. We show that molecular motors create a contractile stress above a critical number of cross-linkers. When passive cross-linkers are allowed to turn over, the stress exerted by the network vanishes due to the formation of clusters. When both filaments and passive cross-linkers turn over, clustering is prevented and the network reaches a dynamic contractile steady state. A maximum stress is reached for an optimum ratio of the filament and cross-linker turnover rates. Taken together, our work reveals conditions for stress generation by molecular motors in a fluid isotropic network of rearranging filaments. PMID:27203344

  7. Role of Active Contraction and Tropomodulins in Regulating Actin Filament Length and Sarcomere Structure in Developing Zebrafish Skeletal Muscle

    PubMed Central

    Mazelet, Lise; Parker, Matthew O.; Li, Mei; Arner, Anders; Ashworth, Rachel

    2016-01-01

    Whilst it is recognized that contraction plays an important part in maintaining the structure and function of mature skeletal muscle, its role during development remains undefined. In this study the role of movement in skeletal muscle maturation was investigated in intact zebrafish embryos using a combination of genetic and pharmacological approaches. An immotile mutant line (cacnb1ts25) which lacks functional voltage-gated calcium channels (dihydropyridine receptors) in the muscle and pharmacological immobilization of embryos with a reversible anesthetic (Tricaine), allowed the study of paralysis (in mutants and anesthetized fish) and recovery of movement (reversal of anesthetic treatment). The effect of paralysis in early embryos (aged between 17 and 24 hours post-fertilization, hpf) on skeletal muscle structure at both myofibrillar and myofilament level was determined using both immunostaining with confocal microscopy and small angle X-ray diffraction. The consequences of paralysis and subsequent recovery on the localization of the actin capping proteins Tropomodulin 1 & 4 (Tmod) in fish aged from 17 hpf until 42 hpf was also assessed. The functional consequences of early paralysis were investigated by examining the mechanical properties of the larval muscle. The length-force relationship, active and passive tension, was measured in immotile, recovered and control skeletal muscle at 5 and 7 day post-fertilization (dpf). Recovery of muscle function was also assessed by examining swimming patterns in recovered and control fish. Inhibition of the initial embryonic movements (up to 24 hpf) resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralyzed fish by 42 hpf. In conclusion, myofibril organization is regulated by a dual mechanism involving movement-dependent and movement-independent processes. The initial contractile event itself drives the localization of Tmod1 to its sarcomeric position

  8. Role of Active Contraction and Tropomodulins in Regulating Actin Filament Length and Sarcomere Structure in Developing Zebrafish Skeletal Muscle.

    PubMed

    Mazelet, Lise; Parker, Matthew O; Li, Mei; Arner, Anders; Ashworth, Rachel

    2016-01-01

    Whilst it is recognized that contraction plays an important part in maintaining the structure and function of mature skeletal muscle, its role during development remains undefined. In this study the role of movement in skeletal muscle maturation was investigated in intact zebrafish embryos using a combination of genetic and pharmacological approaches. An immotile mutant line (cacnb1 (ts25) ) which lacks functional voltage-gated calcium channels (dihydropyridine receptors) in the muscle and pharmacological immobilization of embryos with a reversible anesthetic (Tricaine), allowed the study of paralysis (in mutants and anesthetized fish) and recovery of movement (reversal of anesthetic treatment). The effect of paralysis in early embryos (aged between 17 and 24 hours post-fertilization, hpf) on skeletal muscle structure at both myofibrillar and myofilament level was determined using both immunostaining with confocal microscopy and small angle X-ray diffraction. The consequences of paralysis and subsequent recovery on the localization of the actin capping proteins Tropomodulin 1 & 4 (Tmod) in fish aged from 17 hpf until 42 hpf was also assessed. The functional consequences of early paralysis were investigated by examining the mechanical properties of the larval muscle. The length-force relationship, active and passive tension, was measured in immotile, recovered and control skeletal muscle at 5 and 7 day post-fertilization (dpf). Recovery of muscle function was also assessed by examining swimming patterns in recovered and control fish. Inhibition of the initial embryonic movements (up to 24 hpf) resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralyzed fish by 42 hpf. In conclusion, myofibril organization is regulated by a dual mechanism involving movement-dependent and movement-independent processes. The initial contractile event itself drives the localization of Tmod1 to its sarcomeric

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

    PubMed

    Joo, E Emily; Yamada, Kenneth M

    2016-05-01

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

  11. 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.

  12. Filament networks attached to membranes: cytoskeletal pressure and local bilayer deformation

    NASA Astrophysics Data System (ADS)

    Auth, Thorsten; Safran, S. A.; Gov, Nir S.

    2007-11-01

    Several cell types, among them red blood cells, have a cortical, two-dimensional (2D) network of filaments sparsely attached to their lipid bilayer. In many mammalian cells, this 2D polymer network is connected to an underlying 3D, more rigid cytoskeleton. In this paper, we consider the pressure exerted by the thermally fluctuating, cortical network of filaments on the bilayer and predict the bilayer deformations that are induced by this pressure. We treat the filaments as flexible polymers and calculate the pressure that a network of such linear chains exerts on the bilayer; we then minimize the bilayer shape in order to predict the resulting local deformations. We compare our predictions with membrane deformations observed in electron micrographs of red blood cells. The polymer pressure along with the resulting membrane deformation can lead to compartmentalization, regulate in-plane diffusion and may influence protein sorting as well as transmit signals to the polymerization of the underlying 3D cytoskeleton.

  13. Rapid non-equilibrium turnover fluidizes entangled F-actin solutions

    NASA Astrophysics Data System (ADS)

    McCall, Patrick M.; Kovar, David R.; Gardel, Margaret L.

    The actin cytoskeleton of living cells is a semiflexible polymer network which regulates cell division, motility, and morphogenesis by controlling cell shape. These complex shape-changing processes require both mechanical deformation and remodeling of the actin cytoskeleton. Molecular motors generate internal forces to drive deformation, while cytoskeletal remodeling is regulated by non-equilibrium polymer turnover. Although the mechanical properties of equilibrium actin filament (F-actin) networks are well-described by theories of semiflexible polymers, these theories do not incorporate the effects of non-equilibrium turnover. To address this experimentally, we developed a model system in which both the turnover rate and the length distribution of purified F-actin can be tuned independently at steady-state through the combined action of actin regulatory proteins. Specifically we tune the concentrations of cofilin, profilin, and formin to regulate F-actin severing, recycling, and nucleation, respectively. We find that the actin turnover rate can be tuned by cofilin up to 25-fold (31 +/- 2 subunits/sec/filament). Surprisingly, changes in turnover rate have no effect on the steady-state F-actin length distribution, which is instead set by formin concentration. Passive microrheology measurements show that increased turnover leads to striking fluidization in both entangled and crosslinked networks. Non-equilibrium turnover thus enables modulation of network mechanics, which impacts force transmission and material deformation.

  14. Stress Enhanced Gelation in α-Actinin-4 Cross-linked Actin Networks

    NASA Astrophysics Data System (ADS)

    Yao, Norman; Broedersz, Chase; Depken, Martin; Becker, Daniel; Pollak, Martin; Mackintosh, Frederick; Weitz, David

    2012-02-01

    A hallmark of biopolymer networks is their exquisite sensitivity to stress, demonstrated for example, by pronounced nonlinear elastic stiffening. Typically, they also yield under increased static load, providing a mechanism to achieve fluid-like behavior. In this talk, I will demonstrate an unexpected dynamical behavior in biopolymer networks consisting of F-actin cross-linked by a physiological actin binding protein, α-Actinin-4. Applied stress actually enhances gelation of these networks by delaying the onset of structural relaxation and network flow, thereby extending the regime of solid-like behavior to much lower frequencies. By using human kidney disease-associated mutant cross-linkers with varying binding affinities, we propose a molecular origin for this stress-enhanced gelation: It arises from the increased binding affinity of the cross-linker under load, characteristic of catch-bond-like behavior. This property may have important biological implications for intracellular mechanics, representing as it does a qualitatively new class of material behavior.

  15. 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.

  16. 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.

  17. FMNL2 drives actin-based protrusion and migration downstream of Cdc42.

    PubMed

    Block, Jennifer; Breitsprecher, Dennis; Kühn, Sonja; Winterhoff, Moritz; Kage, Frieda; Geffers, Robert; Duwe, Patrick; Rohn, Jennifer L; Baum, Buzz; Brakebusch, Cord; Geyer, Matthias; Stradal, Theresia E B; Faix, Jan; Rottner, Klemens

    2012-06-01

    Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia. PMID:22608513

  18. A 'hot-spot' mutation alters the mechanical properties of keratin filament networks.

    PubMed

    Ma, L; Yamada, S; Wirtz, D; Coulombe, P A

    2001-05-01

    Keratins 5 and 14 polymerize to form the intermediate filament network in the progenitor basal cells of many stratified epithelia including epidermis, where it provides crucial mechanical support. Inherited mutations in K5 or K14 result in epidermolysis bullosa simplex (EBS), a skin-fragility disorder. The impact that such mutations exert on the intrinsic mechanical properties of K5/K14 filaments is unknown. Here we show, by using differential interference contrast microscopy, that a 'hot-spot' mutation in K14 greatly reduces the ability of reconstituted mutant filaments to bundle under crosslinking conditions. Rheological assays measure similar small-deformation mechanical responses for crosslinked solutions of wild-type and mutant keratins. The mutation, however, markedly reduces the resilience of crosslinked networks against large deformations. Single-particle tracking, which probes the local organization of filament networks, shows that the mutant polymer exhibits highly heterogeneous structures compared to those of wild-type filaments. Our results indicate that the fragility of epithelial cells expressing mutant keratin may result from an impaired ability of keratin polymers to be crosslinked into a functional network. PMID:11331879

  19. Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments

    PubMed Central

    Schevzov, Galina; Kee, Anthony J.; Wang, Bin; Sequeira, Vanessa B.; Hook, Jeff; Coombes, Jason D.; Lucas, Christine A.; Stehn, Justine R.; Musgrove, Elizabeth A.; Cretu, Alexandra; Assoian, Richard; Fath, Thomas; Hanoch, Tamar; Seger, Rony; Pleines, Irina; Kile, Benjamin T.; Hardeman, Edna C.; Gunning, Peter W.

    2015-01-01

    ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor–stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells. PMID:25971798

  20. Insulin-like growth factor binding protein-1 expression in baboon endometrial stromal cells: regulation by filamentous actin and requirement for de novo protein synthesis.

    PubMed

    Kim, J J; Jaffe, R C; Fazleabas, A T

    1999-02-01

    Stromal fibroblasts in the primate endometrium undergo dramatic morphological and biochemical changes in response to pregnancy. This transformation is characterized by the expression of insulin-like growth factor binding protein-1 (IGFBP-1). Stromal cells from the baboon endometrium of nonpregnant animals were cultured and subsequently treated with cytochalasin D to disrupt actin filaments. In response to cytochalasin D treatment, cells contracted and became rounded as early as 10 min after the initiation of treatment. When cytochalasin D was removed, cells reverted back to their original fibroblastic shape within 1 h. After cells were treated with cytochalasin D for 5 h, addition of (Bu)2cAMP and/or hormones (estradiol, medroxyprogesterone acetate, and relaxin) resulted in the expression of IGFBP-1 messenger RNA and protein within 24 h. Cells with an intact cytoskeleton did not express detectable levels of IGFBP-1 in response to hormones and/or (Bu)2cAMP. Furthermore, the addition of cycloheximide inhibited expression of IGFBP-1 in cytochalasin D-treated cells. Stromal cells were also isolated from early pregnant and simulated pregnant animals. Within 48 h, cells from both the pregnant and simulated pregnant animals produced IGFBP-1 in response to hormones and/or (Bu)2cAMP. In these studies, IGFBP-1 expression was also inhibited by cycloheximide. These studies suggest that induction of IGFBP-1 requires an intermediary protein and that alterations in the cytoskeleton may be involved. PMID:9927334

  1. Actin filaments participate in the relocalization of phosphatidylinositol3-kinase to glucose transporter-containing compartments and in the stimulation of glucose uptake in 3T3-L1 adipocytes.

    PubMed Central

    Wang, Q; Bilan, P J; Tsakiridis, T; Hinek, A; Klip, A

    1998-01-01

    Insulin stimulates the rate of glucose uptake into muscle and adipose cells by translocation of glucose transporters from an intracellular storage pool to the plasma membrane. This event requires the prior activation of phosphatidylinositol 3-kinase (PI 3-kinase). Here we report that insulin causes an increase in wortmannin-sensitive PI 3-kinase activity and a gain in the enzyme's regulatory and catalytic subunits p85alpha and p110beta (but not p110alpha) in the intracellular compartments containing glucose transporters. The hormone also caused a marked reorganization of actin filaments, which was prevented by cytochalasin D. Cytochalasin D also decreased significantly the insulin-dependent association of PI 3-kinase activity and the levels of insulin receptor substrate (IRS)-1, p85alpha and p110beta with immunopurified GLUT4-containing compartments. In contrast, the drug did not alter the insulin-induced tyrosine phosphorylation of IRS-1, the association of PI 3-kinase with IRS-1, or the stimulation of PI 3-kinase by insulin in anti-(IRS-1) or anti-p85 immunoprecipitates from whole cell lysates. Cytochalasin D, and the chemically unrelated latrunculin B, which also inhibits actin filament reassembly, prevented the insulin stimulation of glucose transport by approx. 50%. Cytochalasin D decreased by about one-half the insulin-dependent translocation to the plasma membrane of the GLUT1 and GLUT4 glucose transporters. The results suggest that the existence of intact actin filament is correlated with the full recruitment of glucose transporters by insulin. The underlying function of the actin filaments might be to facilitate the insulin-mediated association of the p85-p110 PI 3-kinase with glucose-transporter-containing compartments. PMID:9560323

  2. Excitable actin dynamics in lamellipodial protrusion and retraction.

    PubMed

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

    2012-04-01

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

  3. Actin Rings of Power.

    PubMed

    Schwayer, Cornelia; Sikora, Mateusz; Slováková, Jana; Kardos, Roland; Heisenberg, Carl-Philipp

    2016-06-20

    Circular or ring-like actin structures play important roles in various developmental and physiological processes. Commonly, these rings are composed of actin filaments and myosin motors (actomyosin) that, upon activation, trigger ring constriction. Actomyosin ring constriction, in turn, has been implicated in key cellular processes ranging from cytokinesis to wound closure. Non-constricting actin ring-like structures also form at cell-cell contacts, where they exert a stabilizing function. Here, we review recent studies on the formation and function of actin ring-like structures in various morphogenetic processes, shedding light on how those different rings have been adapted to fulfill their specific roles. PMID:27326928

  4. Localization of cell surface glycoproteins in membrane domains associated with the underlying filament network.

    PubMed

    Roos, E; Spiele, H; Feltkamp, C A; Huisman, H; Wiegant, F A; Traas, J; Mesland, D A

    1985-11-01

    To visualize the localization of cell surface constituents in relation to the plasma membrane-associated filament network, we developed a method based on a combination of immunogold labeling and dry-cleaving. For labeling we used trinitrophenyl-derivatized ligand, anti-TNP antibodies, and protein A-coated colloidal gold. Dry-cleaving (Mesland, D. A. M., H. Spiele, and E. Roos, 1981, Exp. Cell Res., 132: 169-184) involves cleavage of lightly fixed critical point-dried cells by means of adhesive tape. Since cells cleave close to the cell surface, the remaining layer is thin enough to be examined in transmission electron microscopy. Using this method, we studied concanavalin A-binding constituents on the medium-facing surface of H35 hepatoma cells. The distribution of the gold particles, which was partly dispersed and partly patchy, coincided strikingly with membrane-associated filaments, and label was virtually absent from areas overlying openings in the filament network. In stereo pairs we observed the label to be localized to areas of somewhat enhanced electron density at the plane of the membrane. These areas were interconnected in a pattern congruent with the filament network. Preliminary observations on wheat germ agglutinin receptors on the hepatoma cells as well as concanavalin A receptors on isolated hepatocytes yielded comparable results. It thus appears that surface glycoproteins, although seemingly randomly distributed as observed in thin sections, may actually be localized to particular membrane domains associated with underlying filaments. PMID:3902855

  5. 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. PMID:27104747

  6. Concentration profiles of actin-binding molecules in lamellipodia

    NASA Astrophysics Data System (ADS)

    Falcke, Martin

    2016-04-01

    Motile cells form lamellipodia in the direction of motion, which are flat membrane protrusions containing an actin filament network. The network flows rearward relative to the leading edge of the lamellipodium due to actin polymerization at the front. Thus, actin binding molecules are subject to transport towards the rear of the cell in the bound state and diffuse freely in the unbound state. We analyze this reaction-diffusion-advection process with respect to the concentration profiles of these species and provide an analytic approximation for them. Network flow may cause a depletion zone of actin binding molecules close to the leading edge. The existence of such zone depends on the free molecule concentration in the cell body, on the ratio of the diffusion length to the distance bound molecules travel rearward with the flow before dissociating, and the ratio of the diffusion length to the width of the region with network flow and actin binding. Our calculations suggest the existence of depletion zones for the F-actin cross-linkers filamin and α-actinin in fish keratocytes (and other cell types), which is in line with the small elastic moduli of the F-actin network close to the leading edge found in measurements of the force motile cells are able to exert.

  7. 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.

  8. Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis.

    PubMed

    Wollert, Torsten; Weiss, Dieter G; Gerdes, Hans-Hermann; Kuznetsov, Sergei A

    2002-11-25

    It is widely believed that microtubule- and F-actin-based transport of cytoplasmic organelles and membrane fusion is down-regulated during mitosis. Here we show that during the transition of Xenopus egg extracts from interphase to metaphase myosin V-driven movement of small globular vesicles along F-actin is strongly inhibited. In contrast, the movement of ER and ER network formation on F-actin is up-regulated in metaphase extracts. Our data demonstrate that myosin V-driven motility of distinct organelles is differently controlled during the cell cycle and suggest an active role of F-actin in partitioning, positioning, and membrane fusion of the ER during cell division. PMID:12438410

  9. Actin from Saccharomyces cerevisiae.

    PubMed Central

    Greer, C; Schekman, R

    1982-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Theriot, Julie

    2005-03-01

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

  11. The catalytic domain of inositol-1,4,5-trisphosphate 3-kinase-a contributes to ITPKA-induced modulation of F-actin.

    PubMed

    Ashour, Dina Julia; Pelka, Benjamin; Jaaks, Patricia; Wundenberg, Torsten; Blechner, Christine; Zobiak, Bernd; Failla, Antonio Virgilio; Windhorst, Sabine

    2015-02-01

    Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has been considered as an actin bundling protein because its N-terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross-linked actin filaments, here we analyzed if full length-ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F-actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C-terminal ABD, we exclude that ITPKA cross-links actin filaments by simultaneous F-actin binding with two different ABDs. Instead, stimulated-emission-depletion-microscopy and measurement of InsP3 Kinase activity give evidence that that N-terminal ABD-homodimers of ITPKA bind to F-actin while the monomeric C-termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3 Kinase activity is doubled and the product of InsP3 Kinase activity, Ins(1,3,4,5)P4 , inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3 Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3 Kinase domain substantially contributes to this process. PMID:25620569

  12. Co-option of the polarity gene network shapes filament morphology in angiosperms.

    PubMed

    de Almeida, Ana Maria Rocha; Yockteng, Roxana; Schnable, James; Alvarez-Buylla, Elena R; Freeling, Michael; Specht, Chelsea D

    2014-01-01

    The molecular genetic mechanisms underlying abaxial-adaxial polarity in plants have been studied as a property of lateral and flattened organs, such as leaves. In leaves, laminar expansion occurs as a result of balanced abaxial-adaxial gene expression. Over- or under- expression of either abaxializing or adaxializing genes inhibits laminar growth, resulting in a mutant radialized phenotype. Here, we show that co-option of the abaxial-adaxial polarity gene network plays a role in the evolution of stamen filament morphology in angiosperms. RNA-Seq data from species bearing laminar (flattened) or radial (cylindrical) filaments demonstrates that species with laminar filaments exhibit balanced expression of abaxial-adaxial (ab-ad) genes, while overexpression of a YABBY gene is found in species with radial filaments. This result suggests that unbalanced expression of ab-ad genes results in inhibition of laminar outgrowth, leading to a radially symmetric structure as found in many angiosperm filaments. We anticipate that co-option of the polarity gene network is a fundamental mechanism shaping many aspects of plant morphology during angiosperm evolution. PMID:25168962

  13. Reorganization of the cortical actin cytoskeleton during maturation division in the Tubifex egg: possible involvement of protein kinase C.

    PubMed

    Shimizu, T

    1997-08-01

    Tubifex eggs undergo a drastic reorganization of the cortical actin cytoskeleton during metaphase of the second meiosis. At the end of the first meiosis, the egg cortex displays only scattered actin filaments and tiny dots of F-actin; during the following 90 min, cortical F-actin gradually increases in amount, becomes organized into foci that are interlinked by actin bundles, and generates a geodesic dome-like organization. In this study, we have characterized this reorganization of the cortical actin cytoskeleton. In living eggs injected with rhodamine-phalloidin at the beginning of the second meiosis, cortical actin assembly (i.e., formation of actin foci and bundles) proceeds normally, but labeled F-actin is not found to be included significantly in the formed cortical actin network, suggesting that the increase in cortical F-actin is not simply ascribable to the recruitment of preexisting actin filaments. Cortical actin assembly can be induced precociously not only by calcium ionophore A23187 but also by a phorbol ester PMA, an agonist of protein kinase C (PKC). Conversely, the formation of actin foci and bundles is inhibited by PKC antagonists, although cortical F-actin increases to some extent in the presence of these inhibitors. Similar inhibition of the cortical reorganization is elicited in eggs whose intracellular free calcium level ([Ca2+]i) has been clamped low by microinjection of a calcium chelator BAPTA. The treatment of BAPTA-injected eggs with PMA results in the formation of actin foci and bundles. An experiment with eggs injected with fluo-3 shows that [Ca2+]i increases during metaphase of the second meiosis. These results suggest that the reorganization of cortical actin during metaphase of the second meiosis requires activation of PKC, which depends on increases in [Ca2+]i. PMID:9245516

  14. Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath

    NASA Technical Reports Server (NTRS)

    Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1997-01-01

    Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

  15. Monophasic Pulsed 200-μA Current Promotes Galvanotaxis With Polarization of Actin Filament and Integrin α2β1 in Human Dermal Fibroblasts

    PubMed Central

    Uemura, Mikiko; Maeshige, Noriaki; Koga, Yuka; Ishikawa-Aoyama, Michiko; Miyoshi, Makoto; Sugimoto, Masaharu; Terashi, Hiroto

    2016-01-01

    Objective: The monophasic pulsed microcurrent is used to promote wound healing, and galvanotaxis regulation has been reported as one of the active mechanisms in the promotion of tissue repair with monophasic pulsed microcurrent. However, the optimum monophasic pulsed microcurrent parameters and intracellular changes caused by the monophasic pulsed microcurrent have not been elucidated in human dermal fibroblasts. The purpose of this study was to investigate the optimum intensity for promoting galvanotaxis and the effects of electrical stimulation on integrin α2β1 and actin filaments in human dermal fibroblasts. Methods: Human dermal fibroblasts were treated with the monophasic pulsed microcurrent of 0, 100, 200, or 300 μA for 8 hours, and cell migration and cell viability were measured 24 hours after starting monophasic pulsed microcurrent stimulation. Polarization of integrin α2β1 and lamellipodia formation were detected by immunofluorescent staining 10 minutes after starting monophasic pulsed microcurrent stimulation. Results: The migration toward the cathode was significantly higher in the cells treated with the 200-μA monophasic pulsed microcurrent than in the controls (P < .01) without any change in cell viability; treatment with 300-μA monophasic pulsed microcurrent did not alter the migration ratio. The electrostimulus of 200 μA also promoted integrin α2β1 polarization and lamellipodia formation at the cathode edge (P < .05). Conclusion: The results show that 200 μA is an effective monophasic pulsed microcurrent intensity to promote migration toward the cathode, and this intensity could regulate polarization of migration-related intracellular factors in human dermal fibroblasts. PMID:26819649

  16. Monitoring the integrity of filament-wound structures using built-in sensor networks

    NASA Astrophysics Data System (ADS)

    Lin, Mark; Kumar, Amrita; Qing, Xinlin; Beard, Shawn J.; Russell, Samuel S.; Walker, James L.; Delay, Thomas K.

    2003-08-01

    Monitoring the integrity of filament wound composite structures such as solid rocket motors and liquid fuel bottles is important in order to prevent catastrophic failures and to prolong the service life of these structures. To ensure the safety and reliability of rocket components, they require frequent inspection for structural damages that might have occurred during manufacturing, transportation, and storage. The timely and accurate detection, characterization and monitoring of structural cracking, delamination, debonding and other types of damage is a major concern in the operational environment. Utilization of a sensor network system integrated with the structure itself can greatly reduce this inspection burden through fast in-situ data collection and processing. Acellent Technologies, Inc. is currently developing integrated structural monitoring tools for continuous monitoring of composite and metal structures on aircraft and spacecraft. Acellent's integrated structural monitoring system consists of a flexible sensor/actuator network layer called the SMART Layer, supporting diagnostic hardware, and data processing/analysis software. Recently, Acellent has been working with NASA Marshall Space Flight Center to develop ways of embedding the SMART Layer inside filament wound composite bottles. SMART Layers were designed and manufactured for the filament wound bottles and embedded in them during the filament winding process. Acellent has been working on developing a complete structural health monitoring system for the filament wound bottles including data processing tools to interpret the changes in sensor signal caused by changes in the structural condition or material property. A prototype of a filament wound composite bottle with an embedded sensor network has been fabricated and preliminary data analysis tools have been developed.

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

    PubMed

    Ryan, M P; Higgins, P J

    1988-10-01

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

  18. A network of spectrin and plectin surrounds the actin cuffs of apical tubulobulbar complexes in the rat.

    PubMed

    Aristaeus de Asis, Marc; Pires, Manuel; Lyon, Kevin; Vogl, A Wayne

    2013-07-01

    Tubulobulbar complexes (TBCs) are actin-related endocytic structures that internalize intercellular junctions in the seminiferous epithelium. The structures consist of elongate tubular projections of the attached plasma membranes of two adjacent cells that project into Sertoli cells. This double membrane core is cuffed by a dentritic actin network and is capped at its end by a clathrin-coated pit. Here we explore the possibility that elements of the spectrin cytoskeleton are associated with clusters of tubulobulbar complexes that develop at adhesion junctions between late spermatids and Sertoli cells at the apex of the epithelium, and extend what is known about the distribution of plectin at the sites. Cryo-sections of perfusion-fixed testes and apical processes of Sertoli cells mechanically dissociated from perfusion-fixed testes were probed for spectrin, EPB41, and actin and analyzed using conventional fluorescence microscopy and confocal microscopy. Data sets from confocal microscopy were analyzed further in three-dimensional reconstructions using computer software. Additional apical Sertoli cell processes were probed for plectin and analyzed using conventional fluorescence microscopy. Antibodies generated against elements of the spectrin cytoskeleton react with material around and between the actin cuffs of tubulobulbar complexes, but appear excluded from the actin cuffs themselves. A similar staining pattern occurs with a probe for plectin. Immunoelectron microscopy confirmed the staining patterns observed by fluourescence microscopy. Based on our results, we suggest that a network of spectrin and plectin forms a scaffold around tubulobulbar complexes that may provide support for the actin network that cuffs each complex and also link adjacent complexes together. PMID:24381803

  19. A network of spectrin and plectin surrounds the actin cuffs of apical tubulobulbar complexes in the rat

    PubMed Central

    Aristaeus de Asis, Marc; Pires, Manuel; Lyon, Kevin; Vogl, A Wayne

    2013-01-01

    Tubulobulbar complexes (TBCs) are actin-related endocytic structures that internalize intercellular junctions in the seminiferous epithelium. The structures consist of elongate tubular projections of the attached plasma membranes of two adjacent cells that project into Sertoli cells. This double membrane core is cuffed by a dentritic actin network and is capped at its end by a clathrin-coated pit. Here we explore the possibility that elements of the spectrin cytoskeleton are associated with clusters of tubulobulbar complexes that develop at adhesion junctions between late spermatids and Sertoli cells at the apex of the epithelium, and extend what is known about the distribution of plectin at the sites. Cryo-sections of perfusion-fixed testes and apical processes of Sertoli cells mechanically dissociated from perfusion-fixed testes were probed for spectrin, EPB41, and actin and analyzed using conventional fluorescence microscopy and confocal microscopy. Data sets from confocal microscopy were analyzed further in three-dimensional reconstructions using computer software. Additional apical Sertoli cell processes were probed for plectin and analyzed using conventional fluorescence microscopy. Antibodies generated against elements of the spectrin cytoskeleton react with material around and between the actin cuffs of tubulobulbar complexes, but appear excluded from the actin cuffs themselves. A similar staining pattern occurs with a probe for plectin. Immunoelectron microscopy confirmed the staining patterns observed by fluourescence microscopy. Based on our results, we suggest that a network of spectrin and plectin forms a scaffold around tubulobulbar complexes that may provide support for the actin network that cuffs each complex and also link adjacent complexes together. PMID:24381803

  20. Retrograde Flow and Myosin II Activity within the Leading Cell Edge Deliver F-Actin to the Lamella to Seed the Formation of Graded Polarity Actomyosin II Filament Bundles in Migrating Fibroblasts

    PubMed Central

    Anderson, Tom W.; Vaughan, Andrew N.

    2008-01-01

    In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles. PMID:18799629

  1. Redundancy and Cooperativity in the Mechanics of Compositely Crosslinked Filamentous Networks

    PubMed Central

    Das, Moumita; Quint, D. A.; Schwarz, J. M.

    2012-01-01

    The cytoskeleton of living cells contains many types of crosslinkers. Some crosslinkers allow energy-free rotations between filaments and others do not. The mechanical interplay between these different crosslinkers is an open issue in cytoskeletal mechanics. Therefore, we develop a theoretical framework based on rigidity percolation to study a generic filamentous system containing both stretching and bond-bending forces to address this issue. The framework involves both analytical calculations via effective medium theory and numerical simulations on a percolating triangular lattice with very good agreement between both. We find that the introduction of angle-constraining crosslinkers to a semiflexible filamentous network with freely rotating crosslinks can cooperatively lower the onset of rigidity to the connectivity percolation threshold—a result argued for years but never before obtained via effective medium theory. This allows the system to ultimately attain rigidity at the lowest concentration of material possible. We further demonstrate that introducing angle-constraining crosslinks results in mechanical behaviour similar to just freely rotating crosslinked semflexible filaments, indicating redundancy and universality. Our results also impact upon collagen and fibrin networks in biological and bio-engineered tissues. PMID:22590515

  2. Actin Automata with Memory

    NASA Astrophysics Data System (ADS)

    Alonso-Sanz, Ramón; Adamatzky, Andy

    Actin is a globular protein which forms long polar filaments in eukaryotic. The actin filaments play the roles of cytoskeleton, motility units, information processing and learning. We model actin filament as a double chain of finite state machines, nodes, which take states “0” and “1”. The states are abstractions of absence and presence of a subthreshold charge on actin units corresponding to the nodes. All nodes update their state in parallel to discrete time. A node updates its current state depending on states of two closest neighbors in the node chain and two closest neighbors in the complementary chain. Previous models of actin automata consider momentary state transitions of nodes. We enrich the actin automata model by assuming that states of nodes depend not only on the current states of neighboring node but also on their past states. Thus, we assess the effect of memory of past states on the dynamics of acting automata. We demonstrate in computational experiments that memory slows down propagation of perturbations, decrease entropy of space-time patterns generated, transforms traveling localizations to stationary oscillators, and stationary oscillations to still patterns.

  3. The interaction of vinculin with actin.

    PubMed

    Golji, Javad; Mofrad, Mohammad R K

    2013-04-01

    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

  4. HopW1 from Pseudomonas syringae Disrupts the Actin Cytoskeleton to Promote Virulence in Arabidopsis

    PubMed Central

    Cecchini, Nicolas M.; Li, Yujie; Lee, Min Woo; Kovar, David R.; Greenberg, Jean T.

    2014-01-01

    A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis. PMID:24968323

  5. Molecular Architecture of Synaptic Actin Cytoskeleton in Hippocampal Neurons Reveals a Mechanism of Dendritic Spine Morphogenesis

    PubMed Central

    Korobova, Farida

    2010-01-01

    Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility. PMID:19889835

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-07-01

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

  8. 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.

  9. Unveiling a Network of Parallel Filaments in the Infrared Dark Cloud G14.225-0.506

    NASA Astrophysics Data System (ADS)

    Busquet, Gemma; Zhang, Qizhou; Palau, Aina; Liu, Hauyu Baobab; Sánchez-Monge, Álvaro; Estalella, Robert; Ho, Paul T. P.; de Gregorio-Monsalvo, Itziar; Pillai, Thushara; Wyrowski, Friedrich; Girart, Josep M.; Santos, Fábio P.; Franco, Gabriel A. P.

    2013-02-01

    We present the results of combined NH3 (1,1) and (2,2) line emission observed with the Very Large Array and the Effelsberg 100 m telescope of the infrared dark cloud G14.225-0.506. The NH3 emission reveals a network of filaments constituting two hub-filament systems. Hubs are associated with gas of rotational temperature T rot ~ 15 K, non-thermal velocity dispersion σNT ~ 1 km s-1, and exhibit signs of star formation, while filaments appear to be more quiescent (T rot ~ 11 K and σNT ~ 0.6 km s-1). Filaments are parallel in projection and distributed mainly along two directions, at P.A. ~ 10° and 60°, and appear to be coherent in velocity. The averaged projected separation between adjacent filaments is between 0.5 pc and 1 pc, and the mean width of filaments is 0.12 pc. Cores within filaments are separated by ~0.33 ± 0.09 pc, which is consistent with the predicted fragmentation of an isothermal gas cylinder due to the "sausage"-type instability. The network of parallel filaments observed in G14.225-0.506 is consistent with the gravitational instability of a thin gas layer threaded by magnetic fields. Overall, our data suggest that magnetic fields might play an important role in the alignment of filaments, and polarization measurements in the entire cloud would lend further support to this scenario.

  10. A network of 2-4 nm filaments found in sea urchin smooth muscle. Protein constituents and in situ localization.

    PubMed

    Pureur, R P; Coffe, G; Soyer-Gobillard, M O; de Billy, F; Pudles, J

    1986-01-01

    In this report the coisolation of two proteins from sea urchin smooth muscle of apparent molecular weights (Mr) 54 and 56 kD respectively, as determined on SDS-PAGE, is described. Like the intermediate filament proteins, these two proteins are insoluble in high ionic strength buffer solution. On two-dimensional gel electrophoresis and by immunological methods it is shown that these proteins are not related (by these criteria) to rat smooth muscle desmin (54 kD) or vimentin (56 kD). Furthermore, in conditions where both desmin and vimentin assemble in vitro into 10 nm filaments, the sea urchin smooth muscle proteins do not assemble into filaments. Ultrastructural studies on the sea urchin smooth muscle cell show that the thin and thick filaments organization resembles that described in the vertebrate smooth muscle. However, instead of 10 nm filaments, a network of filaments, 2-4 nm in diameter, is revealed, upon removal of the thin and thick filaments by 0.6 M KCl treatment. By indirect immunofluorescence microscopy, and in particular by immunocytochemical electron microscopy studies on the sea urchin smooth muscle cell, it is shown that the antibodies raised against both 54 and 56 kD proteins appear to specifically label these 2-4 nm filaments. These findings indicate that both the 54 and 56 kD proteins might be constituents of this category of filaments. The possible significance of this new cytoskeletal element, that we have named echinonematin filaments, is discussed. PMID:3509996

  11. Nuclear and cytoplasmic actin in dinoflagellates.

    PubMed

    Soyer-Gobillard, M O; Ausseil, J; Géraud, M L

    1996-01-01

    Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the

  12. Actin dynamics and the evolution of the memory trace.

    PubMed

    Rudy, Jerry W

    2015-09-24

    The goal of this essay is to link the regulation of actin dynamics to the idea that the synaptic changes that support long-term potentiation and memory evolve in temporally overlapping stages-generation, stabilization, and consolidation. Different cellular/molecular processes operate at each stage to change the spine cytoarchitecture and, in doing so, alter its function. Calcium-dependent processes that degrade the actin cytoskeleton network promote a rapid insertion of AMPA receptors into the post synaptic density, which increases a spine's capacity to express a potentiated response to glutamate. Other post-translation events then begin to stabilize and expand the actin cytoskeleton by increasing the filament actin content of the spine and reorganizing it to be resistant to depolymerizing events. Disrupting actin polymerization during this stabilization period is a terminal event-the actin cytoskeleton shrinks and potentiated synapses de-potentiate and memories are lost. Late-arriving, new proteins may consolidate changes in the actin cytoskeleton. However, to do so requires a stabilized actin cytoskeleton. The now enlarged spine has properties that enable it to capture other newly transcribed mRNAs or their protein products and thus enable the synaptic changes that support LTP and memory to be consolidated and maintained. This article is part of a Special Issue entitled SI: Brain and Memory. PMID:25498985

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

    PubMed Central

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

    2015-01-01

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

  14. Signalling to actin assembly via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3 complex.

    PubMed Central

    Millard, Thomas H; Sharp, Stewart J; Machesky, Laura M

    2004-01-01

    The assembly of a branched network of actin filaments provides the mechanical propulsion that drives a range of dynamic cellular processes, including cell motility. The Arp2/3 complex is a crucial component of such filament networks. Arp2/3 nucleates new actin filaments while bound to existing filaments, thus creating a branched network. In recent years, a number of proteins that activate the filament nucleation activity of Arp2/3 have been identified, most notably the WASP (Wiskott-Aldrich syndrome protein) family. WASP-family proteins activate the Arp2/3 complex, and consequently stimulate actin assembly, in response to extracellular signals. Structural studies have provided a significant refinement in our understanding of the molecular detail of how the Arp2/3 complex nucleates actin filaments. There has also been much progress towards an understanding of the complicated signalling processes that regulate WASP-family proteins. In addition, the use of gene disruption in a number of organisms has led to new insights into the specific functions of individual WASP-family members. The present review will discuss the Arp2/3 complex and its regulators, in particular the WASP-family proteins. Emphasis will be placed on recent developments in the field that have furthered our understanding of actin dynamics and cell motility. PMID:15040784

  15. Dynamic network morphology and tension buildup in a 3D model of cytokinetic ring assembly.

    PubMed

    Bidone, Tamara C; Tang, Haosu; Vavylonis, Dimitrios

    2014-12-01

    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. PMID:25468341

  16. Structural, Mechanical, and Dynamical Variability of the Actin Cortex in Living Cells

    PubMed Central

    Eghiaian, Frédéric; Rigato, Annafrancesca; Scheuring, Simon

    2015-01-01

    In eukaryotic cells, an actin-based cortex lines the inner leaflet of the plasma membrane, endowing the cells with crucial mechanical and functional properties. Unfortunately, it has not been possible to study the structural dynamics of the actin cortex at high lateral resolution in living cells. Here, we performed atomic force microscopy time-lapse imaging and mechanical mapping of actin in the cortex of living cells at high lateral and temporal resolution. Cortical actin filaments adopted discernible arrangements, ranging from large parallel bundles with low connectivity to a tight meshwork of short filaments. Mixing of these architectures resulted in attuned cortex networks with specific connectivity, mechanical responses, and marked differences in their dynamic behavior. PMID:25809247

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  18. The structure of the cuticular plate, an in vivo actin gel.

    PubMed

    DeRosier, D J; Tilney, L G

    1989-12-01

    The cuticular plate is a network of actin filaments found in hair cells of the cochlea. In the alligator lizard, it consists of rootlets, emanating from the stereocilia, and of cross-connecting actin filaments that anchor these rootlets. In thin sections, this network displays striking patches of 650 +/- 110-A striae. By quantitative analyses of the images, the mystery of the striae can be explained. They are due in part to the rootlets which are sets of flat ribbons of actin filaments. The ribbons in each set are separated by approximately 650 A. Numerous whiskers 30 A in diameter extend from each ribbon's face, interconnecting adjacent ribbons. The nonrootlet filaments, except at the margins of the cell, occur primarily as single filaments. Like the ribbons, they are bristling with whiskers. The patches of striae are explained by ribbons and filaments held at a 650-A separation by the whiskers that project from them. A simple model for regions of bewhiskered filaments is a box crammed full of randomly oriented test-tube brushes. A thin slice through the box will show regions of dark lines or striae due to the wire backbones of the brushes separated from one another by the bristle length. Using the computer instead of test-tube brushes, we have been able to model quantitatively the filament distribution and pattern of striae seen in the cuticular plate of the lizard. The organization of actin filaments we have deduced from our simulations differs from that found in macrophages or in the terminal web of intestinal epithelial cells. PMID:2592408

  19. 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. PMID:25299690

  20. Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis.

    PubMed Central

    Szymanski, D B; Marks, M D; Wick, S M

    1999-01-01

    Actin microfilaments form a three-dimensional cytoskeletal network throughout the cell and constitute an essential throughway for organelle and vesicle transport. Development of Arabidopsis trichomes, unicellular structures derived from the epidermis, is being used as a genetic system in which to study actin-dependent growth in plant cells. The present study indicates that filamentous actin (F-actin) plays an important role during Arabidopsis trichome morphogenesis. For example, immunolocalization of actin filaments during trichome morphogenesis identified rearrangements of the cytoskeletal structure during the development of the mature cell. Moreover, pharmacological experiments indicate that there are distinct requirements for actin- and microtubule-dependent function during trichome morphogenesis. The F-actin-disrupting drug cytochalasin D does not affect the establishment of polarity during trichome development; however, maintenance and coordination of the normal pattern of cell growth are very sensitive to this drug. In contrast, oryzalin, an agent that depolymerizes microtubules, severely inhibits cell polarization. Furthermore, cytochalasin D treatment phenocopies a known class of mutations that cause distorted trichome morphology. Results of an analysis of cell shape and microfilament structure in wild-type, mutant, and drug-treated trichomes are consistent with a role for actin in the maintenance and coordination of an established growth pattern. PMID:10590162

  1. Actinic Keratosis

    MedlinePlus

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

  2. Bidirectional actin transport is influenced by microtubule and actin stability.

    PubMed

    Chetta, Joshua; Love, James M; Bober, Brian G; Shah, Sameer B

    2015-11-01

    Local and long-distance transport of cytoskeletal proteins is vital to neuronal maintenance and growth. Though recent progress has provided insight into the movement of microtubules and neurofilaments, mechanisms underlying the movement of actin remain elusive, in large part due to rapid transitions between its filament states and its diverse cellular localization and function. In this work, we integrated live imaging of rat sensory neurons, image processing, multiple regression analysis, and mathematical modeling to perform the first quantitative, high-resolution investigation of GFP-actin identity and movement in individual axons. Our data revealed that filamentous actin densities arise along the length of the axon and move short but significant distances bidirectionally, with a net anterograde bias. We directly tested the role of actin and microtubules in this movement. We also confirmed a role for actin densities in extension of axonal filopodia, and demonstrated intermittent correlation of actin and mitochondrial movement. Our results support a novel mechanism underlying slow component axonal transport, in which the stability of both microtubule and actin cytoskeletal components influence the mobility of filamentous actin. PMID:26043972

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

    NASA Astrophysics Data System (ADS)

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

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

  4. The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast.

    PubMed

    Li, Yujie; Christensen, Jenna R; Homa, Kaitlin E; Hocky, Glen M; Fok, Alice; Sees, Jennifer A; Voth, Gregory A; Kovar, David R

    2016-06-01

    The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction. PMID:27075176

  5. The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast

    PubMed Central

    Li, Yujie; Christensen, Jenna R.; Homa, Kaitlin E.; Hocky, Glen M.; Fok, Alice; Sees, Jennifer A.; Voth, Gregory A.; Kovar, David R.

    2016-01-01

    The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction. PMID:27075176

  6. Intermediate Filaments and Polarization in the Intestinal Epithelium.

    PubMed

    Coch, Richard A; Leube, Rudolf E

    2016-01-01

    The cytoplasmic intermediate filament cytoskeleton provides a tissue-specific three-dimensional scaffolding with unique context-dependent organizational features. This is particularly apparent in the intestinal epithelium, in which the intermediate filament network is localized below the apical terminal web region and is anchored to the apical junction complex. This arrangement is conserved from the nematode Caenorhabditis elegans to humans. The review summarizes compositional, morphological and functional features of the polarized intermediate filament cytoskeleton in intestinal cells of nematodes and mammals. We emphasize the cross talk of intermediate filaments with the actin- and tubulin-based cytoskeleton. Possible links of the intermediate filament system to the distribution of apical membrane proteins and the cell polarity complex are highlighted. Finally, we discuss how these properties relate to the establishment and maintenance of polarity in the intestine. PMID:27429003

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

    PubMed Central

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

    2011-01-01

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

  8. Dynamic assembly of polymer nanotube networks via kinesin powered microtubule filaments

    NASA Astrophysics Data System (ADS)

    Paxton, Walter F.; Bouxsein, Nathan F.; Henderson, Ian M.; Gomez, Andrew; Bachand, George D.

    2015-06-01

    We describe for the first time how biological nanomotors may be used to actively self-assemble mesoscale networks composed of diblock copolymer nanotubes. The collective force generated by multiple kinesin nanomotors acting on a microtubule filament is large enough to overcome the energy barrier required to extract nanotubes from polymer vesicles comprised of poly(ethylene oxide-b-butadiene) in spite of the higher force requirements relative to extracting nanotubes from lipid vesicles. Nevertheless, large-scale polymer networks were dynamically assembled by the motors. These networks displayed enhanced robustness, persisting more than 24 h post-assembly (compared to 4-5 h for corresponding lipid networks). The transport of materials in and on the polymer membranes differs substantially from the transport on analogous lipid networks. Specifically, our data suggest that polymer mobility in nanotubular structures is considerably different from planar or 3D structures, and is stunted by 1D confinement of the polymer subunits. Moreover, quantum dots adsorbed onto polymer nanotubes are completely immobile, which is related to this 1D confinement effect and is in stark contrast to the highly fluid transport observed on lipid tubules.We describe for the first time how biological nanomotors may be used to actively self-assemble mesoscale networks composed of diblock copolymer nanotubes. The collective force generated by multiple kinesin nanomotors acting on a microtubule filament is large enough to overcome the energy barrier required to extract nanotubes from polymer vesicles comprised of poly(ethylene oxide-b-butadiene) in spite of the higher force requirements relative to extracting nanotubes from lipid vesicles. Nevertheless, large-scale polymer networks were dynamically assembled by the motors. These networks displayed enhanced robustness, persisting more than 24 h post-assembly (compared to 4-5 h for corresponding lipid networks). The transport of materials in and on

  9. Self-organized Gels in DNA/F-Actin mixtures without Crosslinkers

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

    Interactions between flexible chains and rigid rods govern a broad range of soft matter systems. As a model system of like-charged rigid rods and flexible chains, we examine mixtures of DNA and filamentous actin (F-actin). 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 small-angle x-ray scattering (SAXS) indicates that the DNA mesh squeezes the F-actin domains into a nematic state with an inter-actin spacing that decreases with increasing DNA concentration. Salt strongly influences the domain sizes and transitions the system from a counterion-controlled regime to a depletion-controlled regime, both mechanisms of which are entropic in origin.

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    2009-01-01

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

  12. Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

    PubMed Central

    Tang, Haosu; Bidone, Tamara C.

    2015-01-01

    The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

  13. Active microrheology with optical tweezers: a versatile tool to investigate anisotropies in intermediate filament networks

    NASA Astrophysics Data System (ADS)

    Neckernuss, T.; Mertens, L. K.; Martin, I.; Paust, T.; Beil, M.; Marti, O.

    2016-02-01

    Mechanical properties of cells are determined by the cytoskeleton and especially by intermediate filaments (IFs). To measure the contribution of IFs to the mechanics of the cytoskeleton, we determine the shear moduli of in vitro assembled IF networks consisting of keratin 8/18 and MgCl2, serving as a crosslinker. In this study we want to present a new method, a combination of active and passive microrheology, to characterize these networks. We also show the applicability of the new method and discuss new findings on the organization and force transmission in keratin networks gained by the new method. We trap and move embedded polystyrene particles with an optical tweezers setup in an oscillatory manner. The amplitude response of the trapped particle is measured and evaluated with a lock-in approach in order to suppress random motions. With this technique we determine the degree of isotropy of the assembled network and sense preferred directions due to inhomogeneities of the network. Furthermore, we show that we can deliberately create anisotropic networks by adjusting the assembly process and chamber geometry. To determine whether there are local network anisotropies in a globally isotropic network, we altered the evaluation method and included the motion of embedded particles in the vicinity of the trapped one. The correlations of the observed motions enable us to map local network anisotropies. Finally, we compare mechanical properties determined from passive with ones from active microrheology. We find the networks measured with the active technique to be approximately 20% more compliant than the ones from passive measurements.

  14. Dynamic assembly of polymer nanotube networks via kinesin powered microtubule filaments

    SciTech Connect

    Paxton, Walter F.; Bachand, George D.; Gomez, Andrew; Henderson, Ian M.; Bouxsein, Nathan F.

    2015-04-24

    In this study, we describe for the first time how biological nanomotors may be used to actively self-assemble mesoscale networks composed of diblock copolymer nanotubes. The collective force generated by multiple kinesin nanomotors acting on a microtubule filament is large enough to overcome the energy barrier required to extract nanotubes from polymer vesicles comprised of poly(ethylene oxide-b-butadiene) in spite of the higher force requirements relative to extracting nanotubes from lipid vesicles. Nevertheless, large-scale polymer networks were dynamically assembled by the motors. These networks displayed enhanced robustness, persisting more than 24 h post-assembly (compared to 4–5 h for corresponding lipid networks). The transport of materials in and on the polymer membranes differs substantially from the transport on analogous lipid networks. Specifically, our data suggest that polymer mobility in nanotubular structures is considerably different from planar or 3D structures, and is stunted by 1D confinement of the polymer subunits. Moreover, quantum dots adsorbed onto polymer nanotubes are completely immobile, which is related to this 1D confinement effect and is in stark contrast to the highly fluid transport observed on lipid tubules.

  15. Dynamic assembly of polymer nanotube networks via kinesin powered microtubule filaments

    DOE PAGESBeta

    Paxton, Walter F.; Bachand, George D.; Gomez, Andrew; Henderson, Ian M.; Bouxsein, Nathan F.

    2015-04-24

    In this study, we describe for the first time how biological nanomotors may be used to actively self-assemble mesoscale networks composed of diblock copolymer nanotubes. The collective force generated by multiple kinesin nanomotors acting on a microtubule filament is large enough to overcome the energy barrier required to extract nanotubes from polymer vesicles comprised of poly(ethylene oxide-b-butadiene) in spite of the higher force requirements relative to extracting nanotubes from lipid vesicles. Nevertheless, large-scale polymer networks were dynamically assembled by the motors. These networks displayed enhanced robustness, persisting more than 24 h post-assembly (compared to 4–5 h for corresponding lipid networks).more » The transport of materials in and on the polymer membranes differs substantially from the transport on analogous lipid networks. Specifically, our data suggest that polymer mobility in nanotubular structures is considerably different from planar or 3D structures, and is stunted by 1D confinement of the polymer subunits. Moreover, quantum dots adsorbed onto polymer nanotubes are completely immobile, which is related to this 1D confinement effect and is in stark contrast to the highly fluid transport observed on lipid tubules.« less

  16. WAVE binds Ena/VASP for enhanced Arp2/3 complex–based actin assembly

    PubMed Central

    Havrylenko, Svitlana; Noguera, Philippe; Abou-Ghali, Majdouline; Manzi, John; Faqir, Fahima; Lamora, Audrey; Guérin, Christophe; Blanchoin, Laurent; Plastino, Julie

    2015-01-01

    The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex–based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly. PMID:25355952

  17. 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

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

    PubMed

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

    2015-01-01

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

  19. Actin-based propulsion of functionalized hard versus fluid spherical objects

    NASA Astrophysics Data System (ADS)

    Delatour, Vincent; Shekhar, Shashank; Reymann, Anne-Cécile; Didry, Dominique; Diêp Lê, Kim Hô; Romet-Lemonne, Guillaume; Helfer, Emmanuèle; Carlier, Marie-France

    2008-02-01

    The directed polymerization of a branched actin network against a functionalized surface drives cell protrusions and organelle propulsion in living cells. Solid microspheres or giant unilamellar vesicles, functionalized with neural Wiskott Aldrich syndrome protein (N-WASP), initiate the formation of a branched actin array using actin-related protein 2/3 (Arp2/3) complex, when placed in a motility assay reconstituted with pure proteins. These systems are useful biomimetic models of actin-based propulsion that allow to address how the interplay between the physical properties of the functionalized surface and the dynamics of the actin cytoskeleton determines motile behavior. Both solid beads and deformable vesicles display either continuous or saltatory propulsive motions, which are analyzed comparatively; we show that the deformability of liposomes and the mobility of N-WASP at the lipid surface affect the dynamic and structural parameters of the actin meshwork. Our results indicate that beads and vesicles use different mechanisms to translate insertional polymerization of actin at their surface into directed movement: stress relaxation within the actin gel prevents the accumulation of filaments at the front of moving beads, while segregation of nucleators reduces actin polymerization at the front of moving vesicles.

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

    PubMed Central

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

    2003-01-01

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

  1. Immunofluorescent localization of actin in relation to transcription sites in mouse pronuclei.

    PubMed

    Nguyen, E; Besombes, D; Debey, P

    1998-07-01

    Previous biochemical and morphological studies have shown the presence of actin in the nucleus of different cell types where its role remains unclear. In this work, through fluorescence microscopy we studied the localization of actin in the nuclei of early mouse embryos with particular attention to its possible involvement in the onset of transcription occurring at the late one-cell stage. Fluorescent labelling of embryo sections showed that nuclear actin in abundant, in a non-filamentous state, in the whole nucleoplasm excluding the nucleolar precursor bodies. Immunofluorescence on permeabilized embryos revealed that insoluble nuclear actin accumulates in a few large aggregates in transcriptionally inert early one-cell embryos and progressively redistributes into many small aggregates in transcriptionally active late one-cell embryos. Interestingly, these actin aggregates clearly colocalize with transcription sites. Treatment of late one-cell embryos with cytochalasin D induces the formation of actin bundles network in the nucleoplasm but has no apparent effect on the transcriptional activity. In addition, the inhibition of transcription by alpha-amanitin does not modify the nuclear actin distribution. Hence, there does not appear to be a direct causal relationship between transcriptional activity and nuclear actin organization at the one-cell stage although nuclear actin aggregates appear associated with transcription sites. PMID:9621302

  2. The cardiac-restricted protein ADP-ribosylhydrolase-like 1 is essential for heart chamber outgrowth and acts on muscle actin filament assembly.

    PubMed

    Smith, Stuart J; Towers, Norma; Saldanha, José W; Shang, Catherine A; Mahmood, S Radma; Taylor, William R; Mohun, Timothy J

    2016-08-15

    Adprhl1, a member of the ADP-ribosylhydrolase protein family, is expressed exclusively in the developing heart of all vertebrates. In the amphibian Xenopus laevis, distribution of its mRNA is biased towards actively growing chamber myocardium. Morpholino oligonucleotide-mediated knockdown of all Adprhl1 variants inhibits striated myofibril assembly and prevents outgrowth of the ventricle. The resulting ventricles retain normal electrical conduction and express markers of chamber muscle differentiation but are functionally inert. Using a cardiac-specific Gal4 binary expression system, we show that the abundance of Adprhl1 protein in tadpole hearts is tightly controlled through a negative regulatory mechanism targeting the 5'-coding sequence of Xenopus adprhl1. Over-expression of full length (40kDa) Adprhl1 variants modified to escape such repression, also disrupts cardiac myofibrillogenesis. Disarrayed myofibrils persist that show extensive branching, with sarcomere division occurring at the actin-Z-disc boundary. Ultimately, Adprhl1-positive cells contain thin actin threads, connected to numerous circular branch points. Recombinant Adprhl1 can localize to stripes adjacent to the Z-disc, suggesting a direct role for Adprhl1 in modifying Z-disc and actin dynamics as heart chambers grow. Modelling the structure of Adprhl1 suggests this cardiac-specific protein is a pseudoenzyme, lacking key residues necessary for ADP-ribosylhydrolase catalytic activity. PMID:27217161

  3. Solid friction between soft filaments.

    PubMed

    Ward, Andrew; Hilitski, Feodor; Schwenger, Walter; Welch, David; Lau, A W C; Vitelli, Vincenzo; Mahadevan, L; Dogic, Zvonimir

    2015-06-01

    Any macroscopic deformation of a filamentous bundle is necessarily accompanied by local sliding and/or stretching of the constituent filaments. Yet the nature of the sliding friction between two aligned filaments interacting through multiple contacts remains largely unexplored. Here, by directly measuring the sliding forces between two bundled F-actin filaments, we show that these frictional forces are unexpectedly large, scale logarithmically with sliding velocity as in solid-like friction, and exhibit complex dependence on the filaments' overlap length. We also show that a reduction of the frictional force by orders of magnitude, associated with a transition from solid-like friction to Stokes's drag, can be induced by coating F-actin with polymeric brushes. Furthermore, we observe similar transitions in filamentous microtubules and bacterial flagella. Our findings demonstrate how altering a filament's elasticity, structure and interactions can be used to engineer interfilament friction and thus tune the properties of fibrous composite materials. PMID:25730393

  4. 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. PMID:17506863

  5. The Small Heat Shock Protein Hsp27 Affects Assembly Dynamics and Structure of Keratin Intermediate Filament Networks

    PubMed Central

    Kayser, Jona; Haslbeck, Martin; Dempfle, Lisa; Krause, Maike; Grashoff, Carsten; Buchner, Johannes; Herrmann, Harald; Bausch, Andreas R.

    2013-01-01

    The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance. Our knowledge